Respiratory syncytial virus vaccine composition and application thereof

Abstract

The invention relates to a respiratory syncytial virus vaccine composition and application thereof. The recombinant respiratory syncytial virus vaccine comprises RSVpreF protein and a pharmaceutically acceptable composite adjuvant carrier, and the composite adjuvant carrier is formed by combining an aluminum adjuvant with a CpG adjuvant. Experimental results prove that the vaccine composition provided by the invention has good immunogenicity and a better immune effect.

Description

A respiratory syncytial virus vaccine composition and its use Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a respiratory syncytial virus vaccine composition and its use. Background Technology

[0002] Human respiratory syncytial virus (RSV) belongs to the genus Orthopneumovirus of the family Pneumoviridae. It is an enveloped, non-segmented, single-stranded negative-sense RNA virus. RSV is one of the leading pathogens causing acute lower respiratory tract infections in infants, immunocompromised individuals, and the elderly worldwide. It is highly contagious and primarily spreads through droplets and direct contact (bodily fluids and secretions). In recent years, research on RSV vaccines has gradually attracted the attention of the scientific and industrial communities. One of the major challenges is designing a safe and effective vaccine to protect high-risk populations from RSV infection and provide long-term immune protection.

[0003] Adjuvants, also known as immunomodulators or immunopotentiators, are additives used in vaccines and play a crucial role in vaccinology, particularly in enhancing vaccine immunogenicity, improving stability, and promoting longer-lasting protective effects. In the development of RSV vaccines, the use of adjuvants can significantly enhance vaccine immunogenicity, improve its long-term protective efficacy, and potentially reduce the occurrence of adverse reactions.

[0004] However, designing and selecting suitable adjuvants requires a deep understanding of vaccine immunology, the interaction mechanisms of adjuvant components, and the dynamics of the immune response after vaccination. Therefore, the field still needs to further improve the efficacy of RSV vaccines through the rational design and optimization of the composition and ratio of compound adjuvants. Summary of the Invention

[0005] Through extensive and in-depth research, the inventors of this invention have developed a novel respiratory syncytial virus (RSV) vaccine composition that can effectively enhance the immunogenicity of the antigen, stimulate or induce high levels of functional antibodies, improve the immunogenicity of RSV vaccines, and thus effectively prevent RSV infection.

[0006] To achieve the above objectives, the present invention first provides a recombinant respiratory syncytial virus (RSV) vaccine composition comprising RSVpreF protein and a pharmaceutically acceptable composite adjuvant carrier, wherein the composite adjuvant carrier is an aluminum adjuvant combined with a CpG adjuvant.

[0007] Preferably, the aluminum adjuvant is selected from AlPO4 and / or Al(OH)3.

[0008] Preferably, the aluminum adjuvant is selected from AlPO4.

[0009] Preferably, the CpG adjuvant is selected from CpG-7909 and / or CpG-1018.

[0010] Preferably, the CpG adjuvant is CpG-7909.

[0011] Preferably, the RSVpreF protein content in a unit dose of the vaccine composition is 5-300 μg, more preferably 50-300 μg, and even more preferably 150-300 μg.

[0012] Preferably, the aluminum adjuvant content in a unit dose of the vaccine composition is 550–750 μg, more preferably 580–720 μg, and even more preferably 600–700 μg.

[0013] Preferably, the CpG adjuvant content in a unit dose of the vaccine composition is 150–3000 μg, more preferably 300–1200 μg, and even more preferably 300–600 μg.

[0014] Preferably, in a unit dose of the vaccine composition, the content of the RSVpreF protein is 5-300 μg, the content of the aluminum adjuvant is 550-750 μg, and the content of the CpG adjuvant is 150-3000 μg.

[0015] Preferably, the content of RSVpreF protein is 50-300 μg, the content of aluminum adjuvant is 580-720 μg, and the content of CpG adjuvant is 300-1200 μg.

[0016] Preferably, the content of RSVpreF protein is 150-300 μg, the content of aluminum adjuvant is 600-700 μg, and the content of CpG adjuvant is 300-600 μg.

[0017] Preferably, the isoelectric point of AlPO4 is 7.0 to 9.0.

[0018] Preferably, the amino acid sequence of the RSVpreF protein is selected from any one of the following groups:

[0019] (1) The amino acid sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:3;

[0020] (2) An amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and retaining the biological function of the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;

[0021] (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted in the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, and retains the biological function of the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0022] Preferably, the vaccine composition further includes pharmaceutically acceptable excipients.

[0023] Preferably, the excipients include 0.01-0.1 mol / L histidine, 0.05-0.5 mol / L sodium chloride, and 0.001-0.01% polysorbate 80 by mass.

[0024] More preferably, the excipients include 0.02 mol / L histidine, 0.18 mol / L sodium chloride, and 0.006% (w / w) polysorbate 80.

[0025] Secondly, the present invention provides a method for preparing the vaccine composition described in any one of the above claims, the method comprising:

[0026] (1) The RSVpreF protein was mixed with aluminum adjuvant to prepare the adsorption stock solution;

[0027] (2) Add the CpG adjuvant to the adsorption stock solution obtained in step (1) to obtain the vaccine composition.

[0028] Furthermore, the present invention provides a kit comprising the vaccine composition described in any one of the above claims or the vaccine composition prepared by the above methods.

[0029] Preferably, the kit further includes a vaccine administration device, which may include a needle device, a liquid spray device, a powder device, or a spray device. The choice of device is mainly based on different routes of administration, common routes of administration include intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection, or administration via the oral / gastrointestinal tract, respiratory tract, and genitourinary tract mucosa. The vaccine of the present invention can be administered via intramuscular injection, and the administration device is a syringe and an injection needle device.

[0030] Finally, the present invention provides the use of the vaccine composition according to any one of the above claims, the vaccine composition obtained by the preparation method described above, or the kit described above in the preparation of a medicament for the prevention or treatment of respiratory syncytial virus infection-related diseases.

[0031] Compared with the prior art, the RSV vaccine composition provided by the present invention has the following advantages:

[0032] This invention, through the rational design, screening, and optimization of the components and proportions of the compound adjuvant, obtained suitable adjuvant components and effective concentration ranges. The components have a synergistic effect, significantly improving the immunogenicity of the antigen and further enhancing the immune effect of the RSV vaccine. The combined use of the compound adjuvant and RSVpreF protein showed unexpectedly high immune activity, and the vaccine composition had stronger stability. Attached Figure Description

[0033] Figure 1 is a schematic diagram of the immunogenicity test combined with antibody titer results in each group of BALB / c mice.

[0034] Figure 2 is a schematic diagram of the neutralizing antibody titer results in the immunogenicity experiment of each group of BALB / c mice;

[0035] Figure 3 is a schematic diagram of the IFN-γ secretion results in the immunogenicity experiment of each group of BALB / c mice;

[0036] Figure 4 is a schematic diagram of the IL-2 secretion results in the immunogenicity experiment of each group of BALB / c mice;

[0037] Figure 5 is a schematic diagram of the binding antibody levels in BALB / c mice in each group 49 days after a single immunization in Example 4.

[0038] Figure 6 is a schematic diagram of the IFN-γ secretion results in each group of BALB / c mice in Example 4. The significant differences are *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, and ns: no significant difference.

[0039] Figure 7 is a schematic diagram of the IL-2 secretion results in each group of BALB / c mice in Example 4. The significant differences are *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, and ns: no significant difference.

[0040] Figure 8 shows the titer of specific binding antibodies in monkey serum 42 days after immunization in the immunogenicity study. *P<0.05, ****P<0.0001, ns: no significant difference.

[0041] Figure 9 shows the titers of RSVpreF-specific binding antibodies at different time points after immunization in the immunogenicity study of monkeys.

[0042] Figure 10 shows the bar chart of neutralizing antibody titers in monkey serum 42 days after immunization in the immunogenicity study of monkeys. Among them, the significant differences are *P<0.05, **P<0.01, ***P<0.001, and ns: no significant difference.

[0043] Figure 11 is a schematic diagram showing the results of neutralizing antibody titers at different time points after immunization in a study on immunogenicity in monkeys. Detailed Implementation

[0044] To make the technical solution and beneficial effects of the present invention more apparent and understandable, a detailed description is provided below by listing specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application pertains.

[0045] As used herein, the term "adjuvant" refers to a nonspecific immune enhancer that, when delivered to the body along with or before an antigen, can enhance the body's immune response to the antigen or alter the type of immune response. There are many types of adjuvants, including but not limited to aluminum adjuvants (e.g., aluminum phosphate), Freund's adjuvants (e.g., complete and incomplete Freund's adjuvants), Corynebacterium breve, lipopolysaccharides, and cytokines.

[0046] As used herein, the term "pharmaceuticalally acceptable excipient" can be, for example, inert diluents such as water or other solvents, solubilizers and emulsifiers such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, oils (specifically cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, fatty acid esters of sorbitol, and mixtures thereof; in addition to inert diluents, oral compositions may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, coloring agents, flavoring agents, and preservatives; suspension formulations may also contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and sorbitol esters, microcrystalline cellulose, aluminum hydroxide, bentonite, agar and tragali gum, and mixtures thereof; these compositions may also contain excipients such as wetting agents, emulsifiers, and dispersants. It may also be necessary to include isotonic agents in the composition, such as sugars or sodium chloride. Additionally, prolonged absorption of injectable drugs can be achieved by incorporating substances that delay absorption, such as aluminum monostearate and gelatin.

[0047] As used in this article, the term "immunogenicity" refers to the ability to stimulate the body to produce specific antibodies or sensitized lymphocytes. It refers both to the property of an antigen to stimulate specific immune cells, causing them to activate, proliferate, and differentiate, ultimately producing immune effector substances such as antibodies and sensitized lymphocytes, and to the body's immune system's ability to form a specific immune response of antibodies or sensitized T lymphocytes after antigen stimulation. Immunogenicity is the most important property of an antigen; whether an antigen can successfully induce an immune response in the host depends on three factors: the nature of the antigen, the host's reactivity, and the mode of immunization.

[0048] The present invention first provides a recombinant respiratory syncytial virus (RSV) vaccine composition, wherein the recombinant RSV vaccine comprises RSVpreF protein and a pharmaceutically acceptable composite adjuvant carrier, wherein the composite adjuvant carrier is an aluminum adjuvant combined with a CpG adjuvant.

[0049] In some embodiments, the aluminum adjuvant is selected from AlPO4 and / or Al(OH)3.

[0050] In some embodiments, the aluminum adjuvant is selected from AlPO4.

[0051] In some embodiments, the CpG adjuvant is selected from CpG-7909 and / or CpG-1018.

[0052] In some embodiments, the CpG adjuvant is CpG-7909.

[0053] In this application, the CpG adjuvant is well known to those skilled in the art and has been described in the prior art, such as in international applications WO 96 / 02555 and WO 99 / 33488. For example, CpG 7909 or CpG1018 of this application can be obtained through conventional commercial channels. Of course, those skilled in the art can also design artificially synthesized sequences as needed.

[0054] The fundamental principle of vaccine formulation development is that each dose of vaccine should be sufficient to induce a protective immune response in the recipient without significant toxic side effects. Generally, different antigen proteins require different dosages, and the optimal dosage for a specific vaccine can be determined by observing antibody titers and other responses in the recipient.

[0055] In some embodiments, the RSV preF protein content per unit dose of the vaccine composition is 5–300 μg, for example 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, 100 μg, 105 μg, 110 μg, 115 μg, 120 μg, 125 μg, 13 μg, etc. 0μg, 135μg, 140μg, 145μg, 150μg, 155μg, 160μg, 165μg, 170μg, 175μg, 180μg, 185μg, 190μ g, 195μg, 200μg, 210μg, 220μg, 230μg, 240μg, 250μg, 260μg, 270μg, 280μg, 290μg, 300μg, etc.

[0056] In some embodiments, the RSV preF protein content is 50–300 μg per unit dose of the vaccine composition.

[0057] In a preferred embodiment, the RSV preF protein content in a unit dose of the vaccine composition is 150–300 μg.

[0058] In some embodiments, the aluminum adjuvant content in a unit dose of the vaccine composition is 550–750 μg, for example, 560 μg, 570 μg, 580 μg, 590 μg, 605 μg, 610 μg, 615 μg, 620 μg, 625 μg, 630 μg, 635 μg, 640 μg, 645 μg, 650 μg, 655 μg, 660 μg, 665 μg, 670 μg, 675 μg, 680 μg, 685 μg, 690 μg, 695 μg, 700 μg, 705 μg, 710 μg, 715 μg, 720 μg, 725 μg, 730 μg, 735 μg, 740 μg, 745 μg, etc.

[0059] In some embodiments, the aluminum adjuvant content in a unit dose of the vaccine composition is 580–720 μg.

[0060] In a preferred embodiment, the aluminum adjuvant content in a unit dose of the vaccine composition is 600–700 μg.

[0061] In some embodiments, the CpG adjuvant content per unit dose of the vaccine composition is 150–3000 μg, for example, 200 μg, 250 μg, 300 μg, 350 μg, 400 μg, 450 μg, 500 μg, 550 μg, 600 μg, 650 μg, 700 μg, 750 μg, 800 μg, 850 μg, 900 μg, 950 μg, 1000 μg, 1050 μg, 1100 μg, 11 50μg, 1200μg, 1250μg, 1300μg, 1350μg, 1400μg, 1450μg, 1500μg, 1550μg, 1600μg, 1700μg, 1800μg, 1 900μg, 2000μg, 2100μg, 2200μg, 2300μg, 2400μg, 2500μg, 2600μg, 2700μg, 2800μg, 2900μg, 3000μg, etc.

[0062] In some embodiments, the CpG adjuvant content in a unit dose of the vaccine composition is 300–1200 μg.

[0063] In some embodiments, the CpG adjuvant content is 300–600 μg per unit dose of the vaccine composition.

[0064] In some embodiments, the RSV preF protein content is 5–300 μg, the aluminum adjuvant content is 550–750 μg, and the CpG adjuvant content is 150–3000 μg per unit dose of the vaccine composition.

[0065] In some embodiments, the RSV preF protein content is 50–300 μg, the aluminum adjuvant content is 580–720 μg, and the CpG adjuvant content is 300–1200 μg.

[0066] In some embodiments, the RSV preF protein content is 150–300 μg, the aluminum adjuvant content is 600–700 μg, and the CpG adjuvant content is 300–600 μg.

[0067] In some embodiments, the isoelectric point of the AlPO4 is 7.0 to 9.0.

[0068] In some embodiments, the amino acid sequence of the RSVpreF protein is selected from any of the following groups:

[0069] (1) The amino acid sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:3;

[0070] (2) An amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and retaining the biological function of the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;

[0071] (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted in the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, and retains the biological function of the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0072] SEQ ID NO:1:

[0073] MELLIHRSSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYHCVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTCAVGSGSGGSGSGRSLGFLLGVGSAIASGC AVSKVLHLEGEVNKIKNALQLTNKAVVSLSNGVSVLCSRVLDLKNYINNQLLPMVNRQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQ QSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAI VSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSSEFDASISQVNEKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL

[0074] SEQ ID NO:2:

[0075] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTCATGSGSGGSGSGRSLGFLLGVGSAIASGCAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLCSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL

[0076] SEQ ID NO:3:

[0077] MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLSALRTGWYHCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTCAAGSGSGGSGSGRSLGFLLGVGSAIASGC AVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLCSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQ QSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAI VSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL

[0078] In this invention, the above-mentioned antigen can be obtained using conventional techniques of modern molecular biology. A typical method includes expressing the above-mentioned RSVpreF protein in CHO cells, comprising the following steps:

[0079] (1) The RSVpreF protein gene of the present invention was cloned into the expression vector;

[0080] (2) Transfect the expression vector obtained in step (1) into CHO cells;

[0081] (3) By screening mixed clonal cell populations and single clonal cells, an engineered cell line that can stably express RSVpreF protein was obtained.

[0082] (4) Use the cell line described in step (3) to express and obtain RSVpreF protein.

[0083] The proteins obtained above can be processed using conventional methods, such as hydrophobic chromatography, anion exchange chromatography, hydroxyapatite chromatography, ultrafiltration, and nanofiltration, to obtain antigen proteins with a purity of over 95%.

[0084] It should be noted that the method of stably expressing RSVpreF protein using CHO cell lines is a well-known method in the art, and specific details can be found in *Molecular Cloning: A Laboratory Manual* and other literature, such as Haumont M et al., *Virus Research 40 (1996)*, 199-204, "Purification, characterization and immunogenicity of recombinant varicella-zoster virus glycoprotein gE secreted by Chinese hamsterovary cells." Those skilled in the art can also choose other expression systems, such as *Escherichia coli* and *Pichia pastoris*, to obtain RSVpreF protein.

[0085] In some embodiments, the vaccine composition further includes pharmaceutically acceptable excipients.

[0086] In a preferred embodiment, the excipients include 0.01–0.1 mol / L histidine, 0.05–0.5 mol / L sodium chloride, and 0.001–0.01% polysorbate 80 by mass.

[0087] In a preferred embodiment, the concentration of histidine is 0.01 to 0.05 mol / L, for example, 0.02 mol / L, 0.03 mol / L, 0.04 mol / L, 0.05 mol / L, etc.

[0088] In a preferred embodiment, the concentration of sodium chloride is 0.05–0.3 mol / L, for example, 0.06 mol / L, 0.07 mol / L, 0.08 mol / L, 0.1 mol / L, 0.12 mol / L, 0.14 mol / L, 0.15 mol / L, 0.16 mol / L, 0.17 mol / L, 0.18 mol / L, 0.19 mol / L, 0.2 mol / L, 0.22 mol / L, 0.24 mol / L, 0.26 mol / L, 0.28 mol / L, etc.

[0089] In a preferred embodiment, the mass fraction of the polysorbate 80 is 0.001 to 0.008%, for example, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, etc.

[0090] In a specific and preferred embodiment, the excipients include 0.02 mol / L histidine, 0.18 mol / L sodium chloride, and 0.006% (w / w) polysorbate 80.

[0091] The specific preparation method of the vaccine formulation provided by this invention can be found in, for example, the book "Vaccine Design: The Subunit and Adjuvant Approach" (ISBN: 0-306-44867-X). As one of the most conventional methods, the preparation method provided by this invention involves mixing the aforementioned antigen protein with the aforementioned compound adjuvant.

[0092] In some embodiments, the present invention provides a method for preparing the vaccine composition according to any one of the above claims, the method comprising:

[0093] (1) The purified RSVpreF protein was mixed with an aluminum adjuvant (e.g., AlPO4) to prepare the adsorption stock solution;

[0094] (2) Add the CpG adjuvant to the adsorption stock solution obtained in step (1) to obtain the vaccine composition.

[0095] In a preferred embodiment, the RSVpreF protein is first adsorbed onto an aluminum adjuvant (e.g., AlPO4), and then the CpG adjuvant is also adsorbed onto the aluminum adjuvant (e.g., AlPO4) to maintain the stability of the RSVpreF protein and enhance its immunogenicity.

[0096] Furthermore, the present invention provides a kit comprising the vaccine composition described in any one of the above claims or the vaccine composition prepared by the above methods.

[0097] In some embodiments, the kit further includes a vaccine administration device, which may include a needle device, a liquid spray device, a powder device, or a spray device. The choice of device is primarily based on different routes of administration, common routes of administration including intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection, or administration via the oral / gastrointestinal tract, respiratory tract, and genitourinary tract mucosa. The vaccine of the present invention can be administered via intramuscular injection, and the administration device is a syringe and an injection needle device.

[0098] Finally, the present invention provides the use of the vaccine composition according to any one of the above claims, the vaccine composition obtained by the preparation method described above, or the kit described above in the preparation of a medicament for the prevention or treatment of respiratory syncytial virus infection-related diseases.

[0099] The present invention will be further described below through specific embodiments. Unless otherwise specified, "%" represents a mass percentage. The materials and reagents used in the following embodiments are all commonly used materials or reagents in the art, and can be obtained commercially or synthesized by known methods. Experimental methods in the following embodiments without specified conditions are generally performed according to conventional experimental conditions or the conditions recommended by the manufacturer of the relevant reagent (kit).

[0100] Example 1: Preparation and purification of RSVpreF protein

[0101] The coding gene sequence of respiratory syncytial virus (RSVpreF) protein (amino acid sequence as shown in SEQ ID: NO.3) was synthesized and amplified. The RSVpreF gene amplification fragment and the OPM-V02 expression vector were digested with restriction endonucleases EcoRI and HindIII, respectively. The two were then ligated to obtain a recombinant construct plasmid. After confirming the sequence, the plasmid was linearized and electroporated into CHO cells for expression and purification to obtain RSVpreF protein.

[0102] Example 2: Preparation of a vaccine composition containing RSVpreF fusion protein

[0103] This embodiment prepares several vaccine compositions, the components and proportions of which are shown in Table 1. The specific preparation method is as follows: First, the RSVpreF antigen stock solution obtained according to the preparation method in Example 1 is adsorbed onto an aluminum adjuvant (aluminum phosphate adjuvant / aluminum hydroxide) to prepare adsorption samples with different RSVpreF antigen / aluminum adjuvant (w / w) ratios (the aluminum adjuvant content here is essentially the aluminum element content); then, different concentrations of CpG adjuvant are added to the RSVpreF antigen / aluminum adsorption samples. After thorough mixing, the above preparations are stored at 2–8°C protected from light. The isoelectric point (pI) of AlPO4 in groups 1–7 and 10–14 in Table 1 is 7.0–9.0.

[0104] Table 1

[0105]

[0106] Example 3: Mouse Immunization Experiment with Respiratory Syncytial Virus Vaccine

[0107] The components prepared according to the method in Example 2 were taken out and immunogenicity studies were carried out using BALB / c mice as an animal model to determine the immunogenicity of the respiratory syncytial virus vaccine in mice and the effectiveness of different adjuvant systems.

[0108] BALB / c mice were randomly divided into groups of eight. Different vaccine components and negative control samples (groups 15, PBS) were administered intramuscularly, with an injection volume of 0.1 ml per mouse. Group 10 received a single immunization on day 0, while the other groups received two immunizations on days 0 and 21. Blood samples were collected from each group on days 0, 21, and 35, and serum was separated for antibody titer detection. On day 35, eight mice from each group were harvested, and spleen cells were isolated for ELISPOT assay. The immunogenicity evaluation method followed standard technical procedures in this field. For example, a more detailed procedure is as follows:

[0109] (1) Detection of binding antibodies

[0110] Peripheral blood was collected and incubated overnight at 4°C, then centrifuged at 10,000 rpm for 10 min to separate serum, which was then refrigerated at -80°C. Antigen was diluted to 4 μg / ml with PBS, and 100 μl of the diluted antigen was used to coat 96-well plates, incubating overnight at 4°C. After washing the coated 96-well plates with PBST, 200 μl of 5% skim milk was added to each well, and the plates were blocked at 37°C for 2 h. After blocking, the plates were washed with PBST. Serum was diluted 4-fold with 2% skim milk to the initial concentration and added to 96-well plates, incubating at 37°C for 1 h. After washing with PBST, diluted HRP-labeled secondary antibody (using goat anti-mouse secondary antibody) was added, and the plates were incubated at 37°C for 1 h. After incubation, the plates were washed with PBST, and 100 μl of TMB chromogenic solution was added to each well, incubating at room temperature in the dark for 15 min. After chromogenic development, 50 μl of 2M chromogenic solution was added to each well. H2SO4 was used to stop the color development, and the ELISA reader reading was taken. Antibody titer determination: 2.1 times the OD value of the negative control group (calculated as 0.05 if less than 0.05) was used as the cut-off value, and the highest serum dilution with an OD greater than the cut-off value was used as the antibody titer of that serum.

[0111] (2) Detection of neutralizing antibodies

[0112] Cell plating: Hep-2 cells were digested and seeded into 96-well cell culture plates. The cells were placed in a CO2 incubator (37℃, 5% CO2) overnight to ensure cell confluence greater than 95% the next day. Serum dilution: Serum was serially diluted using DMEM maintenance medium. Virus dilution: Virus was diluted using DMEM maintenance medium. Neutralization incubation: Equal volumes of diluted serum and virus were mixed and neutralized at 37℃ for 1 h. Cell incubation: Cell supernatant was removed from the 96-well plates, and 200 μl of the neutralization mixture was added to each well. The 96-well plates were incubated in a CO2 incubator for 48 h before fluorescence detection. Cell fixation: After 48 h, cell supernatant was removed, and 50 μl of ice-cold methanol was added to each well. The culture plates were fixed at -20℃ for 20 min. Primary antibody incubation: The plates were washed 3 times with PBS. Dilute Motavizumab antibody to a final concentration of 6 ng / μl using PBS wash buffer, 50 μl per well, and incubate at 37°C for 1.5 h; Secondary antibody incubation: discard the primary antibody and wash the plate 3 times. Dilute FITC-goat anti-human IgG fluorescent secondary antibody using PBS wash buffer containing 0.02% Evans blue, 50 μl per well, and incubate at 37°C for 50 min; Fluorescence development: discard the secondary antibody and wash the plate 3 times. Read the fluorescent spots using an ELISA reader; Data analysis: Calculate the NT50 or NT90 values.

[0113] (3) ELISPOT experiment

[0114] Fourteen days after the second immunization, eight mice from each group were euthanized by cervical induction and their spleens were harvested. The levels of IFN-γ and IL-2 induced by stimulation of lymphocytes with F protein-derived peptide pools were detected using ELISPOT technology.

[0115] ELISPOT plate blocking: Take a 96-well PVDF plate from the Mouse ELISPOT kit, add 200 μl of 1640 complete medium to each well, and block at room temperature for at least 30 min; Spleen cell thawing: Remove spleen cells from the -80℃ freezer and thaw rapidly at 37℃; Add 0.5 ml of 1640 complete medium to each tube and let rest at 37℃ for 1 h, then count cells and adjust cell concentration; Spleen cell plating: Discard the blocking solution in the 96-well PVDF plate, and add 100 μl of isolated and counted mouse lymphocytes to each well; Stimulation: Dilute RSV-F peptide library, DMSO, PMA, and calcium ionomycin with 1640 complete medium and add 10 μl to each well of the 96-well PVDF plate. Incubate at 37℃ for 48 h in a CO2 incubator; After incubation, discard the cells in the plate and wash the plate 5 times (PBS); Primary antibody incubation: Dilute mouse detection antibodies R4-6A2-Biotin and 5H4-Biotin and mix well. Add 100 μl of antibody dilution to each well and incubate at room temperature for 2 h. Discard the cells in the plate and wash the plate 5 times. Secondary antibody incubation: Dilute Streptavidine-ALP and mix well. Add 100 μl to each well and incubate at room temperature for 1 h. Discard the cells in the plate and wash the plate 5 times. Add 100 μl of chromogenic reagent (BCIP / NBT-plus) to each well and react at room temperature in the dark for 20 min. Remove the chromogenic reagent and peel off the adhesive pad at the bottom of the plate. Wash the plate with ultrapure water to stop the chromogenic reaction. Allow the 96-well plate to dry completely in the dark. Read the spots using an ELISA speckle analyzer, save and analyze the experimental results.

[0116] The specific results are as follows: The results of each group are shown in Table 2 and Figures 1 to 4.

[0117] Table 2

[0118]

[0119] The results showed that, based on groups 1, 2, 3 and 15, the serum antibody titer, neutralizing antibody titer, and the level of cytokine secretion induced by mouse spleen cells were significantly higher in the single-antigen group (group 1) than in the blank control group (group 15, PBS), but significantly lower in the single-adjuvant group (group 2). Furthermore, the serum antibody titer and neutralizing antibody titer in the single-adjuvant group (group 2) were lower than those in the antigen-adjuvant combination group (group 3).

[0120] Comparing the serum binding antibody titers and neutralizing antibody titers of mice in different aluminum adjuvant groups within the combined adjuvant groups revealed that, when the antigen and CpG contents were the same (groups 4 and 8), the effects of aluminum adjuvants AlPO4 and Al(OH)3 were similar, significantly higher than those in group 9 (aluminum adjuvant AlPO4, pI = 4.0). However, in inducing cytokine secretion by mouse spleen cells, the aluminum adjuvant AlPO4 group was better than the aluminum adjuvant Al(OH)3 group.

[0121] Comparison of the effects of different CpG adjuvants in the combined adjuvant group revealed that when the antigen and AlPO4 content were the same (groups 4 and 7), both CpG-7909 and CpG-1018 could increase the titer of binding antibodies. However, when CpG-7909 was used, it was superior to CpG-1018 in terms of neutralizing antibody titer and in inducing cytokine secretion by mouse spleen cells.

[0122] Comparison of the effects of different CpG doses in the combined adjuvant groups revealed that when the antigen and AlPO4 contents were the same (groups 3-6), there was no significant difference in the titers of binding antibodies and neutralizing antibodies in mouse serum at different CpG-7909 concentrations. Compared with group 4, increasing the antigen content led to an increase in the titers of binding antibodies and neutralizing antibodies in mouse serum, with the highest increase being 4-5 times (e.g., group 13). Simultaneously, compared with group 4, the cytokine secretion levels in groups 11 and 13 / 14 were also significantly increased, ranging from 3-7 times that of group 4. Additionally, serum IgG... 2a The results of the / IgG1 antibody ratio detection were basically consistent with the results of cell-mediated immunity. After increasing the antigen dose, IgG... 2a The change in the IgG1 ratio from less than 1 to greater than 1 indicates that the cellular immune response has been enhanced.

[0123] Experimental results show that the RSVpreF antigen / AlPO4 / CpG-7909 combined adjuvant system vaccine can stimulate cellular immunity in mice and has excellent cellular immune effects.

[0124] Example 4: Immunogenicity and Protective Effects in Mice

[0125] This embodiment uses BALB / c mice as an animal model to conduct in vivo immunogenicity and protective effects experiments in mice.

[0126] BALB / c mice were randomly divided into groups of 12. Group 1 served as a blank control and received no treatment. The other five groups received intramuscular injections of PBS, formalin-inactivated vaccine, GSK-AREXVY vaccine, and different batches of vaccine samples (see Table 3), respectively, with an injection volume of 0.05 ml per mouse. Immunization was performed on days 0 and 21, and blood samples were collected on days 0, 21, and 49 for serum separation and antibody titer detection. On day 50, all mice except Group 1 were intranasally infected with RSVA long at a challenge dose of 2.5 × 10⁻⁶. 7 PFU was administered via nasal drops at a volume of 116 μl per mouse. On day 55, the first 8 mice from each group were euthanized, blood was collected, and lymphocytes were isolated from the spleen for ELISPOT assay.

[0127] Table 3

[0128]

[0129] Twenty-eight days after the second immunization, the titer of binding antibodies in mouse serum was measured (using the same method as in Example 3). The level of binding antibodies in mice 49 days after the first immunization is shown in Figure 5. The experimental results indicate that the titers of binding antibodies in the serum of mice treated with the low, medium, and high doses of this invention are higher than those of the GSK-AREXVY vaccine.

[0130] Five days after challenge with the virus, mice were sacrificed and their spleens were harvested. The levels of IFN-γ induced by stimulation of lymphocytes with an F-based peptide pool were detected using ELISPOT technology (the method was the same as in Example 3). The results of IFN-γ secretion are shown in Figure 6, and the results of IL-2 secretion are shown in Figure 7. The results showed that the levels of immune cytokines secreted by the low, medium, and high dose vaccines of this invention were all higher than those of the GSK-AREXVY vaccine.

[0131] Example 5: Stability Comparison Study with Marketed Vaccine Antigens

[0132] (1) Sample information and source

[0133] RSVpreF, amino acid sequence as shown in SEQ ID: NO.3, is the original solution produced by our company.

[0134] DS-Cav1: The amino acid sequence of the DS-Cav1 protein was obtained from a previously reported literature (Jason S McLellan et al., Science. 2013 Nov 1; 342(6158):592-8), and was obtained through affinity purification after transient transfection expression in mammalian cells. The DS-Cav1 protein is the antigen used in GSK's marketed RSV vaccine AREXVY.

[0135] (2) Sample processing

[0136] Sample adsorption was performed according to Table 6 (each sample volume was 0.5 ml / dose); samples were placed in a dark environment at 25℃±2℃ for 0 days and 7 days, respectively. The samples were mixed with the desorption buffer at a ratio of 1:3 (volume ratio), placed in a constant temperature incubator, and inverted overnight at 25℃.

[0137] Table 6

[0138] Sample Information: Antigen Content (μg / dose) AlPO4 (μg / dose) CpG-7909 (μg / dose) RSVpreF+AlPO4+CpG-7909 150 700 600 DS-Cav1+AlPO4+CpG-7909 150 700 600 surface

[0139] (3) Sample testing

[0140] Affinity was measured for each group of samples using the Octet molecular interaction analyzer. Probes: Octet AHC2 Biosensors; Antibody: D25 10 μg / mL; Regeneration buffer: 10 mM Gly (pH 1.5); Detection procedures are shown in Table 7.

[0141] Table 7

[0142] Program timings: Baseline (PBST) 60s, Loading (antibody) 180s, Baseline 2 (PBST) 60s, Association (sample) 90s, Dissociation (PBST) 120s, Regeneration (regeneration solution) 30s surface

[0143] (4) Test results

[0144] The D25 antibody used in this assay specifically binds to the pre-fusion conformational epitope Φ (critical neutralizing epitope) of the RSVF protein. Affinity was measured on samples 0 days and 7 days after desorption, determining the KD affinity between the RSVpreF sample and antibody D25. The results are shown in Table 8. The results indicate that under the formulation conditions described in this patent, the RSVpreF stock solution maintained an affinity of -12 gamma after 7 days at 25°C, demonstrating conformational stability. In contrast, the DS-Cav1 sample showed a significant decrease in affinity, decreasing from -11 gamma to -10 gamma. This demonstrates that under the formulation conditions described in this patent, the RSVpreF protein in this product exhibits superior structural stability compared to the DS-Cav1 protein.

[0145] Table 8

[0146] Sample Information 0 days 7 days RSVpreF++AlPO4+CpG-79091.39E-121.58E-12DS-Cav1+AlPO4+CpG-79099.83E-112.97E-10 surface

[0147] Example 6: Immunogenicity Study in Monkeys

[0148] Rhesus monkeys were randomly divided into three groups (G1–G3), with four monkeys in each group, half male and half female. The drugs were administered via intramuscular injection. Groups G1, G2, and G3 received a single dose on Day 0. Specific grouping and administration protocols are shown in Table 9. The amino acid sequence of the antigen is shown in SEQ ID: NO.3. Blood samples were collected from all animals on days -4, 21, 42, and 70. Serum and PBMCs were separated, and the titers of specific binding antibodies, neutralizing antibodies, and Th1 / Th2 cytokine levels were measured.

[0149] Table 9

[0150]

[0151] (1) The method for detecting serum RSVpreF / postF antigen-specific binding antibodies was the same as in Example 3. Serum samples were collected from each group of monkeys 42 days after immunization, and the titers of binding antibodies against pre-fusion F protein (RSVpreF) and post-fusion F protein (RSVpostF) in the monkey serum were detected. The results showed that both the medium-dose and high-dose groups induced high titers (>10) 42 days after immunization. 4 The titer of binding antibodies against RSV preF was higher in the medium-dose and high-dose groups than in the GSK-AREXVY group. Meanwhile, the RSV preF / postF specific antibody ratio was slightly higher in the medium-dose and high-dose groups than in the GSK-AREXVY group (see Figure 8).

[0152] Twenty-one days post-immunization, the RSVpreF-specific binding antibody titers in the medium-dose group, high-dose group, and GSK-AREXVY group reached peak values, with GMT values ​​of 16384, 92682, and 10321, respectively. At 70 days post-immunization, the antibody titers in the medium- and high-dose groups remained higher than those in the GSK-AREXVY group (see Figure 9). This indicates that the vaccine composition of the present invention can induce high levels of RSVpreF-specific IgG binding antibodies in rhesus monkeys and exhibits good immunogenicity, superior to the marketed vaccine AREXVY.

[0153] (2) The method for detecting serum neutralizing antibodies was the same as in Example 3. Serum from monkeys in each group was collected 42 days after immunization, and the titers of neutralizing antibodies against different respiratory syncytial virus strains (RSV-A2, RSV-A-Long, RSV-B-18537, and RSV-B-9320) were detected. The results showed that GSK-AREXVY, the medium-dose group, and the high-dose group all showed strong neutralizing effects against the four respiratory syncytial virus strains, with the best neutralizing effect against RSV-A2. The neutralizing antibody titers in the high-dose group and the medium-dose group were higher than those in the marketed vaccine AREXVY (see Figure 10). The duration of immunity of the vaccine was further evaluated (see Figure 11). The results showed that the neutralizing antibody titers in the medium-dose group and the high-dose group were higher than those in the marketed vaccine AREXVY at different time points.

[0154] (3) The detection method for Th1 / Th2 cytokines in monkey PBMCs was the same as in Example 3. The ELISPOT kit (ELISpot Plus:Monkey IFN-γALP, ELISpot Plus:Monkey IL-2ALP) was used to detect Th1 / Th2 cytokines produced by monkey PBMC stimulation 42 days after immunization. The results showed that after stimulation with the RSV F peptide library, the levels of cytokines (IL2, IFN-γ) induced by the vaccine in the medium-dose and high-dose groups were higher than those of the marketed vaccine AREXVY, as shown in Table 10.

[0155] Table 10

[0156]

[0157] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations included in the claims. Various modifications and changes can be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments can be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only illustrate several implementations of the present invention and do not limit the scope of protection of this patent.

Claims

1. A recombinant respiratory syncytial virus vaccine composition, characterized in that, The recombinant respiratory syncytial virus vaccine composition comprises RSVpreF protein and a pharmaceutically acceptable complex adjuvant carrier, wherein the complex adjuvant carrier is an aluminum adjuvant combined with a CpG adjuvant, preferably, the aluminum adjuvant is selected from AlPO4 and / or Al(OH)3; preferably, the CpG adjuvant is selected from CpG-7909 and / or CpG-1018.

2. The vaccine composition according to claim 1, characterized in that, In a unit dose of the vaccine composition, the content of the RSVpreF protein is 5-300 μg, preferably 50-300 μg, and more preferably 150-300 μg.

3. The vaccine composition according to claim 1, characterized in that, In a unit dose of the vaccine composition, the content of the aluminum adjuvant is 550–750 μg, preferably 580–720 μg, and more preferably 600–700 μg.

4. The vaccine composition according to claim 1, characterized in that, In a unit dose of the vaccine composition, the content of the CpG adjuvant is 150–3000 μg, preferably 300–1200 μg, and more preferably 300–600 μg.

5. The vaccine composition according to claim 1, characterized in that, In a unit dose of the vaccine composition, the content of the RSVpreF protein is 5-300 μg, the content of the aluminum adjuvant is 550-750 μg, and the content of the CpG adjuvant is 150-3000 μg; preferably, the content of the RSVpreF protein is 50-300 μg, the content of the aluminum adjuvant is 580-720 μg, and the content of the CpG adjuvant is 300-1200 μg; preferably, the content of the RSVpreF protein is 150-300 μg, the content of the aluminum adjuvant is 600-700 μg, and the content of the CpG adjuvant is 300-600 μg.

6. The vaccine composition according to claim 1, characterized in that, The aluminum adjuvant is selected from AlPO4, and the isoelectric point of AlPO4 is 7.0 to 9.0; preferably, the CpG adjuvant is CpG-7909; preferably, the amino acid sequence of the RSVpreF protein is selected from any one of the following groups: (1) the amino acid sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:3; (2) an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and retaining the biological function of the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; (3) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted, or inserted in the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and retaining the biological function of the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; Biological function of the amino acid sequence shown in NO:

3.

7. The vaccine composition according to any one of claims 1 to 6, characterized in that, The vaccine composition further includes pharmaceutically acceptable excipients; preferably, the excipients include 0.01–0.1 mol / L histidine, 0.05–0.5 mol / L sodium chloride, and 0.001–0.01% polysorbate 80 by mass.

8. A method for preparing the vaccine composition according to any one of claims 1 to 7, characterized in that, The preparation method includes: (1) mixing RSVpreF protein with aluminum adjuvant to prepare an adsorption stock solution; (2) adding the CpG adjuvant to the adsorption stock solution obtained in step (1) to obtain the vaccine composition.

9. A reagent kit, characterized in that, The vaccine composition comprises any one of claims 1 to 7 or a vaccine composition prepared by the method of claim 8.

10. Use of the vaccine composition according to any one of claims 1 to 7, the vaccine composition obtained by the preparation method according to claim 8, or the kit according to claim 9 in the preparation of a medicament for the prevention or treatment of respiratory syncytial virus infection-related diseases.

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