Method for inactivating and preserving respiratory syncytial virus
阅读说明：本技术 灭活及保存呼吸道合胞病毒的方法 (Method for inactivating and preserving respiratory syncytial virus ) 是由 郑子峥 张伟 张璐婧 孙永鹏 陈莉 夏宁邵 于 2018-07-06 设计创作，主要内容包括：本申请涉及病毒学与免疫学领域。具体而言,本申请涉及一种灭活分离的呼吸道合胞病毒(Respiratory Syncytial Virus,RSV)且稳定所述RSV中的pre-F蛋白的方法。此外,本申请还涉及一种保存RSV病毒且稳定所述RSV中的pre-F蛋白的方法。本申请还涉及,含有灭活的RSV病毒的疫苗,所述灭活的RSV病毒通过本发明的方法制备和/或保存,以及所述疫苗用于预防或治疗RSV感染或与RSV感染相关的疾病的用途。(The present application relates to the fields of virology and immunology. In particular, the present application relates to a method of inactivating an isolated Respiratory Syncytial Virus (RSV) and stabilizing the pre-F protein in said RSV. In addition, the present application relates to a method for preserving RSV virus and stabilizing the pre-F protein in said RSV. The present application also relates to vaccines comprising inactivated RSV virus, which inactivated RSV virus is produced and/or preserved by the methods of the invention, and the use of the vaccines for preventing or treating RSV infection or a disease associated with RSV infection.)
1. A method of inactivating an isolated Respiratory Syncytial Virus (RSV) and stabilizing a pre-F protein in the RSV virus, comprising the steps of:
(1) providing an isolated live RSV virus;
(2) immobilizing and inactivating the live RSV virus using a fixative selected from the group consisting of: formaldehyde solution and paraformaldehyde solution; wherein the formaldehyde is present in a concentration of about 0.015% to about 0.27% by weight (w/w, the same applies hereinafter); the concentration of paraformaldehyde is from about 0.02% to about 0.3% by weight (w/w, the same applies below);
(3) removing the fixative from the product of step (2) to obtain an inactivated RSV virus.
2. The method of claim 1, wherein, in step (2), the fixing agent is a formaldehyde solution and the concentration of formaldehyde is from about 0.015% to about 0.27%, such as from about 0.0156% to about 0.2667%; for example, from about 0.0156% to about 0.0234%, from about 0.0234% to about 0.0244%, from about 0.0244% to about 0.0351%, from about 0.0351% to about 0.0527%, from about 0.0527% to about 0.079%, from about 0.079% to about 0.0977%, from about 0.0977% to about 0.1185%, or from about 0.1185% to about 0.1778%, or from about 0.1778% to about 0.2667%;
preferably, the live RSV virus is fixed and inactivated using a formaldehyde solution at a temperature of about 0 ℃ to about 40 ℃ (e.g., about 0 ℃ to about 4 ℃, about 4 ℃ to about 10 ℃, about 10 ℃ to about 15 ℃, about 15 ℃ to about 20 ℃, about 20 ℃ to about 25 ℃, about 25 ℃ to about 30 ℃, about 30 ℃ to about 35 ℃, about 35 ℃ to about 37 ℃, or about 37 ℃ to about 40 ℃; e.g., about 4 ℃, about 25 ℃, or about 37 ℃);
preferably, the live RSV viruses are fixed and inactivated using formaldehyde solution for a period of about 6h to about 36h (e.g., about 6h to about 12h, about 12h to about 24h, or about 24h to about 36 h; e.g., about 6h, about 12h, about 24h, or about 36 h); preferably, the live RSV virus is fixed and inactivated using formaldehyde solution for about 12 hours.
3. The method of claim 1, wherein, in step (2), the fixing agent is a paraformaldehyde solution and the concentration of paraformaldehyde is from about 0.02% to about 0.3%, such as from about 0.026% to about 0.2963%; for example, from about 0.026% to about 0.039%, from about 0.039% to about 0.0585%, from about 0.0585% to about 0.0625%, from about 0.0625% to about 0.0878%, from about 0.0878% to about 0.1317%, from about 0.1317% to about 0.1975%, from about 0.1975% to about 0.25%, or from about 0.25% to about 0.2963%;
preferably, the live RSV virus is fixed and inactivated using a paraformaldehyde solution at a temperature of from about 10 ℃ to about 40 ℃ (e.g., from about 10 ℃ to about 15 ℃, from about 15 ℃ to about 20 ℃, from about 20 ℃ to about 25 ℃, from about 25 ℃ to about 30 ℃, from about 30 ℃ to about 35 ℃, from about 35 ℃ to about 37 ℃, or from about 37 ℃ to about 40 ℃; e.g., from about 10 ℃, about 25 ℃, about 37 ℃, or about 40 ℃);
preferably, the live RSV viruses are fixed and inactivated using paraformaldehyde solution for about 6h to about 36h (e.g., about 6h to about 12h, about 12h to about 24h, or about 24h to about 36 h; e.g., about 6h, about 12h, about 24h, or about 36 h); preferably, the live RSV virus is fixed and inactivated using paraformaldehyde solution for about 12 hours;
preferably, the live RSV viruses are fixed and inactivated using a paraformaldehyde solution at a concentration of about 0.015% to about 0.25% (e.g., about 0.0156% to about 0.25%; e.g., about 0.0156% to about 0.0625%, or about 0.0625% to about 0.25%) for about 6 hours to about 36 hours (e.g., about 6 hours to about 12 hours, about 12 hours to about 24 hours, or about 24 hours to about 36 hours; e.g., about 12 hours) at a temperature of about 20 ℃ to about 30 ℃ (e.g., about 20 ℃, about 25 ℃, or about 30 ℃); alternatively, the live RSV viruses are fixed and inactivated using a paraformaldehyde solution at a concentration of about 0.0156% to about 0.0625% for about 6 hours to about 36 hours (e.g., about 6 hours to about 12 hours, about 12 hours to about 24 hours, or about 24 hours to about 36 hours; e.g., about 12 hours) at a temperature of about 35 ℃ to about 40 ℃ (e.g., about 35 ℃, about 37 ℃ or about 40 ℃).
4. The method of any one of claims 1-3, wherein in step (1), the isolated 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) collecting and lysing the cultured host cells obtained in step (1b), and recovering the RSV virus from its lysate;
preferably, the product of step (1c) is free of host cells.
5. The method of any one of claims 1-4, wherein, in step (3), the fixative is removed by dialysis, filtration, or centrifugation;
preferably, in step (3), the product of step (2) is dialyzed against a salt solution to remove the fixative;
preferably, the salt solution has an ionic concentration of about 100 to about 1000mM (e.g., about 150 to about 1000mM, e.g., about 150 to about 200mM, about 200 to about 250mM, about 250 to about 300mM, about 300 to about 350mM, about 350 to about 400mM, about 400 to about 450mM, about 450 to about 500mM, about 500 to about 550mM, about 550 to about 600mM, about 600 to about 650mM, about 650 to about 700mM, about 700 to about 750mM, about 750 to about 800mM, about 800 to about 850mM, about 850 to about 900mM, about 900 to about 950mM, or about 950 to about 1000mM, e.g., about 150mM, about 330mM, about 550mM, about 880 mM);
preferably, the salt solution has an ionic concentration of about 300 to about 1000mM (e.g., about 300 to about 900mM, about 300 to about 350mM, about 350 to about 400mM, about 400 to about 450mM, about 450 to about 500mM, about 500 to about 550mM, about 550 to about 600mM, about 600 to about 650mM, about 650 to about 700mM, about 700 to about 750mM, about 750 to about 800mM, about 800 to about 850mM, or about 850 to about 900 mM; e.g., about 330mM, about 550mM, or about 880 mM).
6. A method of preserving an RSV virus and stabilizing the pre-F protein in said RSV virus, comprising the step of placing the RSV virus in a stock solution having an ionic concentration of about 150 to about 1000mM (e.g., about 200 to about 1000mM, or about 300 to about 1000 mM; e.g., about 150 to about 200mM, about 200 to about 250mM, about 250 to about 300mM, about 300 to about 350mM, about 350 to about 400mM, about 400 to about 450mM, about 450 to about 500mM, about 500 to about 550mM, about 550 to about 600mM, about 600 to about 650mM, about 650 to about 700mM, about 700 to about 750mM, about 750 to about 800mM, about 800 to about 850mM, about 850 to about 900mM, about 900 to about 950mM, or about 950 to about 1000mM, e.g., about 150mM, about 330mM, about 550mM, or about 880mM) salt solution;
preferably, the stock solution is a salt solution having an ionic concentration of about 300 to about 1000mM (e.g., about 300 to about 900mM, about 300 to about 350mM, about 350 to about 400mM, about 400 to about 450mM, about 450 to about 500mM, about 500 to about 550mM, about 550 to about 600mM, about 600 to about 650mM, about 650 to about 700mM, about 700 to about 750mM, about 750 to about 800mM, about 800 to about 850mM, or about 850 to about 900 mM; e.g., about 330mM, about 550mM, or about 880 mM);
preferably, the inactivated RSV virus is placed in a stock solution by dialysis, filtration, or centrifugation.
7. The method of claim 6, wherein the RSV virus is dialyzed against a salt solution to place the RSV virus in a stock solution; wherein the salt solution has an ionic concentration of about 150 to about 1000mM (e.g., about 200 to about 1000mM, or about 300 to about 1000 mM; e.g., about 150 to about 200mM, about 200 to about 250mM, about 250 to about 300mM, about 300 to about 350mM, about 350 to about 400mM, about 400 to about 450mM, about 450 to about 500mM, about 500 to about 550mM, about 550 to about 600mM, about 600 to about 650mM, about 650 to about 700mM, about 700 to about 750mM, about 750 to about 800mM, about 800 to about 850mM, about 850 to about 900mM, about 900 to about 950mM, or about 950 to about 1000mM, e.g., about 150mM, about 330mM, about 550mM, or about 880 mM);
preferably, the salt solution has an ionic concentration of about 300 to about 1000mM (e.g., about 300 to about 900mM, about 300 to about 350mM, about 350 to about 400mM, about 400 to about 450mM, about 450 to about 500mM, about 500 to about 550mM, about 550 to about 600mM, about 600 to about 650mM, about 650 to about 700mM, about 700 to about 750mM, about 750 to about 800mM, about 800 to about 850mM, or about 850 to about 900 mM; e.g., about 330mM, about 550mM, or about 880 mM);
preferably, the RSV virus is dialyzed against the salt solution for about 6 hours to about 24 hours (e.g., about 12 hours to about 24 hours, about 12 hours to about 20 hours, or about 16 hours to about 20 hours; e.g., about 18 hours).
8. The method of claim 6 or 7, wherein the RSV virus is an inactivated virus;
preferably, the method is used to preserve an inactivated RSV virus and stabilize the pre-F protein in the inactivated RSV virus;
preferably, the inactivated RSV virus is provided by:
(i) providing an isolated live RSV virus;
(ii) immobilizing and inactivating the live RSV virus using a fixative;
(iii) (iii) removing the fixative from the product of step (ii) thereby obtaining an inactivated RSV virus;
preferably, in step (ii), the fixing agent is selected from the group consisting of formaldehyde solution, paraformaldehyde solution, glutaraldehyde solution and beta propiolactone solution; preferably, the fixing agent is formaldehyde solution or paraformaldehyde solution;
preferably, the inactivated RSV virus is provided by the method of any one of claims 1-5.
9. The method of claim 8, comprising the steps of:
(1) providing an isolated live RSV virus;
(2) immobilizing and inactivating the live RSV virus using a fixative;
(3) dialyzing the product of step (2) against a salt solution, thereby obtaining a stock solution comprising inactivated RSV virus;
wherein, in step (3), the salt solution has an ionic concentration of about 150 to about 1000mM (e.g., about 200 to about 1000mM, or about 300 to about 1000 mM; e.g., about 150 to about 200mM, about 200 to about 250mM, about 250 to about 300mM, about 300 to about 350mM, about 350 to about 400mM, about 400 to about 450mM, about 450 to about 500mM, about 500 to about 550mM, about 550 to about 600mM, about 600 to about 650mM, about 650 to about 700mM, about 700 to about 750mM, about 750 to about 800mM, about 800 to about 850mM, about 850 to about 900mM, about 900 to about 950mM, or about 950 to about 1000mM, e.g., about 150mM, about 330mM, about 550mM, or about 880 mM);
preferably, the salt solution has an ionic concentration of about 300 to about 1000mM (e.g., about 300 to about 900mM, about 300 to about 350mM, about 350 to about 400mM, about 400 to about 450mM, about 450 to about 500mM, about 500 to about 550mM, about 550 to about 600mM, about 600 to about 650mM, about 650 to about 700mM, about 700 to about 750mM, about 750 to about 800mM, about 800 to about 850mM, or about 850 to about 900 mM; e.g., about 330mM, about 550mM, or about 880 mM).
10. The method of claim 9, wherein, in step (2), the fixing agent is a formaldehyde solution;
preferably, the concentration of formaldehyde is no greater than about 0.27%, such as no greater than about 0.2667% by weight;
preferably, the live RSV virus is fixed and inactivated using a formaldehyde solution at a temperature of about 0 ℃ to about 40 ℃ (e.g., about 0 ℃ to about 4 ℃, about 4 ℃ to about 10 ℃, about 10 ℃ to about 15 ℃, about 15 ℃ to about 20 ℃, about 20 ℃ to about 25 ℃, about 25 ℃ to about 30 ℃, about 30 ℃ to about 35 ℃, about 35 ℃ to about 37 ℃, or about 37 ℃ to about 40 ℃; e.g., about 4 ℃, about 25 ℃, or about 37 ℃);
preferably, the live RSV viruses are fixed and inactivated using formaldehyde solution for a period of about 6h to about 36h (e.g., about 6h to about 12h, about 12h to about 24h, or about 24h to about 36 h; e.g., about 6h, about 12h, about 24h, or about 36 h).
11. The method of claim 9, wherein, in step (2), the fixing agent is a paraformaldehyde solution;
preferably, the concentration of paraformaldehyde is no greater than about 0.3%, for example no greater than about 0.2963%;
preferably, the live RSV virus is fixed and inactivated using a paraformaldehyde solution at a temperature of from about 10 ℃ to about 40 ℃ (e.g., from about 10 ℃ to about 15 ℃, from about 15 ℃ to about 20 ℃, from about 20 ℃ to about 25 ℃, from about 25 ℃ to about 30 ℃, from about 30 ℃ to about 35 ℃, from about 35 ℃ to about 37 ℃, or from about 37 ℃ to about 40 ℃; e.g., from about 10 ℃, about 25 ℃, about 37 ℃, or about 40 ℃);
preferably, the live RSV viruses are fixed and inactivated using paraformaldehyde solution for about 6h to about 36h (e.g., about 6h to about 12h, about 12h to about 24h, or about 24h to about 36 h; e.g., about 6h, about 12h, about 24h, or about 36 h).
12. The method of any of claims 6-11, further comprising the step of storing the stock solution comprising RSV virus at a temperature of from about 0 ℃ to about 40 ℃ (e.g., from about 0 ℃ to about 4 ℃, from about 4 ℃ to about 10 ℃, from about 10 ℃ to about 15 ℃, from about 15 ℃ to about 20 ℃, from about 20 ℃ to about 25 ℃, from about 25 ℃ to about 30 ℃, from about 30 ℃ to about 35 ℃, from about 35 ℃ to about 37 ℃, or from about 37 ℃ to about 40 ℃; e.g., about 4 ℃, about 25 ℃, or about 37 ℃).
13. An inactivated RSV virus produced by the method of any one of claims 1-5 and/or preserved by the method of any one of claims 6-12.
14. A vaccine comprising the inactivated RSV virus of claim 13, and optionally, a pharmaceutically acceptable carrier and/or excipient (e.g., adjuvant).
15. A method of making a vaccine comprising mixing the inactivated RSV virus of claim 13 with a pharmaceutically acceptable carrier and/or excipient (e.g., adjuvant).
16. Use of an inactivated RSV virus of claim 13 in the manufacture of a vaccine for preventing, treating or inhibiting RSV infection or a disease associated with RSV infection (e.g., pneumonia, such as pediatric pneumonia) in a subject;
preferably, the subject is a mammal, such as a human.
Technical Field
The present application relates to the fields of virology and immunology. In particular, the present application relates to a method of inactivating an isolated Respiratory Syncytial Virus (RSV) and stabilizing the pre-F protein in said RSV. In addition, the present application relates to a method for preserving RSV virus and stabilizing the pre-F protein in said RSV. The present application also relates to vaccines comprising inactivated RSV virus, which inactivated RSV virus is produced and/or preserved by the methods of the invention, and the use of the vaccines for preventing or treating RSV infection or a disease associated with RSV infection.
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 HPIV3pre-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, neutralizing epitopes on pre-F and post-F protein conformations have also been 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 as
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, stabilize and maintain methods for inactivating the pre-F protein in isolated 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 the RSV F proteins known so far are mainly directed 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 aa422-438 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.
The results of previous studies (McLellan JS, Chen M, et al science 2013,340: 1113-1117) have shown,
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., epitomeping 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 "KD"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 than about 10-6M、10-7M、10-8M、10- 9M or 10-10Dissociation equilibrium constant (K) of M or lessD) 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 "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 cells, 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 β 5 cells, Vero cells, BHK-MKL cells, RK-13 cells, HeLa cells, TbL cells, SK-OV-3 cells, U2-OS cells, 143B cells, MCF-7 cells, MDA-MB-231 cells, T-47D cells, RD cells, BGC-AGA cells, AGS cells, MeaS 431 cells, WoS NS cells, WoS 431 cells, WoS 3 cells, WoS-mK cells, and Wo, 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 residual), 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,19the d. pennsylvania: machine 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.
As used herein, the term "about" when used to describe a measurable value (e.g., concentration of a substance, time, temperature, etc.) is meant to encompass a range of ± 10%, ± 5%, or ± 1% of the given value. In certain exemplary embodiments, the term "about" refers to plus or minus 10% of a given value, e.g., about 0.015% refers to a range of 0.0135% to 0.0165%.
The inventor has found, surprisingly, through a large number of experimental studies: in fixing/inactivating an RSV virus, by using a specific fixing agent (e.g., formaldehyde, or paraformaldehyde) and using specific fixing/inactivating conditions (e.g., a specific fixing agent concentration), an inactivated RSV virus that is particularly advantageous can be obtained, which contains a higher amount of pre-F protein (i.e., more F protein exists in the pre-F conformation in the inactivated RSV virus obtained by the present invention) than an inactivated virus obtained by a conventional method. Meanwhile, when an RSV virus is preserved, by using a specific preservation condition (e.g., a specific stock solution ion concentration), the pre-F protein in the RSV virus can be maintained and stabilized, and even the content of the pre-F protein can be maintained at a level close to that of a fresh live virus. 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 a first aspect, the present invention provides a method of inactivating an isolated Respiratory Syncytial Virus (RSV) and stabilizing the pre-F protein in said RSV virus, comprising the steps of:
(1) providing an isolated live RSV virus;
(2) immobilizing and inactivating the live RSV virus using a fixative selected from the group consisting of: formaldehyde solution and paraformaldehyde solution; wherein the formaldehyde is present in a concentration of about 0.015% to about 0.27% by weight (w/w, the same applies hereinafter); the concentration of paraformaldehyde is from about 0.02% to about 0.3% by weight (w/w, the same applies below);
(3) removing the fixative from the product of step (2) to obtain an inactivated RSV virus.
In the present invention, the expression "isolated" means that the RSV virus is not contained in or otherwise provided within a cell (e.g., a host cell).
In certain preferred embodiments, in step (2), the fixing agent is a formaldehyde solution and the concentration of formaldehyde is from about 0.015% to about 0.27%, for example from about 0.0156% to about 0.2667%; for example, from about 0.0156% to about 0.0234%, from about 0.0234% to about 0.0244%, from about 0.0244% to about 0.0351%, from about 0.0351% to about 0.0527%, from about 0.0527% to about 0.079%, from about 0.079% to about 0.0977%, from about 0.0977% to about 0.1185%, or from about 0.1185% to about 0.1778%, or from about 0.1778% to about 0.2667%. 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 preferred embodiments, the live RSV virus is immobilized and inactivated using a formaldehyde solution at a temperature of from about 0 ℃ to about 40 ℃ (e.g., from about 0 ℃ to about 4 ℃, from about 4 ℃ to about 10 ℃, from about 10 ℃ to about 15 ℃, from about 15 ℃ to about 20 ℃, from about 20 ℃ to about 25 ℃, from about 25 ℃ to about 30 ℃, from about 30 ℃ to about 35 ℃, from about 35 ℃ to about 37 ℃, or from about 37 ℃ to about 40 ℃; e.g., from about 4 ℃, about 25 ℃, or about 37 ℃).
In certain preferred embodiments, formaldehyde solution is used to fix and inactivate the live RSV virus for about 6h to about 36h (e.g., about 6h to about 12h, about 12h to about 24h, or about 24h to about 36 h; e.g., about 6h, about 12h, about 24h, or about 36 h). In certain preferred embodiments, formaldehyde solution is used to fix and inactivate the live RSV virus for about 12 hours.
In certain preferred embodiments, in step (2), the fixing agent is a paraformaldehyde solution, and the concentration of paraformaldehyde is from about 0.02% to about 0.3%, for example from about 0.026% to about 0.2963%; for example, from about 0.026% to about 0.039%, from about 0.039% to about 0.0585%, from about 0.0585% to about 0.0625%, from about 0.0625% to about 0.0878%, from about 0.0878% to about 0.1317%, from about 0.1317% to about 0.1975%, from about 0.1975% to about 0.25%, or from about 0.25% to about 0.2963%. 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 preferred embodiments, the live RSV virus is immobilized and inactivated using a paraformaldehyde solution at a temperature of from about 10 ℃ to about 40 ℃ (e.g., from about 10 ℃ to about 15 ℃, from about 15 ℃ to about 20 ℃, from about 20 ℃ to about 25 ℃, from about 25 ℃ to about 30 ℃, from about 30 ℃ to about 35 ℃, from about 35 ℃ to about 37 ℃, or from about 37 ℃ to about 40 ℃; e.g., about 10 ℃, about 25 ℃, about 37 ℃, or about 40 ℃).
In certain preferred embodiments, the live RSV viruses are fixed and inactivated using paraformaldehyde solution for about 6h to about 36h (e.g., about 6h to about 12h, about 12h to about 24h, or about 24h to about 36 h; e.g., about 6h, about 12h, about 24h, or about 36 h). In certain preferred embodiments, the live RSV virus is fixed and inactivated using paraformaldehyde solution for about 12 hours.
In certain preferred embodiments, the live RSV virus is fixed and inactivated using a paraformaldehyde solution at a concentration of about 0.015% to about 0.25% (e.g., about 0.0156% to about 0.25%; e.g., about 0.0156% to about 0.0625%, or about 0.0625% to about 0.25%) for about 6 hours to about 36 hours (e.g., about 6 hours to about 12 hours, about 12 hours to about 24 hours, or about 24 hours to about 36 hours; e.g., about 12 hours) at a temperature of about 20 ℃ to about 30 ℃ (e.g., about 20 ℃, about 25 ℃, or about 30 ℃).
In certain preferred embodiments, the live RSV virus is fixed and inactivated using a paraformaldehyde solution at a concentration of about 0.0156% to about 0.0625% for about 6 hours to about 36 hours (e.g., about 6 hours to about 12 hours, about 12 hours to about 24 hours, or about 24 hours to about 36 hours; e.g., about 12 hours) at a temperature of about 35 ℃ to about 40 ℃ (e.g., about 35 ℃, about 37 ℃, or about 40 ℃).
In certain preferred embodiments, in step (1), the isolated 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) collecting and lysing the cultured host cells obtained in step (1b), and recovering the RSV virus from its lysate. In the present invention, the product of step (1c) is free of host cells.
In certain preferred embodiments, in step (1c), the RSV virus is recovered by centrifugation or filtration, and the host cells are removed.
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 cells, 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 β 5 cells, Vero cells, BHK-MKL cells, RK-13 cells, HeLa cells, TbL cells, SK-OV-3 cells, U2-OS cells, 143B cells, MCF-7 cells, MDA-MB-231 cells, T-47D cells, RD cells, BGC-AGA cells, AGS cells, MeaS 431 cells, WoS NS cells, WoS 431 cells, WoS 3 cells, WoS-mK cells, and Wo, 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 lysed by sonication.
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 dialyzing the product of step (2) into a fixative-free solution.
In certain preferred embodiments, in step (3), the product of step (2) is dialyzed against a salt solution to remove the fixative. In certain preferred embodiments, the salt solution has an ionic concentration of about 100 to about 1000mM (e.g., about 150 to about 1000mM, e.g., about 100 to about 150mM, about 150 to about 200mM, about 200 to about 250mM, about 250 to about 300mM, about 300 to about 350mM, about 350 to about 400mM, about 400 to about 450mM, about 450 to about 500mM, about 500 to about 550mM, about 550 to about 600mM, about 600 to about 650mM, about 650 to about 700mM, about 700 to about 750mM, about 750 to about 800mM, about 800 to about 850mM, about 850 to about 900mM, about 900 to about 950mM, about 950 to about 1000mM, e.g., about 150mM, about 330mM, about 550mM, about 880 mM).
In certain preferred embodiments, in step (3), the product of step (2) is dialyzed against a salt solution having an ionic concentration of about 300 to about 1000mM (e.g., about 300 to about 900mM, about 300 to about 350mM, about 350 to about 400mM, about 400 to about 450mM, about 450 to about 500mM, about 500 to about 550mM, about 550 to about 600mM, about 600 to about 650mM, about 650 to about 700mM, about 700 to about 750mM, about 750 to about 800mM, about 800 to about 850mM, or about 850 to about 900 mM; e.g., about 330mM, about 550mM, or about 880 mM).
In certain exemplary embodiments, the salt solution comprises a sodium salt (e.g., sodium chloride, disodium hydrogen phosphate, or a combination thereof) and/or a potassium salt (e.g., potassium chloride, potassium dihydrogen phosphate, or a combination thereof). In certain exemplary embodiments, the salt solution is Phosphate Buffered Saline (PBS). In certain exemplary embodiments, the salt solution is a sodium salt solution (e.g., sodium chloride, disodium hydrogen phosphate, or combinations thereof).
In certain preferred embodiments, the pre-F protein in an inactivated RSV virus obtained by the methods of the invention can be retained by at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to the pre-F protein in a live, freshly harvested virus. In certain preferred embodiments, the pre-F protein in an inactivated RSV virus obtained by the methods of the invention can be retained by at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to the pre-F protein in a live, freshly harvested virus, and the above ratio can be maintained for at least 24 hours, e.g., at least 48 hours, or at least 72 hours.
In certain preferred embodiments, the pre-F protein in the inactivated RSV virus is capable of maintaining its conformation for at least 24 hours, such as at least 48 hours, or at least 72 hours.
In a second aspect, the invention provides a method of preserving an RSV virus and stabilizing the pre-F protein in said RSV virus, comprising the step of placing the RSV virus in a stock solution having an ionic concentration of about 150 to about 1000mM (e.g., about 200 to about 1000mM, or about 300 to about 1000 mM; e.g., about 150 to about 200mM, about 200 to about 250mM, about 250 to about 300mM, about 300 to about 350mM, about 350 to about 400mM, about 400 to about 450mM, about 450 to about 500mM, about 500 to about 550mM, about 550 to about 600mM, about 600 to about 650mM, about 650 to about 700mM, about 700 to about 750mM, about 750 to about 800mM, about 800 to about 850mM, about 850 to about 900mM, about 900 to about 950mM, about 950 to about 1000mM, e.g., about 150mM, about 330mM, about 550mM, or about 880 mM).
In certain preferred embodiments, the stock solution is a salt solution having an ionic concentration of about 300 to about 1000mM (e.g., about 300 to about 900mM, about 300 to about 350mM, about 350 to about 400mM, about 400 to about 450mM, about 450 to about 500mM, about 500 to about 550mM, about 550 to about 600mM, about 600 to about 650mM, about 650 to about 700mM, about 700 to about 750mM, about 750 to about 800mM, about 800 to about 850mM, or about 850 to about 900 mM; e.g., about 330mM, about 550mM, or about 880 mM).
In certain preferred embodiments, the storage liquid is a salt solution comprising a sodium salt (e.g., sodium chloride, disodium hydrogen phosphate, or combinations thereof) and/or a potassium salt (e.g., potassium chloride, potassium dihydrogen phosphate, or combinations thereof). In certain exemplary embodiments, the stock solution is Phosphate Buffered Saline (PBS), or alternatively, the stock solution is a sodium salt solution (e.g., sodium chloride, disodium hydrogen phosphate, or a combination thereof).
In certain preferred embodiments, the inactivated RSV virus is placed in a stock solution by dialysis, filtration, or centrifugation.
In certain preferred embodiments, the RSV virus is dialyzed against a salt solution, thereby placing the RSV virus in a stock solution; wherein the salt solution has an ionic concentration of about 150 to about 1000mM (e.g., about 200 to about 1000mM, or about 300 to about 1000 mM; e.g., about 150 to about 200mM, about 200 to about 250mM, about 250 to about 300mM, about 300 to about 350mM, about 350 to about 400mM, about 400 to about 450mM, about 450 to about 500mM, about 500 to about 550mM, about 550 to about 600mM, about 600 to about 650mM, about 650 to about 700mM, about 700 to about 750mM, about 750 to about 800mM, about 800 to about 850mM, about 850 to about 900mM, about 900 to about 950mM, about 950 to about 1000mM, e.g., about 150mM, about 330mM, about 550mM, or about 880 mM).
In certain preferred embodiments, the salt solution has an ionic concentration of about 300 to about 1000mM (e.g., about 300 to about 900mM, about 300 to about 350mM, about 350 to about 400mM, about 400 to about 450mM, about 450 to about 500mM, about 500 to about 550mM, about 550 to about 600mM, about 600 to about 650mM, about 650 to about 700mM, about 700 to about 750mM, about 750 to about 800mM, about 800 to about 850mM, or about 850 to about 900 mM; e.g., about 330mM, about 550mM, or about 880 mM).
In certain preferred embodiments, the salt solution comprises a sodium salt (e.g., sodium chloride, disodium hydrogen phosphate, or combinations thereof) and/or a potassium salt (e.g., potassium chloride, potassium dihydrogen phosphate, or combinations thereof); for example, the salt solution is Phosphate Buffered Saline (PBS), or the salt solution is a sodium salt solution (e.g., sodium chloride, disodium hydrogen phosphate, or a combination thereof).
In certain preferred embodiments, the RSV virus is dialyzed against the salt solution for a period of about 6 hours to about 24 hours (e.g., about 12 hours to about 24 hours, about 12 hours to about 20 hours, or about 16 hours to about 20 hours; e.g., about 18 hours).
In certain preferred embodiments, the RSV virus is a live virus. In such embodiments, the methods are used to preserve live RSV virus and stabilize the pre-F protein in the live RSV virus. In certain preferred embodiments, the RSV virus is a live virus freshly harvested from a host cell.
In certain preferred embodiments, the RSV virus is an inactivated virus. In such embodiments, the methods are used to preserve an inactivated RSV virus and stabilize the pre-F protein in the inactivated RSV virus. In the present invention, methods of inactivating a virus to destroy its ability to infect cells in a subject (e.g., a mammal, such as a human) are well known to those skilled in the art, and include chemical means and physical means. Suitable means for inactivating the virus include, but are not limited to, treatment with an effective amount of one or more selected from the group consisting of: fixatives (e.g., alcohols (e.g., methanol), aldehydes (e.g., formaldehyde, glutaraldehyde), beta propiolactone, phenol, etc.), heat, radiation (e.g., electromagnetic radiation, X-ray radiation, gamma radiation, ultraviolet radiation, etc.), psoralea fruit, methylene blue, ozone, etc.
In certain preferred embodiments, the inactivated RSV virus is provided by:
(i) providing an isolated live RSV virus;
(ii) immobilizing and inactivating the live RSV virus using a fixative;
(iii) (iii) removing the fixative from the product of step (ii) thereby obtaining an inactivated RSV virus.
In certain preferred embodiments, in step (ii), the fixing agent is selected from the group consisting of formaldehyde solution, paraformaldehyde solution, glutaraldehyde solution, and beta propiolactone solution. In certain preferred embodiments, the fixing agent is a formaldehyde solution or a paraformaldehyde solution.
In certain preferred embodiments, the inactivated RSV virus is provided by a method of inactivating an RSV virus as described in the first aspect.
In certain preferred embodiments, the method comprises the steps of:
(1) providing an isolated live RSV virus;
(2) immobilizing and inactivating the live RSV virus using a fixative;
(3) dialyzing the product of step (2) against a salt solution, thereby obtaining a stock solution comprising inactivated RSV virus;
wherein, in step (3), the salt solution has an ionic concentration of about 150 to about 1000mM (e.g., about 200 to about 1000mM, or about 300 to about 1000 mM; e.g., about 150 to about 200mM, about 200 to about 250mM, about 250 to about 300mM, about 300 to about 350mM, about 350 to about 400mM, about 400 to about 450mM, about 450 to about 500mM, about 500 to about 550mM, about 550 to about 600mM, about 600 to about 650mM, about 650 to about 700mM, about 700 to about 750mM, about 750 to about 800mM, about 800 to about 850mM, about 850 to about 900mM, about 900 to about 950mM, about 950 to about 1000mM, e.g., about 150mM, about 330mM, about 550mM, or about 880 mM).
In certain preferred embodiments, the salt solution has an ionic concentration of about 300 to about 1000mM (e.g., about 300 to about 900mM, about 300 to about 350mM, about 350 to about 400mM, about 400 to about 450mM, about 450 to about 500mM, about 500 to about 550mM, about 550 to about 600mM, about 600 to about 650mM, about 650 to about 700mM, about 700 to about 750mM, about 750 to about 800mM, about 800 to about 850mM, or about 850 to about 900 mM; e.g., about 330mM, about 550mM, or about 880 mM).
In certain preferred embodiments, the storage liquid is a salt solution comprising a sodium salt (e.g., sodium chloride, disodium hydrogen phosphate, or combinations thereof) and/or a potassium salt (e.g., potassium chloride, potassium dihydrogen phosphate, or combinations thereof); for example, the stock solution is Phosphate Buffered Saline (PBS), or the stock solution is a sodium salt solution (e.g., sodium chloride, disodium hydrogen phosphate, or a combination thereof).
In certain preferred embodiments, in step (2), the fixing agent is a formaldehyde solution.
In certain preferred embodiments, the concentration of formaldehyde is no greater than about 0.27%, such as no greater than about 0.2667%, by weight. In certain preferred embodiments, the live RSV virus is immobilized and inactivated using a formaldehyde solution at a temperature of from about 0 ℃ to about 40 ℃ (e.g., from about 0 ℃ to about 4 ℃, from about 4 ℃ to about 10 ℃, from about 10 ℃ to about 15 ℃, from about 15 ℃ to about 20 ℃, from about 20 ℃ to about 25 ℃, from about 25 ℃ to about 30 ℃, from about 30 ℃ to about 35 ℃, from about 35 ℃ to about 37 ℃, or from about 37 ℃ to about 40 ℃; e.g., from about 4 ℃, about 25 ℃, or about 37 ℃). In certain preferred embodiments, formaldehyde solution is used to fix and inactivate the live RSV virus for about 6h to about 36h (e.g., about 6h to about 12h, about 12h to about 24h, or about 24h to about 36 h; e.g., about 6h, about 12h, about 24h, or about 36 h).
In certain preferred embodiments, in step (2), the fixing agent is a paraformaldehyde solution.
In certain preferred embodiments, the concentration of paraformaldehyde is no greater than about 0.3%, for example no greater than about 0.2963%, by weight. In certain preferred embodiments, the live RSV virus is immobilized and inactivated using a paraformaldehyde solution at a temperature of from about 10 ℃ to about 40 ℃ (e.g., from about 10 ℃ to about 15 ℃, from about 15 ℃ to about 20 ℃, from about 20 ℃ to about 25 ℃, from about 25 ℃ to about 30 ℃, from about 30 ℃ to about 35 ℃, from about 35 ℃ to about 37 ℃, or from about 37 ℃ to about 40 ℃; e.g., about 10 ℃, about 25 ℃, about 37 ℃, or about 40 ℃). In certain preferred embodiments, the live RSV viruses are fixed and inactivated using paraformaldehyde solution for about 6h to about 36h (e.g., about 6h to about 12h, about 12h to about 24h, or about 24h to about 36 h; e.g., about 6h, about 12h, about 24h, or about 36 h).
In certain preferred embodiments, the method further comprises the step of storing the stock solution comprising RSV virus at a temperature of from about 0 ℃ to about 40 ℃ (e.g., from about 0 ℃ to about 4 ℃, from about 4 ℃ to about 10 ℃, from about 10 ℃ to about 15 ℃, from about 15 ℃ to about 20 ℃, from about 20 ℃ to about 25 ℃, from about 25 ℃ to about 30 ℃, from about 30 ℃ to about 35 ℃, from about 35 ℃ to about 37 ℃, or from about 37 ℃ to about 40 ℃; e.g., from about 4 ℃, about 25 ℃, or about 37 ℃).
In certain preferred embodiments, the pre-F protein in an inactivated RSV virus preserved by the methods of the invention can be retained by at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) compared to the pre-F protein in a live, freshly harvested virus. In certain preferred embodiments, the pre-F protein in an inactivated RSV virus preserved by the methods of the invention can be retained by at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) compared to the pre-F protein in a live, freshly harvested virus, and the above ratio can be maintained for at least 24 hours, e.g., at least 48 hours, or at least 72 hours.
In another aspect, the invention provides an inactivated RSV virus prepared by the method of the first aspect and/or preserved by the method of the second aspect.
In another aspect, the invention provides a vaccine comprising an inactivated RSV virus according to the invention, and optionally, a pharmaceutically acceptable carrier and/or excipient (e.g. an adjuvant).
In certain preferred embodiments, the inactivated RSV virus is prepared by a method as described in the first aspect; alternatively, the inactivated RSV virus is preserved by a method according to the second aspect; alternatively, the inactivated RSV virus is prepared by a method as described in the first aspect and preserved by a method as described in the second aspect.
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.
The vaccines of the present invention may be used alone or in combination with other pharmaceutically active agents (e.g., interferons, such as interferon or pegylated interferon).
In another aspect, the invention provides a method of preparing a vaccine comprising admixing an inactivated RSV virus according to the invention with a pharmaceutically acceptable carrier and/or excipient (e.g. adjuvant).
In certain preferred embodiments, the inactivated RSV virus is prepared by a method as described in the first aspect; alternatively, the inactivated RSV virus is preserved by a method according to the second aspect; alternatively, the inactivated RSV virus is prepared by a method as described in the first aspect and preserved by a method as described in the second aspect.
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 a vaccine according to the invention.
In certain preferred embodiments, the inactivated RSV virus is prepared by a method as described in the first aspect; alternatively, the inactivated RSV virus is preserved by a method according to the second aspect; alternatively, the inactivated RSV virus is prepared by a method as described in the first aspect and preserved by a method as described in the second aspect.
In another aspect, there is provided the use of an inactivated RSV virus of the invention in the manufacture of a vaccine 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 certain preferred embodiments, the inactivated RSV virus is prepared by a method as described in the first aspect; alternatively, the inactivated RSV virus is preserved by a method according to the second aspect; alternatively, the inactivated RSV virus is prepared by a method as described in the first aspect and preserved by a method as described in the second aspect.
In another aspect, an inactivated RSV virus or vaccine of the invention is provided 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.
Advantageous effects of the invention
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
(1) the method of inactivating an RSV of the present invention can be used to prepare an inactivated RSV virus comprising a pre-F protein and maintain and stabilize the conformation of the pre-F protein.
(2) The method for preserving an RSV of the present invention can maintain and stabilize the conformation of the pre-F protein, and can even maintain the content of the pre-F protein at a level close to that of a fresh live virus, and can be used for preparing an inactivated RSV virus containing the pre-F protein.
(3) The inactivated RSV viruses of the present invention, which contain a higher amount of pre-F protein than those obtained by conventional inactivation or storage methods, induce a more effective immune response in vivo, and are therefore particularly useful for the development of vaccines against RSV viruses for the prevention or treatment of RSV infection or diseases associated with RSV infection (e.g., pneumonia, such as infantile pneumonia).
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. Result display, prethe-F and post-F proteins share about 50% of the protein surface and have epitopes with high neutralizing activity (strongly neutralizing epitopes) such as
Fig. 2 shows an image of a gel of a sample incubated with 9F7 antibody, 5C4 antibody, or 8C2 antibody, wherein the sample is, from left to right: inactivated viruses obtained by using a formaldehyde solution having a concentration of 6.25%, 1.5625%, 0.3906%, 0.0977%, 0.0244%, 0.0061% as a fixative, and viruses immobilized/inactivated without adding a fixative.
Figure 3 shows the relative proportion of virus surface pre-F protein retention (figure 3A, incubated with 5C4 antibody) and the relative proportion of total F protein (pre-F conformation and/or post-F conformation) retention (figure 3B, incubated with 8C2 antibody) in samples treated with the indicated concentrations of beta-propiolactone for the indicated times.
Figure 4 shows the relative proportion of virus surface pre-F protein retention (figure 4A, incubated with 5C4 antibody) and the relative proportion of total F protein (pre-F conformation and/or post-F conformation) retention (figure 4B, incubated with 8C2 antibody) in samples treated with glutaraldehyde at the indicated concentrations for the indicated times.
Figure 5 shows the relative proportions of virus surface pre-F protein retention (figures 5A, 5C, 5E, 5G, incubated with 5C4 antibody) and total F protein (pre-F conformation and/or post-F conformation) retention (figures 5B, 5D, 5F, 5H, incubated with 8C2 antibody) in samples treated with formaldehyde at the indicated concentrations for the indicated times.
Figure 6 shows the relative proportion of virus surface pre-F protein retention (figure 6A, incubated with 5C4 antibody) and the relative proportion of total F protein (pre-F conformation and/or post-F conformation) retention (figure 6B, incubated with 8C2 antibody) in samples treated with formaldehyde at the indicated concentrations for 12h and left for the indicated times.
Fig. 7 shows the relative proportion of virus surface pre-F protein retention (fig. 7A, incubated with 5C4 antibody) and the relative proportion of total F protein (pre-F conformation and/or post-F conformation) retention (fig. 7B, incubated with 8C2 antibody) in samples treated with paraformaldehyde at the indicated concentrations for 12h and left for the indicated times.
Fig. 8 shows the relative proportion of virus surface pre-F protein retention (fig. 8A, incubated with 5C4 antibody) and the relative proportion of total F protein (pre-F conformation and/or post-F conformation) retention (fig. 8B, incubated with 8C2 antibody) in samples treated with formaldehyde at the indicated concentrations for 12h and left for the indicated times.
Fig. 9 shows the relative proportion of virus surface pre-F protein retention (fig. 9A, incubated with 5C4 antibody) and the relative proportion of total F protein (pre-F conformation and/or post-F conformation) retention (fig. 9B, incubated with 8C2 antibody) in samples treated with paraformaldehyde at the indicated concentrations for 12h and left for the indicated times.
FIG. 10 shows the relative proportion of virus surface pre-F protein retention (FIGS. 10A-10C, incubated with 5C4 antibody) and total F protein (pre-F conformation and/or post-F conformation) retention (FIGS. 10D-10F, incubated with 8C2 antibody) in samples treated with paraformaldehyde at the indicated concentrations for 12h and placed for the indicated times at 4 deg.C, 25 deg.C or 37 deg.C.
FIG. 11 shows the relative proportion of pre-F protein retained on the surface of the virus as a function of time in the formaldehyde-treated samples and the samples that were not treated with the fixative while stored in PBS solution.
FIG. 12 shows the relative proportion of pre-F protein retained on the surface of the virus (FIG. 12A-12D, incubated with 5C4 antibody) and the relative proportion of total F protein (pre-F conformation and/or post-F conformation) retained (FIG. 12E-12H, incubated with 8C2 antibody) as the time of standing after dialysis of inactivated/fixed virus in 150mM, 330mM, 550mM, 880mM salt solutions, respectively.
FIG. 13 shows the results of detection of RSV-specific neutralizing antibody levels in mice immunized with the preferred concentration of formalin and the non-preferred concentration of formalin inactivated virus as immunogens.
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)
MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPV TLSKDQLSGINNIAFSN
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.
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