Method for stabilizing respiratory syncytial virus fusion protein

文档序号：431895 发布日期：2021-12-24 浏览：100次中文

阅读说明：本技术 稳定呼吸道合胞病毒融合蛋白的方法 (Method for stabilizing respiratory syncytial virus fusion protein ) 是由郑子峥张伟张璐婧孙永鹏夏宁邵于 2017-12-27 设计创作，主要内容包括：本申请涉及稳定呼吸道合胞病毒(RSV)的融合蛋白的方法。具体而言,本申请涉及一种灭活RSV且稳定所述RSV中的pre-F蛋白的方法,以及通过该方法获得的经灭活的RSV病毒。此外,本申请还涉及一种制备含有pre-F蛋白的免疫原性组合物的方法,以及通过所述方法获得的免疫原性组合物。本申请还涉及,所述经灭活的RSV病毒和所述免疫原性组合物的用途。(The present application relates to methods of stabilizing fusion proteins of Respiratory Syncytial Virus (RSV). In particular, the present application relates to a method for inactivating RSV and stabilizing the pre-F protein in said RSV, as well as to an inactivated RSV virus obtained by the method. Furthermore, the present application relates to a method for preparing an immunogenic composition comprising a pre-F protein, and to an immunogenic composition obtained by said method. The application also relates to uses of the inactivated RSV virus and the immunogenic composition.)

1. A method of inactivating Respiratory Syncytial Virus (RSV) and stabilizing the pre-F protein in said RSV virus, comprising the steps of:

(1) providing a host cell comprising a live RSV virus;

(2) immobilizing and inactivating said host cell comprising live RSV virus using a fixative selected from the group consisting of: formaldehyde solutions, and paraformaldehyde solutions; wherein the concentration of formaldehyde is 0.0069% -0.1185% (w/w); the concentration of paraformaldehyde is 0.0173% -1% (w/w) by weight; and

(3) removing the fixative from the product of step (2), thereby obtaining an inactivated RSV virus;

preferably, in step (1), live RSV virus is provided by: (1a) infecting host cells with RSV virus; (1b) culturing the infected host cell obtained in step (1a) under conditions that allow proliferation of the RSV virus; and (1c) harvesting the cultured host cells obtained in step (1b) comprising live RSV virus; preferably, in step (1c), the cultured host cells are washed prior to collecting the cultured host cells;

preferably, the host cell is an adherent cell or a suspension cell, such as a respiratory epithelial cell, a liver cell, a lung cell, a kidney cell, a cervical cell, an ovarian cell, a bone cell, a breast cell, a striated muscle cell, a gastric epithelial cell, a skin epidermal cell, a fibroblast cell, and a prostate cell of a mammal (e.g., rodents and primates, such as mice, monkeys, and humans);

preferably, the live RSV virus is located on the surface of the host cell.

2. The method of claim 1, wherein, in step (2), the fixing agent is a formaldehyde solution and the concentration of formaldehyde is from 0.0069% to 0.1185%, such as from 0.0069% to 0.0104%, from 0.0104% to 0.0156%, from 0.0156% to 0.0234%, from 0.0234% to 0.0244%, from 0.0244% to 0.0351%, from 0.0351% to 0.0527%, from 0.0527% to 0.079%, from 0.079% to 0.0977%, or from 0.0977% to 0.1185%;

preferably, the host cells comprising live RSV virus are fixed and inactivated using formaldehyde solution at a temperature of 0-40 ℃ (e.g., 0-4 ℃, 4-10 ℃, 10-15 ℃, 15-20 ℃, 20-25 ℃, 25-30 ℃, 30-35 ℃, 35-37 ℃, or 37 ℃ -40 ℃; e.g., 4 ℃, 25 ℃ or 37 ℃);

preferably, the host cells comprising live RSV virus are fixed and inactivated using formaldehyde solution for 0.5-48h (e.g., 0.5-1h, 1-5h, 5-12h, 12-24h, or 24-48 h);

preferably, the live RSV virus-containing host cells are fixed and inactivated for 0.5-24 hours using a formaldehyde solution at a concentration of 0.0069% -0.1185% (e.g., 0.0069% -0.0104%, 0.0104% -0.0156%, 0.0156% -0.0234%, 0.0234% -0.0244%, 0.0244% -0.0351%, 0.0351% -0.0527%, 0.0527% -0.079%, 0.079% -0.0977%, or 0.0977% -0.1185%); alternatively, the live RSV virus-containing host cells are fixed and inactivated for no more than 48 hours using a formaldehyde solution at a concentration of 0.0104% to 0.1185% (e.g., 0.0104% to 0.0156%, 0.0156% to 0.0234%, 0.0234% to 0.0244%, 0.0244% to 0.0351%, 0.0351% to 0.0527%, 0.0527% to 0.079%, 0.079% to 0.0977%, or 0.0977% to 0.1185%).

3. The method of claim 1, wherein, in step (2), the fixing agent is a paraformaldehyde solution and the concentration of paraformaldehyde is 0.0173% -1%, such as 0.0173% -0.0585%, 0.0585% -0.0625%, 0.0625% -0.878%, 0.878% -0.1317%, 0.1317% -0.1975%, 0.1975% -0.25%, 0.25% -0.2963%, 0.2963% -0.4444%, or 0.4444% -1%;

preferably, the host cells comprising live RSV virus are fixed and inactivated using a paraformaldehyde solution at a temperature of 0-40 ℃ (e.g., 0-4 ℃, 4-10 ℃, 10-15 ℃, 15-20 ℃, 20-25 ℃, 25-30 ℃, 30-35 ℃, 35-37 ℃, or 37 ℃ -40 ℃; e.g., 4 ℃, 25 ℃ or 37 ℃);

preferably, the host cells comprising live RSV virus are fixed and inactivated using paraformaldehyde solution for no more than 48 hours, such as 0.5-48 hours (e.g., 0.5-1 hour, 1-5 hours, 5-12 hours, 12-24 hours, or 24-48 hours);

preferably, the host cells comprising live RSV virus are fixed and inactivated for no more than 24 hours at a temperature of 0-10 ℃ (e.g., 0 ℃,4 ℃, or 10 ℃) using a paraformaldehyde solution at a concentration of 0.0585% -1% (e.g., 0.0585% -0.0625%, 0.0625% -0.878%, 0.878% -0.1317%, 0.1317% -0.1975%, 0.1975% -0.25%, 0.25% -0.2963%, 0.2963% -0.4444%, or 0.4444% -1%); alternatively, the host cells comprising live RSV virus are fixed and inactivated for no more than 48 hours at a temperature of 0-10 ℃ (e.g., 0 ℃,4 ℃, or 10 ℃) using a paraformaldehyde solution at a concentration of 0.0585% -0.4444% (e.g., 0.0585% -0.0625%, 0.0625% -0.878%, 0.878% -0.1317%, 0.1317% -0.1975%, 0.1975% -0.25%, 0.25% -0.2963%, or 0.2963% -0.4444%); alternatively, the host cells comprising live RSV virus are fixed and inactivated for no more than 48 hours at a temperature of 20-30 ℃ (e.g., 20 ℃, 25 ℃ or 30 ℃) using paraformaldehyde solution at a concentration of 0.0173% -0.1975% (e.g., 0.0173% -0.0585%, 0.0585% -0.0625%, 0.0625% -0.878%, 0.878% -0.1317%, or 0.1317% -0.1975%); alternatively, the host cells comprising live RSV virus are fixed and inactivated for no more than 48 hours using a paraformaldehyde solution at a concentration of 0.0173% -0.1317% (e.g., 0.0173% -0.0585%, 0.0585% -0.0625%, 0.0625% -0.878%, or 0.878% -0.1317%) at a temperature of 35-40 ℃ (e.g., 35 ℃,37 ℃, or 40 ℃).

4. The method of claim 1, wherein, in step (3), the fixative is removed by dialysis, filtration, or centrifugation;

for example, in step (3), the fixative is removed by: (3a) filtering or centrifuging the product of step (2) and collecting the immobilized host cells comprising inactivated RSV virus; (3b) washing the immobilized host cells collected in step (3a) with a buffer; and, (3c) recovering the washed host cells of step (3b) comprising inactivated RSV virus (e.g., by filtration or centrifugation); alternatively, in step (3), the fixative is removed by dialyzing the product of step (2) into a fixative-free solution.

5. An inactivated RSV virus produced by the method of any one of claims 1-4.

6. A method of preparing an immunogenic composition comprising a pre-F protein, comprising the steps of:

(1) providing a host cell comprising a live RSV virus;

(2) immobilizing and inactivating said host cell comprising live RSV virus using a fixative selected from the group consisting of: formaldehyde solutions, and paraformaldehyde solutions; wherein the concentration of formaldehyde is 0.0069% -0.1185% (w/w, the same below); the concentration of paraformaldehyde is 0.0173% -1% (w/w, the same below) by weight; and

(3) removing the fixative from the product of step (2) to obtain an immunogenic composition comprising a pre-F protein;

preferably, the live RSV virus is located on the surface of the host cell.

7. The method of claim 6, wherein, in step (2), the fixing agent is a formaldehyde solution and the concentration of formaldehyde is from 0.0069% to 0.1185%, such as from 0.0069% to 0.0104%, from 0.0104% to 0.0156%, from 0.0156% to 0.0234%, from 0.0234% to 0.0244%, from 0.0244% to 0.0351%, from 0.0351% to 0.0527%, from 0.0527% to 0.079%, from 0.079% to 0.0977%, or from 0.0977% to 0.1185%;

preferably, the host cells comprising live RSV virus are fixed and inactivated using formaldehyde solution for 0.5-48h (e.g., 0.5-1h, 1-5h, 5-12h, 12-24h, or 24-48 h);

8. The method of claim 6, wherein, in step (2), the fixing agent is a paraformaldehyde solution and the concentration of paraformaldehyde is 0.0173% -1%, such as 0.0173% -0.0585%, 0.0585% -0.0625%, 0.0625% -0.878%, 0.878% -0.1317%, 0.1317% -0.1975%, 0.1975% -0.25%, 0.25% -0.2963%, 0.2963% -0.4444%, or 0.4444% -1%;

9. The method of claim 6, wherein, in step (3), the fixative is removed by dialysis, filtration or centrifugation;

10. An immunogenic composition prepared by the method of any one of claims 6-9.

11. A vaccine comprising an inactivated RSV virus according to claim 5 or an immunogenic composition according to claim 10, and optionally a pharmaceutically acceptable carrier and/or excipient (e.g. an adjuvant).

12. A method of making a vaccine comprising mixing an inactivated RSV virus according to claim 5 or an immunogenic composition according to claim 10 with a pharmaceutically acceptable carrier and/or excipient (e.g. an adjuvant).

13. A method for preventing, treating or inhibiting RSV infection or a disease associated with RSV infection (e.g. pneumonia, such as pediatric pneumonia) in a subject, comprising administering to a subject in need thereof an effective amount of an inactivated RSV virus according to claim 5, or an immunogenic composition according to claim 10, or a vaccine according to claim 11.

Technical Field

The present application relates to the fields of virology and immunology. In particular, the present application relates to a method of inactivating Respiratory Syncytial Virus (RSV) and stabilizing the pre-F protein in said RSV, and to an inactivated RSV Virus obtained by the method. Furthermore, the present application relates to a method for preparing an immunogenic composition comprising a pre-F protein, and to an immunogenic composition obtained by said method. The application also relates to vaccines comprising the inactivated RSV viruses or the immunogenic compositions, and the use of the inactivated RSV viruses, immunogenic compositions and vaccines for the prevention or treatment of RSV infection or a disease associated with RSV infection. Furthermore, the present application relates to methods of preventing or treating RSV infection or a disease associated with RSV infection comprising the use of an inactivated RSV virus, immunogenic composition or vaccine of the invention.

Background

Since the discovery in the 50's of the 20 th century, human Respiratory Syncytial Virus (RSV) has been the leading cause of lower respiratory tract infections in infants and young children. In the united states, RSV is the leading cause of hospitalization in infants under 1 year of age (d.k.shay, r.c.holman. et al, JAMA,282(1999) 1440-. Over 3000 million lower respiratory tract infections worldwide per year are caused by RSV, with over 300 million people requiring hospitalization. RSV is the most common cause of hospitalization in children younger than 5 years of age (h.nair, w.a.brooks, et al., Lancet,378(2011) 1917-. Premature infants, broncho-and pulmonary dysplasia, congenital heart disease, and immunodeficient infants have RSV infection rates as high as 50-70% (a.c. cooper, n.c. banasiaak, p.j. allen, Pediatr Nurs,29(2003) 452-. Each year, 16-60 ten thousand cases of childhood death are associated with RSV (T.S. Howard, L.H.Hoffman, et al.J. Pediatr,137 (2000)) 227-. Hospitalization of infants and young children with RSV infection can take up to 2.5 months, and the associated medical costs can be as high as $ 3.6 to $ 5.7 billion per year in the united states (e.a. simoes. lancet,354(1999) 847-. Elderly persons are also susceptible to RSV, and more than 12000 elderly people die annually from RSV infection, which is about influenza mortality in the same population at 1/3(A.R.Falsey, P.A.Hennessey, et al.N Engl J Med,352 (2005): 1749-. In China, due to the lack of RSV diagnostic reagents developed in China, RSV detection cannot be popularized due to high cost, so that the prevalence and harmfulness of RSV in China are not completely clear. However, studies in some areas of our country have shown that RSV infection is also an important cause of lower respiratory tract infection in children in China (Xuguanren, Sun Song Wen, Xuxu Qing et al. J. disease control, 4(2000) 37-39; Xie Jian Screen, He Cui Juan et al. Chinese J. pediatrics, 35(1997) 402-403; Zhu Ru nan, Deng Jie, Wang Fang et al. 21(2003) 25-28).

In the 60's of the 19th century, the protective efficacy of FI-RSV (formalin inactivated whole virus vaccine, intramuscular injection, aluminum adjuvant) has been evaluated in infants and children. However, the results show that this vaccine lacks protection in subsequent RSV natural infection and even results in increased disease severity. The development of RSV vaccines is severely retarded by the phenomenon of increased disease severity caused by the vaccine. To date, there is no vaccine against RSV that provides effective protection. Currently, only one neutralizing antibody (Palivizumab, trade name: Synagis) for recognizing RSV fusion protein can generate passive immunity effect on neonates, and the incidence rate of the neonates is reduced. The use of Syangis suggests that neutralizing mabs that bind the RSV-F protein may be used for clinical protection and that there is an effective neutralizing active site on the F protein. The F protein is located on the surface of the virus and is essential for virus entry and syncytia formation. Therefore, the F protein is an important target protein for developing anti-RSV vaccines and screening for prophylactic and protective antibodies.

RSV is a single-stranded, non-segmented RNA virus of the genus Pneumovirus of the family Paramyxoviridae having a genome of 15222 nucleotides encoding 10 major proteins; among them, the F protein (Fusion protein) is an N-glycosylated type I transmembrane glycoprotein, has 574 amino acids in its entire length, and is an important surface molecule during RSV infection as a major transmembrane protein. The mechanism and process of membrane fusion triggered by the F protein is unclear. McLellan et al (J.S.McLellan, M.Chen, J.S.Chang, et al.J Virol,84(2010) 12236-. In the case of pre-F proteins, it is difficult to study the structure of the pre-F protein by preparing crystals because the structure is unstable and various intermediates exist. McLellan et al (supra) have modeled and predicted the structure of the RSV pre-F protein using the structurally known HPIV3 pre-F protein, and suggest that the RSV F protein may exist in the pre-F conformation. In addition, McLellan et al (supra) also suggest that after binding of the F protein to the target cell, the conformation changes from a pre-fusion F protein conformation in a high-energy, metastable state to a highly stable post-fusion F protein conformation, resulting in fusion of the viral membrane with the cell membrane. The free energies of the metastable pre-F conformation and the stable post-F conformation differ greatly, which results in a membrane fusion process that is irreversible.

In addition, neutralization of the pre-F and post-F protein conformations has also been performedEpitopes were identified. The results showed that the pre-F and post-F proteins share about 50% of the protein surface and that epitopes with high neutralizing activity (strongly neutralizing epitopes) such asMainly distributed on the pre-F conformation, while the post-F conformation mainly contains epitopes with weaker neutralizing activity (weakly neutralizing epitopes), such as site II and site IV (see FIG. 1).

These findings indicate that the pre-F protein has more, stronger neutralizing epitopes than the post-F protein, and thus has a higher potential for use as a vaccine. However, the development of pre-F proteins as effective vaccines remains extremely difficult and challenging, since they are in a metastable state and are highly susceptible to conversion to stable post-F proteins. There is a need in the art to develop methods for stabilizing and maintaining the pre-F protein in inactivated RSV viruses to improve the efficacy of the inactivated RSV viruses for use as vaccines.

Disclosure of Invention

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, cell culture, molecular genetics, nucleic acid chemistry, immunology laboratory procedures, as used herein, are conventional procedures that are widely used in the relevant art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

As used herein, the term "RSV Fusion protein" or "F protein" refers to a Fusion protein (F protein) of Respiratory Syncytial Virus (RSV), which is well known to those skilled in the art, and exemplary amino acid sequences thereof can be found, for example, in NCBI GENBANK database accession No.: p03420. Herein, "RSV fusion protein", "F protein" are used interchangeably.

As used herein, when referring to the amino acid sequence of the F protein, it uses the amino acid sequence of SEQ ID NO:1, is described. For example, the expression "amino acid residue 196-209 of the F protein" means that the amino acid sequence of SEQ ID NO:1 at amino acid residue 196-209 of the polypeptide. However, it is understood by those skilled in the art that mutations or variations (including, but not limited to, substitutions, deletions and/or additions, such as F proteins of different genotypes or gene subtypes) may be naturally occurring or artificially introduced in the amino acid sequence of the F protein without affecting its biological function. Thus, in the present invention, the term "F protein" shall include all such sequences, including for example SEQ ID NO:1 and natural or artificial variants thereof. And, when describing the sequence fragment of the F protein, it includes not only SEQ ID NO:1, and also includes the corresponding sequence fragments in natural or artificial variants thereof. For example, the expression "amino acid residues 196-209 of the F protein" includes the amino acid sequences shown in SEQ ID NO:1, and the corresponding fragment in a variant (natural or artificial) thereof. According to the invention, the expression "corresponding sequence fragment" or "corresponding fragment" refers to the fragments at equivalent positions in the sequences being compared when the sequences are optimally aligned, i.e. when the sequences are aligned to obtain the highest percentage identity.

Previous studies have shown that the F protein exists in at least 1 defined conformation, post-F. The results of studies of McLellan et al on the binding of the F protein of parainfluenza virus (PIV) suggest that the F protein of RSV may also exist in the pre-F conformation (McLellan et al (2010), J Vriol, 84: 12236-12244). In general, the pre-F conformation is unstable, which will spontaneously convert to a stable post-F conformation. Thus, the F protein expressed and purified from the cell exists mainly in the post-F conformation; also, in inactivated RSV virus, the F protein is also present predominantly in the post-F conformation.

As used herein, the term "pre-F protein" refers to an F protein that exists in a pre-F conformation. As used herein, the term "post-F protein" refers to an F protein that exists in a post-F conformation. For a more detailed description of pre-F proteins, post-F proteins and their conformations see, McLellan et al (2010), J Vriol, 84: 12236-12244; McLellan et al (2013), Science, 340: 1113-; McLellan et al (2015), Curr Opin Virol,11: 70-75; chinese patent application 201480013927.7, and PCT international application PCT/CN2014/073505 (which are incorporated herein by reference in their entirety for all purposes). "pre-F" is used interchangeably with "pre-Fusion" herein; "post-F" is used interchangeably with "post-Fusion".

As used herein, the expression "stabilizing a pre-F protein" refers to at least partially inhibiting, reducing or delaying the conversion of a pre-F protein to a post-F protein. Furthermore, the expression also means that the pre-F conformation of the F protein is maintained as far as possible, avoiding its conversion into the post-F conformation.

As one of the most major surface structural proteins of viruses, there are a large number of neutralizing antibody recognition epitopes present on the surface of the F protein. Neutralizing antibodies to RSV F proteins known to date are directed predominantly against the following epitopes (J.S. McLellan, Y.Yang, et al.J Virol,85(2011) 7788-:

site II epitope: antibodies against Site II epitopes include the marketed preventive mAb Synagis and its equivalent derivatives motavizumab and 47F; they mainly recognize aa 255-275 of the F protein. McLellan et al (J.S. McLellan, M.Chen, J.S. Chang, et al.J Virol,84(2010)12236-12244) confirmed by analysis of the crystal structure of the complex of motavizumab monoclonal antibody and F protein peptide segment aa 254-277, that this region forms a "helix-turn-helix" secondary structure. The crystal structure shows that motavizumab binds at one end of the "helix-turn-helix" structure and allows hydrogen and ionic bonding to the Asn at position 268 and Lys at position 272. Further studies have shown that mutations at these two points can cause antibody escape. The structure of the motavizumab-bound Site ii epitope remains very intact in the post-F conformation and the antibody binding Site is fully exposed. The structures of motavizumab and post-F protein reveal the mechanism of neutralizing activity of the Synagis and motavizumab monoclonal antibodies. The mimicry of the RSV pre-F protein, however, shows that the epitope is within the conformation of the pre-F protein and cannot be exposed on the surface of the pre-F protein. Graham et al demonstrated that Synagis and motavizumab only inhibited RSV fusion with cells, but not RSV adsorption (J.S.McLellan, Y.Yang, et al.J Virol,85(2011) 7788-.

Site I epitope: the antibody recognizing Site I epitope has 131-2a, which recognizes cysteine-rich region of F protein. Such antibodies block up to 50% of RSV viral infections, indicating that the epitope is posttranslationally heterogeneous, or that the antibodies neutralize through indirect effects (e.g., viral coagulation). In addition, these antibodies partially block virus adsorption to target cells. The Site I epitope is close to the viral cell membrane in the conformation of the pre-F protein, but is at the vertex in the conformation of the post-F protein.

Site iv epitope: the Site IV epitope is the target of monoclonal antibody antibodies such as 19 and 101F, and mainly relates to aa 422 and 438 of F protein. This epitope is located in a region of the F protein that is conformationally relatively conserved. McLellan et al (J.S.McLellan, Y.Yang, et al.J Virol,85(2011) 7788-. The results show that the core region of the Site IV epitope is aa 427-437.

Epitope:epitopes are targets for pre-F specific antibodies D25, AM22 and 5C 4. McLellan et al (McLellan JS, Chen M, et al science 2013,340:1113-1117) discovered by structural analysis of the complex of pre-F specific antibodies with pre-F that involves the loose region in the F protein (aa 62-69) and the α 4 helix in the F protein (aa 196-209). Furthermore, the results of the study show that the epitope is at least present when the F protein is converted from pre-F to post-F conformationAnd the alpha 4 helix is shifted by 180 deg.. Therefore, an antibody recognizing this epitope is a pre-F-specific antibody, and cannot recognize the post-F protein.

The results of previous studies (McLellan JS, Chen M, et al science 2013,340:1113-1117) have shown,epitopes have high neutralizing activity and are distributed mainly in pre-F conformation; the neutralizing activity of Site II epitopes and Site IV epitopes was relatively weak and there was a distribution in both pre-F and post-F conformations (FIG. 1).

As used herein, the term "epitope" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. An "epitope" is also referred to in the art as an "antigenic determinant". Epitopes or antigenic determinants usually consist of chemically active surface groups of molecules such as amino acids or carbohydrates or sugar side chains and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. For example, an epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation, which can be "linear" or "conformational". See, e.g., epitopic Mapping Protocols in Methods in Molecular Biology, vol 66, g.e. morris, Ed. (1996). In a linear epitope, the points of all interactions between a protein and an interacting molecule (e.g., an antibody) are linearly present along the primary amino acid sequence of the protein. In conformational epitopes, the point of interaction exists across protein amino acid residues that are separated from each other.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. In certain embodiments, an antibody that specifically binds to (or is specific for) an antigen means that the antibody is present in an amount less than about 10^-5M, e.g. less than about 10^-6M、10^-7M、10^-8M、10^-9M or 10^-10M or less affinity (K)_D) Binding the antigen.

As used herein, the term "K_D"refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the more tight the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, the antibody is present in an amount less than about 10^-5M, e.g. less thanAbout 10^-6M、10^-7M、10^-8M、10^- ⁹M or 10^-10Dissociation equilibrium constant (K) of M or less_D) Binding to the antigen, e.g., as determined in a BIACORE instrument using Surface Plasmon Resonance (SPR).

As used herein, the term "neutralizing epitope" refers to an epitope capable of inducing the neutralizing activity of the body against viruses. Such epitopes are not only involved in the recognition of viral proteins by the immune system (e.g., antibodies), but can often induce the immune system of the body to produce antibodies with neutralizing activity (i.e., neutralizing antibodies). As used herein, "neutralizing antibody" refers to an antibody that is capable of significantly reducing or completely inhibiting the virulence (e.g., the ability to infect cells) of a target virus. Generally, neutralizing antibodies are capable of recognizing and binding neutralizing epitopes on a target virus and preventing the target virus from entering/infecting cells of a subject. Herein, neutralizing activity of an epitope refers to the ability of an epitope to induce the body to produce neutralizing activity against a virus. The higher the neutralizing activity of an epitope, the stronger its ability to induce the body to produce neutralizing activity against the virus.

As used herein, the term "immunogenicity" refers to the ability of a body to be stimulated to form specific antibodies or to sensitize lymphocytes. It refers to the characteristic that an antigen can stimulate specific immune cells to activate, proliferate and differentiate the immune cells and finally produce immune effector substances such as antibodies and sensitized lymphocytes, and also refers to the characteristic that the immune system of the organism can form specific immune response of the antibodies or sensitized T lymphocytes after the antigen stimulates the organism. Immunogenicity is the most important property of an antigen, and the success of an antigen in inducing an immune response in a host depends on three factors: the nature of the antigen, the reactivity of the host and the mode of immunization.

As used herein, the term "isolated" or "isolated" refers to a product obtained from a natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be altered from its natural environment, or it may be isolated from its natural environment, or both. For example, a polynucleotide or polypeptide that is not isolated naturally occurs in a living animal, and a polynucleotide or polypeptide that is the same in high purity and that is isolated from such a natural state is said to be isolated. The term "isolated" or "isolated" does not exclude the presence of substances mixed artificially or synthetically or other impurities which do not affect the activity of the substance.

As used herein, the term "host cell" refers to a cell capable of being infected with an RSV virus and allowing the RSV virus to proliferate therein. Such host cells may be adherent cells or suspension cells, and include primary cells and established cell lines. Examples of such host cells include, but are not limited to, respiratory epithelial cells, liver cells, lung cells, kidney cells, cervical cells, ovarian cells, bone cells, breast cells, striated muscle cells, gastric epithelial cells, skin epidermal cells, fibroblasts, and prostate cells of mammals (e.g., rodents and primates, such as mice, monkeys, and humans); for example Hep-2 cells, CNE1 cells, CNE2 cells, BEL-7404 cells, BEL-7402 cells, QSG-7701 cells, PLC/PRF/5 cells, Huh7 cells, Huh7.5.1 cells, SSMC-7721 cells, BNL-HCC cells, Hep3B, SNU-739 cells, TIB75 cells, A549 cells, H480 cells, H1299 cells, H441 cells, H368 cells, H1335 cells, H23 cells, L929 cells, 293FT cells, 293T cells, 293 beta 5 cells, Vero cells, BHK-MKL cells, RK-13 cells, HeLa cells, TZM-bl cells, SK-OV-3 cells, U2-OS cells, 143B cells, MCF-7 cells, MDA-MB-231 cells, T-47D cells, RD cells, BGC-823 cells, AGS cells, A431NS cells, MeWo cells, LNCap cells, RM1 cells and PC-3 cells.

As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both of the sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of the total 6 positions match). Typically, the comparison is made when the two sequences are aligned to yield maximum identity. Such alignments can be performed by using, for example, Needleman et al (1970) j.mol.biol.48: 443-453. The algorithm of E.Meyers and W.Miller (Compout.appl biosci., 4:11-17(1988)) which has been incorporated into the ALIGN program (version 2.0) can also be used to determine percent identity between two amino acid sequences using a PAM120 weight residue table (weight residue table), a gap length penalty of 12, and a gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI biol.48: 444-.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the essential characteristics of the protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., a substitution with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., of similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., Brummell et al, biochem.32:1180-1187 (1993); Kobayashi et al Protein Eng.12(10):879-884 (1999); and Burks et al, Proc. Natl Acad. set USA 94:412-417(1997), which are incorporated herein by reference).

As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations as is well known in the art. For example, alanine can be represented by A or Ala.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical sciences. edited by geno AR,19th ed. pennsylvania: mach Publishing Company,1995), and include, but are not limited to: pH regulator, surfactant, adjuvant, and ionic strength enhancer. For example, pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

As used herein, the term "adjuvant" refers to a non-specific immunopotentiator which, when delivered with or prior to an antigen into the body, enhances the body's immune response to the antigen or alters the type of immune response. Adjuvants are of various types, including, but not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, and the like. Freund's adjuvant is currently the most commonly used adjuvant in animal testing. Aluminum hydroxide adjuvants are used more often in clinical trials.

As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, a desired effect. For example, a prophylactically effective amount of a disease (e.g., RSV infection or a disease associated with RSV infection) refers to an amount sufficient to prevent, or delay the onset of a disease (e.g., RSV infection or a disease associated with RSV infection); a therapeutically effective amount for a disease is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. It is well within the ability of those skilled in the art to determine such effective amounts. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently, and the like.

As used herein, the term "subject" refers to a mammal, e.g., a primate, e.g., a human.

The inventor has found, surprisingly, through a large number of experimental studies: in fixing/inactivating RSV virus on the surface of a host cell, by using a specific fixing agent (e.g., methanol, formaldehyde, paraformaldehyde, etc.) and using specific fixing/inactivating conditions (e.g., a specific fixing agent concentration), it is possible to obtain particularly advantageous inactivated RSV virus that contains a higher amount of pre-F protein (i.e., more F protein is present in the pre-F conformation in the inactivated RSV virus obtained by the present invention) than in the inactivated virus obtained by the conventional method. This is particularly advantageous as such inactivated RSV viruses will exhibit more of the strong neutralizing epitopes present only in the pre-F protein and not in the post-F protein, and thus be able to induce the body to produce a stronger neutralizing activity against RSV virus, and thus be particularly suitable for use in the development of vaccines against RSV virus for the prevention or treatment of RSV infection or diseases associated with RSV infection (e.g. pneumonia, such as pediatric pneumonia).

Accordingly, in one aspect, the present invention provides a method of inactivating Respiratory Syncytial Virus (RSV) and stabilizing the pre-F protein in said RSV virus, comprising the steps of:

(1) providing a host cell comprising a live RSV virus;

(2) immobilizing and inactivating said host cell comprising live RSV virus using a fixative selected from the group consisting of: methanol solution, formaldehyde solution, and paraformaldehyde solution; wherein the concentration of methanol is 0.3125-5% (w/w, the same applies below); the concentration of formaldehyde is 0.0069% -0.1185% (w/w, the same below); the concentration of paraformaldehyde is 0.0173% -1% (w/w, the same below) by weight; and

(3) removing the fixative from the product of step (2) to obtain an inactivated RSV virus.

In certain preferred embodiments, in step (1), live RSV virus is provided by: (1a) infecting host cells with RSV virus; (1b) culturing the infected host cell obtained in step (1a) under conditions that allow proliferation of the RSV virus; and (1c) harvesting the cultured host cells obtained in step (1b) comprising live RSV virus. In certain preferred embodiments, the host cell is an adherent cell. In certain preferred embodiments, the host cell is a suspension cell. In certain preferred embodiments, the host cell is a primary cell. In certain preferred embodiments, the host cell is an established cell line. In certain preferred embodiments, the host cell is selected from the group consisting of respiratory epithelial cells, liver cells, lung cells, kidney cells, cervical cells, ovarian cells, bone cells, breast cells, striated muscle cells, gastric epithelial cells, skin epidermal cells, fibroblasts, and prostate cells of a mammal (e.g., rodents and primates, e.g., mice, monkeys, and humans); for example Hep-2 cells, CNE1 cells, CNE2 cells, BEL-7404 cells, BEL-7402 cells, QSG-7701 cells, PLC/PRF/5 cells, Huh7 cells, Huh7.5.1 cells, SSMC-7721 cells, BNL-HCC cells, Hep3B, SNU-739 cells, TIB75 cells, A549 cells, H480 cells, H1299 cells, H441 cells, H368 cells, H1335 cells, H23 cells, L929 cells, 293FT cells, 293T cells, 293 beta 5 cells, Vero cells, BHK-MKL cells, RK-13 cells, HeLa cells, TZM-bl cells, SK-OV-3 cells, U2-OS cells, 143B cells, MCF-7 cells, MDA-MB-231 cells, T-47D cells, RD cells, BGC-823 cells, AGS cells, A431NS cells, MeWo cells, LNCap cells, RM1 cells and PC-3 cells. In certain preferred embodiments, in step (1c), the cultured host cells are collected by scraping with a spatula or by digestion with pancreatin or by filtration or centrifugation. In certain preferred embodiments, in step (1c), the cultured host cells are washed prior to collecting the cultured host cells. In certain preferred embodiments, in step (1c), the cultured host cells are washed with a buffer (e.g., PBS) and subsequently recovered (e.g., by filtration or centrifugation). In certain preferred embodiments, the live RSV virus is located on the surface of the host cell.

In certain preferred embodiments, in step (2), the fixing agent is a methanol solution and the concentration of methanol is 0.3125% to 5%. In certain preferred embodiments, the concentration of methanol is from 0.3125% to 0.625%, from 0.625% to 1.25%, from 1.25% to 2.5%, or from 2.5% to 5%. In certain preferred embodiments, the methanolic solution is a solution of methanol in an inorganic solvent. Preferably, the inorganic solvent is selected from the group consisting of water, medium and buffer. In certain exemplary embodiments, the buffer is Phosphate Buffered Saline (PBS). In certain exemplary embodiments, the buffer is Phosphate Buffered Saline (PBS) and the salt concentration is 100-600mM, e.g., 100-150mM, 150-200mM, 200-250mM, 250-300mM, 300-350mM, 350-400mM, 400-450mM, 450-500mM, 500-550mM, 550-600mM, e.g., 150mM, 330mM, 550 mM. In certain preferred embodiments, the host cells comprising live RSV virus are immobilized and inactivated using a methanol solution at a temperature of 0-40 deg.C (e.g., 0-4 deg.C, 4-10 deg.C, 10-15 deg.C, 15-20 deg.C, 20-25 deg.C, 25-30 deg.C, 30-35 deg.C, 35-37 deg.C, or 37 deg.C-40 deg.C; e.g., 4 deg.C, 25 deg.C, or 37 deg.C). In certain preferred embodiments, the host cells comprising live RSV virus are fixed and inactivated using a methanol solution for no more than 12 hours, such as 0.5-12 hours (e.g., 0.5-1 hour, 1-5 hours, or 5-12 hours).

In certain preferred embodiments, in step (2), the fixing agent is a formaldehyde solution and the concentration of formaldehyde is from 0.0069% to 0.1185%. In certain preferred embodiments, the concentration of formaldehyde is from 0.0069% to 0.0104%, from 0.0104% to 0.0156%, from 0.0156% to 0.0234%, from 0.0234% to 0.0244%, from 0.0244% to 0.0351%, from 0.0351% to 0.0527%, from 0.0527% to 0.079%, from 0.079% to 0.0977%, or from 0.0977% to 0.1185%. In certain preferred embodiments, the formaldehyde solution is a solution of formaldehyde in an inorganic solvent. Preferably, the inorganic solvent is selected from the group consisting of water, medium and buffer. In certain exemplary embodiments, the buffer is Phosphate Buffered Saline (PBS). In certain exemplary embodiments, the buffer is Phosphate Buffered Saline (PBS) and the salt concentration is 100-600mM, e.g., 100-150mM, 150-200mM, 200-250mM, 250-300mM, 300-350mM, 350-400mM, 400-450mM, 450-500mM, 500-550mM, 550-600mM, e.g., 150mM, 330mM, 550 mM. In certain preferred embodiments, the host cells comprising live RSV virus are fixed and inactivated using formaldehyde solution at a temperature of 0-40 ℃ (e.g., 0-4 ℃, 4-10 ℃, 10-15 ℃, 15-20 ℃, 20-25 ℃, 25-30 ℃, 30-35 ℃, 35-37 ℃, or 37 ℃ -40 ℃; e.g., 4 ℃, 25 ℃ or 37 ℃). In certain preferred embodiments, the host cells comprising live RSV virus are fixed and inactivated using formaldehyde solution for 0.5-48h (e.g., 0.5-1h, 1-5h, 5-12h, 12-24h, or 24-48 h). In certain preferred embodiments, the live RSV virus-containing host cells are fixed and inactivated for 0.5 to 24 hours (e.g., 0.5 to 1 hour, 1 to 5 hours, 5 to 12 hours, or 12 to 24 hours) using a formaldehyde solution at a concentration of 0.0069% to 0.1185% (e.g., 0.0069% to 0.0104%, 0.0104% to 0.0156%, 0.0156% to 0.0234%, 0.0234% to 0.0244%, 0.0244% to 0.0351%, 0.0351% to 0.0527%, 0.0527% to 0.079%, 0.079% to 0.0977%, or 0.0977% to 0.1185%). In certain preferred embodiments, the live RSV virus-containing host cells are fixed and inactivated for no more than 48 hours, e.g., 0.5-48 hours (e.g., 0.5-1 hour, 1-5 hours, 5-12 hours, 12-24 hours, or 24-48 hours), using formaldehyde solutions at concentrations of 0.0104% to 0.1185% (e.g., 0.0104% to 0.0156%, 0.0156% to 0.0234%, 0.0234% to 0.0244%, 0.0244% to 0.0351%, 0.0351% to 0.0527%, 0.0527% to 0.079%, 0.079% to 0.0977%, or 0.0977% to 0.1185%).

In certain preferred embodiments, in step (2), the fixing agent is a paraformaldehyde solution, and the concentration of paraformaldehyde is 0.0173% -1%. In certain preferred embodiments, the concentration of paraformaldehyde is from 0.0173% to 0.0585%, from 0.0585% to 0.0625%, from 0.0625% to 0.878%, from 0.878% to 0.1317%, from 0.1317% to 0.1975%, from 0.1975% to 0.25%, from 0.25% to 0.2963%, from 0.2963% to 0.4444%, or from 0.4444% to 1%. In certain preferred embodiments, the paraformaldehyde solution is a solution of paraformaldehyde in an inorganic solvent. Preferably, the inorganic solvent is selected from the group consisting of water, medium and buffer. In certain exemplary embodiments, the buffer is Phosphate Buffered Saline (PBS). In certain exemplary embodiments, the buffer is Phosphate Buffered Saline (PBS) and the salt concentration is 100-600mM, e.g., 100-150mM, 150-200mM, 200-250mM, 250-300mM, 300-350mM, 350-400mM, 400-450mM, 450-500mM, 500-550mM, 550-600mM, e.g., 150mM, 330mM, 550 mM. In certain preferred embodiments, the host cells comprising live RSV virus are fixed and inactivated using a paraformaldehyde solution at a temperature of 0-40 deg.C (e.g., 0-4 deg.C, 4-10 deg.C, 10-15 deg.C, 15-20 deg.C, 20-25 deg.C, 25-30 deg.C, 30-35 deg.C, 35-37 deg.C, or 37 deg.C-40 deg.C; e.g., 4 deg.C, 25 deg.C, or 37 deg.C). In certain preferred embodiments, the host cells comprising live RSV virus are fixed and inactivated using paraformaldehyde solution for no more than 48 hours, such as 0.5-48 hours (e.g., 0.5-1 hour, 1-5 hours, 5-12 hours, 12-24 hours, or 24-48 hours).

In certain preferred embodiments, the host cells comprising live RSV virus are fixed and inactivated for no more than 24 hours, e.g., 0.5-24 hours (e.g., 0.5-1 hour, 1-5 hours, 5-12 hours, or 12-24 hours), using a paraformaldehyde solution at a concentration of 0.0585% to 1% (e.g., 0.0585% to 0.0625%, 0.0625% to 0.878%, 0.878% to 0.1317%, 0.1317% to 0.1975%, 0.1975% to 0.25%, 0.25% to 0.2963%, 0.2963% to 0.4444%, or 0.4444% to 1%) at a temperature of 0-10 ℃ (e.g., 0 ℃,4 ℃, or 10 ℃).

In certain preferred embodiments, the host cells comprising live RSV virus are fixed and inactivated for no more than 48 hours, e.g., 0.5-1 hour, 1-5 hours, 5-12 hours, 12-24 hours, or 24-48 hours, using a paraformaldehyde solution at a concentration of 0.0585% to 0.4444% (e.g., 0.0585% to 0.0625%, 0.0625% to 0.878%, 0.878% to 0.1317%, 0.1317% to 0.1975%, 0.1975% to 0.25%, 0.25% to 0.2963%, or 0.2963% to 0.4444%) at a temperature of 0-10 ℃ (e.g., 0 ℃,4 ℃, or 10 ℃).

In certain preferred embodiments, the host cells comprising live RSV virus are fixed and inactivated for no more than 48 hours, such as 0.5-48 hours (e.g., 0.5-1 hour, 1-5 hours, 5-12 hours, 12-24 hours, or 24-48 hours), using paraformaldehyde solution at a concentration of 0.0173% -0.1975% (e.g., 0.0173% -0.0585%, 0.0585% -0.0625%, 0.0625% -0.878%, 0.878% -0.1317%, or 0.1317% -0.1975%) at a temperature of 20-30 ℃ (e.g., 20 ℃, 25 ℃, or 30 ℃).

In certain preferred embodiments, the host cells comprising live RSV virus are fixed and inactivated with a paraformaldehyde solution at a concentration of 0.0173% to 0.1317% (e.g., 0.0173% to 0.0585%, 0.0585% to 0.0625%, 0.0625% to 0.878%, or 0.878% to 0.1317%) for no more than 48 hours, e.g., 0.5 to 48 hours (e.g., 0.5 to 1 hour, 1 to 5 hours, 5 to 12 hours, 12 to 24 hours, or 24 to 48 hours), at a temperature of 35 ℃ to 40 ℃ (e.g., 35 ℃,37 ℃, or 40 ℃).

In certain preferred embodiments, in step (3), the fixative is removed by dialysis, filtration or centrifugation. In certain preferred embodiments, in step (3), the fixative is removed by: (3a) filtering or centrifuging the product of step (2) and collecting the immobilized host cells comprising inactivated RSV virus; (3b) washing the immobilized host cells collected in step (3a) with a buffer; and, (3c) recovering the washed host cells of step (3b) comprising inactivated RSV virus (e.g., by filtration or centrifugation). In certain preferred embodiments, in step (3), the fixative is removed by dialyzing the product of step (2) into a fixative-free solution. For example, in certain preferred embodiments, in step (3), the immobilization agent is removed by dialyzing the product of step (2) into a salt solution having a salt concentration of 100-.

In certain preferred embodiments, a significant amount of the pre-F protein (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or more of the pre-F protein) in the inactivated RSV virus is capable of maintaining its conformation for at least 24 hours, e.g., at least 48 hours, or at least 96 hours.

In another aspect, the invention provides an inactivated RSV virus prepared by the method as described above. In certain preferred embodiments, the inactivated RSV virus comprises a pre-F protein. In certain preferred embodiments, a significant amount of the pre-F protein (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or more of the pre-F protein) in the inactivated RSV virus is capable of maintaining its conformation for at least 24 hours, e.g., at least 48 hours, or at least 96 hours.

In another aspect, the present invention provides a method of making an immunogenic composition comprising a pre-F protein, comprising the steps of:

(1) providing a host cell comprising a live RSV virus;

(3) removing the fixative from the product of step (2) to obtain an immunogenic composition comprising a pre-F protein.

In certain preferred embodiments, in step (1), live RSV virus is provided by: (1a) infecting host cells with RSV virus; (1b) culturing the infected host cell obtained in step (1a) under conditions that allow proliferation of the RSV virus; and (1c) harvesting the cultured host cells obtained in step (1b) comprising live RSV virus. In certain preferred embodiments, the host cell is an adherent cell. In certain preferred embodiments, the host cell is a suspension cell. In certain preferred embodiments, the host cell is a primary cell. In certain preferred embodiments, the host cell is an established cell line. In certain preferred embodiments, the host cell is selected from the group consisting of respiratory epithelial cells, liver cells, lung cells, kidney cells, cervical cells, ovarian cells, bone cells, breast cells, striated muscle cells, gastric epithelial cells, skin epidermal cells, fibroblasts, and prostate cells of a mammal (e.g., rodents and primates, e.g., mice, monkeys, and humans); for example Hep-2 cells, CNE1 cells, CNE2 cells, BEL-7404 cells, BEL-7402 cells, QSG-7701 cells, PLC/PRF/5 cells, Huh7 cells, Huh7.5.1 cells, SSMC-7721 cells, BNL-HCC cells, Hep3B, SNU-739 cells, TIB75 cells, A549 cells, H480 cells, H1299 cells, H441 cells, H368 cells, H1335 cells, H23 cells, L929 cells, 293FT cells, 293T cells, 293 beta 5 cells, Vero cells, BHK-MKL cells, RK-13 cells, HeLa cells, TZM-bl cells, SK-OV-3 cells, U2-OS cells, 143B cells, MCF-7 cells, MDA-MB-231 cells, T-47D cells, RD cells, BGC-823 cells, AGS cells, A431NS cells, MeWo cells, LNCap cells, RM1 cells and PC-3 cells. In certain preferred embodiments, in step (1c), the cultured host cells are recovered by scraping with a spatula or by digestion with pancreatin or by filtration or centrifugation. In certain preferred embodiments, in step (1c), the cultured host cells are washed prior to recovering the cultured host cells. In certain preferred embodiments, in step (1c), the cultured host cells are washed with a buffer (e.g., PBS) and subsequently recovered (e.g., by filtration or centrifugation). In certain preferred embodiments, the live RSV virus is located on the surface of the host cell.

In certain preferred embodiments, in step (2), the fixing agent is a paraformaldehyde solution, and the concentration of paraformaldehyde is 0.0173% -1%. In certain preferred embodiments, the concentration of paraformaldehyde is from 0.0173% to 0.0585%, from 0.0585% to 0.0625%, from 0.0625% to 0.878%, from 0.878% to 0.1317%, from 0.1317% to 0.1975%, from 0.1975% to 0.25%, from 0.25% to 0.2963%, from 0.2963% to 0.4444%, or from 0.4444% to 1%. In certain preferred embodiments, the paraformaldehyde solution is a solution of paraformaldehyde in an inorganic solvent. Preferably, the inorganic solvent is selected from the group consisting of water, medium and buffer. In certain exemplary embodiments, the buffer is Phosphate Buffered Saline (PBS). In certain exemplary embodiments, the buffer is Phosphate Buffered Saline (PBS) and the salt concentration is 100-600mM, such as 100-150mM, 150-200mM, 200-250mM, 250-300mM, 300-350mM, 350-400mM, 400-450mM, 450-500mM, 500-550mM, 550-600mM, 600-650mM, such as 150mM, 330mM, 550 mM. In certain preferred embodiments, the host cells comprising live RSV virus are fixed and inactivated using a paraformaldehyde solution at a temperature of 0-40 deg.C (e.g., 0-4 deg.C, 4-10 deg.C, 10-15 deg.C, 15-20 deg.C, 20-25 deg.C, 25-30 deg.C, 30-35 deg.C, 35-37 deg.C, or 37 deg.C-40 deg.C; e.g., 4 deg.C, 25 deg.C, or 37 deg.C). In certain preferred embodiments, the host cells comprising live RSV virus are fixed and inactivated using paraformaldehyde solution for no more than 48 hours, such as 0.5-48 hours (e.g., 0.5-1 hour, 1-5 hours, 5-12 hours, 12-24 hours, or 24-48 hours).

In certain preferred embodiments, a significant amount of pre-F protein (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or more of the pre-F protein) in the immunogenic composition is capable of maintaining its conformation for at least 24 hours, e.g., at least 48 hours, or at least 96 hours.

In another aspect, the invention provides an immunogenic composition prepared by the method as described above. In certain preferred embodiments, the immunogenic composition comprises a pre-F protein. In certain preferred embodiments, a significant amount of pre-F protein (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or more of the pre-F protein) in the immunogenic composition is capable of maintaining its conformation for at least 24 hours, e.g., at least 48 hours, or at least 96 hours.

In another aspect, the invention provides a vaccine comprising an inactivated RSV virus according to the invention or an immunogenic composition according to the invention, and optionally a pharmaceutically acceptable carrier and/or excipient (e.g. an adjuvant). The vaccines of the invention are useful for preventing, treating or inhibiting RSV infection or a disease associated with RSV infection (e.g., pneumonia, such as pediatric pneumonia) in a subject.

In another aspect, the invention provides a method of making a vaccine comprising admixing an inactivated RSV virus according to the invention or an immunogenic composition according to the invention with a pharmaceutically acceptable carrier and/or excipient (e.g. an adjuvant).

In another aspect, the invention provides a method for preventing, treating or inhibiting RSV infection or a disease associated with RSV infection (e.g. pneumonia, such as pediatric pneumonia) in a subject, comprising administering to a subject in need thereof an effective amount of an inactivated RSV virus according to the invention, or an immunogenic composition according to the invention, or a vaccine according to the invention.

In another aspect, there is provided the use of an inactivated RSV virus or immunogenic composition of the invention in the manufacture of a vaccine for the prevention, treatment or inhibition of RSV infection or a disease associated with RSV infection (e.g., pneumonia, such as pediatric pneumonia) in a subject.

In another aspect, there is provided an inactivated RSV virus or immunogenic composition of the invention for use in preventing, treating or inhibiting RSV infection or a disease associated with RSV infection (e.g., pneumonia, such as pediatric pneumonia) in a subject.

The inactivated RSV viruses, immunogenic compositions and vaccines provided herein can be used alone or in combination, or in combination with other pharmaceutically active agents (e.g., interferon-based drugs, such as interferon or pegylated interferon).

Advantageous effects of the invention

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

(1) the methods of the invention can be used to prepare inactivated RSV viruses and immunogenic compositions comprising the pre-F protein, and to maintain and stabilize the conformation of the pre-F protein.

(2) The inactivated RSV viruses and immunogenic compositions of the invention comprise higher levels of pre-F protein than inactivated viruses obtained by conventional methods.

Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.

Drawings

FIG. 1 shows the distribution of neutralizing epitopes on pre-F and post-F proteins. The results showed that the pre-F and post-F proteins share about 50% of the protein surface and that epitopes with high neutralizing activity (strongly neutralizing epitopes) such asMainly distributed in the pre-F conformation, while the post-F conformation mainly contains epitopes with weaker neutralizing activity (weakly neutralizing epitopes), such as site II and site IV.

FIG. 2 shows the results of flow cytometry analysis of samples incubated with 9F7 antibody (FIG. 2A), 5C4 antibody (FIG. 2B) or 8C2 antibody (FIG. 2C), wherein the samples were Hep-2 cells (resuspended in PBS) without fixed hRSV infection; the abscissa (GaM-FITC (FITC-labeled goat anti-mouse antibody)) represents the signal intensity of FITC; the ordinate (count) represents the cell count. In fig. 2A, a threshold value for FITC signal (indicated by a dotted line) was set based on the flow cytometric analysis results of the negative control samples; at this threshold, the percentage of positive cells in the negative control sample was 0.472%. In fig. 2B and 2C, the percentage of positive cells in the sample incubated with 5C4 antibody was 53.9%, as determined by the threshold set in fig. 2A; the percentage of positive cells in the sample incubated with the 8C2 antibody was 53.0%.

FIG. 3 shows the positive rate of cells containing pre-F protein (FIG. 3A, incubated with 5C4 antibody) and cells containing F protein (pre-F conformation and/or post-F conformation) (FIG. 3B, incubated with 8C2 antibody) in samples treated with a specified concentration of beta-propiolactone for a specified time at 4 ℃; wherein the abscissa represents the concentration (%) of β -propiolactone and the ordinate represents the positive rate (%) of the cells.

FIG. 4 shows the positive rate of cells containing pre-F protein (FIG. 4A, incubated with 5C4 antibody) and cells containing F protein (pre-F conformation and/or post-F conformation) (FIG. 4B, incubated with 8C2 antibody) in samples treated with glutaraldehyde at the indicated concentrations for the indicated times at 4 ℃; wherein the abscissa represents the concentration (%) of glutaraldehyde and the ordinate represents the positive rate (%) of cells.

FIG. 7 shows the positive rate of cells containing pre-F protein (FIG. 7A, incubated with 5C4 antibody) and cells containing F protein (pre-F conformation and/or post-F conformation) (FIG. 7B, incubated with 8C2 antibody) in samples treated with paraformaldehyde at the indicated concentrations for the indicated times at 4 ℃; wherein the abscissa represents the concentration (%) of paraformaldehyde, and the ordinate represents the positive rate (%) of cells.

FIG. 8 shows the positive rate of cells containing pre-F protein (FIG. 8A, incubated with 5C4 antibody) and the positive rate of cells containing F protein (pre-F conformation and/or post-F conformation) (FIG. 8B, incubated with 8C2 antibody) in samples treated with formaldehyde at the indicated concentrations for the indicated times at 37 ℃; wherein the abscissa represents the concentration (%) of formaldehyde and the ordinate represents the positive rate (%) of cells.

FIG. 9 shows the positive rate of cells containing pre-F protein (FIG. 9A, incubated with 5C4 antibody) and cells containing F protein (pre-F conformation and/or post-F conformation) (FIG. 9B, incubated with 8C2 antibody) in samples treated with paraformaldehyde at the indicated concentrations for the indicated times at 4 ℃; wherein the abscissa represents the concentration (%) of paraformaldehyde, and the ordinate represents the positive rate (%) of cells.

FIG. 10 shows the positive rate of cells containing pre-F protein (FIGS. 10A-10C, incubated with 5C4 antibody) and the positive rate of cells containing F protein (pre-F conformation and/or post-F conformation) (FIGS. 10D-10F, incubated with 8C2 antibody) in samples treated with paraformaldehyde at specified concentrations for specified times at 4 deg.C, 25 deg.C or 37 deg.C; wherein the abscissa represents the concentration (%) of paraformaldehyde, and the ordinate represents the positive rate (%) of cells.

FIG. 11 shows the time-dependent positive rate of pre-F protein-containing cells in formaldehyde-treated and non-fixative samples stored in 1 × PBS buffer; wherein the abscissa represents the time (hours) of storage in 1 × PBS buffer, and the ordinate represents the positive rate (%) of the cells.

FIG. 12 shows the level of RSV neutralizing antibodies in the serum of mice immunized with samples treated with different fixed conditions. Wherein, the ordinate represents the neutralizing capacity of the serum sample to be tested of each immune group relative to the 8C2 monoclonal antibody (1 neutralizing unit of 8C2 antibody of 1 mg/mL); the abscissa indicates various fixation conditions, F formaldehyde, PF paraformaldehyde, and no immuno mice not immunized; the line pattern shows the average neutralizing capacity (i.e., mean and standard error) of multiple mouse serum samples from each immunization group.

Fig. 13 shows HE staining results of tissue sections of lung tissue of mice immunized with samples treated with different fixation conditions before challenge on day 5 after challenge with hRSVA2, where F denotes formaldehyde, PF denotes paraformaldehyde, No immuno denotes mice that were not immunized but challenged, and No infection denotes mice that were not challenged.

FIG. 14 shows the inflammation scores of tissue sections of lung tissue of mice immunized with formaldehyde (FIGS. 14A-14C) or paraformaldehyde (FIGS. 14D-14F) treated samples prior to challenge at day 5 after challenge with hRSVA2, where FIGS. 14A and 14D show the scores for perivascular phenomenon (perivascular occlusion); FIGS. 14B and 14E show the results of scoring interstitial pneumonia or alveolitis (Intermental pulmonary Or alveolitis); FIGS. 14C and 14F show the scoring results for bronchiolitis (bronchinolitis); f represents formaldehyde, PF represents paraformaldehyde, No immunolo represents a mouse which is not immunized but is attacked, and No infection represents a mouse which is not attacked.

Description of sequences

The information of the sequences referred to in this application is as follows:

SEQ ID NO:1 (amino acid sequence of F protein)

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

Unless otherwise indicated, the molecular biological experimental methods and immunoassay methods used in the present invention are essentially described by reference to j.sambrook et al, molecular cloning: a laboratory manual, 2 nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, eds. molecular biology laboratory Manual, 3 rd edition, John Wiley & Sons, Inc., 1995; the use of restriction enzymes follows the conditions recommended by the product manufacturer. The examples are given by way of illustration and are not intended to limit the scope of the invention as claimed.

Example 1 immobilization of host cells and inactivation of RSV Virus

1. Materials and instruments:

hep-2 cells: (CCL-23^TM): obtained from ATCC.

hRSV (psynklsv a2D 46F): human respiratory syncytial virus standard strain, obtained from national institute of health, NIH.

5C4 antibody: the method is self-made in a laboratory. The 5C4 antibody specifically recognizes and binds to the pre-F protein, but not to the post-F protein. Recognition of the pre-F protein by the 5C4 antibodyAn epitope, and which is a strong neutralizing antibody, the neutralizing activity being significantly higher than Palivizumab. For detailed information on the 5C4 antibody, see, chinese patent application 201480013927.7 and PCT international application PCT/CN 2014/073505.

8C2 antibody: the method is self-made in a laboratory. The 8C2 antibody is capable of specifically binding to both the pre-F protein and the post-F protein. The 8C2 antibody recognizes Site II epitopes on pre-F and post-F proteins and is a neutralizing antibody with neutralizing activity substantially comparable to Palivizumab.

9F7 antibody: the method is self-made in a laboratory. The 9F7 antibody is an antibody that specifically recognizes hepatitis E virus and is not specifically reactive with either the pre-F protein or the post-F protein. For details on the 9F7 antibody, see, e.g., Min Zhao et al J Biol Chem,2015,290: 19910-.

GaM-FITC: FITC-labeled goat anti-mouse antibody obtained from Sigma.

Facsaria III flow cytometer: obtained from BD company.

2. Preparation of virus-infected cells

Hep-2 cells (c)CCL-23^TM) Seeded in cell culture plates and plated with a seed containing 10% FBS (Gibco, cat #: 10099141) and 100U/ml penicillin-streptomycin (Gibco, cat #: 15140122) (Gibco, cat #: 11095072) was cultured. When the cell density reached 80% -90% confluence, cells were infected with hRSV (psynk rsv A2D46F) with an MOI of 0.3. After infection, cells were cultured for an additional 72 h. After the culture, the cells were collected with a cell scraper.

3. Immobilization of host cells and inactivation of RSV viruses

The fixing solution of the specified type and concentration was added to each EP tube. Thawing virus at 37 deg.C, mixing virus solution and each fixative uniformly, and fixing at the designated temperature for the designated time. The fixative solution used was as follows:

methanol (CH)₃OH, AR, chemical industry of Shigaku Kagaku K.K.: 1030003AR 500);

beta-propiolactone (SERVA, cat # 57-57-8);

formaldehyde (CH)₂O, CP, chemical industry of juju, cat #: 50-00-0);

paraformaldehyde (HO (CH)₂O)_nH, n-10-100, SIGMA-ALDRICH, cat #: 16005).

After fixation, dialysis was performed at 25 ℃ for 18h in 1 × PBS (0.27g/L potassium dihydrogen phosphate, 1.42g/L disodium hydrogen phosphate, 8g/L sodium chloride, 0.2g/L potassium chloride; pH 7.4) to remove residual fixative. After dialysis, the sample was removed and placed in a 1.5ml EP tube for use.

Detection of Pre-F and post-F proteins

The virus and cells in the EP tubes were incubated with 100 μ l primary antibody (20ng/μ l, diluted in 1 × PBS) for 20min at room temperature. The primary antibodies used included: a 5C4 antibody that specifically binds to pre-F protein; 8C2 antibody that binds to pre-F protein and post-F protein; and, 9F7 antibodies that do not bind to the pre-F protein and post-F protein. After incubation, the EP tube was centrifuged at 200g for 3min at 25 ℃ and the supernatant was discarded. The virus and cells in the EP tubes were washed once with 100 μ l 1 × PBS. Subsequently, the virus and cells in the EP tube were incubated with 100. mu.l of the secondary antibody GaM-FITC (Sigma, cat # F5387-2 mL; diluted in 1 × PBS) for 20min at room temperature. The obtained sample was examined using a Facsaria III flow cytometer (BD Co., Ltd., product No. P648200155), and experimental data was recorded.

5. Data processing

For each fixative, samples incubated with an equal volume of 9F7 antibody as the primary antibody were used as negative controls. The threshold for FITC signal was set based on the flow cytometric analysis of the negative control samples. At this threshold, the percentage of cells in the negative control sample that are judged to be positive (i.e., cells for which the FITC signal is above the threshold) is no greater than 0.5%. Then, based on the threshold, the percentage of cells judged to be positive in the sample treated with the same fixative, incubated with 5C4 antibody or 8C2 antibody, was determined.

By a similar method, the percentage of positive cells in each test sample can be determined. Subsequently, the percentage of positive cells in each sample that was fixed and inactivated under different fixing conditions was normalized (i.e., the relative proportion of positive cells (positive rate) in each sample was calculated) with reference to the percentage of positive cells in a sample that was not treated with a fixing solution (i.e., the cells obtained in the "preparation of virus-infected cells" step, which were resuspended in 1 × PBS and were not fixed and inactivated but were directly used for antibody incubation and subsequent flow cytometry analysis; hereinafter also referred to simply as "0 h group").

The positive rate of a certain sample is the percentage of positive cells in the sample/the percentage of positive cells in the sample not treated with the fixative × 100%

6. Results of the experiment

6.1 selection of fixative and concentration thereof

We first evaluated the fixation and inactivation effects of several fixatives at the indicated concentrations. For these fixatives, we have adopted the conditions (including temperature) recommended for fixation in the pharmacopoeia or references.

Briefly, methanol was formulated with 1 × PBS to the indicated concentration (80%, 20%, 5%, 1.25%, 0.3125%, or 0%) and left to stand at 25 ℃ for 30 min. Subsequently, the prepared methanol solution was used to resuspend (fix) the sample at 25 ℃ for the indicated time (1h, 5h, 12h or 24 h). The concentration of 0% indicates that the samples were resuspended in 1 × PBS and incubated for the indicated time (same below). Subsequently, the fixative is removed and the fixed sample is used for antibody incubation and flow cytometry analysis as described above.

Beta-propiolactone was formulated with 1 × PBS to the indicated concentration (1.6%, 0.4%, 0.025%, 0.00625%, 0.001563% or 0.000391%) and left to stand at 4 ℃ for 30 min. Subsequently, the formulated β -propiolactone solution was used to resuspend (fix) the sample at 4 ℃ for the indicated time (1h, 5h, 12h or 24 h). Subsequently, the fixative is removed and the fixed sample is used for antibody incubation and flow cytometry analysis as described above.

Glutaraldehyde was formulated with 1 × PBS to the indicated concentration (0.15625%, 0.039063%, 0.009766%, 0.002441%, 0.00061%, 0.000153%, 0.000038%, 0.00001%, or 0%) and left to stand at 4 ℃ for 30 min. Subsequently, the formulated glutaraldehyde solution was used to resuspend (fix) the sample at 4 ℃ for the indicated time (0.25h, 0.5h, 1h, 5h or 12 h). Subsequently, the fixative is removed and the fixed sample is used for antibody incubation and flow cytometry analysis as described above.

Formaldehyde was formulated with 1 × PBS to the indicated concentration (25%, 6.25%, 1.5625%, 0.3906%, 0.0977%, 0.0244%, 0.0061%, or 0.0015%) and left to stand at 37 ℃ for 30 min. Subsequently, the prepared formaldehyde solution was used to resuspend (fix) the sample at 37 ℃ for the indicated time (1h, 5h, 12h, 24h or 48 h). Subsequently, the fixative is removed and the fixed sample is used for antibody incubation and flow cytometry analysis as described above.

Paraformaldehyde was formulated with 1 × PBS to the indicated concentration (4%, 1%, 0.25%, 0.0625%, 0.0156%, 0.0039%, or 0.001%) and left to stand at 4 ℃ for 30 min. Subsequently, the prepared paraformaldehyde solution was used to resuspend (fix) the sample at 4 ℃ for the indicated time (1h, 12h, 24h or 48 h). Subsequently, the fixative is removed and the fixed sample is used for antibody incubation and flow cytometry analysis as described above.

The results of the experiments are shown in FIGS. 3-7. FIG. 3 shows the positive rate of cells containing pre-F protein (FIG. 3A, incubated with 5C4 antibody) and the positive rate of cells containing F protein (pre-F conformation and/or post-F conformation) (FIG. 3B, incubated with 8C2 antibody) in samples treated with a specified concentration of beta-propiolactone for a specified time; wherein the abscissa represents the concentration (%) of β -propiolactone and the ordinate represents the positive rate (%) of the cells. The results show that the cells in the samples contained almost no pre-F protein after 12h or 24h treatment of the samples with the indicated concentrations of beta-propiolactone. This result indicates that beta-propiolactone is not capable of stabilizing or maintaining the conformation of the pre-F protein and is not suitable for inactivating RSV virus.

FIG. 4 shows the positive rate of cells containing pre-F protein (FIG. 4A, incubated with 5C4 antibody) and the positive rate of cells containing F protein (pre-F conformation and/or post-F conformation) (FIG. 4B, incubated with 8C2 antibody) in samples treated with glutaraldehyde at the indicated concentrations for the indicated times; wherein the abscissa represents the concentration (%) of glutaraldehyde and the ordinate represents the positive rate (%) of cells. The results show that the cells in the sample contained almost no pre-F protein after 12h treatment with glutaraldehyde at the indicated concentration. This result indicates that glutaraldehyde is not able to stabilize or maintain the conformation of the pre-F protein and is not suitable for inactivating RSV virus.

FIG. 5 shows the positive rate of cells containing pre-F protein (FIG. 5A, incubated with 5C4 antibody) and the positive rate of cells containing F protein (pre-F conformation and/or post-F conformation) (FIG. 5B, incubated with 8C2 antibody) in samples treated with methanol at the indicated concentrations for the indicated times; wherein the abscissa represents the concentration (%) of methanol and the ordinate represents the positive rate (%) of cells. The results show that after 1, 5 or 12h treatment of the samples with methanol at a concentration of 0.3125% -5%, the samples still contain significant amounts of pre-F protein positive cells (i.e., the conformation of the pre-F protein in the sample is stabilized and maintained). This result indicates that methanol at concentrations ranging from 0.3125% to 5% stabilizes and maintains the conformation of the pre-F protein in the presence of immobilization and inactivation for up to 12 hours, and is thus particularly suitable for inactivating RSV virus.

FIG. 6 shows the positive rate of cells containing pre-F protein (FIG. 6A, incubated with 5C4 antibody) and the positive rate of cells containing F protein (pre-F conformation and/or post-F conformation) (FIG. 6B, incubated with 8C2 antibody) in samples treated with formaldehyde at the indicated concentrations for the indicated times; wherein the abscissa represents the concentration (%) of formaldehyde and the ordinate represents the positive rate (%) of cells. The results show that after 1, 5, 12h, 24h or 48h treatment of the samples with formaldehyde at concentrations of 0.0244% to 0.0977%, a significant amount of pre-F protein positive cells were still contained in the samples (i.e., the conformation of the pre-F protein in the samples was stabilized and maintained). This result indicates that formaldehyde concentrations in the range of 0.0244% to 0.0977% can stabilize and maintain the conformation of the pre-F protein in the presence of fixation and inactivation for up to 48 hours, and is thus particularly suitable for inactivating RSV viruses.

FIG. 7 shows the positive rate of cells containing pre-F protein (FIG. 7A, incubated with 5C4 antibody) and the positive rate of cells containing F protein (pre-F conformation and/or post-F conformation) (FIG. 7B, incubated with 8C2 antibody) in samples treated with paraformaldehyde at the indicated concentrations for the indicated times; wherein the abscissa represents the concentration (%) of paraformaldehyde, and the ordinate represents the positive rate (%) of cells. The results show that after treatment of samples with paraformaldehyde at a concentration of 0.0625% -1% for 1, 5, 12, or 24h, the samples still contain significant amounts of pre-F protein positive cells (i.e., the conformation of the pre-F protein in the sample is stabilized and maintained). This result indicates that paraformaldehyde at a concentration in the range of 0.0625% -1% is capable of stabilizing and maintaining the conformation of the pre-F protein in the case of fixation and inactivation for up to 24 hours, and thus is particularly suitable for inactivating RSV virus. Furthermore, the results also show that paraformaldehyde at a concentration in the range of 0.0625% -0.25% is still able to stabilize and maintain the conformation of the pre-F protein in the case of fixation and inactivation for up to 48 hours, and is thus particularly suitable for inactivating RSV viruses.

Furthermore, the results of fig. 4-5 also show that in the case of incubating the sample without fixative, but with only 1 × PBS, the reactivity of the sample with antibody 5C4 was significantly reduced or even disappeared after 12 hours of incubation, but still retained high reactivity with the 8C2 antibody. This result indicates that the pre-F protein in the sample is unstable and that allosteric during 1 × PBS incubation; also, after 12 hours of incubation, pre-F protein was almost no longer present in the samples.

The results in FIGS. 3-7 also show that different fixatives have different effects on the pre-F protein. In particular, the results of fig. 3-4 show that treatment with a high concentration of β -propiolactone (or glutaraldehyde) results in a more rapid decrease in the reactivity of the sample with 5C4 antibody (i.e., the allosteric progression of the pre-F protein proceeds more rapidly) than treatment with a low concentration of β -propiolactone (or glutaraldehyde). These results indicate that β -propiolactone and glutaraldehyde contribute to the allosteric profile of the pre-F protein. Beta-propiolactone and glutaraldehyde are detrimental to the maintenance and stabilization of the pre-fusion conformation of the RSV F protein.

In contrast, the results of the experiments in FIGS. 5-7 show that the effect of methanol, formaldehyde and paraformaldehyde on the pre-F conformation of the F protein is related to its concentration; each of which has an optimal concentration range suitable for stabilizing the pre-F protein. In particular, when fixation is performed with methanol at a concentration of 0.3125% -5%, the fixation time can be as long as 12 hours, and a significant amount of pre-F protein still remains in the fixed sample. When fixation is carried out with formaldehyde at a concentration of 0.0244% to 0.0977%, the fixation time can be as long as 48 hours, and a significant amount of pre-F protein still remains in the fixed sample. When fixation is performed with paraformaldehyde at a concentration of 0.0625% -1%, the fixation time can be as long as 24 hours, and a significant amount of pre-F protein still remains in the fixed sample. When fixation is performed with paraformaldehyde at a concentration of 0.0625% -0.25%, the fixation time can be as long as 48 hours, and a significant amount of pre-F protein still remains in the fixed sample.

In addition, the experimental results of fig. 5-7 also show that when methanol, formaldehyde or paraformaldehyde are used at a concentration lower than the optimum concentration range, the reactivity of the sample with 5C4 antibody (pre-F protein) is significantly reduced after 12 hours of treatment, tending to disappear completely; but the reactivity of the sample with the 8C2 antibody did not change significantly. When methanol, formaldehyde or paraformaldehyde were used at concentrations above the optimal concentration range, the reactivity of the samples with both 5C4 antibody and 8C2 antibody (pre-F protein and post-F protein) was significantly reduced after 12 hours of treatment, indicating that the epitopes of the F proteins (pre-F and post-F) were easily destroyed completely by high concentrations of fixative.

The pre-F conformation of the F protein has been shown to be the preferred conformation for inducing a protective antibody response against RSV. Also, previous studies have shown that pre-F protein induces neutralizing antibody titers that are 1-2 LOG higher than post-F protein. As analyzed above, the inactivated RSV virus (immunogenic composition) obtained by the method of the invention retains a significant amount of the pre-F protein and is therefore particularly suitable for use as an antiviral vaccine for preventing or treating RSV infection or a disease associated with RSV infection.

6.2 selection of the concentration of Formaldehyde and Paraformaldehyde

We further investigated the optimum concentration ranges for formaldehyde and paraformaldehyde. Briefly, formaldehyde was formulated with 1 × PBS to the indicated concentrations (0.4%, 0.2667%, 0.1778%, 0.1185%, 0.079%, 0.0527%, 0.0351%, 0.0234%, 0.0156%, 0.0104%, 0.0069%, 0.0046%, or 0.0031%) and left to stand at 37 ℃ for 30 min. Subsequently, the prepared formaldehyde solution was used to resuspend (fix) the sample at 37 ℃ for the indicated time (24h or 48 h). Subsequently, the fixative is removed and the fixed sample is used for antibody incubation and flow cytometry analysis as described above.

In addition, paraformaldehyde was formulated with 1 × PBS to the indicated concentration (1%, 0.6667%, 0.4444%, 0.2963%, 0.1975%, 0.1317%, 0.0878%, 0.0585%, 0.039%, 0.026%, 0.0173%, 0.0116%, or 0%) and left to stand at 4 ℃ for 30 min. Subsequently, the prepared paraformaldehyde solution was used to resuspend (fix) the sample at 4 ℃ for the indicated time (24h or 48 h). Subsequently, the fixative is removed and the fixed sample is used for antibody incubation and flow cytometry analysis as described above.

The results of the experiment are shown in FIGS. 8-9. FIG. 8 shows the positive rate of cells containing pre-F protein (FIG. 8A, incubated with 5C4 antibody) and the positive rate of cells containing F protein (pre-F conformation and/or post-F conformation) (FIG. 8B, incubated with 8C2 antibody) in samples treated with formaldehyde at the indicated concentrations for the indicated times; wherein the abscissa represents the concentration (%) of formaldehyde and the ordinate represents the positive rate (%) of cells.

The results show that after 24h treatment of the sample with formaldehyde at a concentration of 0.0069% -0.1185%, the sample still contains a significant amount of pre-F protein positive cells (i.e., the conformation of the pre-F protein in the sample is stabilized and maintained). This result indicates that formaldehyde concentrations in the range of 0.0069% -0.1185% stabilize and maintain the conformation of the pre-F protein in the case of fixation and inactivation for up to 24 hours, and is thus particularly suitable for inactivation of RSV virus. Furthermore, the results also show that paraformaldehyde at a concentration in the range of 0.0104% to 0.1185% is still able to stabilize and maintain the conformation of the pre-F protein in the presence of fixation and inactivation for up to 48 hours, and is thus particularly suitable for the inactivation of RSV viruses. In addition, the results also show that the pre-F protein positive cells were highest in the samples treated with formaldehyde at a concentration of 0.0156% to 0.079% when the treatment time was 24 h; and, when the treatment time was 48 hours, the content of pre-F protein-positive cells was the highest in the sample treated with formaldehyde at a concentration of 0.0234% to 0.0527%.

FIG. 9 shows the positive rate of cells containing pre-F protein (FIG. 9A, incubated with 5C4 antibody) and the positive rate of cells containing F protein (pre-F conformation and/or post-F conformation) (FIG. 9B, incubated with 8C2 antibody) in samples treated with paraformaldehyde at the indicated concentrations for the indicated times; wherein the abscissa represents the concentration (%) of paraformaldehyde, and the ordinate represents the positive rate (%) of cells.

The results show that after treatment of the samples with paraformaldehyde at a concentration of 0.0585% -0.4444% for 24h or 48h, the samples still contain significant amounts of pre-F protein positive cells (i.e., the conformation of the pre-F protein in the sample is stabilized and maintained). This result indicates that paraformaldehyde at a concentration in the range of 0.0585% to 0.4444% stabilizes and maintains the conformation of the pre-F protein in the presence of fixation and inactivation for up to 48 hours, and is thus particularly suitable for inactivating RSV virus. In addition, the results also show that the pre-F protein positive cells were highest in the samples treated with paraformaldehyde at a concentration of 0.0585% to 0.4444% when the treatment time was 24 h; and, when the treatment time was 48 hours, the content of pre-F protein-positive cells was the highest in the sample treated with formaldehyde at a concentration of 0.0878% -0.2963%.

6.3 selection of temperature

We further investigated the effect of temperature on the immobilization of the fixative. In general, methanol solutions and formaldehyde solutions are relatively stable fixatives, the fixing of which is substantially unaffected by temperature changes. Our experimental results have also shown that both methanol and formaldehyde solutions in the optimal concentration range can be used to immobilize and inactivate RSV virus and stabilize and maintain the pre-F protein in the inactivated virus at temperatures between 0-40 ℃.

Paraformaldehyde is relatively stable at low temperatures (0-10 c), but can degrade at higher temperatures to form formaldehyde. Therefore, paraformaldehyde is generally used under low temperature conditions (e.g., 4 ℃). To investigate the effect of temperature on the effect of paraformaldehyde, we further performed the following experiments.

Briefly, paraformaldehyde was formulated with 1 × PBS to the indicated concentration (0.44%, 0.1975%, 0.1317%, 0.0585%, 0.0173%, or 0.0116%) and allowed to stand at the indicated temperature (4 ℃, 25 ℃, or 37 ℃) for 30 min. Subsequently, the formulated paraformaldehyde solution was used to resuspend (fix) the sample at the indicated temperature for the indicated time (24h or 48 h). Subsequently, the fixative is removed and the fixed sample is used for antibody incubation and flow cytometry analysis as described above.

The results of the experiment are shown in FIG. 10. FIG. 10 shows the positive rate of cells containing pre-F protein (FIGS. 10A-10C, incubated with 5C4 antibody) and the positive rate of cells containing F protein (pre-F conformation and/or post-F conformation) (FIGS. 10D-10F, incubated with 8C2 antibody) in samples treated with paraformaldehyde at specified concentrations for specified times at 4 deg.C, 25 deg.C or 37 deg.C; wherein the abscissa represents the concentration (%) of paraformaldehyde, and the ordinate represents the positive rate (%) of cells.

The results show that after treatment of the samples with paraformaldehyde at a concentration of 0.0585% -0.44% for 24h or 48h at 4 ℃, the samples still contain significant amounts of pre-F protein positive cells (i.e., the conformation of the pre-F protein in the sample is stabilized and maintained). After treatment of the sample with paraformaldehyde at a concentration of 0.0173% -0.1975% for 24h or 48h at 25 ℃, the sample still contains a significant amount of pre-F protein positive cells (i.e., the conformation of the pre-F protein in the sample is stabilized and maintained). After treatment of the sample with paraformaldehyde at a concentration of 0.0173% -0.1317% for 24h or 48h at 37 ℃, the sample still contains a significant amount of pre-F protein positive cells (i.e., the conformation of the pre-F protein in the sample is stabilized and maintained).

These results indicate that paraformaldehyde can exert its effect at different temperatures (i.e., inactivate RSV virus and stabilize and maintain the pre-F protein in the virus), but that its optimum concentration range needs to be appropriately adjusted depending on the temperature at which it is actually used. Most preferably, however, paraformaldehyde is used to fix and inactivate RSV viruses and stabilize and maintain the pre-F protein under cryogenic conditions (0-10 ℃).

Example 2 stability of the Pre-F protein in the immobilized samples

In this example, we further investigated the stability of pre-F protein in the immobilized sample. Briefly, formaldehyde was formulated with 1 × PBS to a concentration of 0.0351% and allowed to stand at 37 ℃ for 30 min. Subsequently, the prepared formaldehyde solution was used to resuspend (fix) the sample at 37 ℃ for 24 h. Subsequently, the fixative was removed and the fixed sample was stored in 1 × PBS buffer as described above. In addition, samples not treated with fixative were also stored in 1 × PBS buffer for use as controls. Subsequently, after storage at room temperature for the indicated time (48h or 96h), the samples were used for antibody incubation and flow cytometry analysis as described above.

The results of the experiment are shown in FIG. 11. FIG. 11 shows the time-dependent positive rate of pre-F protein-containing cells in formaldehyde-treated and non-fixative samples stored in 1 × PBS buffer; wherein the abscissa represents the time (hours) of storage in 1 × PBS buffer, and the ordinate represents the positive rate (%) of the cells.

The results show that after storage for up to 96h, the formaldehyde-treated samples still contain significant amounts of pre-F protein positive cells (i.e., the pre-F protein in the sample remains stable and no allosteric event occurs). In contrast, the amount of pre-F protein positive cells in the non-fixative treated samples decreased significantly and tended to disappear after 48h of storage. These results indicate that the method for immobilizing and inactivating an RSV virus according to the present invention is effective in stabilizing the pre-F protein in the RSV virus from conversion to post-F protein.

Example 3 detection of immunoprotection

In this example, we investigated the immunoprotection of samples (containing host cells of inactivated RSV virus) after treatment with formaldehyde fixative. Briefly, formaldehyde was formulated with 1 × PBS to a concentration of 25%, 0.0527%, 0.0351% and allowed to stand at 37 ℃ for 30 min. Subsequently, each of the prepared formaldehyde solutions was used to resuspend (fix) the sample at 37 ℃ for 24 h. Subsequently, the fixative was removed and the fixed sample was stored in saline buffer for subcutaneous immunization of SPF grade Balb/C mice (n ═ 14) as described above. The immunization dose was 1 x 10⁷Each cell/mouse, without adjuvant, had an immune cycle of once every 10 days, 4 times total. 10 days after the completion of immunization, blood of mice was collected by blood sampling from eyeballs, and the level of neutralizing antibodies in serum was measured.

Furthermore, we investigated the immunoprotection of samples (containing host cells of inactivated RSV virus) after treatment with paraformaldehyde fixative. Briefly, paraformaldehyde was formulated with 1 × PBS to concentrations of 4%, 0.2963%, 0.1975% and left to stand at 4 ℃ for 30 min. Subsequently, each prepared paraformaldehyde solution was used to resuspend (fix) the sample at 4 ℃ for 24 h. Subsequently, the fixative was removed and the fixed sample was stored in saline buffer for subcutaneous immunization of SPF grade Balb/C mice (n ═ 14) as described above. The immunization dose was 1 x 10⁷Each cell/mouse, without adjuvant, had an immune cycle of once every 10 days, 4 times total. 10 days after the completion of immunization, blood of mice was collected by blood sampling from eyeballs, and the level of neutralizing antibodies in serum was measured.

By the following schemeSerum is tested for the level of neutralizing antibodies. In a 96-well plate (SIGMA-ALDRICH), mouse serum was diluted with medium. Initial concentrations were 10-fold dilutions (i.e., 90 μ l media +10 μ l serum), followed by 4-fold serial dilutions for a 9-fold gradient. 75 μ L of diluted serum was mixed with 75 μ L RSV-A mkate virus (titer 9 x 10)⁴An FFU; the virus expressed fluorescent protein mkate in the cells after infecting the cells, whereby the infection intensity could be judged by the mkate fluorescence intensity), mixed well, and incubated at 37 ℃ for 1 hour. Subsequently, 100. mu.l of a mixture of serum and virus was added to the pre-plated Hep-2 cells (ATCC) (3X 10)⁴Each cell per well) in a 96-well cell plate, and the cell plate was cultured at 37 ℃ for 24 hours. The fluorescence values of each well were then read using a PARADIGM multifunctional plate reader (BECKMAN COULTER). Statistical analysis of the assay results was performed using Graph Prism software, and virus-neutralizing IC was calculated by curve fitting for each serum sample₅₀. In addition, 8C2 monoclonal antibody was used as a positive control in the experiment, and IC was utilized₅₀Data, the neutralizing capacity of each serum sample was calculated relative to 8C2 mab (1 neutralizing unit of 8C2 antibody at 1 mg/mL).

The results of the experiment are shown in FIG. 12. FIG. 12 shows the level of RSV neutralizing antibodies in the serum of mice immunized with samples treated with different fixed conditions. Wherein, the ordinate represents the neutralizing capacity of the serum sample to be tested of each immune group relative to the 8C2 monoclonal antibody (1 neutralizing unit of 8C2 antibody of 1 mg/mL); the abscissa indicates various fixation conditions, F formaldehyde, PF paraformaldehyde, and no immuno mice not immunized; the line pattern shows the average neutralizing capacity (i.e., mean and standard error) of multiple mouse serum samples from each immunization group.

The results show that samples fixed with either 0.0351% or 0.0527% formaldehyde solutions or 0.1975% or 0.2963% paraformaldehyde solutions were able to elicit higher levels of neutralizing antibodies in mice. In contrast, samples treated with 25% formaldehyde solution and 4% paraformaldehyde solution had a lower ability to elicit neutralizing antibody levels in mice.

Furthermore, on day 10 after completion of immunization, each immunization was also performed using hRSVA2The mice of the group were challenged. The virus counteracting scheme is that 100 mu L virus suspension is applied to the nasal cavity, and the virus counteracting dose is 1 x 10⁷PFU/mouse. On day 5 after challenge, mouse lung tissue was harvested and dehydrated for embedding (laika ASP200) and tissue sectioning (laika paraffin slicer RM 2235). Subsequently, the obtained tissue sections were HE stained and observed under a microscope. Tissue sections were scored for inflammation according to the previously reported inflammation scoring criteria (Mucosal delivery of a vectored RSV vaccine and antibiotics protective immunity in nerves and nonhuman matrices). The results of the experiment are shown in FIGS. 13 and 14.

The results in fig. 13-14 show that mice in the immunized group treated with samples treated with either 0.0351% or 0.0527% formaldehyde solution or 0.1975% or 0.2963% paraformaldehyde solution had significantly lower severity of lung tissue inflammation, inflammatory cell infiltration and thickening of the vascular, tracheal and bronchial walls after challenge, compared to the immunized group treated with samples treated with 25% formaldehyde solution and 4% paraformaldehyde solution and the control group (not immunized). This further demonstrates that samples treated by the method of the invention have greater immunoprotective potential and can better help mice to fight infection by RSV virus and the resulting conditions. Thus, samples treated using the methods of the invention can be used as vaccines for preventing or treating RSV infection or diseases associated with RSV infection.

While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Sequence listing

<110> university of mansion

Xiamen Innovax Biotech Co.,Ltd.

<120> method for stabilizing respiratory syncytial virus fusion protein

<130> IDC210334

<150> 201710020673.7

<151> 2017-01-12

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 574

<212> PRT

<213> respiratory syncytial virus (respiratory syncytial virus)

<400> 1

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gln Ser Thr Pro Pro Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro

100 105 110

Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr

115 120 125

Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val

130 135 140

Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu

145 150 155 160

Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys

165 170 175

Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val

180 185 190

Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn

195 200 205

Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln

210 215 220

Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn

225 230 235 240

Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu

245 250 255

Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys

260 265 270

Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile

275 280 285

Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro

290 295 300

Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro

305 310 315 320

Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg

325 330 335

Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe

340 345 350

Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp

355 360 365

Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Ile Asn Leu Cys Asn Val

370 375 380

Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr

385 390 395 400

Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys

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Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile

420 425 430

Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Met Asp

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Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly

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Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro

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Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn

485 490 495

Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu

500 505 510

Leu His Asn Val Asn Ala Gly Lys Ser Thr Thr Asn Ile Met Ile Thr

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Thr Ile Ile Ile Val Ile Ile Val Ile Leu Leu Ser Leu Ile Ala Val

530 535 540

Gly Leu Leu Leu Tyr Cys Lys Ala Arg Ser Thr Pro Val Thr Leu Ser

545 550 555 560

Lys Asp Gln Leu Ser Gly Ile Asn Asn Ile Ala Phe Ser Asn

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Method for stabilizing respiratory syncytial virus fusion protein

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