Respiratory syncytial virus mRNA vaccine and preparation method and use thereof

Abstract

In order to further reduce the risk of respiratory syncytial virus in immunocompromised populations, novel mRNA vaccines for the prevention of RSV are provided that are superior in stability and significantly enhance humoral immunity after vaccination.SOLUTION: A respiratory syncytial virus (RSV) vaccine comprising RNA encoding an RSVF protein or mutant thereof, and methods of making the same. The vaccine can prevent RSV infection and its complications.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure is in the field of nucleic acid vaccines, and specifically relates to respiratory syncytial virus mRNA vaccines and their preparation methods and uses. [Background technology]

[0002] Respiratory syncytial virus (RSV) is a negative-stranded single-stranded RNA virus in the genus Pneumovirus and family Paramyxoviridae. In 1956, scientists isolated RSV from a chimpanzee's respiratory tract. The virus was named RSV because it induces fusion of adjacent cells in cell culture, transforming them into syncytial-like structures.

[0003] RSV is an important pathogen causing lower respiratory tract infections in infants, young children, the elderly, and immunocompromised patients. RSV is transmitted through the air and enters the trachea and lungs via inhalation through the mouth and nose. It invades epithelial cells, causing airway damage, and respiratory mucus secreted from the surface of the airways blocks the lumen, resulting in respiratory distress. Common symptoms of RSV infection include runny nose, fever, cough, and asthma. Severe cases can lead to respiratory distress or airway obstruction, leading to respiratory failure and even death.

[0004] RSV is a common respiratory virus. RSV infection, which typically causes cold-like symptoms, occurs primarily in the fall, winter, and spring and is one of the leading causes of serious respiratory illness in infants and young children under 5 years of age and elderly people over 65 years of age. In most young people, RSV causes mild, cold-like symptoms; however, in infants, young children, and elderly people, RSV infection can become fatal.

[0005] Respiratory syncytial virus (RSV) infection has become a global public health problem. RSV is the most common viral pathogen causing acute lower respiratory tract infections (ALRTIs) in children under 5 years of age worldwide and is the leading cause of hospitalization due to viral respiratory infections in infants and young children. Data show that RSV infections account for 28% of all ALRTIs, with hospital deaths due to RSV accounting for 13–22% of ALRTI deaths. According to a World Health Organization (WHO) report, in 2019, there were approximately 33 million cases of RSV-related acute lower respiratory tract infections (ALRTIs) in children under 5 years of age worldwide, of which approximately 3.57 million children required hospitalization, approximately 1.4 million of which occurred in infants aged 0–6 months. Over 100,000 cases and deaths occurred, with over 95% occurring in low- and middle-income countries.

[0006] RSV infection is characterized by global epidemics, which are influenced by factors such as geographic location, temperature, and humidity. For example, the RSV epidemic season in northern China begins in mid-October and ends in mid-May of the following year, while in southern China, the frequency of outbreaks is higher in winter and spring, closely related to temperature. RSV has only one serotype, which is divided into two subtypes, A and B, and in northern China, epidemics tend to alternate.

[0007] Vaccines are the most cost-effective and effective preventative measure against RSV infection. The WHO has identified RSV vaccine development as one of its urgent priorities. However, since RSV vaccine development began in the 1960s, no effective vaccine has been approved for sale by a regulatory authority. Recently, there have been a series of major breakthroughs in RSV vaccines. On May 3, 2023, Arexvy, a respiratory syncytial virus (RSV) vaccine developed by GSK, received marketing approval from the FDA, becoming the first RSV vaccine approved for sale worldwide. This vaccine is primarily used to prevent lower respiratory tract (bronchial and pulmonary) disease caused by RSV infection in patients aged 60 years and older. On June 1, 2023, the US FDA approved the Pfizer RSV protein vaccine, Abrysvo.TM It is used to prevent acute respiratory and lower respiratory tract disease caused by RSV in adults 60 years of age and older.

[0008] RSV vaccines currently under development include live-attenuated vaccines, subunit vaccines, recombinant vector vaccines, and mRNA vaccines. Conventional vaccines are typically based on inactivated vaccines, live-attenuated influenza vaccines, or pathogen-derived subunit proteins. While these vaccines produce effective immune responses, they also have numerous safety issues. For example, the inactivated RSV vaccines developed early on not only failed to protect recipients but also resulted in enhanced respiratory disease (ERD), meaning that they not only failed to prevent RSV infection in recipients but also worsened their condition after infection. In clinical trials of the inactivated RSV vaccine, 16 of 20 infants in the test group developed severe symptoms and required hospitalization, with an 80% hospitalization rate, and two infants died.

[0009] mRNA vaccines involve the introduction of mRNA encoding viral antigens into the body, where it is expressed in the body to produce the antigens and subsequently stimulate an immune response. The mRNA exists only in the cytoplasm, is not integrated into the host genome, and is naturally degraded in the body, making them safer. mRNA vaccines themselves function as adjuvants to induce natural immunity. They can induce strong humoral and cellular immunity, generally demonstrating stronger immune effects than traditional inactivated vaccines and subunit protein vaccines. Because mRNA vaccines do not require the use of other vaccine formulations, they can reduce side effects associated with other formulations. Compared to traditional vaccines, mRNA vaccines have a relatively simple manufacturing process and a short development cycle, offering technical advantages such as rapid response to the development of new infectious diseases. mRNA vaccines have been successfully developed for the novel coronavirus, and the safety and efficacy of mRNA vaccine technology have been demonstrated through large-scale vaccination.

[0010] Neutralizing antibodies generated by RSV infection primarily target the F protein. The RSV F protein is a type I fusion glycoprotein that is relatively conserved among RSV types and is an ideal vaccine antigen target. The F protein switches between two conformations: a metastable prefusion conformation (pre-F) and a stable postfusion conformation (post-F). Although antigenic epitopes targeted by neutralizing antibodies are present in both conformations of the F protein, characteristics of the human innate immune response to RSV infection indicate that most RSV neutralizing antibody epitopes are located in the prefusion conformation of the F protein. Due to the instability of the prefusion conformation of the F protein, all vaccines based on inactivated RSV vaccines or F protein subunit vaccines extracted directly from RSV have failed.

[0011] Given the above issues, in order to further reduce the risk of respiratory syncytial virus infection in immunocompromised populations (including children and the elderly), it is urgent to provide a novel mRNA vaccine for preventing RSV that is highly stable and significantly enhances humoral immunity after vaccination. Summary of the Invention

[0012] The present invention provides an mRNA vaccine encoding the RSV F protein, particularly the pre-fusion conformation pre-F of the F protein. The wild-type protein sequence of the RSV A2 subtype is shown in SEQ ID NO: 1 (GenBank ID: 138251). Based on this wild-type, it contains three naturally occurring mutations (SEQ ID NO: 210) of P102A, I379V, and M447V. The mutated sequences of these three amino acid residues are also conserved in other RSV A strains. Considering the differences in the F protein sequences of current mainstream strains, mutations were made based on this. Design candidate sequences for alloantigens.

[0013] RSV viruses include subtypes A and B, and their F protein amino acid sequences are approximately 90% identical. The F precursor polypeptide sequences of subtype A, e.g., strain A2 (GenBank GI: 138251, Swiss Prot P03420), and subtype B, e.g., strain 18537 (GenBank GI: 138250, Swiss Prot P13843), are both 574 amino acid sequences, and the signal peptide sequences of both subtypes are reported as amino acids 1 to 25 (GenBank and UniProt). Due to the high homology between the F protein sequences of both RSV types A and B in the two sequences, the RSV pre-F mRNA designed according to the present invention and vaccines produced therefrom are effective in preventing infection with both RSV types A and B.

[0014] As described herein, the three-dimensional structure of pre-F is used as a guide to engineer and construct a stabilized form of the pre-fusion F protein (the "pre-F" antigen) and use it to generate an RSV-neutralizing immune response in an animal model, which immune response is expressed in Abrysvo. TM The immune response achieved by the RSV F protein-based immunogen is significantly improved compared to that achieved by other RSV F protein-based immunogens and provides protection against RSV challenge in animal models. The pre-F antigen can be used as a potential vaccine and diagnostic molecule for RSV.

[0015] In some embodiments, the recombinant RSV F protein contains one or more amino acid substitutions that stabilize the protein in a pre-fusion conformation, such as stabilizing the membrane distal portion of the F protein (including the N-terminal region of the F1 polypeptide) in a pre-fusion conformation, specifically binding to one or more pre-fusion conformation antibodies and / or presenting an antigenic site, such as antigenic site Φ, present in the pre-fusion conformation but not in the post-fusion conformation of the RSV F protein.

[0016] In some other embodiments, the recombinant RSV F protein may include one or more modifications (e.g., truncations and amino acid substitutions) to the C-terminus of the F1 polypeptide, which, in combination with modifications that stabilize the membrane distal region of the F polypeptide, can enhance the stability of the recombinant F protein in the prefusion conformation.

[0017] The present application relates to the following embodiments: 1. Respiratory syncytial virus (RSV) ribonucleic acid (RNA), wherein the RNA encodes a wild-type RSV F protein or a variant thereof; wherein the sequence of the wild-type RSV F protein is set forth in SEQ ID NO: 1.

[0018] 2. The composition of embodiment 1, wherein the RSV F protein variant comprises one or more mutations selected from sequence truncations, sequence deletions, and site mutations.

[0019] 3. The sequence truncation includes a deletion of the cytoplasmic domain, for example, the sequence truncation is RSV The composition of embodiment 2, wherein the truncation comprises amino acids 550 to 574 at the C-terminus of the F protein, for example, compared to the wild-type F protein, and the truncation comprises amino acids 1 to 549 of the RSV F protein.

[0020] 4. The composition of embodiment 2 or 3, wherein the sequence deletion is selected from one or more of an amino acid deletion at positions 104 to 136 and an amino acid deletion at positions 104 to 144, and optionally, the amino acids of the sequence deletion are replaced by a GSGS short peptide or a GS short peptide.

[0021] 5. The composition of any one of embodiments 2 to 4, wherein the site mutations comprise one or more selected from P102A, I379V, and M447V, for example, the site mutations comprise P102A and I379V, P102A and M447V, I379V and M447V, P102A, I379V, and M447V.

[0022] 6. The composition of embodiment 5, wherein the site mutations are selected from one or more of S46G, S215P, L373R, S213P and N216P, such as selected from S46G, S215P and L373R site mutations.

[0023] 7. The site mutation is (i)Q26C, N27C, T29C, E31C, F32C, Q34C, T36C, S41C, Y44C, Y53C, T54C, S55C, T100C, T103C, L138C, G139C , F140C, L141C, L142C, G145C, S146C, A147C, I148C, A149C, S150C, V152C, A153C, V154C, S155C, N183GC, S 186C, L188C, S287C, I288C, S290C, I291C, I292C, V300C, L305C, Y306C, S319C, N325C, S330C, T337C, R33 9C, Q354C, T369C, M370C, N371C, S377C, N428C, Y458C, K461C, Q462C, K465C, L467C, Y468C, K470C, G471C, (ii)T54H, T58L, S190I, S190F, V207L, T219I, V296I, (iii) E92D, K465Q, D486S, and (iv)V152I, C69Y, D73E, A74V, N88S 7. The composition of embodiment 5 or 6, further comprising one or more selected from:

[0024] 8. The site mutation is (i)E31C, Q34C, T54C, S55C, T100C, I148C, A149C, V152C, V154C, S155C, L188C, I288C, S290C, R339C, S377C, Y458C, L467C, G471C, (ii)S190F, V296I, V207L, T219I, S190I, (iii) E92D, K465Q, D486S, and (iv)V152I, C69Y, D73E, A74V, N88S 7. The composition of embodiment 5 or 6, further comprising one or more combinations selected from:

[0025] 9. The site mutation is (i)T54C, S55C, T100C, I148C, A149C, V152C, V154C, S155C, L188C, I288C, S290C, R339C, S377C, Y458C, (ii)S190F, V296I, V207L, T219I, S190I, (iii) E92D, K465Q, D486S, and (iv)V152I, C69Y, D73E, A74V, N88S 7. The composition of embodiment 5 or 6, further comprising one or more combinations selected from:

[0026] 10. The site mutation is (i)T54C, I148C, A149C, V152C, S155C, I288C, S290C, Y458C, (ii) S190F, V296I, V207L, (iii) E92D, K465Q, D486S, and (iv)V152I, C69Y, D73E, A74V, N88S 7. The composition of embodiment 5 or 6, further comprising one or more combinations selected from:

[0027] 11. The composition of any one of embodiments 1 to 10, wherein the RSV F protein variant comprises a P102A, I379V, and M447V mutation, and the RSV F protein variant is a truncated protein in which amino acids 550 to 574 of SEQ ID NO: 1 are truncated, and amino acids 104 to 144 of SEQ ID NO: 1 are substituted with a GS linker, wherein the RSV F protein variant further comprises a combination of site mutations selected from the group consisting of:

[0028] [Table 1] TIFF2026010683000003.tif146170

[0029] 12. The composition of any one of embodiments 1 to 10, wherein the RSV F protein variant comprises a P102A, I379V, and M447V mutation, wherein the RSV F protein variant is a truncated protein in which amino acids 550 to 574 of SEQ ID NO: 1 are truncated, and amino acids 104 to 144 of SEQ ID NO: 1 are substituted with a GS linker, and wherein the RSV F protein variant further comprises a combination of site mutations selected from the group consisting of:

[0030] [Table 2] TIFF2026010683000005.tif160169

[0031] 13. The composition of any one of embodiments 1 to 10, wherein the RSV F protein variant comprises a P102A, I379V, and M447V mutation, wherein the RSV F protein variant is a truncated protein in which amino acids 550 to 574 of SEQ ID NO: 1 are truncated, and amino acids 104 to 144 of SEQ ID NO: 1 are substituted with a GS linker, and wherein the RSV F protein variant further comprises a combination of site mutations selected from the group consisting of:

[0032] [Table 3]

[0033] 14. The RSV F protein variants are selected from the group consisting of SEQ ID NO: 204 (YK-RSV-061), SEQ ID NO: 12 (YK-RSV-003), SEQ ID NO: 208 (YK-RSV-062), SEQ ID NO: 176 (YK-RSV-054), SEQ ID NO: 116 (YK-RSV-039), SEQ ID NO: 136 (YK-RSV-044), SEQ ID NO: 40 (YK-RSV-012), SEQ ID NO: 52 (YK-RSV-015), SEQ ID NO: 4 ( 14. The composition according to any one of embodiments 1 to 13, comprising an amino acid sequence having at least 80%, 85%, 90%, 95% or at least 98% or 100% identity to the sequence set forth in SEQ ID NO: 104 (YK-RSV-036), SEQ ID NO: 200 (YK-RSV-060), SEQ ID NO: 36 (YK-RSV-011) or SEQ ID NO: 80 (YK-RSV-030). .

[0034] 15. The composition of embodiment 14, wherein the RSV F protein variant comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or at least 98% or 100% identity to the sequence set forth in SEQ ID NO: 204 (YK-RSV-061), SEQ ID NO: 12 (YK-RSV-003), SEQ ID NO: 208 (YK-RSV-062), SEQ ID NO: 176 (YK-RSV-054), SEQ ID NO: 116 (YK-RSV-039), SEQ ID NO: 136 (YK-RSV-044), SEQ ID NO: 40 (YK-RSV-012), SEQ ID NO: 52 (YK-RSV-015), SEQ ID NO: 4 (YK-RSV-001), SEQ ID NO: 104 (YK-RSV-036), or SEQ ID NO: 200 (YK-RSV-060).

[0035] 16. The composition of embodiment 15, wherein the RSV F protein variant comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or at least 98% or 100% identity to the sequence set forth in SEQ ID NO: 204 (YK-RSV-061), SEQ ID NO: 12 (YK-RSV-003), SEQ ID NO: 208 (YK-RSV-062), SEQ ID NO: 176 (YK-RSV-054), or SEQ ID NO: 116 (YK-RSV-039).

[0036] 17. The composition of any one of embodiments 1 to 16, wherein the open reading frame (ORF) encoding the RSV F protein or variant thereof comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, or at least 98% or 100% identity to SEQ ID NO:203, SEQ ID NO:11, SEQ ID NO:207, SEQ ID NO:175, SEQ ID NO:115, SEQ ID NO:135, SEQ ID NO:39, SEQ ID NO:51, SEQ ID NO:3, SEQ ID NO:103, SEQ ID NO:199, SEQ ID NO:35, or SEQ ID NO:79.

[0037] 18. The composition of embodiment 17, wherein the ORF comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, or at least 98% or 100% identity to SEQ ID NO:203, SEQ ID NO:11, SEQ ID NO:207, SEQ ID NO:175, SEQ ID NO:115, SEQ ID NO:135, SEQ ID NO:39, SEQ ID NO:51, SEQ ID NO:3, SEQ ID NO:103, or SEQ ID NO:199.

[0038] 19. The composition of embodiment 18, wherein the ORF comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, or at least 98% or 100% identity to SEQ ID NO: 203, SEQ ID NO: 11, SEQ ID NO: 207, SEQ ID NO: 175, or SEQ ID NO: 115.

[0039] 20. The composition of any one of embodiments 1 to 19, wherein the RSV RNA further comprises a 5' untranslated region (UTR).

[0040] 21. The composition of embodiment 20, wherein the sequence of the 5'UTR is SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220 or SEQ ID NO: 221.

[0041] 22. The composition of embodiment 21, wherein the sequence of the 5'UTR is SEQ ID NO: 217.

[0042] 23. The composition of any one of embodiments 1 to 22, wherein the RSV RNA further comprises a 3' untranslated region (UTR).

[0043] 24. The composition of embodiment 23, wherein the sequence of the 3'UTR is SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224 or SEQ ID NO: 225.

[0044] 25. The composition of embodiment 24, wherein the sequence of the 3'UTR is SEQ ID NO: 222. .

[0045] 26. The composition of any one of embodiments 1 to 25, wherein the RSV RNA further comprises a poly(A) tail.

[0046] 27. The composition of embodiment 26, wherein the poly(A) tail length is 50 to 150 nucleotides.

[0047] 28. The composition of any one of embodiments 1 to 27, wherein the RSV RNA further comprises a 5' end cap structure.

[0048] 29. The composition of embodiment 28, wherein the 5' end cap structure is 7mG(5')ppp(5')NlmpNp.

[0049] 30. The composition of any one of embodiments 1 to 29, wherein the sequence of the open reading frame (ORF) encoding the RSV F protein or variant thereof in the RSV RNA is codon-optimized.

[0050] 31. The composition of embodiment 30, wherein the sequence of the ORF comprises at least one base modification.

[0051] 32. The composition of embodiment 31, wherein the base modifications comprise any one or more selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyluridine.

[0052] 33. The composition of embodiment 32, wherein the base modification comprises replacing uracil with pseudouridine and / or N1-methylpseudouridine.

[0053] 34. The composition of embodiment 31, wherein the base modifications are 1 to 100% base modifications, for example, 100% base modifications.

[0054] 35. The composition of any one of embodiments 1 to 34, wherein the RSV RNA sequence has at least 80%, 85%, 90%, 95%, or at least 98% or 100% identity to SEQ ID NO:205, SEQ ID NO:13, SEQ ID NO:209, SEQ ID NO:177, SEQ ID NO:117, SEQ ID NO:137, SEQ ID NO:41, SEQ ID NO:53, SEQ ID NO:5, SEQ ID NO:105, SEQ ID NO:201, SEQ ID NO:37, or SEQ ID NO:81.

[0055] 36. The composition of embodiment 35, wherein the RSV RNA sequence has at least 80%, 85%, 90%, 95%, or at least 98% or 100% identity to SEQ ID NO:205, SEQ ID NO:13, SEQ ID NO:209, SEQ ID NO:177, SEQ ID NO:117, SEQ ID NO:137, SEQ ID NO:41, SEQ ID NO:53, SEQ ID NO:5, SEQ ID NO:105, or SEQ ID NO:201.

[0056] 37. The RSV RNA sequence is SEQ ID NO: 205, SEQ ID NO: 13, SEQ ID NO: 209, 37. The composition of embodiment 36, having at least 80%, 85%, 90%, 95% or at least 98% or 100% identity to SEQ ID NO: 177 or SEQ ID NO: 117.

[0057] 38. The composition of any one of embodiments 1 to 37, wherein the RSV RNA is mRNA.

[0058] 39. The composition of any one of embodiments 1 to 38, wherein the sequence encoding the RSV F protein or variant thereof has at least 80%, 85%, 90%, 95%, or at least 98% or 100% identity to SEQ ID NO:202, SEQ ID NO:10, SEQ ID NO:206, SEQ ID NO:174, SEQ ID NO:114, SEQ ID NO:134, SEQ ID NO:38, SEQ ID NO:50, SEQ ID NO:2, SEQ ID NO:102, SEQ ID NO:198, SEQ ID NO:34, or SEQ ID NO:78.

[0059] 40. The composition of embodiment 39, wherein the sequence encoding the RSV F protein or a variant thereof has at least 80%, 85%, 90%, 95%, or at least 98% or 100% identity to SEQ ID NO:202, SEQ ID NO:10, SEQ ID NO:206, SEQ ID NO:174, SEQ ID NO:114, SEQ ID NO:134, SEQ ID NO:38, SEQ ID NO:50, SEQ ID NO:2, SEQ ID NO:102, or SEQ ID NO:198.

[0060] 41. The composition of embodiment 40, wherein the sequence encoding the RSV F protein or a variant thereof has at least 80%, 85%, 90%, 95%, or at least 98% or 100% identity to SEQ ID NO: 202, SEQ ID NO: 10, SEQ ID NO: 206, SEQ ID NO: 174, or SEQ ID NO: 114.

[0061] 42. Providing a transcriptional template for the RSV RNA; transcribing using said template under conditions suitable for transcription of said RNA. 42. A method for producing the composition of any one of embodiments 1 to 41, comprising:

[0062] 43. The method of embodiment 42, further comprising a purification step selected from lithium chloride precipitation, affinity chromatography, solution exchange by ultrafiltration, and cellulose chromatography.

[0063] 44. The composition of any one of embodiments 1-41, which is a vaccine and further comprises a pharmaceutically acceptable carrier.

[0064] 45. The composition of embodiment 44, wherein the pharmaceutically acceptable carrier comprises a lipid mixture, the lipid mixture being, for example, a lipid nanoparticle (LNP).

[0065] 46. ​​The composition of embodiment 44 or 45, wherein the vaccine is an mRNA vaccine.

[0066] 47. The composition of embodiment 45, wherein the lipid nanoparticles comprise, for example, cationic lipids, neutral lipids, structured lipids, and polymer-conjugated lipids.

[0067] 48. The cationic lipid is a compound of formula I, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein G1 is C 1~6 alkylene, and G2 is C 2~8 alkylene, and G3 is C 1~3 alkylene, and L1 is C 6~15 is a straight chain alkyl, and L2 is C 12~25 is a branched alkyl; For example, the composition of embodiment 47, wherein the structure of formula II is YK-009.

[0068] [ka]

[0069] 49. The cationic lipid is a compound of formula II, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein G1 is C 2~8 alkylene, and G2 is C 2~8 alkylene, L1 is -C(O)O- or -OC(O)-, L2 is -C(O)O- or -OC(O)-, and R1 is C 6~25 is a straight or branched chain alkyl; R2 is C 6~25 is a straight or branched chain alkyl, G3 is HO(CH2)2- or HO(CH2)3-, G4 is HO(CH2)2- or HO(CH2)3-, and L is (CH2)2- or -(CH2)3- or -(CH2)4-; For example, the composition of embodiment 47, which is YK-401 having the structure of formula II-I or YK-402 having the structure of formula II-II.

[0070] [ka]

[0071] 50. The cationic lipid is a compound of formula III, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein G1 is C 1~6 alkylene, and G2 is C 2~8 alkylene, and R1 is C 6~20 is a straight or branched chain alkyl; R2 is C 12~25 Branched chain alkyl, G3 is HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)( CH2)2-, (CH3)2N(CH2)3SC(O)O(CH2)2-, (CH3)2N(CH2)3SC(O)-, CH3NH(CH2)2N(CH3)(CH2)2- or CH3CH2NH(CH2)2-, For example, the composition of embodiment 47, which is YK-201 having the structure of formula III-I or YK-202 having the structure of formula III-II.

[0072] [ka]

[0073] 51. The cationic lipid is a compound of formula IV, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein G1 is C 1~8 alkylene, and G2 is C 2~8 alkylene, and R1 is C 6~25 is a straight or branched chain alkyl; R2 is C 12~25 is a straight or branched chain alkyl; G3 is HO(CH2)2N(R3)CH2CH(OH)CH2-, where R3 is -CH3 or -CH2CH3 or -CH2CH2OH; The composition of embodiment 47, which is YK-305 having the structure of formula IV-I or YK-310 having the structure of formula IV-II.

[0074] [ka]

[0075] 52. The cationic lipid is a compound of formula V, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein G1 and G2 are each independently an unsubstituted C6-C 10 alkylene, and G3 is an unsubstituted C1-C 12 alkylene, and R1 and R2 are each independently C6 to C 24 Alkyl or C6-C 24 alkenyl, R3 is OR5, N, -C(=O)OR4, -OC(=O)R4 or -NR5C(=O)R4, and R4 is C1-C 12 hydrocarbyl and R5 is H or C1-C6 alkyl; For example, the composition of embodiment 47, which is ALC0315, of the structure of formula VI.

[0076] [ka]

[0077] 53. The cationic lipid is a compound of formula VI, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein R4 is -(CH2) n Q and -(CH2) n CHQR, where Q is -OR, -OH, or -O(CH2) n and heterocyclyl, wherein n is 1, 2 or 3; and R is hydrogen, C, C, C(S)N(R), -C(S)N(R), -C(S)N(R), -C(S)N(R), -C(S)N(R), -C(S)N(R), -C(S)N(R), -C(S)N(R), -C(S)N(R), -C(S)N(R), -C(S)N(R), -C(S)N(R), -C(S)N(R), and heterocyclyl, wherein n is 1, 2 or 3; and R is hydrogen, C, C, C(S)N(R), -C(S)N(R), -C(S)N(R), -C(S)N(R), -C(S)N(R), and heterocyclyl, wherein n is 1, 2 or 3; and 1~3 Alkyl, C 2~3 Alkenyl or (CH2) q OR * where q is 1, 2, or 3; R * is C 1~12 Alkyl or C 2~12 alkenyl, X is fluorine, chlorine, bromine or iodine, and R is C 3~6 is cycloalkyl or heterocyclyl, For example, the composition of embodiment 47, which is SM102 having the structure of formula VI-I.

[0078] [ka]

[0079] 54. The composition of embodiment 47, wherein the cationic lipid is a compound of formula VII, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof.

[0080] [ka]

[0081] 55. The composition of embodiment 47, wherein the cationic lipid comprises the compounds YK-009, YK-401, YK-305, ALC0315, SM102, DLIN-MC3.

[0082] [ka]

[0083] 56. The composition according to any one of embodiments 47 to 55, wherein the molar ratio of the cationic lipid to the neutral lipid is (1-10):1.

[0084] 57. The composition of any one of embodiments 47-55, wherein the molar ratio of the cationic lipid to the structural lipid is (1-5):1.

[0085] 58. The cationic lipid, the neutral lipid, the structured lipid, and the polymer-conjugated lipid 56. The composition of any one of embodiments 47 to 55, wherein the molar ratio is (25-75):(5-25):(15-65):(0.5-10).

[0086] 59. The composition of embodiment 58, wherein the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (35-49):(7.5-15):(35-55):(1-5), for example, 49:10:39.5:1.5 or 45:10:43.5:1.5.

[0087] 60. The composition of any one of embodiments 47 to 59, wherein the neutral lipid is selected from one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and derivatives thereof.

[0088] 61. The neutral lipid may be 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16).0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoylphosphatidylethanolamine 61. The composition of any one of embodiments 47 to 60, wherein the phosphatidylcholine is selected from one or more of: sphingomyelin, dipalmitoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine (LPE).

[0089] 62. The composition of any one of embodiments 47-61, wherein the neutral lipid is DOPE and / or DSPC.

[0090] 63. The composition of any one of embodiments 47 to 62, wherein the structured lipid is selected from one or more of cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, and corticosteroids.

[0091] 64. The composition of embodiment 63, wherein the structured lipid is cholesterol.

[0092] 65. The composition of any one of embodiments 47-64, wherein the polymer-conjugated lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0093] 66. The composition of any one of embodiments 65, wherein the polymer-conjugated lipid is selected from one or more of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), 1,2-dimyristoyl-rac-glycero-methoxypolyethylene glycol-2000 (DMG-PEG2000), and methoxypoly(ethylene glycol) ditetradecylacetamide (ALC-0159).

[0094] 67. The composition of any one of embodiments 44 to 66, wherein the effective amount of RSV RNA is 25 μg to 200 μg, preferably 50 μg to 100 μg.

[0095] 68. The composition of any one of embodiments 44 to 67, wherein the vaccine is in the form of an injectable dosage form.

[0096] 69. A method for producing a composition according to any one of embodiments 44 to 68, comprising mixing the RSV RNA with the pharmaceutically acceptable carrier, e.g., encapsulating at least a portion of the RNA within a lipid nanoparticle.

[0097] 70. Use of a composition according to any one of embodiments 1 to 41 and 44 to 68 in the manufacture of a vaccine for inducing a protective immune response, particularly a humoral immune response, against RSV in a subject, wherein the protective immune response comprises, for example, the production of neutralizing antibodies, and the subject is 40 years of age or older or under 10 years of age, e.g., 50 years of age or older, 55 years of age or older, 60 years of age or older, 65 years of age or older, 70 years of age or older, 75 years of age or older, or 80 years of age or older, or under 8 years of age, 7 years of age or older, 6 years of age or older, 5 years of age or older, 4 years of age or older, 3 years of age or younger, or 2 years of age or younger.

[0098] 71. Use of a composition according to any one of embodiments 1 to 41 and 44 to 68 in the manufacture of a vaccine for preventing RSV infection.

[0099] The beneficial effects of the present invention are at least one of the following: The present application provides at least 11 ribonucleic acids encoding variants of the RSV F protein based on the wild-type RSV F protein, with specific variant forms (including sequence truncation, site mutation, and / or sequence deletion), thereby providing a novel mRNA vaccine for preventing RSV, which has high stability and significantly enhances the effect of humoral immunity after vaccination, including at least one of the following effects: The effect of increasing the binding rate of the pre-fusion Φ epitope in RSV F mutants to the corresponding antibody The effect of improving the stability of the prefusion conformation of RSV F mutants after treatment with different temperature, osmolality and pH conditions. The effect of improving the binding amount and binding ability of antibodies against RSV F mutant prefusion conformation-specific epitopes (Φ epitopes, quaternary epitopes, and / or V epitopes), The effect of increasing the titer of neutralizing antibodies produced after immunization with RSV F protein, Significantly reducing the inflammatory response and viral load in the body (including the lungs and nasal turbinates), Effective against multiple RSV subtypes simultaneously.

[0100] Specifically, the results and technical advantages of the present invention are as follows: (1) Western blot detection revealed that the following 50 RSV F mutants were identified: YK-RSV-001, YK-RSV-002, YK-RSV-003, YK-RSV-004, YK-RSV-005, YK-RSV-008, YK-RSV-009, YK-RSV-010, YK-RSV-011, YK-RSV-012, YK-RSV-013, YK-RSV-014, YK-RSV- 015, YK-RSV-016, YK-RSV-017, YK-RSV-018, YK-RSV-019, YK-RSV-022, YK-RSV-029, YK -RSV-030, YK-RSV-031, YK-RSV-032, YK-RSV-033, YK-RSV-034, YK-RSV-035, YK-RSV-03 6, YK-RSV-037, YK-RSV-038, YK-RSV-039, YK-RSV-040, YK-RSV-041, YK-RSV-042, YK-R SV-043, YK-RSV-044, YK-RSV-045, YK-RSV-046, YK-RSV-048, YK-RSV-050, YK-RSV-051, It is shown that YK-RSV-052, YK-RSV-053, YK-RSV-054, YK-RSV-055, YK-RSV-056, YK-RSV-057, YK-RSV-058, YK-RSV-059, YK-RSV-060, YK-RSV-061 and YK-RSV-062 all have significant protein expression.

[0101] (2) By detecting the binding rate of the antibody corresponding to the epitope on the RSV F mutant, the following 42 RSV F mutant (YK-RSV-061, YK-RSV-003, YK-RSV-062, YK-RSV-054, YK-RSV-039, YK-RS V-044, YK-RSV-012, YK-RSV-015, YK-RSV-001, YK-RSV-036, YK-RSV-060, YK-RSV- 005, YK-RSV-010, YK-RSV-017, YK-RSV-030, YK-RSV-038, YK-RSV-043, YK-RSV-0 55, YK-RSV-056, YK-RSV-057, YK-RSV-048, YK-RSV-052, YK-RSV-013, YK-RSV-002 , YK-RSV-009, YK-RSV-050, YK-RSV-032, YK-RSV-019, YK-RSV-040, YK-RSV-046, YK-RSV-008, YK-RSV-059, YK-RSV-034, YK-RSV-004, YK-RSV-058, YK-RSV-016, YK-RSV-029, YK-RSV-035, YK-RSV-037, YK-RSV-011, YK-RSV-045, YK-RSV-051) were screened, and the binding rates of the Φ epitope and II epitope with the corresponding antibodies were about 80% or more, and both binding rates were good.

[0102] (3) Stability tests were performed on the following 20 RSV F mutants (including YK-RSV-061, YK-RSV-003, YK-RSV-062, YK-RSV-054, YK-RSV-039, YK-RSV-044, YK-RSV-012, YK-RSV-015, YK-RSV-001, YK-RSV-036, YK-RSV-060, YK-RSV-009, YK-RSV-010, YK-RSV-011, YK-RSV-030, YK-RSV-043, YK-RSV-048, YK-RSV-050, YK-RSV-055, and YK-RSV-057) after stress treatment under different temperature, osmotic pressure, and pH conditions. After rinsing and storage at 4°C for 1 week, all stress tolerance parameters were above 0.44 and could reach 0.68. After incubation at 50°C for 60 minutes, all stress tolerance parameters were above 0.64. After treatment under high osmotic pressure for 60 minutes, all stress tolerance parameters were above 0.61. After treatment under low osmotic pressure for 60 minutes, all stress tolerance parameters were above 0.70. After treatment under pH=3.5 and pH=10 for 60 minutes, all stress tolerance parameters were above 0.5. 6 or more. The stress tolerance parameters after treatment under the various conditions above are superior to or comparable to the control group.

[0103] (4) ELISA was used to detect the binding levels of different epitopes and corresponding antibodies. The following 15 mutants (including YK-RSV-061, YK-RSV-003, YK-RSV-062, YK-RSV-054, YK-RSV-039, YK-RSV-044, YK-RSV-012, YK-RSV-015, YK-RSV-001, YK-RSV-036, YK-RSV-060, YK-RSV-011, YK-RSV-030, YK-RSV-048, and YK-RSV-050) were screened, and the Φ epitope, quaternary epitope, and V epitope all bound well to the corresponding antibodies, with a larger number of epitopes and stronger binding ability.

[0104] For example, the binding level of the Φ epitope on YK-RSV-061, YK-RSV-003, and YK-RSV-062 to the RSV pre-F antibody (3C12) was 1.83 to 2.00 times higher than that of the control PC-RSV-064, and the binding level of the quaternary epitope to the RSV pre-F antibody (7H11) was 2.01 to 2.12 times higher than that of the control PC-RSV-064.

[0105] The binding level of the Φ epitope on YK-RSV-054 to the RSV pre-F antibody (3C12) was 2.52-fold higher than that of the control PC-RSV-064, the binding level of the quaternary epitope to the RSV pre-F antibody (7H11) was 1.82-fold higher than that of the control PC-RSV-064, the binding level of the V epitope to the RSV F antibody (7E11) was 3.30-fold higher than that of the control PC-RSV-064, the binding level of the II epitope to the RSV F antibody (11A9) was 1.79-fold higher than that of the control PC-RSV-064, and the binding level of the III epitope to the RSV F antibody (4B9) was 2.27-fold higher than that of the control PC-RSV-064.

[0106] (5) The affinity of different antibodies to the target protein was analyzed using BLI. The results showed that the Φ epitope and quaternary epitope of 13 mutants (including YK-RSV-061, YK-RSV-003, YK-RSV-062, YK-RSV-054, YK-RSV-039, YK-RSV-044, YK-RSV-012, YK-RSV-015, YK-RSV-001, YK-RSV-036, YK-RSV-060, YK-RSV-011, and YK-RSV-030) had the strongest binding affinity with the corresponding antibodies.

[0107] The binding affinity constant K between the Φ epitope on the mutant and the RSV pre-F antibody (3C12) D is 3.26E-12~7.79E-11M, and the control K D Value (K D is approximately 1E-10 M, which is one to two orders of magnitude lower than the RI. This indicates a high affinity between the antigen and the antibody.

[0108] Binding affinity constant K for RSV pre-F antibody (7H11) with quaternary epitopes on the mutant D The values ​​were 3.05E-12 to 6.13E-10M, and YK-RSV-044, YK-RSV-060, and YK-RSV-030 were the control K D Other than the corresponding values, the K values ​​of other mutants D All values ​​were significantly lower than the control (with a difference of 1 to 2 orders of magnitude), indicating high affinity between the antigen and antibody.

[0109] (6) Mouse: Binding antibody ELISA was used to detect pre-fusion and post-fusion F protein-specific IgG titers in the serum of mice 35 days after immunization. The results showed that 11 test substances (YK-RSV-061, YK-RSV-003, YK-RSV-062, YK-RSV-054, YK-RSV-066) were significantly higher than those of the control group (YK-RSV-061, YK-RSV-003, YK-RSV-062, YK-RSV-054, YK-RSV-066). -039, YK-RSV-044, YK-RSV-012, YK-RSV-015, YK-RSV-001, YK-RSV-036, and YK-RSV-060), and pre-F IgG antibody titers were measured using Abrysvo TM The post-F IgG antibody titers were 2.52 to 5.31 times higher than those of the positive control. TM 1.13 to 5.21 times that of the positive control.

[0110] Among all the tested compounds detected, the ratio of pre-F / post-F was all higher than 1, indicating that the pre-fusion conformation F protein produced higher IgG titers than the post-fusion conformation F protein.

[0111] Mouse: Neutralizing antibody 1) RSV type A virus: A spot reduction microneutralization test was used to detect serum neutralizing antibody titers 35 days after immunization of mice. 11 test articles (including YK-RSV-061, YK-RSV-003, YK-RSV-062, YK-RSV-054, YK-RSV-039, YK-RSV-044, YK-RSV-012, YK-RSV-001, YK-RSV-036, and YK-RSV-060) were screened and the neutralizing antibody titers IC 50 Abrysvo TM The IC was 3.72 to 12.67 times that of the positive control. 90 Abrysvo TM The results were 2.34 to 14.41 times those of the positive control, and all were superior to the positive control.

[0112] 2) RSV type B virus: IC of neutralizing antibody titers of 11 test articles (including YK-RSV-061, YK-RSV-003, YK-RSV-062, YK-RSV-054, YK-RSV-039, YK-RSV-044, YK-RSV-012, YK-RSV-015, YK-RSV-001, YK-RSV-036, and YK-RSV-060) 50 Abrysvo TM The IC was 2.85 to 10.63 times that of the positive control. 90 Abrysvo TM The neutralizing antibody titers were 2.88 to 14.23 times higher than the positive control, and all were significantly higher than the positive control.

[0113] (7) Cotton rat: conjugated antibody ELISA was used to detect pre-fusion and post-fusion F protein-specific IgG titers in the serum of cotton rats on days 42 and 48 post-immunization, and the 11 test articles (including YK-RSV-061, YK-RSV-003, YK-RSV-062, YK-RSV-054, YK-RSV-039, YK-RSV-044, YK-RSV-012, YK-RSV-015, YK-RSV-001, YK-RSV-036, and YK-RSV-060) were used. On day 42, the pre-F IgG antibody titer was Abrysvo TMThe post-F IgG antibody titers were 3.05 to 13.10 times higher than the positive control. TM The pre-F / post-F ratios for all test samples ranged from 1.39 to 3.95, indicating that the IgG antibody titers produced by the pre-fusion F protein were significantly higher than those produced by the post-fusion F protein.

[0114] The pre-F IgG antibody titer on day 48 was Abrysvo TM The post-F IgG antibody titer was 4.44 to 12.35 times higher than the positive control. TM The pre-F / post-F ratios for all test samples ranged from 1.78 to 4.29 times, indicating that the IgG antibody titers produced by the pre-fusion F protein were still significantly higher than those produced by the post-fusion F protein after immunization on day 48.

[0115] Cotton rat: neutralizing antibodies The neutralizing antibody titer against RSV A2 virus in the serum of cotton rats was detected, and the detection results were compared for 11 test products (YK-RSV-061, YK-RSV-003, YK-RSV -062, YK-RSV-054, YK-RSV-039, YK-RSV-044, YK-RSV-012, YK-RSV-015, YK-RSV-001, YK-RSV-036, YK-RSV-060) and indicates the following: (1) IC of neutralizing antibodies against RSV A2 virus 42 days after immunization 50 Abrysvo TM The IC was 2.10 to 14.30 times that of the positive control. 90 Abrysvo TM The values ​​were 2.26 to 22.33 times those of the positive control, and all were significantly higher than those of the positive control.

[0116] IC of neutralizing antibody titers produced against RSV B virus 50 Abrysvo TM The IC was 2.16 to 14.63 times that of the positive control.90 Abrysvo TM The values ​​were 2.49 to 17.26 times those of the positive control, and all were significantly higher than those of the positive control.

[0117] 2) IC of neutralizing antibodies produced against RSV A2 virus 48 days after immunization 50 Abrysvo TM The IC was 1.70 to 7.57 times that of the positive control. 90 Abrysvo TM The values ​​were 1.46 to 11.56 times higher than the positive control, and all were higher than the positive control.

[0118] (8) Pathological section results of the lungs of cotton rats showed that the peribronchial infiltration scores of 11 test substances (including YK-RSV-061, YK-RSV-003, YK-RSV-062, YK-RSV-054, YK-RSV-039, YK-RSV-044, YK-RSV-012, YK-RSV-015, YK-RSV-001, YK-RSV-036, and YK-RSV-060) ranged from 0.33 to 0.83, indicating mild inflammatory infiltration. The interstitial infiltration scores of all test substances were significantly higher than those of the positive control, Abrysvo. TM The scores were better, ranging from 0.67 to 1.17, indicating mild inflammatory infiltration. All test specimens had low overall inflammatory response scores, with overall pathological scores ranging from 1.00 to 2.00, both lower than control LNP. The scores for perivascular and alveolar infiltration were all 0 for both the control and test specimens, indicating no visible lesions in the tissue.

[0119] (9) The results of measuring the viral load in the lungs and nasal turbinates of cotton rats showed that the lung viral load after immunization with 11 test substances (including YK-RSV-061, YK-RSV-003, YK-RSV-062, YK-RSV-054, YK-RSV-039, YK-RSV-044, YK-RSV-012, YK-RSV-015, YK-RSV-001, YK-RSV-036, and YK-RSV-060) was 1.61 to 2.08, and the positive control Abrysvo TM The viral load in the nasal turbinates was 0.62-1.51, compared with the positive control AbrysvoTM The viral load was only 0.19 to 0.45 times that of the control, indicating a significant reduction in the viral load and excellent antiviral effect of the test substance. [Brief explanation of the drawings]

[0120] [Figure 1] Antigen expression status of YK-RSV-001 to YK-RSV-062. [Figure 2] (A) and (B) show mouse serum RSV A2 pre-F binding antibody titers on day 35, respectively. [Figure 3] (A) and (B) show mouse serum RSV A2 post-F binding antibody titers on day 35, respectively. [Figure 4] (A) and (B) show the IC50 of mouse serum RSV A2 neutralizing antibody titers on day 35, respectively. [Figure 5] (A) and (B) show the IC90 of mouse serum RSV A2 neutralizing antibody titers on day 35, respectively. [Figure 6] (A) and (B) show the IC50 of mouse serum RSV B neutralizing antibody titers on day 35, respectively. [Figure 7] (A) and (B) show the IC90 of mouse serum RSV B neutralizing antibody titers on day 35, respectively. [Figure 8] (A) and (B) show cotton rat serum RSV A2 pre-F binding antibody titers on day 42, respectively. [Figure 9] (A) and (B) show cotton rat serum RSV A2 post-F binding antibody titers on day 42, respectively. [Figure 10] (A) and (B) show cotton rat serum RSV A2 pre-F binding antibody titers on day 48, respectively. [Figure 11] (A) and (B) show cotton rat serum RSV A2 post-F binding antibody titers on day 48, respectively. [Figure 12] (A) and (B) show the cotton rat serum RSV A2 neutralizing antibody titer IC50 on day 42, respectively. [Figure 13] (A) and (B) show the cotton rat serum RSV A2 neutralizing antibody titer IC90 on day 42, respectively. [Figure 14] (A) and (B) show the cotton rat serum RSV B neutralizing antibody titer IC50 on day 42, respectively. [Figure 15] (A) and (B) show the cotton rat serum RSV B neutralizing antibody titer IC90 on day 42, respectively. [Figure 16] (A) and (B) show the IC50 of cotton rat serum RSV A2 neutralizing antibody titers on day 48, respectively. [Figure 17] (A) and (B) show the cotton rat serum RSV A2 neutralizing antibody titer IC90 on day 48, respectively. [Figure 18] 1 shows the results of pathological sections of lungs from cotton rats treated with test articles of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0121] The present invention will be further described below by way of examples. It should be understood that the examples of the present invention are only used to illustrate the present invention and do not limit the present invention, and any simple improvements of the present invention based on the technical solutions of the present invention are all within the protection scope of the present invention.

[0122] The abbreviations for amino acid residues used herein are as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0123] As used herein, the following terms have the following meanings:

[0124] vaccine A vaccine refers to a substance containing an immunogen capable of inducing a prophylactic or therapeutic immune response in an individual, and may be in the form of a pharmaceutical composition. Typically, a vaccine induces an antigen-specific immune response against an antigen of a pathogen, such as a viral pathogen.

[0125] nucleic acid Nucleic acids are polymers containing nucleotides (nucleotide monomers). Therefore, nucleic acids are also called polynucleotides. Nucleic acids include, for example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), threose nucleic acid (TNA), glycol ribonucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA with a β-D-ribose configuration, α-LNA (a diastereomer of LNA) with an α-L-ribose configuration, 2′-amino-LNA with a 2′-amino functionalization, and 2′-amino-LNA with a 2′-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), and / or chimeras. and / or any combination thereof.

[0126] Messenger RNA (mRNA) is any RNA that encodes (at least one) protein (naturally occurring, non-naturally occurring, or modified amino acid polymer) and is translated in vitro, in vivo, in situ, or ex vivo to produce the encoded protein. Unless otherwise specified, the nucleic acid sequences described in this application may recite a "T" in a representative DNA sequence, provided that one of skill in the art will understand that when the sequence represents RNA (e.g., mRNA), the "T" is replaced with a "U." Thus, any DNA disclosed and identified herein by a specific sequence identification number also discloses the corresponding RNA (e.g., mRNA) sequence complementary to the DNA, in which each "T" in the DNA sequence is replaced with a "U."

[0127] An open reading frame (ORF) is a contiguous DNA or RNA sequence that begins with a start codon (e.g., methionine (ATG or AUG)) and ends with a stop codon (e.g., TAA, TAG, or TGA, or UAA, UAG, or UGA). ORFs typically encode proteins. The sequences disclosed herein may also include additional elements, such as 5' and 3' UTRs, although it should be understood that, unlike ORFs, these elements are not necessarily present in the RNA polynucleotides of the present disclosure.

[0128] The compositions of the present disclosure include RNA having an open reading frame (ORF) encoding a respiratory syncytial virus antigen. In some embodiments, the RNA is messenger RNA (mRNA). In some embodiments, the RNA (e.g., mRNA) also includes a 5' UTR, a 3' UTR, a poly(A) tail, and / or a 5' cap analog (or a 5' cap end-cap structure).

[0129] It should also be understood that the mRNA vaccines of the present disclosure may include any 5' untranslated region (UTR) and / or any 3' UTR. Exemplary UTR sequences are provided in the sequence listing. However, other UTR sequences may be used or substituted with any of the UTR sequences described herein. UTRs may also be omitted from the RNA polynucleotides provided herein.

[0130] antigen An antigen is a protein that can induce an immune response (e.g., cause the immune system to produce antibodies against the antigen). As used herein, unless otherwise specified, the use of the term "antigen" covers immunogenic proteins and immunogenic fragments of an immune response (e.g., an immunogenic fragment that induces (or is capable of inducing) an immune response against at least one respiratory syncytial virus, e.g., RSV A2 (138251). It is understood that the term "protein" includes peptides, and the term "antigen" includes antigenic fragments.

[0131] Exemplary sequences of respiratory syncytial virus antigens of the compositions of the disclosure and RNA encoding the respiratory syncytial virus antigens are provided in the Sequence Listing.

[0132] epitope Antigenic determinant clusters, also known as antigenic determinant clusters, are specific regions on antigen molecules that can be recognized by specific antibodies; one or more epitopes may exist on a single antigen molecule. Antigenic epitopes are recognized by B-cell receptors (BCRs) on the surface of B cells, leading to subsequent antibody synthesis and secretion, which can mediate humoral immune responses. The pre-fusion pre-F conformation contains six major antigenic sites / epitopes (including Φ, I, II, III, IV, and V epitopes). Analysis of the crystal structure of the RSV F protein revealed that the AM14 antibody extensively interacts with the F protein during the pre-fusion to post-fusion transition. It has been shown that antibodies specifically recognize quaternary epitopes that span both protomers, including regions that undergo significant structural changes. These epitopes are highly antigenic but relatively unstable. On the other hand, the post-fusion conformation has only four antigenic sites / epitopes (including epitopes I, II, III, and IV), which are more stable but less antigenic. While epitopes I, II, III, and IV are present in both pre-fusion and post-fusion conformations, epitopes Φ and V are present only in the pre-F conformation, and studies have shown that these epitopes are sensitive to neutralizing antibodies and induce stronger neutralizing antibody responses. The antibody-recognized epitopes in this disclosure are as follows: (1) The RSV pre-F antibody (3C12), which recognizes the Φ epitope, specifically binds only to the F protein in the prefusion conformation.

[0133] (2) The RSV pre-F antibody (7H11) recognizes a quaternary epitope (spanning both the IV and V epitopes) and specifically binds to the prefusion conformation of the F protein.

[0134] (3) The RSV F antibody (11A9) that recognizes the II epitope recognizes both the pre- and post-fusion conformations of the F protein.

[0135] (4) The RSV F antibody (4B9) that recognizes the III epitope preferentially recognizes the prefusion conformation and exhibits weak binding activity to the postfusion conformation.

[0136] (5) The RSV pre-F antibody (7E11) recognizes the V epitope and specifically binds to the prefusion conformation of the F protein.

[0137] It has been shown that the ability of a new vaccine to specifically bind to the Φ epitope of a prefusion antigen and its binding capacity are closely related to its efficacy in preventing RSV infection. Φ and II epitopes account for approximately 35% and <10% of the neutralizing activity, respectively. RSV neutralizing activity in human serum is primarily derived from prefusion-specific antibodies. Therefore, the use of prefusion antigens that retain the Φ epitope is useful for inducing or enhancing neutralizing activity through vaccination.

[0138] The antigenic epitopes of the RSV F protein change between the pre-fusion and post-fusion conformations, with the Φ epitope retained only in the pre-fusion conformation and the II epitope retained in both the pre-fusion and post-fusion conformations. Antigen-specific Φ epitope-specific antibodies specifically recognize only the pre-F conformation and are therefore distinct from neutralizing antibodies targeting other known antigenic epitopes on the RSV F protein. Research has shown that the ability to specifically bind to the Φ epitope of the pre-fusion antigen and its binding ability are closely related to the efficacy of novel vaccines in preventing RSV infection, and this will be considered as a key indicator. The Φ epitope is located at the top of the pre-F trimer conformation, providing less steric hindrance and favoring antibody binding. Therefore, the stronger the antibody's ability to bind to the Φ epitope, the greater the protective effect against RSV.

[0139] Epitope binding analysis using monoclonal antibodies (mAbs) showed that the ability to bind to antibodies specific for the Φ epitope (e.g., number, binding rate, binding strength, and degree of binding) correlated with neutralizing activity, whereas the degree of binding to the II epitope correlated poorly with neutralizing activity. Furthermore, the neutralizing activity of RSV in human serum is primarily derived from pre-F-specific antibodies; therefore, vaccination enhances neutralizing activity by utilizing the Φ epitope in the prefusion conformation.

[0140] Prefusion conformation of RSV F protein This is the structural conformation adopted by the RSV F protein before undergoing a fusion event in which RSV F transitions to a post-fusion conformation and becomes the mature RSV F protein in the secretory system after processing. In some embodiments, the antigen encoded by the mutant nucleic acid Whether a mutant is stable in a pre-fusion form can be determined by binding to a specific epitope. The stable pre-fusion RSV F mutants of the present invention are in a pre-fusion form and contain at least one epitope specific to the F protein in the pre-fusion form (as shown). An epitope specific to the pre-fusion form F protein is an epitope that is not present in the post-fusion form. The pre-fusion form of the RSV F protein may contain the same epitope as the RSV F protein expressed on native RSV virions, which can provide the advantage of being able to induce protective neutralizing antibodies.

[0141] In some embodiments, the stable pre-fusion RSV F mutant can specifically bind to the RSV pre-F antibody (3C12) that recognizes the Φ epitope and the RSV pre-F antibody (7H11) that recognizes the quaternary epitope.

[0142] Post-fusion conformation of RSV F protein The structural conformation adopted by the RSV F protein is not the prefusion conformation, but the RSV The N-terminus and C-terminus of the F protein are adjacent in a stable helix-helix. The post-fusion conformation of the RSV F protein has been described at the atomic level (see, for example, McLellan et al., J. Virol., 85, 7788, 2011; Swanson et al., Proc. Natl. Acad. Sci. USA, 108, 9619, 2011; and the structural coordinates deposited under PDB accession number 3RRR, each of which is incorporated herein by reference). In the post-fusion conformation, the RSV F protein cannot bind to certain species of antibodies, such as antibodies that recognize the Φ epitope.

[0143] Mutants In some embodiments, compositions of the present disclosure include RNA encoding an antigenic variant of a respiratory syncytial virus. An antigenic variant or other polypeptide variant refers to a molecule whose amino acid sequence differs from a wild-type, native, or reference sequence. The antigen / polypeptide variant antigen may have substitutions, deletions, and / or insertions at specific positions within the amino acid sequence compared to the native or reference sequence. Generally, a variant has at least 50% identity to a wild-type, native, or reference sequence. In some embodiments, a variant has at least 80% or at least 90% identity to a wild-type, native, or reference sequence, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% or 100% identity. In this document, the terms "mutant" and "variant" can be used interchangeably.

[0144] mutation The term "mutation" refers to a phenomenon in which the amino acid sequence changes relative to the wild-type protein sequence. In the present invention, an antigen mutant has one or more base mutations compared to the wild-type RSV F protein. After mutation, the amino acid sequence of a protein or polypeptide is shortened, an amino acid residue is deleted, or an amino acid residue is substituted compared to the amino acid sequence of a reference protein or polypeptide. As used herein, an amino acid substitution at a specific position in a protein sequence is indicated using the notation "(amino acid residue of wild-type protein) (amino acid position) (amino acid residue of genetically modified protein)". For example, Y44C means that the tyrosine (Y) residue at position 44 of the amino acid sequence of the reference protein is replaced with a cysteine ​​(C) residue (in the mutant of the reference protein). If there is a mutation in the amino acid residue at the same position between different wild-type sequences, the amino acid code before the position number, such as "44C", can be omitted from the annotation.

[0145] Sequence shortening Sequence truncation refers to a partial C-terminal amino acid sequence based on the wild-type RSV F protein. For example, in the present invention, sequence shortening refers to truncating the amino acids 550 to 574 at the C-terminus of the RSV F protein.

[0146] Sequence deletion The sequence deletion refers to a partial deletion of bases at an amino acid position based on the wild-type RSV F protein. For example, in the present invention, the sequence deletion is selected from one or more of the amino acid deletions at positions 104 to 136 and 104 to 144.

[0147] Site mutation Site mutation refers to the alteration of one or more amino acid positions based on the wild-type RSV F protein to stabilize the RSV F protein in the pre-fusion conformation. For example, in the present invention, the site mutations include P102A, I379V and M447V, S46G, S215P, L373R, S213P and N216P, Q26C, N27C, T29C, E31C, F32C, Q34C, T36C, S41C, Y44C, Y53C, T54C, S55C, T100C, T103C, L138C, G139C, F140C, L141C, L142C, G145C, S146C, A147C, I148C, A149C, S150C, V152C, A153C, V154C, S155C, N183GC, S186C, L188C, S287C, I288C, S290C, I291C, I292C, V300C, L305C, Y306C, S319C, N325C, S33 0C, T337C, R339C, Q354C, T369C, M370C, N371C, S377C, N428C, Y458C, K461C, The amino acids selected from the group consisting of Q462C, K465C, L467C, Y468C, K470C, G471C; T54H, T58L, S190I, S190F, V207L, T219I, V296I; E92D, K465Q, D486S; V152I, C69Y, D73E, A74V, and N88S are selected from the group consisting of Q462C, K465C, L467C, Y468C, K470C, G471C; T54H, T58L, S190I, S190F, V207L, T219I, V296I; E92D, K465Q, D486S; V152I, C69Y, D73E, A74V, and N88S. In the above selectable site mutations, amino acids at the same position may be mutated at one of the sites, for example, the above mutations include T54C or T54H, which means that T at position 54 may be mutated to C or H, but not simultaneously.

[0148] Absence of a cytoplasmic domain The cytoplasmic domain consists of residues 550 to 574 of the polypeptide chain of the mature RSV F protein. For example, in the present invention, the absence of the cytoplasmic domain means that the C-terminal amino acid sequence 550 to 574 of the antigen mutant is truncated.

[0149] "One or more" / "Selected from" As used herein, "one or more" means selecting one, two, or more of the listed objects or elements. Similarly, "selected from" means selecting one, two, or more of the listed objects or elements, including any combination of the listed objects or elements, such as any two combinations, any three combinations, ..., any multiple combinations, including all combinations of the opposite. In this sense, "plurality" may include two, three, or more.

[0150] Variant antigens / polypeptides encoded by the nucleic acids of the present disclosure may contain amino acid changes that confer any of a variety of desirable properties, for example, improving their stability or increasing their immunogenicity in an animal. Variant antigens / polypeptides may be produced using conventional mutagenesis techniques and, if necessary, assayed to determine whether they possess the desired properties. Assays for determining expression levels and immunogenicity are well known in the art, and exemplary such assays are provided in the Examples.

[0151] In some embodiments, the composition comprises RNA or an RNA ORF, as described herein. (See Sequence Listing) or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or 100% identity to the nucleotide sequence of any of the sequences provided herein.

[0152] The term "identity" refers to the relationship between two or more polypeptide (e.g., antigen) or polynucleotide (nucleic acid) sequences, as determined by comparing the sequences. Identity also refers to the degree of sequence relatedness between or within sequences, as determined, for example, by the number of matches between strings of two or more amino acid or nucleic acid residues. The identity index measures the percentage of identity matches between the smaller of two or more sequences with gapped alignments (if any) processed by a particular mathematical model or computer program (e.g., an "algorithm"). The identity of related antigens or nucleic acids can be readily calculated using known methods. "Percent identity" when applied to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in a candidate amino acid or nucleic acid sequence that are identical to those in an amino acid or nucleic acid sequence of a second sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percent identity. Methods and computer programs for comparison are well known in the art. While identity is dependent on the calculation of percent identity, it should be understood that the introduction of gaps and penalties in the calculation may result in different identity values. Generally, variants of a particular polynucleotide or polypeptide (e.g., antigen) will have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, but less than 100%, sequence identity with the particular reference polynucleotide or polypeptide, as determined by sequence alignment programs and parameters described herein and known to those of skill in the art. Tools for such comparisons include the BLAST suite (Stephen F. Altschul et al. (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Res. 25:3389-3402), and the like.Another commonly used local comparison technique is based on the Smith-Waterman algorithm (Smith, T.F. and Terman, M.S. (1981) "Identification of common molecular subsequences." J. Mol. Biol. 147:195-197). A common global comparison technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, S.B. and Nsch, C.D. (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins." J. Mol. Biol. 48:443-453). Recently, the Fast Optimal Sequence Alignment Algorithm (FOGSAA) has been developed, which is said to be able to generate global alignments of nucleotide and protein sequences faster than other optimal global alignment methods (including the Needleman-Wunsch algorithm).

[0153] Thus, polynucleotides encoding peptides or polypeptides containing substitutions, insertions and / or additions, deletions, and covalent modifications to reference sequences, particularly the peptide (e.g., antigen) sequences disclosed herein, are included within the scope of this disclosure. In some embodiments, sequences such as signal sequences, termination sequences, transmembrane domains, linkers (or sequences encoding them), etc., may be replaced with alternative sequences that perform the same or similar function. In some embodiments, sequences such as signal sequences, termination sequences, transmembrane domains, linkers, etc. may be replaced with alternative sequences that perform the same or similar function. For example, larger amino acids may be introduced to fill cavities in the protein core. In other embodiments, the buried hydrogen bond network can be replaced with hydrophobic residues to improve stability. Such sequences can be readily identified by one of skill in the art.

[0154] The RSV F mutants provided herein include an F1 polypeptide and an F2 polypeptide, wherein both the F1 polypeptide and the F2 polypeptide contain multiple amino acid mutations introduced into the corresponding native F protein amino acid sequence. Introducing such amino acid mutations into the RSV F mutant confers beneficial properties to the mutant, such as improved immunogenicity, improved stability, or formation of a particular desired physical form or conformation of the mutant, or improved stability.

[0155] Mutant structure of the RSV F protein The human RSV F glycoprotein is first translated from mRNA into a single polypeptide precursor (F0) of 574 amino acids containing an N-terminal signal peptide sequence. After translation, the signal peptide is removed from the endoplasmic reticulum by signal peptidases. F0 is truncated at two sites (between amino acid residues 109 / 110 and 136 / 137) by cellular proteases (specifically, furin protease) during Golgi transport, removing a short glycosylation intervening sequence (also known as the pep27 region, amino acid residues 110-136), generating two domains or subunits: F1 (C-terminal portion, amino acids 137-574) and F2 (N-terminal portion, amino acids 26-109). F2 is linked to F1, and the F1-F2 heterodimer assembles into a homotrimer within the viral particle.

[0156] signal peptide The term "signal peptide" typically refers to a short amino acid sequence (e.g., approximately 18-25 amino acids in length) required for transmembrane transport in the secretory pathway, and controls entry of most proteins into the secretory pathway in eukaryotes and prokaryotes. Signal peptides are often, but not always, present at the N-terminus of a polypeptide and are often truncated by signal peptidases after the protein has crossed the membrane.

[0157] To enhance production, the F2 domain may be preceded by a signal peptide, such as the native F protein signal peptide or a heterologous signal peptide, to enhance production and secretion in host cells expressing the recombinant pre-f antigen.

[0158] F1 polypeptide The term "F1 polypeptide" (F1) refers to the polypeptide chain of the mature RSV F protein. Native F1 consists of approximately residues 137-574 of the RSV F0 precursor. As used herein, the term includes both native F1 polypeptides and F1 polypeptides derived from native sequences containing modifications (e.g., amino acid substitutions, insertions, or deletions), such as modifications designed to stabilize the F mutant or enhance the immunogenicity of the F mutant.

[0159] F1 contains a hydrophobic fusion peptide (FP) and a heptapeptide repeat region A (HRA) at the N-terminus 137-216, and a heptapeptide repeat region B (HRB), a transmembrane (TM, amino acid residues 530-550), and a cytoplasmic region (amino acid residues 551-574) at the C-terminus. The structure of an RSV F protein mutant does not necessarily include the entire F1 domain. Typically, at least a partial sequence (or fragment) of the F1 domain is selected and designed to maintain a stable conformation containing the immunodominant epitopes of the F protein. F1 contains subsequences of many epitopes recognized by neutralizing antibodies. For example, amino acid residues 262-275 are a subsequence of the epitope recognized by palizumab, and amino acid residues 423-436 are a subsequence of the epitope recognized by Centocor's ch101F monoclonal antibody. The F1 domain polypeptide comprises at least about amino acids 262-436 of the RSV F protein polypeptide, and amino acid residues 328-355 comprise a T cell epitope recognized by the RSV F protein.

[0160] Linker One or two of the furin recognition sites can be removed by deleting or substituting one or more amino acids in the furin recognition sites. This design prevents the fusion peptide from being cleaved from F2, preventing the prefusion conformer from being released from the globular head structure and accessing the adjacent membrane. Predicting the interaction of the fusion peptide with the membrane interface is a major issue in the instability of the prefusion state. During the fusion process, the fusion peptide is exposed from within the globular head structure upon interaction with the target membrane, increasing the instability of the prefusion state and folding into the postfusion conformer. This conformational change achieves the membrane fusion process. Removal of one or both of the two furin cleavage sites is predicted to inhibit membrane access to the N-terminal portion of the fusion peptide and stabilize the prefusion state. Optionally, the intervening pep27 peptide can be removed or replaced with, for example, a linker peptide. Optionally, non-furin cleavage sites adjacent to the fusion peptide (e.g., the metalloprotease site at positions 112-113) can also be removed or replaced.

[0161] In this application, the intervening pep27 peptide is replaced with a linker peptide. Analysis of the structural model of the RSV F protein in the pre-fusion state suggests that pep27 generates a large, unconstrained loop between F1 and F2, which does not contribute to the stabilization of the pre-fusion state and is removed after furin cleavage of the native protein. Commonly used protein linkers include flexible linkers, rigid linkers, and shearable linkers, and the linker used herein is a flexible linker.

[0162] The linkers used in this application can be used in this context without disrupting the pre-F antigen conformation.

[0163] F2 polypeptide The term "F2 polypeptide" (F2) refers to the polypeptide chain of the mature RSV F protein. Native F2 comprises approximately 26-109 residues of the RSV F0 precursor. As used herein, the term includes both native F2 polypeptides and F2 polypeptides derived from native sequences containing modifications (e.g., amino acid substitutions, insertions, or deletions), such as modifications designed to stabilize the F mutant or enhance the immunogenicity of the F mutant.

[0164] Those skilled in the art will recognize that the entire F2 domain need not necessarily be included. Typically, conformational considerations are important when selecting a subsequence (or fragment) of the F2 domain. Thus, the F2 domain typically includes portions of the F2 domain that promote assembly and stability of the polypeptide.

[0165] Stabilizing element Naturally occurring eukaryotic mRNA molecules may contain stabilizing elements, including, but not limited to, untranslated regions (UTRs) at their 5'-end (5'UTR) and / or their 3'-end (3'UTR), in addition to other structural features, such as a 5'-cap structure or a 3'-poly(A) tail. Both the 5'UTR and the 3'UTR are typically transcribed from genomic DNA and are components of premature mRNAs. Typically, structural features characteristic of mature mRNAs (e.g., a 5'-cap and a 3'-poly(A) tail) are added to the transcribed (premature) mRNA during mRNA processing.

[0166] In some embodiments, the composition comprises an RNA polynucleotide containing an open reading frame encoding an antigenic polypeptide with at least one modification and at least one 5'-end cap, formulated within a lipid nanoparticle. The 5'-capping of the polynucleotide can be performed simultaneously during the in vitro transcription reaction using the following chemical RNA cap analogs to generate a 5'-guanosine cap structure, according to the manufacturer's protocol:

[0167] A 3'-poly(A) tail, or poly(A) tail, is a series of adenine nucleotides typically added to the 3' end of a transcribed mRNA, located downstream of the 3' UTR, e.g., in the positive downstream (i.e., 3') region, containing multiple consecutive adenosine monophosphates. In some circumstances, it may contain approximately 10 to 400 adenine nucleotides. In some embodiments, the length of the 3'-poly(A) tail is essential for the stability of individual mRNAs. In relevant biological contexts (e.g., intracellular, in vivo), poly(A) tails are used, for example, to protect mRNAs from enzymatic degradation in the cytoplasm and facilitate the termination of transcription and / or transport of mRNAs from the nucleus and translation.

[0168] Untranslated Regions (UTRs) The mRNA of the present disclosure may contain one or more regions or portions that act or function as untranslated regions. When the mRNA is designed to encode at least one target antigen, the nucleic acid may contain one or more of these untranslated regions (UTRs). Wild-type untranslated regions of a nucleic acid are transcribed but not translated. In mRNA, the 5' UTR begins at the transcription initiation site and continues up to, but not including, the start codon, while the 3' UTR begins immediately after the stop codon and continues until the transcription termination signal. There is growing evidence regarding the regulatory role of UTRs in the stability and translation of nucleic acid molecules. The regulatory properties of UTRs can be incorporated into the polynucleotides of the present disclosure to specifically enhance the stability of the molecule. Specific functions can also be incorporated to ensure controlled downregulation of the transcription product if it is misdirected to an undesired organ site.

[0169] The 5' UTR is the region immediately upstream (5') of the start codon of an mRNA (the first codon of an mRNA transcript translated by ribosomes) that does not encode a polypeptide. The 5' UTR is non-coding. Natural 5' UTRs have characteristics that play a role in translation initiation. They commonly contain a Kozak sequence, which is known to be involved in ribosome-initiated translation of many genes. The Kozak sequence shares the sequence CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) located three bases upstream of the start codon (AUG) and is followed by another "G." The 5' UTR is also known to form secondary structures involved in elongation factor binding.

[0170] The 3'UTR refers to the region immediately downstream (3') of the stop codon (the codon that signals the end of translation of the mRNA transcript) of an mRNA that does not code for a polypeptide. The 3'UTR does not code for a protein (it is non-coding). Native or wild-type 3'UTRs are known to contain embedded adenosine and uridine segments.

[0171] Those skilled in the art will appreciate that heterologous or synthetic 5'UTRs can be used with any desired 3'UTR sequence, for example, heterologous 5'UTRs can be used with synthetic 3'UTRs or heterologous 3'UTRs.

[0172] In vitro transcription of RNA cDNA encoding the polynucleotides described herein can be transcribed using an in vitro transcription (IVT) system. In vitro transcription of RNA is accomplished using this technology. It is known in the art and described in International Publication WO / 2014 / 152027, the disclosure of which is incorporated herein by reference in its entirety.

[0173] In some embodiments, the RNA transcripts are produced in an in vitro transcription reaction using a linearized DNA template to produce the RNA transcripts.

[0174] In some embodiments, the in vitro transcription template encodes a 5' untranslated (UTR) region, contains an open reading frame, encodes a 3' UTR, and a poly(A) tail. The specific nucleic acid sequence composition and length of the in vitro transcription template depend on the mRNA encoded by the template.

[0175] In vitro transcription systems typically include a transcription buffer, nucleotide triphosphates (NTPs), an RNAase inhibitor, and a polymerase.

[0176] The NTPs may be produced in-house or purchased from a supplier and may be selected from, but not limited to, the NTPs described herein, including natural and non-natural (modified) NTPs.

[0177] In some embodiments, the RNA transcript is capped by enzymatic capping. In some embodiments, the RNA comprises a 5' end cap.

[0178] Lipid nanoparticles (LNPs) In some embodiments, the RNA (e.g., mRNA) of the present disclosure is formulated in lipid nanoparticles (LNPs). Lipid nanoparticles typically contain an ionizable cationic lipid, a non-cationic lipid, a sterol and PEG lipid fraction, and a target nucleic acid material. The lipid nanoparticles of the present disclosure can be produced using ingredients, compositions, and methods commonly known in the art.

[0179] Stability of RSV F protein mutants In some embodiments, mutant antigens encoded by the nucleic acids of the present disclosure have superior properties to the wild-type, such as enhanced stability of the pre-fusion conformation, and the present embodiments provide detailed descriptions of assay methods for determining the stability of the pre-fusion conformation.

[0180] In some embodiments, the stability of the antigen encoded by the mutant nucleic acid can be detected by measuring thermostability and cold stability. In this embodiment, "thermostable" refers to the mutant still exhibiting binding to at least one line fusion-specific epitope after 60 minutes at an elevated temperature (i.e., 50 ° C), e.g., in this application, binding to the Φ epitope is measured. In this embodiment, "cold stability" refers to the mutant still exhibiting binding to at least one line fusion-specific epitope after 1 week of cold storage at 4 ° C, e.g., in this application, binding to the Φ epitope is measured. Some pre-fusion RSV F variants according to the present invention have increased stability when stored at low temperatures or exposed to heat compared to RSV F polypeptides that do not have one or more of the above mutations.

[0181] In some embodiments, the stability of the antigen encoded by the mutant nucleic acid can be detected by measuring stability under hypotonic and hypertonic conditions.

[0182] In this embodiment, "hypotonic stability" refers to the fact that the mutant still exhibits binding to at least one prefusion-specific epitope, e.g., binding to the Φ epitope, is measured in this application, even after incubation for 60 minutes under hypotonic conditions of 10 mM NaCl. Some pre-fusion RSV F variants of the present invention have increased stability under hypotonic conditions compared to RSV F polypeptides that do not have one or more of the above mutations.

[0183] In this embodiment, "high osmotic stability" refers to the mutant still exhibiting binding to at least one pre-fusion specific epitope, for example, binding to the Φ epitope in this application, even after 60 minutes of incubation under high osmotic conditions of 3.0 M MgCl. Some pre-fusion RSV F variants according to the present invention have increased stability under high osmotic conditions compared to RSV F polypeptides that do not have one or more of the above mutations.

[0184] In some embodiments, the stability of the antigen encoded by the mutant nucleic acid can be detected by measuring stability under acid and base conditions.

[0185] In this embodiment, "acid and base stability" refers to the RSV F mutants being incubated at room temperature for 60 minutes at pH 3.5 and pH 10, and then neutralized to pH 7.4, and still exhibiting binding to at least one line fusion-specific epitope, for example, binding to the Φ epitope in this application. Some line fusion RSV F mutants according to the present invention have increased stability under acid and base conditions compared to RSV F polypeptides that do not have one or more of the above mutations.

[0186] Biofilm interference technology Bio-Layer Interferometry (BLI) is a label-free, real-time monitoring optical detection technique primarily used for comprehensive quantitative analysis of biomolecular interactions and protein concentration measurement. BLI monitors the entire intermolecular binding process in real time and determines the intermolecular affinity constant (K D ), binding rate constant (K a ), dissociation rate constant (K d ) and other important data can be calculated.

[0187] In some embodiments, the affinity of the RSV F protein mutant for an antibody to a different epitope is determined by BLI. In this embodiment, the affinity of the RSV F protein mutant for an antibody to a different epitope is primarily determined. The affinity of the RSV F protein mutant for an antibody to a RSV pre-F antibody (3C12) and an RSV pre-F antibody (7H11) is primarily determined.

[0188] Vaccine immunogenicity The term "immunogenicity" refers to the ability to elicit, induce, stimulate, or induce an immune response in an animal against a particular antigen, with or without the presence of adjuvants. In the sense of the present invention, an immunogen is a translation product of a provided artificial nucleic acid, preferably RNA, that contains at least one coding sequence encoding at least one antigenic peptide, protein, derived from RSV as defined herein.

[0189] The term "neutralizing antibody" refers to an antibody that prevents the pathogen from binding to a receptor on the surface of a host cell during the pathogen infection process, thereby preventing the pathogen from attaching to, invading, replicating, and multiplying in the target cell. Generally, the level of "neutralizing antibody" intuitively reflects the effectiveness of a vaccine.

[0190] In some embodiments, the efficacy of an immune composition (e.g., an RNA vaccine) is measured in a mouse model. For example, an immune composition can be administered to a mouse model and the induction of neutralizing antibody titers in the mouse model can be measured. In some embodiments, the antigen-specific immune response is measured as the geometric mean titer (GMT) of neutralizing antibody titers in mouse or cotton rat serum. The geometric mean titer (GMT) is the average antibody titer of a group of subjects, calculated by multiplying all values ​​and taking the nth root of that number, where where n is the number of subjects with available data.

[0191] Viral challenge studies can also be used to assess the efficacy of the vaccines of the present disclosure, for example, by administering an immune composition to a mouse model, challenging the mouse model with a virus, and measuring survival and / or immune response (e.g., neutralizing antibody response) of the mouse model.

[0192] The terms "heterocyclic group" and "heterocyclyl" can be used interchangeably herein and refer to a non-aromatic heterocyclyl in which one or more ring-forming atoms are heteroatoms such as oxygen, nitrogen, or sulfur atoms, and include monocyclic, fused, bridged, and spirocyclic rings. Preferably, the heterocyclyl has a 5- to 7-membered monocyclic ring or a 7- to 10-membered bicyclic or tricyclic ring, optionally containing 1, 2, or 3 atoms selected from nitrogen, oxygen, and / or sulfur. Examples of "heterocyclyl" include, but are not limited to, morpholinyl, oxetidinyl, thiomorpholinyl, tetrahydropyranyl, morpholinyl, piperidinyl, 2-oxo-piperidinyl, pyrrolidinyl, 2-oxo-pyrrolidinyl, piperazin-2-one, and piperazinyl. The heterocyclyl may be substituted or unsubstituted.

[0193] 1.1 Example 1: Design of RSV F antigen mutants The present invention primarily refers to the RSV A2 (138251) protein sequence, based on which the P102A, I379V, and M447V mutations were implemented (52 candidate mutant antigen sequences (SEQ ID NOS: 1-52) and 6 control sequences (SEQ ID NOS: 53-58) all contain the above three mutations, and other mutation forms are shown in the table below).

[0194] In the form of combining multiple mutations, 52 candidate mutant antigen sequences (SEQ ID NOs: 1 to 52), 6 control sequences (SEQ ID NOs: 53 to 58), 1 wild-type sequence (SEQ ID NO: 59), and 1 positive control (SEQ ID NO: 60) were designed, and the specific mutation combinations of the mutants are as shown in the table below.

[0195] The positive control was Pfizer's commercially available vaccine, Abrysvo. TM (NDC0069-0344-05), and differs from the mRNA vaccine designed in the present invention in that it is a protein vaccine.

[0196] Control sequence design idea: Based on the mutants designed in this application, different control sequences were designed to explore the effects of mutations at different sites. For example, (1) To explore the effects of group 2 and 3 mutations on site mutagenesis, PC-RSV-063 was designed based on a combination of YK-RSV-003 mutations.

[0197] (2) To explore the effect of group 1 mutations on site mutagenesis, we designed PC-RSV-067 and PC-RSV-068 by varying the type of group 1 mutations based on the combination of YK-RSV-061 mutations. (3) To explore the effects of group 2 and 3 mutations on site mutation, PC-RSV-064, PC-RSV-065, and PC-RSV-066 were designed based on combinations of YK-RSV-061 mutations.

[0198] 1, in "M Gordon Joyce et al., Iterative structure-based improvement of a fusion-glycoprotein vaccine against RSV, Nature Structural & Molecular Biology volume 23, 811-820 (2016), doi:10.1038 / nsmb.3267" Mutant PC-RSV-063 was designed with reference to "Design cycle 4, RSV F variant: sc9-10 DS-Cav1 A149C Y458C S46G K465Q S215P E92D; Mutations: A149C, S155C, S190F, V207L, S290C, L373R, Y458C, S46G, K465Q, S215P, E92D; F1-F2 Linker residues: 103-145; Linker sequence: GS; End residue: 513".

[0199] 2. The mutant PC-RSV-064 was designed with reference to SEQ ID NO: 6 in the patent application WO2023 / 166079 A1.

[0200] [Table 4] TIFF2026010683000016.tif223170TIFF2026010683000017.tif224170TIFF2026010683000018.tif216170TIFF2026010683000019.tif223170 TIFF2026010683000020.tif216170TIFF2026010683000021.tif223170TIFF2026010683000022.tif222170TIFF2026010683000023.tif187170

[0201] 1.2 Example 2: Production of RSV F antigen mRNA vaccine After designing the antigen sequence, it was synthesized into the vector pVaX1 with optimized 5'-UTR, 3'-UTR, and poly(A) tail. All plasmids were synthesized by Assunta Bio. The plasmids were synthesized and linearized, and mRNA stock solutions were prepared by in vitro transcription. LNP coating was then performed to obtain mRNA formulation samples.

[0202] Here, mRNA transcribed using pVaX1 as the base scaffold was used for subsequent Western blot analysis, flow cytometry analysis of antigen-epitope binding, ELISA analysis of antigen-epitope binding, and BLI analysis of antigen-antibody affinity. A HIS-tag and T4 fibritin folding were added to the scaffold to detect the stability of trimers formed by the secreted protein.

[0203] The specific manufacturing process of the mRNA sample is as follows.

[0204] 1.2.1 Preparation of mRNA stock solution 1) Experimental materials NEB® Golden Gate Assembly Kit (BsaI-HFv2) (NEB, Cat. No.: R3733), DNA product purification kit (purchased from Tiangen Biotech (Beijing) Co., Ltd. (abbreviated as Tiangen), Cat. No.: DP205), ATP (purchased from Hongene Biotech Corporation (abbreviated as Hongene), Cat. No.: R1-051), CTP (Hongene, Cat.No.:R3-056), GTP (Hongene, Cat.No.:R2-057), N1-Me-pUTP (Hongene, Cat.No.:R5-064), Cap1-GAG m7G(5')ppp(5')(2'OMeA)pG (Hongene, Cat.No.:ON-134), T7 RNA polymerase (Hongene, Cat. No.: ON-004), RNase Inhibitor (Hongene, Cat. No.: ON-039), Pyrophosphatase (Hongene, Cat. No.: ON-025), 10×IVT reaction buffer (Hongene, Cat No: ON-062), DNA enzyme I (DNase I) (Hongene, Cat No: ON-109).

[0205] 2) Experimental process (1) Plasmid linearization enzyme cleavage Plasmid DNA was linearized using BsaI-HFv2 endonuclease. The enzyme digestion reaction system contained plasmid DNA with the target gene inserted, 10x rCutSmart buffer, BsaI-HFv2, and sterile enzyme-free water (RNase-free ddH2O). The enzyme digestion reaction temperature was 37°C, and the enzyme digestion time was 15-30 minutes. The enzyme digestion reaction system is shown in the table below.

[0206] [Table 5]

[0207] After the enzyme cleavage reaction, the linearized product was recovered using a DNA product purification kit, and the concentration of the product was measured using an ultramicro UV spectrophotometer (Denovix). The length and state of the linearized plasmid template were detected through an agarose gel.

[0208] (2) Co-transcriptional capping The linearized plasmid prepared in the above step was used as a template, and NTP solution (NTPs) and cap1 capped analogs were used as starting materials. The resulting DNA was transcribed with T7 RNA polymerase to form m The cap1-capped analogue was Cap1-GAG, which has the structure m7G(5')ppp(5')(2'-OMeA)pG and the molecular formula is C 32 H 43 N 15 O 24 It was P4.

[0209] The specific reaction system can be seen in the table below, and the prepared reaction system was incubated with shaking in a constant temperature incubator at 37°C for 3 hours to react.

[0210] [Table 6]

[0211] After the reaction was completed, 1 μl of DNase I was added to 20 μl of the co-transcriptional capping reaction mixture, mixed uniformly, and then digested at 37°C for 30 minutes to obtain the co-transcriptional capped product. Got it.

[0212] (3) Purification by LiCl precipitation method The co-transcriptionally capped product obtained above was purified by lithium chloride precipitation, as follows. Addition of LiCl: LiCl was added to the co-transcription capped product to a final concentration of 2.8 M and precipitated in the cold for 2 hours.

[0213] Precipitation: High speed centrifugation was performed at 12,000 rpm for 15 minutes, and the precipitate was retained.

[0214] Washing: The sample was washed twice with 75% ethanol and dissolved in sterile enzyme-free water to obtain an mRNA stock solution.

[0215] Detection: After purification, the concentration of the transcription product was measured using an ultramicro UV spectrophotometer (Denovix), and the integrity was detected by capillary electrophoresis.

[0216] 1.2.2 Production of mRNA-LNP (mRNA vaccine) Cationic lipid, DSPC, cholesterol, and DMG-PEG2000 were each accurately weighed and dissolved in absolute ethanol to prepare a carrier mixture solution according to the molar ratio (the molar ratio of cationic lipid, neutral lipid, structural lipid, and polymer-conjugated lipid was controlled to 49:10:39.5:1.5), which was then reserved as the organic phase for later use.

[0217] The mRNA was dissolved in citrate buffer and diluted to prepare a solution, which was then reserved as the aqueous phase. TM The organic and aqueous phases were aspirated using syringes, and the organic phase was placed in the left pump and the aqueous phase in the right pump to produce an LNP intermediate solution within the microfluidic device.

[0218] The LNP intermediate solution was diluted with PBS solution, concentrated by ultrafiltration using an ultrafiltration device, and concentrated to a predetermined volume. After concentrating, the solution was diluted with PBS solution and concentrated using an ultrafiltration device. Ultrafiltration was stopped when the final concentrated volume was less than 5 ml.

[0219] 1.3 Example 3: Detection of RSV F antigen variant expression by Western Blot To confirm whether the designed antigen mRNA can be expressed in cells, the mRNA was transfected into cells, and the expression of the antigen mRNA in the cells was detected using Western blotting.

[0220] 1) Experimental materials 293T cells (purchased from ATCC Cell Bank), Primary antibody: HRSV-F (purchased from AbMax Biotechnology Co., Ltd., hereafter referred to as AbMax, Cat. No.: 11049-R302), Secondary antibody: Goat anti-mouse IgG (H+L) secondary antibody IgG (H+L) Secondary Antibody), HRP (purchased from Shanghai Beyotime Biotechnology Co., Ltd., hereinafter abbreviated as Beyotime, Cat. No.: A0286), Experimental group: a mixture of protein samples obtained by transfecting cells with the series of mRNA vaccines prepared in Example 2 and then dissolving them; Internal reference protein: glyceraldehyde-3-phosphate dehydrogenase GAPDH.

[0221] 2) Experimental process

[0222] Cell culture and transfection: 1.3 × 10 293T cells 5 The cells were seeded at 1000 μg / well into a 24-well plate. The next day, the mRNA-LNP samples (containing 300 ng of mRNA) were added directly to the cells and mixed evenly.

[0223] Protein sample preparation: (1) 16 hours after transfection, the cells were removed from the incubator, pipetted repeatedly to remove all cells from the bottom of the dish, transferred to a 1.5 ml centrifuge tube, and centrifuged at 1,500 rpm at 25°C for 3 minutes. (2) The supernatant was discarded, and 1 ml of PBS was added. The cells were resuspended by pipetting, centrifuged, and centrifuged again at 1,500 rpm for 3 minutes. (3) The above steps were repeated twice. (4) 50 μl of RIPA lysis solution (0.5 μl of 100x protein inhibitor was added before using the RIPA lysis solution) was added to the cells in each tube, and the mixture was left on ice for 30 minutes, during which time the samples were vortexed for 30 seconds every 5 minutes. (5) The centrifuge was pre-cooled, and after the cells were completely lysed, the samples were centrifuged at 12,000 rpm at 4°C for 10 minutes. The supernatant was aspirated and transferred to a new 1.5 ml centrifuge tube to obtain the test protein samples.

[0224] Development and analysis: Protein sample concentrations were detected using a BCA quantification kit. The gel was spotted, and 4 μl of marker was spotted, followed by electrophoresis at 200 V for 30 minutes. The film was transferred using a dry film transfer device, the gel was removed, and a transfer film "sandwich" was created. The negative plate was then covered, and voltages of 21 V, 23 V, and 25 V were applied for 1 minute, 4 minutes, and 2 minutes, respectively, followed by blocking at room temperature for 1 hour. Primary antibody incubation: Incubated at room temperature for 1 hour. Membrane washing: Washed with 1x TBST buffer, with three washes for 5 minutes each. Secondary antibody incubation: Incubated at room temperature for 1 hour. Membrane washing: Washed with 1x TBST buffer, with three washes for 5 minutes each. Color development: 1 ml of developer and fixer were mixed at a 1:1 ratio and added dropwise to the membrane to develop the color, which was then photographed.

[0225] 1.4 Detection of RSV F antigen variant Φ and II epitope binding by flow cytometry

[0226] To determine whether the RSV F antigen mutants have pre-fusion epitopes, in this example, flow cytometry detection was performed using the RSV pre-F antibody (3C12) (specifically binding to the RSV pre-F Φ epitope) and the RSV F antibody (11A9) (binding to pre-F and post-F II epitopes), and it was expected that the RSV F protein mutants in the pre-F conformation would bind to the tested antibodies.

[0227] 1) Experimental materials 293T cells (purchased from ATCC Cell Bank), RSV pre-F antibody (3C12), Φ epitope (Epitope Φ) (Novoprotein, Cat.No.:DA101), RSV F antibody (11A9), II epitope (Epitope Φ) II) (Novoprotein, Cat.No.:DA091), Experimental group: mRNA vaccine corresponding to the expressed antigen in Example 3.

[0228] 2) Experimental process Cell transfection: 1.3 × 10 293T cells 5 The cells were seeded at 1000 μg / well into a 24-well plate. The next day, the mRNA-LNP samples (containing 300 ng of mRNA) were added directly to the cells and mixed evenly.

[0229] (2) Cell collection: After 16 hours of transfection, the cells were removed from the incubator, and the medium was removed by pipetting. After carefully removing the medium from the well plate, 500 μl of 0.1% BSA solution was added to each well, and the cells were pipetted and transferred to a 1.5 ml centrifuge tube.

[0230] (3) Centrifugation: Cells were collected by centrifugation at 400 g and 4°C for 5 minutes in the dark, and the supernatant was discarded.

[0231] (4) Staining: 50.6 μl of RSV fluorescent antibody dilution was added to each of the control and detection samples, and the cells were resuspended and incubated at 4°C for 2 hours in the dark.

[0232] (5) Washing: After incubation, 500 μl of 0.1% BSA solution was added to each sample tube, and the tubes were centrifuged at 400 g and 4°C for 5 minutes in the dark to collect the cells. The supernatant was discarded. This procedure was repeated three times.

[0233] (6) Cell resuspension: 200 μl of 0.1% BSA solution was added to each sample, and the cells were carefully pipetted with a pipette to prepare a cell suspension.

[0234] (7) Flow cytometry detection: The binding kinetics of RSV F mutants to antibodies was measured using a flow cytometer, with a concentration of 1 x 10 per measurement. 4 cells were detected.

[0235] 1.5 Example 5: Detection of RSV F antigen mutant stability by ELISA To investigate the stability of RSV F antigen mutants under different pH, temperature, and osmolality conditions, stress tests were performed on the mutants in this example under different treatment conditions.

[0236] 1) Experimental materials 293T cells (purchased from ATCC Cell Bank), Primary antibody: RSV pre-F antibody (3C12), Φ epitope (purchased from Novoprotein Scientific Inc. (abbreviated as Novoprotein), Cat. No.: DA101); Standard product: RSV pre-F Trimer Protein (Novoprotein, Cat.No.: DRA230), Secondary antibody: Peroxidase-labeled goat anti-mouse IgG (H+L) (Peroxidase AffiniPure Goat Anti-Human IgG (H+L)) (Yeasen Biotechnology (Shanghai) Co., Ltd. (abbreviated as Yeasen), Cat. No.: N / A), Experimental group: mRNA vaccine screened in Example 4.

[0237] 2) Experimental process: (1) Cell transfection and harvesting: The transfection steps were the same as in Example 3, and the cell supernatant was collected.

[0238] (2) Different pH conditions: The RSV F protein solution was adjusted to pH 3.5 and pH 10 with 10% NaOH and 1 M HCl solutions, respectively, incubated at room temperature for 60 minutes, and then neutralized to pH 7.4.

[0239] (3) High temperature treatment: The RSV F protein solution was incubated at 50°C for 60 minutes.

[0240] (4) Low-temperature treatment: The RSV F protein solution was stored at 4°C for 1 week.

[0241] (5) Hypoosmotic treatment: The cell supernatant contained 110 mM NaCl and was diluted to 10 mM NaCl with 2.5 mM Tris buffer (pH 7.5) and incubated at room temperature for 60 minutes.

[0242] (6) Hyperosmotic treatment: The cell supernatant was adjusted to 3.0 M MgCl2 with 4.5 M MgCl2 and incubated at room temperature for 60 minutes.

[0243] (7) Detection of antigen expression by ELISA:

[0244] Encapsulation: The encapsulation working solution was added to a 96-well ELISA microplate and left at 2°C to 8°C for 16 to 20 hours.

[0245] Washing the plate: Phosphate buffered saline (PBST) was added to each well and the plate was washed five times using a plate washer and tapped dry on absorbent paper.

[0246] Blocking: After adding the blocking solution, the plate was placed on a thermostatic shaker for ELISA plates and incubated at 37°C and 400 rpm for 1.5 hours.

[0247] Washing the plate: Add PBST wash solution to each well and wash the plate using a plate washer. The sample was washed five times and patted dry on absorbent paper.

[0248] Primary antibody incubation: Diluted primary antibody solution was added to each well, the plate was sealed with a plate sealing membrane, and the plate was incubated at 37°C, 400 rpm for 1.5 hours.

[0249] Washing the plate: PBST washing solution was added to each well, and the plate was washed five times using a plate washer and tapped dry on absorbent paper.

[0250] Secondary antibody incubation: Secondary antibody dilution was added to each well, the plate was sealed with a plate sealing membrane, and incubated at 37°C, 400 rpm for 45 minutes.

[0251] Washing the plate: PBST washing solution was added to each well, and the plate was washed five times using a plate washer and tapped dry on absorbent paper.

[0252] Color development: A color development solution was added, the plate was sealed with a plate sealing membrane, and placed in an ELISA plate thermostatic shaker and incubated at 37°C and 400 rpm for 25 minutes.

[0253] Termination of reaction: After the color development was completed, 50 μl / well of a reaction stop solution was added to the ELISA plate to terminate the reaction.

[0254] Plate reading: Using a multi-function microplate reader, two wavelengths, 450 nm and 630 nm, were set and the absorbance values ​​(OD values) were read within 5 minutes.

[0255] 1.6 Example 6: Detection of Different Epitope Binding of RSV F Antigen Mutants by ELISA To examine the binding of RSV F antigen mutants to antibodies recognizing different epitopes, binding assays were performed in this example using ELISA.

[0256] 1) Experimental materials 293T cells (purchased from ATCC Cell Bank), Primary antibody: RSV pre-F antibody (3C12), Φ epitope (Epitope Φ) (Novoprotein, Cat. No.: DA101), RSV pre-F antibody (7H11), Quaternary epitope (Novoprotein, Cat. No.: DA109), RSV F antibody (11A9), II epitope (Epitope II) (Novoprotein, Cat. No.: DA091), RSV F antibody (4B9), III epitope (Epitope III) (Novoprotein, Cat. No.: DA110), RSV pre-F antibody (7E11), V epitope (Epitope V) (Cat.No:DA119), Standard: RSV prefusion F-Trimer Protein (Novoprotein, Cat. No.: DRA230), Secondary antibody: Peroxidase-labeled goat anti-mouse IgG (H+L) (Peroxidase AffiniPure Goat Anti-Human IgG (H+L)) (Yeasen, Cat. No.: N / A), Experimental group: mRNA vaccine screened in Example 5.

[0257] 2) Experimental process Cell culture and transfection, preparation of protein samples: The experimental process was the same as in Example 3. Detection of antigen expression by ELISA: The experimental process was the same as in Example 5.

[0258] 1.7 Example 7: Detection of the affinity of RSV F antigen mutants with different antibodies by BLI To investigate the affinity of RSV F antigen mutants with antibodies that specifically recognize prefusion conformational epitopes, affinity measurements were performed in this example using Bio-Layer Interferometry (BLI).

[0259] 1) Experimental materials 293T cells (purchased from ATCC Cell Bank), RSV pre-F antibody (3C12), Φ epitope (Novoprotein, Cat. No.: DA101), RSV pre-F antibody (7H11), Quaternary epitope (Novoprotein, Cat. No.: DA109), Experimental group: mRNA vaccine screened in Example 6.

[0260] 2) Experimental process: Protein sample preparation: The experimental process was the same as in Example 3. Each antibody was diluted to 10 μg / ml using 1×PBST solution. The RSV pre-F antibody (3C12) and the RSV pre-F antibody (7H11) were captured with the ProA probe in 1x PBST solution, respectively, with the capture level controlled at 4.0 nM. The antibodies were then reacted sequentially with diluted target proteins, and the fully reacted solid-state binding products were subjected to dissociation analysis in 1x PBST buffer. The protein interaction was detected using a protein interaction meter, and the results were analyzed with Data Analysis 12.0 software to obtain the binding rate, dissociation rate and affinity constant.

[0261] 1.8 Example 8: Immunogenicity study of RSV F antigen mutants in mice In this example, mice were immunized in vivo to determine the antibody titer produced by RSV F antigen in mice.

[0262] 1.8.1 Immunization of Balb / c mice Experimental materials Animals: Balb / c mice, female, 6-8 weeks old Negative control: LNP sample Positive control: Abrysvo TM , Test object: YK-RSV-001, YK-RSV-011, YK-RSV-030, YK-RSV-036, YK-RSV-044, YK-RSV-060, YK- RSV-062, YK-RSV-061, YK-RSV-003, YK-RSV-012, YK-RSV-015, YK-RSV-039, YK-RSV-054.

[0263] 2) Mouse vaccination protocol

[0264] [Table 7] TIFF2026010683000027.tif54170

[0265] 3) Sample collection and pretreatment Approximately 0.2 ml of blood was collected from the orbital venous plexus of each animal, and serum was separated (centrifugation at 4000 rpm / min for 10 min at 4°C) and stored in a refrigerator at 4°C until processing.

[0266] 4) Health checkup Once daily, including but not limited to mortality, morbidity, respiratory discharge, secretions, feces, and eating and drinking habits.

[0267] 5) Humane endpoint According to IACUC protocol, mice that lost more than 20% of their body weight during the experiment (before infection, based on the weight on day 0; after infection, based on the weight on the day of inoculation) and / or showed signs of moribundity were euthanized and recorded as dead animals in the results.

[0268] 1.8.2 Detection of RSV-neutralizing antibody titers in mouse serum 1) Purpose of detection To measure serum anti-respiratory syncytial virus (RSV) A2 and RSV B18357 neutralizing antibody titers using a spot reduction-based microneutralization test.

[0269] 2) Main reagents Primary antibody: Palivizumab Research Grade Palivizumab (purchased from Wuhan Chemstan Biotechnology Co., Ltd., abbreviated as Chemstan, Cat. No.: CSD00024), Secondary antibody: rabbit anti-human IgG H&L (HRP) (Abcam, Cat.No.:ab6759),

[0270] 3) Data calculation formula: (1) Sample inhibition rate: Calculated using Excel (single well calculation), sample inhibition rate = (1 - (number of sample spots / number of virus control spots)) × 100%. (2) The dilution ratio of the sample was plotted on the X-axis and the inhibition rate on the Y-axis. The data was entered into GraphPad software, and the IC value of the sample was calculated using four-parameter fitting. 50 The linear correlation coefficient R2 was calculated with the IC 50 The values ​​were the neutralizing antibody titers of the samples. The titers were retained to the integer part, and the linear correlation coefficient R2 was retained to two decimal places. (3)Average value:

[0271]

number

[0272] 4) Experimental process The test serum samples were diluted in a gradient, and then a predetermined amount of RSV virus was added. After the neutralization reaction, the mixture was added to the cell plate and cultured for 22-26 hours. Finally, the neutralizing antibody titer of the serum sample was calculated based on the spot formation. The operation process is as follows:

[0273] Cell preparation and plating: HEp-2 cells were purchased from Yuchi (Shanghai) Biotechnology Co., Ltd. They were resuscitated and cultured before the experiment, and were used for measurements 3 to 20 generations after resuscitation. Cells were digested in advance and plated at 2.5 × 10 cells per well in a 96-well plate. 5 The cells were seeded at 0.1 ml / well and cultured at 37°C in a 5% CO2 incubator for 17 to 23 hours. After confirming that the cells were normal, serum samples were collected and diluted according to a gradient.

[0274] Control group setup: Controls were set up in a 96-well plate (neutralization plate), and cell control (CC) and virus control (VC) dilutions were added respectively, with duplicate wells set up.

[0275] Virus addition: The virus diluted with diluent was added to the sample wells and VC wells of the 96-well plate, and the 96-well plate was gently tapped to mix the sample and virus evenly. The plate was then placed in an incubator at 37°C and 5% CO2 to carry out the neutralization reaction.

[0276] Cell preparation, infiltration, and culture: The 96-well plate on which the cells had been seeded was removed, the original medium was aspirated, and the neutralization plate mixture was aspirated and added to the 96-well plate on which the cells had been seeded. The plate was then placed in an incubator at 37°C with 5% CO2 for culture.

[0277] Fixation and staining: After incubation, the original culture medium was aspirated and discarded, and a predetermined volume of fixative was added to each well. After fixation, the fixative was discarded and the wells were washed twice with 1x PBS. After blocking with blocking solution, the blocking solution was discarded and the wells were washed twice with 1x PBS. After incubation with the primary antibody working solution, the wells were discarded and washed twice with 1x PBS. After incubation with the HRP secondary antibody working solution, the wells were discarded and washed twice with 1x PBS. After incubation with the color developing solution at room temperature for 5-20 minutes, the color developing solution was discarded and the wells were gently tapped dry. The spots were then counted using an enzyme-linked immunospot analyzer.

[0278] Calculation of results: Neutralizing antibody titer: Calculate the inhibition rate of each gradient of the sample. Enter the sample dilution ratio - inhibition rate into GraphPad software for fitting, and calculate the IC of the sample. 50 Value and IC 90 Calculate the IC value 50 Value and IC 90 The values ​​were the neutralizing antibody titers of the samples.

[0279] 1.8.3 Detection of RSV-binding antibody titers in mouse serum 1) Purpose of detection Serum samples from the RSV mRNA vaccine immunogenicity study were tested using a validated indirect ELISA method developed in-house, and test endpoints included RSV pre-F and post-F protein-specific antibody titers, providing data support for the analysis of vaccine immunogenicity and protection against viral infection.

[0280] 2) Main reagents Coating solution: RSV-pre-F-11 protein (Vazyme, Cat. No.: RM2187), RSV-post-F-11 protein (Vazyme, Cat. No.: RM2188), Positive quality controls: RSV-pre-F0 specific monoclonal antibody (Monoclonal Anti-RSV-pre-F0 specific antibody*) (Acro, Cat. No.: RS0-Y132), RSV-post-F0 specific monoclonal antibody (Monoclonal Anti-RSV-post-F0 specific antibody*) (Acro, Cat. No.: RSV-Y180), Secondary antibody: HRP-labeled rabbit anti-mouse IgG secondary antibody (Rabbit Anti-Mouse IgG HL (HRP)) (Abcam, Ab6728).

[0281] 3) Data calculation formula Average value (Mean): Mean = Average (OD value X1: OD value Xn), Signal-to-noise ratio (SNR): SNR = sample mean OD value / NC mean OD value, Coefficient of variation (CV%): CV% = STDEV (OD value X1:OD value Xn) / mean OD value × 100, Binding antibody titer calculation: The titer value of each analytical batch was calculated using the FORECAST(x, known_y's, known_x's) function, where x is the cutoff value, known_y's are two dilution factors across the cutoff value, known_x are two OD values ​​across the cutoff value, and the resulting corresponding dilution factors were the titer values ​​of each sample.

[0282] 4) Experimental process In the primary antibody incubation step, diluted test serum samples were added, the plates were sealed with a sealing film, and the plates were incubated in an enzyme-labeled thermostatic shaker at 37°C and 400 rpm for 1.5 hours, in the same manner as in the ELISA detection in Example 5.

[0283] 1.9 Example 9: Immunogenicity Study of RSV F Antigen Mutants in Cotton Rats In this example, cotton rats were immunized in vivo to assess the antibody titers produced by RSV F antigen in cotton rats and the immunoprotective effect after challenge.

[0284] 1.9.1 Immunization of cotton rats 1) Experimental materials Animal: Cotton rat, female, 6-8 weeks old Negative control: LNP sample, Positive control: Abrysvo TM , Test items: YK-RSV-001, YK-RSV-061, YK-RSV-062, YK-RSV-003, YK-RSV-012, YK-RSV-015, YK-RSV-036, YK-RSV-039, YK-RSV-044, YK-RSV-054, YK-RSV-060.

[0285] 2) Cotton Rat Vaccination Protocol

[0286] [Table 8] TIFF2026010683000030.tif33169

[0287] 3) Collection of tissue samples On day 54, lungs and nasal turbinates were aseptically collected.

[0288] 4) Virus attack information The challenge was performed by intranasal instillation on day 49, the strain was RSV A2 virus, and the challenge volume was 50 μl (5 × 10 5 PFU).

[0289] 1.9.2 Measurement of serum RSV-binding and neutralizing antibody titers in cotton rats The methods for detecting binding and neutralizing antibody titers were the same as in Examples 4.8.2 and 4.8.3.

[0290] 1.9.3 Detection of Respiratory Syncytial Virus (RSV) Titers in Cotton Rat Tissue Samples by Plaque Assay 1) Purpose of detection Respiratory syncytial virus (RSV) titers were measured in left lung and nasal turbinate tissue samples from cotton rats using the plaque method, and detection was completed for all biological samples.

[0291] 2) Main reagents Primary antibody: Palivizumab (Research Grade Palivizumab) (Chemstan, Cat. No.: CSD00024), Secondary antibody: rabbit anti-human IgG H&L (HRP) (Abcam, Cat.No.:ab6759), Quality control virus RSV A2.

[0292] 3) Experimental process After gradient dilution of the test sample, the cells were infiltrated, and after removing the infiltration fluid, a cover solution was added and then cultured until plaques formed. The virus titer of the tissue sample was finally calculated based on the presence of plaques or lesions. The operation process is as follows: Cell preparation and plating: HEp-2 cells were purchased from Yuchi (Shanghai) Biotechnology Co., Ltd. They were resuscitated and cultured before the experiment, and were used for measurements 3 to 20 generations after resuscitation. Cells were digested in advance and plated at 2 × 10 cells per well in a 24-well plate. 5 The cells were seeded at 1 ml / well and cultured at 37°C in an incubator with 5% CO2 for 17 to 23 hours.

[0293] Tissue sample processing: After obtaining left lung and nasal turbinate tissue samples, virus maintenance solution was added, tissue grinding was performed, the samples were centrifuged, and the supernatant was collected and gradient diluted.

[0294] Cell treatment: The pre-seeded cell plates were removed from the 37°C carbon dioxide incubator, the culture supernatant was aspirated off, and the cells were washed twice with 1x PBS, and the residual liquid was aspirated off. Viral infiltration and adsorption: The supernatant of the diluted tissue homogenate was added to the corresponding wells of the cell plate, and the 24-well plate was placed in a 37°C, 5% CO2 incubator to allow the virus to completely adsorb to the cells. Addition of the coating layer: After the adsorption was completed, the virus solution was removed by aspiration, and the coating layer was added to each well. The wells were then cultured in a 37°C, 5% CO2 incubator, and the formation of lesions and plaques was observed daily. Fixation: After typical plaques appeared in the virus wells, the covering layer was removed by aspiration, and 4% paraformaldehyde was added to each well for fixation. Staining: After fixing the cell plate, discard the fixative, wash three times with 1x PBS, add blocking solution to block, discard the blocking solution, wash three times with 1x PBS, add primary antibody working solution and incubate, discard the antibody, wash three times with 1x PBS, add HRP secondary antibody working solution and incubate, discard the antibody, wash three times with 1x PBS, add color developing solution and incubate at room temperature for 10-20 minutes, discard the color developing solution, and film the plaques. Observed with the device. Calculating the result: Virus titer [PFU / mL] = (average number of plaques × sample dilution factor) / inoculation volume (mL), Tissue viral titer [PFU / g] 1* = [(average number of plaques × tissue sample dilution ratio) / inoculum volume (mL) × tissue grinding liquid volume (mL)] / tissue weight (g).

[0295] Tissue viral Lg titer [Lg PFU / g] is the logarithm of the tissue viral titer [PFU / g] based on 10. 1*: All plaques at dilutions that could be accurately counted were selected for statistical calculation, and the arithmetic mean value was used for calculation.

[0296] 1.9.4 Detection of pulmonary inflammation indicators by HE staining 1) Purpose of detection HE staining is used to detect inflammation in lung tissue.

[0297] 2) Main reagents Xylene solution (Shanghai Zhanyun Chemical Co., Ltd., Cat. No.: 1330-20-7), hematoxylin solution (Mayer) (Baso, Cat. No.: BASO-BA-4021), eosin (alcoholic) (Baso, Cat. No.: BASO-BA-4022).

[0298] 3) Experimental process After perfusion, the lung tissue was fixed in 4% paraformaldehyde for more than 24 hours, dehydrated, and sliced ​​at the maximum parenchymal region to a thickness of 4 μm. The staining rack on which the sections were placed was placed in an oven at 60-65°C for 1 hour to bake the sections and melt the paraffin wax until it became a transparent liquid. The staining rack was then placed at room temperature for 10 minutes to cool. After cooling, it was stained with HE and finally the sections were sealed.

[0299] 4) Results Semi-quantitative scoring was performed manually, and the scoring criteria were as follows: Data were statistically analyzed using Mean+SEM and one-way ANOVA.

[0300] [Table 9]

[0301] 2Result analysis 2.1 Expression results of RSV F antigen mutants YK-RSV-001, YK-RSV-002, YK-RSV-003, YK-RSV-004, YK-RSV-005, YK-RSV-008, YK-RSV-0 09, YK-RSV-010, YK-RSV-011, YK-RSV-012, YK-RSV-013, YK-RSV-014, YK-RSV-015, YK-RSV -016, YK-RSV-017, YK-RSV-018, YK-RSV-019, YK-RSV-022, YK-RSV-029, YK-RSV-030, YK-R SV-031, YK-RSV-032, YK-RSV-033, YK-RSV-034, YK-RSV-035, YK-RSV-036, YK-RSV-037, YK -RSV-038, YK-RSV-039, YK-RSV-040, YK-RSV-041, YK-RSV-042, YK-RSV-043, YK-RSV-044, YK-RSV-045, YK-RSV-046, YK-RSV-048, YK-RSV-050, YK-RSV-051, YK-RSV-052, YK-RSV-05 3. YK-RSV-054, YK-RSV-055, YK-RSV-056, YK-RSV-057, YK-RSV-058, YK-RSV-059, YK-RSV-060, YK-RSV-061 and YK-RSV-062 mutants all showed significant protein expression with a size of approximately 75 kDa, which is consistent with the theoretical size of the antigen. These mutants could be used as effective candidate antigens for the next step of immunogenicity screening and evaluation. Therefore, the above 50 mutants were further screened by measuring antigen epitope binding by flow cytometry.

[0302] 2.2 Measurement of epitope binding of RSV F antigen mutants Among the major neutralizing-active antigenic epitopes of the RSV F protein, the Φ epitope was located only in the prefusion conformation, whereas the II epitope was retained in both the prefusion and postfusion conformations.

[0303] The ability of an antibody to specifically bind to the Φ epitope is related to neutralizing activity, and the low binding rate of the Φ epitope affected the neutralizing activity, resulting in a decrease in neutralizing activity.

[0304] The RSV pre-F antibody (3C12) used in this example specifically binds to the Φ epitope of RSV pre-F, and the RSV F antibody (11A9) binds to the II epitope of pre-F and post-F. Flow cytometry detected that the Φ epitope and II epitope on the mutants bound to the epitopes of the corresponding antibodies 3C12 and 11A9, respectively, which further indicated whether the pre-fusion conformation of the RSV F mutant was stable. The epitope binding data are shown in the table below.

[0305] [Table 10] TIFF2026010683000033.tif219169TIFF2026010683000034.tif33169

[0306] There were 20 RSV F mutants with Φ epitope binding rates of 90% or higher for antibody 3C12, and the binding rates of the Φ epitopes on these mutants to antibody 3C12 were all superior to those of the controls PC-RSV-064, PC-RSV-065, PC-RSV-066, PC-RSV-067, PC-RSV-068, and wild-type RSV A2(138251)-wt.

[0307] 1) The binding rate of the Φ epitope of the following 11 RSV F mutants to antibody 3C12 exceeded 90% with a distribution of 91.6% to 94.9%, which was significantly better than the control. Mutants within this range are as shown in the table below and include YK-RSV-061, YK-RSV-003, YK-RSV-062, YK-RSV-054, YK-RSV-039, YK-RSV-044, YK-RSV-012, YK-RSV-015, YK-RSV-001, YK-RSV-036, and YK-RSV-060.

[0308] [Table 11]

[0309] 2) The binding rates of the Φ epitopes on the following nine RSV F mutants and antibody 3C12 were all greater than 90%, significantly superior to the control. The binding rates of the Φ epitopes on the nine mutants in the table below with antibody 3C12 ranged from 90.3% to 94.6%, and the mutants within this range were as shown in the table below.

[0310] [Table 12]

[0311] 3) The binding rate of antibody 3C12 to the Φ epitope of the following 15 RSV F mutants was 80% to 90%, which was superior to or comparable to the control. The mutants within this range are shown in the table below. The binding rates of the Φ epitope on these mutants with antibody 3C12 were 81.2% to 89.6%, and the binding rates of the II epitope on these mutants with antibody 11A9 were 88.7% to 95.1%, with Φ / II ratios ranging from 90.7% to 95.4%.

[0312] [Table 13]

[0313] A total of 15 mutants had less than 80% binding of antibody 3C12 to the Φ epitope on the RSV F mutant.

[0314] 4) The binding rate of the Φ epitope of seven RSV F mutants to antibody 3C12 was less than 80%.

[0315] The binding rates of the Φ epitope of the following seven mutants with antibody 3C12 were 76.8% to 79.8%, the binding rates of the II epitope with antibody 11A9 were 83.4% to 87.1%, and the Φ / II ratios were 88.2% to 95.7%. The mutants within this range are as shown in the table below.

[0316] [Table 14]

[0317] In summary, the Φ epitopes on the 42 mutants screened above (see Tables 8, 9, 10 and 11) showed higher binding rates with the corresponding antibodies.

[0318] 2.3 Stability screening results of RSV F antigen mutants The prefusion conformation of RSV F is metastable and can spontaneously rearrange into a highly stable postfusion conformation. The triggering factor for the transition from the prefusion conformation to the postfusion conformation upon cell entry is unknown. However, when F protein is extracted from membranes using detergents such as Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween 20, Tween 80, octyl glucoside, octyl thioglucoside, SDS, CHAPS, or CHAPSO, or when expressed as an extracellular domain, the F glycoprotein is prone to convert to the postfusion conformation after physical or chemical stress or storage (J.S. McLellan et al., 2013; J.O. Ngwuta et al., 2015). While the prefusion conformation of RSV F is useful for inducing or enhancing vaccine neutralizing activity, physical stability directly affects the conformation of the protein. Therefore, in this example, we screened for antigens with better stability under various treatment conditions.

[0319] In this example, the stress test was defined as screening the stability of mutants under different treatment conditions. The stress test included storing the mutants at 4°C for 1 week, incubating them at 50°C for 60 minutes, treating them under hypotonic and hypertonic conditions at room temperature for 60 minutes, and treating them under acid and base conditions at room temperature for 60 minutes. Binding of the mutants to the RSV pre-F antibody (3C12) that recognizes the Φ epitope was detected by ELISA. The stress resistance parameter in this example was calculated as follows: Stress resistance parameter = binding amount of sample and antibody after stress ÷ binding amount of sample and antibody without stress The amount of body binding. More stable mutants are expected to have higher stress resistance. The results of the stability screening are as follows:

[0320] 1) All mutants retained their antibody-binding ability under different stress tests. The ELISA results showed that the mutants still maintained their affinity with the RSV pre-F antibody (3C12), because the RSV pre-F antibody (3C12) specifically binds to the Φ epitope of the pre-fusion conformation of the RSV F protein, and that the mutants remained stable in the pre-fusion conformation after different stress tests.

[0321] 2) Some mutants were highly stable after stress testing. The stability results of all mutants were analyzed between groups, and the mutants with stress resistance parameters less than 0.4 after storage at 4°C for 1 week, stress resistance parameters less than 0.5 after incubation at 50°C for 60 min, stress resistance parameters less than 0.5 after 60 min of hyperosmotic treatment, and stress resistance parameters less than 0.5 after 60 min of hypoosmotic treatment showed relatively significant differences between groups and were therefore used as standards for antigen screening.

[0322] [Table 15] TIFF2026010683000040.tif218169TIFF2026010683000041.tif218169TIFF2026010683000042.tif89169

[0323] After storing the following mutants at 4°C for one week, the stress tolerance parameters were 0.44 or higher, reaching as high as 0.86; after culturing at 50°C for 60 minutes, the stress tolerance parameter was 0.64 or higher; after treatment under high osmotic pressure conditions for 60 minutes, the stress tolerance parameter was 0.61 or higher; after treatment under low osmotic pressure conditions for 60 minutes, the stress tolerance parameter was 0.70 or higher; after treatment under pH=3.5 and pH=10 conditions for 60 minutes, the stress tolerance parameters were all 0.56 or higher. The stress tolerance parameters after treatment under different conditions were superior to or equivalent to the control.

[0324] [Table 16] TIFF2026010683000044.tif74169

[0325] [Table 17] TIFF2026010683000046.tif47169

[0326] 3) Summary: (1) The physical stability screening results showed that 20 mutants (YK-RSV-061, YK-RSV-003, YK-RSV-062, YK-RSV-054, YK-RSV-039, YK-RSV-044, YK-RSV-012, YK-RSV-015, YK-RSV-016, YK-RSV-017, YK-RSV-018, YK-RSV-019, YK-RSV-020, YK-RSV-023, YK-RSV-024, YK-RSV-025, YK-RSV-026, YK-RSV-027, YK-RSV-028, YK-RSV-029, YK-RSV-030, YK-RSV-031, YK-RSV-032, YK-RSV-033 It was found that the following antibodies (including YK-RSV-001, YK-RSV-036, YK-RSV-060, YK-RSV-009, YK-RSV-010, YK-RSV-011, YK-RSV-030, YK-RSV-043, YK-RSV-048, YK-RSV-050, YK-RSV-055, and YK-RSV-057) showed better stability under each stress test, indicating strong overall stability. The binding ability of these antibodies to different epitopes was detected by ELISA as described below.

[0327] 2.4 ELISA results between RSV F antigen mutants and antibodies that recognize different epitopes Among the Φ, Quaternary, V, II, and III epitopes involved in this application, compared with epitopes II and III present in both the pre-fusion and post-fusion conformations, the Φ, Quaternary, and V epitopes specifically bind only to the pre-fusion conformation. Research has confirmed that certain epitopes present only in the pre-fusion conformation are sensitive to neutralizing antibodies and can induce a stronger neutralizing antibody response.

[0328] In this application, the binding of different mutant antigens to antibodies recognizing different epitopes was detected by ELISA, and the binding strength between the antigen and antibody was measured by the binding strength. Here, (1) the RSV pre-F antibody (3C12) recognizing the Φ epitope only binds to the F protein in the pre-fusion conformation, (2) the RSV pre-F antibody (7H11) recognizes a quaternary epitope (spanning both the IV and V epitopes) and specifically binds to the F protein in the pre-fusion conformation, (3) the RSV F antibody (11A9) recognizing the II epitope can simultaneously recognize both the pre-fusion and post-fusion conformations of the F protein, (4) the RSV F antibody (4B9) recognizing the III epitope preferentially recognizes the pre-fusion conformation and has weak binding activity to the post-fusion conformation, and (5) the RSV pre-F antibody (7E11) recognizing the V epitope only binds to the F protein in the pre-fusion conformation. The ELISA detection results are shown below.

[0329] [Table 18] TIFF2026010683000048.tif202170TIFF2026010683000049.tif202170TIFF2026010683000050.tif202170TIFF2026010683000051.tif89170

[0330] 1) The Φ epitope, quaternary epitope, and V epitope of the following 11 mutants all bind well to the corresponding antibodies with strong binding ability, and the binding level of the II epitope and / or III epitope to the corresponding antibodies is better than or the same as that of the control antibody.

[0331] For example, the binding level of the Φ epitope on YK-RSV-061, YK-RSV-003, and YK-RSV-062 with the RSV pre-F antibody (3C12) was 1.83 to 2.00 times higher than that of the control PC-RSV-064, and the binding level of the quaternary epitope with the RSV pre-F antibody (7H11) was 2.01 to 2.12 times higher than that of the control PC-RSV-064.

[0332] The binding level of the Φ epitope on YK-RSV-054 with the RSV pre-F antibody (3C12) was 2.52-fold higher than that of the control PC-RSV-064, the binding level of the quaternary epitope with the RSV pre-F antibody (7H11) was 1.82-fold higher than that of the control PC-RSV-064, the binding level of the V epitope with the RSV F antibody (7E11) was 3.30-fold higher than that of the control PC-RSV-064, the binding level of the II epitope with the RSV F antibody (11A9) was 1.79-fold higher than that of the control PC-RSV-064, and the binding level of the III epitope with the RSV F antibody (4B9) was 2.27-fold higher than that of the control PC-RSV-064.

[0333] [Table 19] TIFF2026010683000053.tif130170

[0334] 2) The Φ epitope, quaternary epitope, and V epitope of the following four mutants bound well to the corresponding antibodies, and the binding ability was also stronger.

[0335] For example, the binding level of the Φ epitope on YK-RSV-011 with the RSV pre-F antibody (3C12) was 2.90-fold higher than that of the control PC-RSV-064, the binding level of the quaternary epitope with the RSV pre-F antibody (7H11) was 1.81-fold higher than that of the control PC-RSV-064, the binding level of the V epitope with the RSV F antibody (7E11) was 2.70-fold higher than that of the control PC-RSV-064, the binding level of the II epitope with the RSV F antibody (11A9) was 2.09-fold higher than that of the control PC-RSV-064, and the binding level of the III epitope with the RSV F antibody (4B9) was 2.47-fold higher than that of the control PC-RSV-064.

[0336] [Table 20] TIFF2026010683000055.tif33170

[0337] In summary, the above describes the binding of five different epitopes on the mutant with the corresponding antibodies. The binding levels were measured, and mutants with better binding levels (see Tables 16 and 17) were further screened as candidate antigens.

[0338] 2.5 Affinity detection results between RSV F antigen mutants and antibodies recognizing different epitopes Bio-Layer Interferometry (BLI) is a label-free, real-time monitoring optical detection technique primarily used for comprehensive quantitative analysis of biomolecular interactions and protein concentration measurement. BLI monitors the binding process between molecules in real time and determines the affinity constant (K D ), binding rate constant (K on or k a ), dissociation rate constant (K off , K. dis or k d ) and other important data can be calculated.

[0339] K D =Koff ÷K on , K D : refers to the affinity constant, which indicates the strength of the bond between the two molecules, K D The larger the bond, the weaker the bond; conversely, the smaller the bond, the stronger the bond. K on : refers to the binding rate constant, K dis (K off ): refers to the dissociation rate constant, K on , K. off mainly reflects the mode of interaction between molecules, such as fast binding and fast dissociation, or slow binding and slow dissociation.

[0340] In this application, the BLI method was used to analyze the affinity of two antibodies to the target protein, where the RSV pre-F antibody (3C12) recognizes the Φ epitope and the RSV pre-F antibody (7H11) recognizes the quaternary epitope. The detection results are shown below:

[0341] [Table 21] TIFF2026010683000057.tif146170

[0342] 1) The following mutants had the strongest affinity for antibody binding, significantly superior to the control: The binding affinity constant K between the Φ epitope on the following mutant and the RSV pre-F antibody (3C12) D is 3.26E-12~7.79E-11M, and the control K D Value (K D was 1E-10 M, which is one to two orders of magnitude lower than the previous value), indicating a high affinity between the antigen and antibody.

[0343] Binding affinity constant K for RSV pre-F antibody (7H11) with quaternary epitopes on the mutant DThe values ​​were 3.05E-12 to 6.13E-10M, and YK-RSV-044, YK-RSV-060, and YK-RSV-030 were the control K D Other than the corresponding values, the K values ​​of other mutants D All of these were significantly lower than the control (one to two orders of magnitude difference), indicating high affinity between the antigen and antibody.

[0344] The following mutants were further screened in animal experiments.

[0345] [Table 22]

[0346] 2.6 Immunogenicity results of RSV F antigen mutants in Balb / c mice Mice are currently the most commonly used animal model for RSV infection due to their small size, rapid growth, low feed costs, and abundant commercially available detection reagents. In 1979, Prince first reported the use of mice as an animal model for hRSV infection, with Balb / c mice being the most commonly used mouse model, and the RSV titer for intravenous infection was 10 5 ~10 7 In PFU, animals showed significant inflammatory changes in the lower airways, and several cytokines, such as TNF-α, IL-6, and IFN-γ, and CC / CXC chemokines, were significantly increased in the tracheal irrigation fluid. Histopathology showed peribronchiolar and perivascular mononuclear cell (lymphocytes, macrophages) infiltration or interstitial pneumonia. Mice infected with different RSV subtypes (e.g., Along, A2, and clinical isolates) exhibited different symptoms. RSV-infected mice developed airway obstruction and increased respiratory rate accompanied by airway hyperresponsiveness, which persisted for a long period of time, similar to RSV infection in children. Furthermore, mice were able to demonstrate pharmacological and immunological responses. It is one of the most commonly used model animals in toxicology research, has a well-defined genetic background, and can meet specific experimental needs through targeted gene transfection or knockout techniques. Therefore, in the following experiments, mice were used as an animal model for detection.

[0347] 2.6.1 Test results for binding antibody titers ELISA was used to detect pre-fusion and post-fusion F protein-specific IgG titers in the serum of mice 35 days after immunization, and the detection results are shown in the table below:

[0348] 1. The pre-F IgG antibody titers of the following 11 test items were Abrysvo TM The post-F IgG antibody titers were 2.52 to 5.31 times higher than those of the positive control. TM The results were 1.13-5.21 times higher than those of the positive control.

[0349] The pre-F IgG antibody titers of the three antibodies, YK-RSV-061, YK-RSV-003, and YK-RSV-062, were determined by Abrysvo TM The post-F IgG antibody titer was 4.29-5.31 times higher than the positive control. TM The results were 4.32-5.21 times higher than those of the positive control.

[0350] Here, the YK-RSV-061 mutant had the highest pre-F IgG antibody titer, and the pre-F IgG antibody titer was higher than that of Abrysvo. TM The post-F IgG antibody titer was 5.31-fold higher than the positive control. TM The positive control was 5.21.

[0351] For all the tested compounds, the ratio of pre-F / post-F was higher than 1, indicating that the pre-fusion conformation F protein produced higher IgG titers than the post-fusion conformation F protein.

[0352] [Table 23] TIFF2026010683000060.tif19170

[0353] 2. The pre-F IgG antibody titers of the two YK-RSV-011 and YK-RSV-030 strains were TM The post-F IgG antibody titer was 2.38-2.50 times higher than the positive control. TM The results were 1.30-2.81 times higher than those of the positive control.

[0354] [Table 24]

[0355] The immunogenicity of the above test articles was further assessed by measuring the levels of neutralizing antibodies produced.

[0356] 2.6.2 Neutralizing antibody titer test results A spot reduction-based microneutralization test was used to detect neutralizing antibody titers in the serum of mice 35 days after immunization, and the detection results are shown in the following table and Figures 4 to 7 (including Figure (A) and Figure (A) respectively):

[0357] IC of neutralizing antibody titers of the following 11 test substances 50 Abrysvo TM The IC was 3.72 to 12.67 times that of the positive control. 90 Abrysvo TM The values ​​were 2.34 to 14.41 times those of the positive control, and all were significantly higher than those of the positive control.

[0358] IC of neutralizing antibody titers for three samples: YK-RSV-061, YK-RSV-003, and YK-RSV-062 50 Abrysvo TM The IC was 6.34 to 12.67 times that of the positive control. 90 Abrysvo TM The neutralizing antibody titers were significantly higher, ranging from 9.56 to 14.41 times that of the positive control.

[0359] Here, YK-RSV-061 had the highest neutralizing antibody titer, IC 50 Abrysvo TM 12.67-fold higher than the positive control, and IC90 Abrysvo TM This was 14.41 times that of the positive control.

[0360] [Table 25]

[0361] 2. RSV type B virus-neutralizing antibody titer results The 11 test substances obtained in the above screening were used to determine the RSV they produce. The neutralizing antibody levels against B18357 virus were further tested, and the detection results are shown in the table below:

[0362] IC of neutralizing antibody titers for the following 11 mutations 50 Abrysvo TM The IC was 2.85 to 10.63 times that of the positive control. 90 Abrysvo TM The neutralizing antibody titers were 2.88 to 14.23 times higher than those of the positive control, and all of the neutralizing antibody titers were significantly higher than those of the positive control.

[0363] The neutralizing antibody titers of the three antibodies, YK-RSV-061, YK-RSV-003, and YK-RSV-062, were IC 50 Abrysvo TM The IC was 5.68 to 10.63 times that of the positive control. 90 Abrysvo TM The neutralizing antibody titers were very high, ranging from 6.87 to 14.23 times higher than those of the positive control.

[0364] Here, YK-RSV-061 had the highest neutralizing antibody titer, IC 50 Abrysvo TM 10.63-fold higher than the positive control, and IC 90 Abrysvo TM This was 14.23 times that of the positive control.

[0365] [Table 26] TIFF2026010683000064.tif34170

[0366] In summary, the above 11 test articles (including YK-RSV-061, YK-RSV-003, YK-RSV-062, YK-RSV-054, YK-RSV-039, YK-RSV-044, YK-RSV-012, YK-RSV-015, YK-RSV-001, YK-RSV-036 and YK-RSV-060) are RSV A2 and RSV B18 All 357 different subtypes were able to generate high neutralizing antibody titers.

[0367] 2.7 Immunogenicity Results of RSV F Antigen Mutants in Cotton Rats In 1971, Dreizin first reported cotton rats as an animal model of hRSV infection. After hRSV infection in cotton rats, the virus replicated and amplified primarily in the lungs and lower respiratory tract. Pathological examination revealed mild to moderate bronchiolitis or pneumonia, airway epithelial cell loss, and atelectasis. Symptoms of upper respiratory tract infection were significant, but associated cytokine changes were not. Cotton rats are a standard animal model for evaluating the efficacy of vaccines, antiviral drugs, and monoclonal antibodies (e.g., palivizumab). Therefore, cotton rats were used as an experimental animal model in the following experiments.

[0368] 2.7.1 Binding antibody titer test results Using ELISA, pre-fusion and post-fusion F protein-specific IgG titers were detected in the serum of cotton rats on days 42 and 48 after immunization (see Figures 8 to 11 (including Figures (A) and (B) respectively)). The IgG titer detection results on day 42 are shown in the table below:

[0369] (1) On day 42, the pre-F IgG antibody titers of the following 11 test articles were Abrysvo TM The post-F IgG antibody titers were 3.05 to 13.10 times higher than those of the TM positive control. TMThe values ​​were 2.36 to 6.38 times those of the positive control, and all were significantly higher than the positive control.

[0370] The pre-F IgG antibody titers of the three antibodies, YK-RSV-061, YK-RSV-003, and YK-RSV-062, were determined by Abrysvo TM The post-F IgG antibody titer was 9.76 to 13.10 times higher than the positive control. TM The results were 4.66-6.33 times higher than those of the positive control.

[0371] Here, the pre-F IgG antibody titer was the highest in the YK-RSV-061 test sample, and the pre-F IgG antibody titer was TM The post-F IgG antibody titer was 13.10 times higher than the positive control. TM The positive control was 6.33.

[0372] The pre-F / post-F ratios for all test samples ranged from 1.39 to 3.95, indicating that the pre-fusion F protein produced significantly higher IgG antibody titers than the post-fusion F protein.

[0373] [Table 27]

[0374] At the same time, the IgG titer in the cotton rat serum on day 48 was detected, and the detection results are shown in the table below:

[0375] (2) On day 48, the pre-F IgG antibody titers of the following 11 test articles were Abrysvo TM The post-F IgG antibody titer was 4.44 to 12.35 times higher than the positive control. TM The values ​​were 1.51 to 6.83 times those of the positive control, and all were significantly higher than the positive control.

[0376] The pre-F IgG antibody titers of the three antibodies, YK-RSV-061, YK-RSV-003, and YK-RSV-062, were determined by Abrysvo TMThe post-F IgG antibody titer was 9.02 to 12.35 times higher than the positive control. TM The concentrations were 4.03-6.83 times higher than those of the positive control.

[0377] Here, the IgG antibody titer of the YK-RSV-061 test sample was the highest, and the IgG antibody titer of pre-F was the highest. TM The post-F IgG antibody titer was 12.35-fold higher than the positive control. TM The positive control was 6.83.

[0378] The pre-F / post-F ratios for all test samples ranged from 1.78 to 4.29 times, indicating that after immunization on day 48, the IgG antibody titers produced by the pre-fusion F protein were still significantly higher than those produced by the post-fusion F protein.

[0379] [Table 28]

[0380] 2.7.2 Neutralizing antibody titer test results The neutralizing antibody titers produced against RSV A2 virus in the serum of cotton rats were detected on days 42 and 48, respectively (see Figures 12(A) and (B), Figures 13(A) and (B), Figures 16(A) and (B), Figures 17(A) and (B)). The results of the neutralizing antibody titer detection on day 42 are shown in the table below:

[0381] (1) IC of neutralizing antibody titers of the following 11 test compounds detected in the serum of cotton rats 42 days after immunization. 50 Abrysvo TM The IC was 2.10 to 14.30 times that of the positive control. 90 Abrysvo TM The values ​​were 2.26 to 22.33 times those of the positive control, and all were significantly higher than the positive control.

[0382] IC of neutralizing antibody titers for three samples: YK-RSV-061, YK-RSV-003, and YK-RSV-062 50Abrysvo TM The IC was 8.84 to 14.30 times that of the positive control. 90 Abrysvo TM The results were 16.46-22.33 times those of the positive control.

[0383] Here, the YK-RSV-061 IC 50 Abrysvo TM 14.30-fold higher than the positive control, and IC 90 Abrysvo TM The neutralizing antibody titer was the highest, 22.33 times that of the positive control.

[0384] [Table 29]

[0385] At the same time, the neutralizing antibody titer produced against RSV B virus in the serum of cotton rats was detected on day 42, and the detection results are shown in the following table, Figures 14(A) and (B) and Figures 15(A) and (B):

[0386] IC of neutralizing antibody titers of the following 11 test substances 50 Abrysvo TM The IC was 2.16 to 14.63 times that of the positive control. 90 Abrysvo TM The values ​​were 2.49 to 17.26 times those of the positive control, and all were significantly higher than those of the positive control.

[0387] IC of neutralizing antibody titers for three samples: YK-RSV-061, YK-RSV-003, and YK-RSV-062 50 Abrysvo TM The IC was 11.52 to 14.63 times that of the positive control. 90 Abrysvo TM The results were 13.44-17.26 times higher than those of the positive control.

[0388] Here, the YK-RSV-061 IC 50 Abrysvo TM 14.63-fold higher than the positive control, and IC90 Abrysvo TM The neutralizing antibody titer was 17.26 times higher than that of the positive control, which was the highest.

[0389] [Table 30]

[0390] (3) 48 days after immunization, the neutralizing antibody titer produced against RSV A2 virus in the cotton rat serum was detected, and the detection results are shown in the table below: IC of neutralizing antibody titers of the following 11 test substances 50 Abrysvo TM The IC was 1.70 to 7.57 times that of the positive control. 90 Abrysvo TM The values ​​were 1.46 to 11.56 times those of the positive control, and all were significantly higher than those of the positive control.

[0391] IC of neutralizing antibody titers for three samples: YK-RSV-061, YK-RSV-003, and YK-RSV-062 50 Abrysvo TM The IC was 4.51 to 7.57 times that of the positive control. 90 Abrysvo TM The results were 8.75-11.56 times higher than those of the positive control.

[0392] Here, the YK-RSV-061 IC 50 Abrysvo TM 7.57-fold higher than the positive control, and IC 90 Abrysvo TM The neutralizing antibody titer was 11.56 times higher than that of the positive control, which was the highest.

[0393] [Table 31] TIFF2026010683000070.tif19170

[0394] In summary, at 42 and 48 days after immunization of cotton rats, the binding and neutralizing antibody titer levels produced by all test articles were significantly higher than those of the control Abrysvo. TM The humoral immune effect was significantly improved compared to the control group. The immune effect of the test product YK-RSV-061 was the most effective. All test products had excellent immune effects against different subtypes (including RSV types A and B).

[0395] 2.7.3 Pathological section results of cotton rat lungs Pathological sections of the cotton rat lungs are shown in Figure 18 and the scoring results are shown in the table below:

[0396] [Table 32]

[0397] From the above results, it was found that the peribronchiolar infiltration scores of all test specimens were between 0.33 and 0.83, indicating mild inflammatory infiltration.

[0398] Interstitial infiltration scores for all test articles were significantly higher than those of the positive control Abrysvo TM The results were more favorable, with scores ranging from 0.67 to 1.17, and mild inflammatory infiltrates.

[0399] All test specimens had low overall inflammatory response scores, and the overall pathological scores ranged from 1.00 to 2.00, all of which were lower than those of control LNP. Both the control and test specimens had scores of 0 for perivascular infiltration and alveolar infiltration, indicating no visible lesions in the tissue.

[0400] 2.7.4 Virus burden results in lungs and nasal turbinates of cotton rats On day 54, the cotton rats were sacrificed and lung and nasal turbinate tissues were aseptically removed for virus titration, with the detection results shown in the table below:

[0401] [Table 33]

[0402] The viral load in the lungs of cotton rats was measured, and the results showed that the tissue viral load after immunization with the negative control LNP was 4.52, and the tissue viral load after immunization with the positive control Abrysvo TM and the other test substances. TM The tissue virus load after immunization with Abrysvo was 2.14, the tissue virus load after immunization with the other test substances was 1.61-2.08, and the positive control Abrysvo TM It was lower.

[0403] The viral load in the nasal turbinates of cotton rats was measured, and the results showed that the tissue viral load after immunization with the negative control LNP was 4.12, which was significantly higher than that of the other test substances. TM The tissue virus load after immunization with Abrysvo was 3.35, the tissue virus load after immunization with the other test substances was 0.62-1.51, and the positive control Abrysvo TM The viral load was only 0.19 to 0.45 times that of the control, which significantly reduced the viral load, indicating that the test material had good antiviral effect.

[0404] 2.8 Mutant Difference Analysis In summary, the present invention designed 52 antigen sequences encoding RSV F protein mutants and screened at least 11 (YK-RSV-061, YK-RSV-003, YK-RSV-062, YK-RSV-054, YK-RSV-039, YK-RSV-044, YK-RSV-012, YK-RSV-015, YK-RSV-001, YK-RSV-036, YK-RSV-060), among which at least five were identified. (YK-RSV-061, YK-RSV-003, YK-RSV-062, YK-RSV-054, YK-RSV-039) are positive control Abrysvo TMThis antigen sequence was superior to the effects of the design controls PC-RSV-063, PC-RSV-064, PC-RSV-065, PC-RSV-066, PC-RSV-067, and PC-RSV-068.

[0405] [Table 34]

[0406] [Table 35]

[0407] [Table 36]

[0408] [Table 37]

[0409] Compared with the YK-RSV-003 mutant, YK-RSV-061 added one site mutation V152I thereon, and YK-RSV-062 added five site mutations C69Y, D73E, A74V, N88S, and V152I thereon.

[0410] The epitopes on the three mutants, YK-RSV-003, YK-RSV-061, and YK-RSV-062, all had good binding levels with the corresponding antibodies. For example, the binding levels of the quaternary epitopes on YK-RSV-061, YK-RSV-003, and YK-RSV-062 with the RSV pre-F antibody (7H11) were 2.01- to 2.12-fold higher than those of the control PC-RSV-064.

[0411] However, the neutralizing antibody titer IC of YK-RSV-061 against RSV type A virus was 50 and IC 90 Abrysvo TMThe IC of neutralizing antibody titers of YK-RSV-003 was 12.67-fold and 14.41-fold higher than that of the positive control, respectively. 50 and IC 90 are Abrysvo TM An unexpected result was that the neutralizing antibody titers of YK-RSV-061 were significantly elevated, 7.39-fold and 9.59-fold higher than the positive control, despite the difference being only a single mutation, V152I. The neutralizing antibody titers of YK-RSV-062 were the same as those of YK-RSV-003. Similarly, the neutralizing antibody titer of YK-RSV-061 had a significant advantage over RSV B virus.

[0412] [Table 38] TIFF2026010683000078.tif100170

[0413] [Table 39] TIFF2026010683000080.tif33170

[0414] [Table 40]

[0415] Compared to the YK-RSV-003 mutant, the control PC-RSV-063 had one altered mutation type in site mutation group 2, replacing V296I with V207L, and deleted the D486S site mutation in site mutation group 3. However, the binding level of the quaternary epitope on YK-RSV-003 with the RSV pre-F antibody (7H11) was 2.04-fold higher than that of the control PC-RSV-063, a significant difference in binding level.

[0416] Compared with the YK-RSV-061 mutant, the controls PC-RSV-065 and PC-RSV-066 each had one site mutation (D486S or V296I) reduced, the control PC-RSV-064 had two site mutations (D486S or V296I) reduced, and the controls PC-RSV-067 and PC-RSV-068 had an altered mutation type in site mutation group 1. The results showed that the affinity constant K for the Φ epitope on the YK-RSV-061 mutant to bind to the RSV pre-F antibody (3C12) was significantly lower. D is 6.21E-11M, and the affinity constant K for the quaternary epitope binding to the RSV pre-F antibody (7H11) D is 3.05E-12M, and the other control K D All values ​​were E-10M, which indicated a difference of 1 to 2 orders of magnitude, demonstrating that the affinity of the antigen YK-RSV-061 with the antibody was higher than that of the other controls.

[0417] The above comparison shows that the binding level and avidity between the epitope on the YK-RSV-003 mutant and the corresponding antibody are both significantly higher than those of the controls PC-RSV-063 to PC-RSV-068. At the same time, combined with the comparison results in part (1), it can be seen that the effect of the YK-RSV-061 mutant is significantly better than that of the YK-RSV-003 mutant, and the effect of YK-RSV-062 is the same as that of YK-RSV-003. Therefore, it can be seen that YK-RSV-061, YK-RSV-003 and YK-RSV-062 are all superior to the controls PC-RSV-063 to PC-RSV-068.

[0418] The following table shows the complete sequence information and SEQ ID NOs of the present application. Take the YK-RSV-001 antigen sequence as an example, where SEQ ID NOs: 3 and 4 are the DNA sequence and amino acid sequence of the open reading frame of the antigen YK-RSV-001, respectively, and SEQ ID NOs: 2 and 5 are , and are the DNA sequence and mRNA sequence of antigen YK-RSV-001, respectively.

[0419] [Table 41] TIFF2026010683000083.tif219170TIFF2026010683000084.tif219170TIFF2026010683000085.tif218170TIFF2026010683000086.tif219170TIFF2026010683000087.tif219170TIFF2026010683000088.tif219170TIFF2026010683000089.tif147170

Claims

1. Respiratory syncytial virus (RSV) ribonucleic acid (RNA), wherein the RNA encodes a variant of a wild-type RSV F protein; wherein the sequence of the wild-type RSV F protein is set forth in SEQ ID NO: 1; The RSV F protein variants include P102A, I379V, and M447V variants, and the RSV F protein variants are truncated proteins in which amino acids 550 to 574 of SEQ ID NO: 1 are truncated, and amino acids 104 to 144 are substituted with a GS linker. At the same time, the RSV F protein variants further comprise a combination of site mutations selected from any of Groups 1 to 5 below, thereby obtaining an RSV F protein variant selected from YK-RSV-061, YK-RSV-003, and / or YK-RSV-062. Table 1

2. The amino acid sequence of the RSV F protein variant is SEQ ID NO:204, SEQ ID NO:12, or SEQ ID NO:208; or An open reading frame (ORF) encoding the RSV F protein variant is F) the nucleotide sequence is SEQ ID NO: 203, SEQ ID NO: 11 or SEQ ID NO: 207; or the RSV RNA sequence is SEQ ID NO:205, SEQ ID NO:13, or SEQ ID NO:209; or 2. The composition of claim 1, wherein the DNA sequence encoding the mutant RSV F protein is SEQ ID NO:202, SEQ ID NO:10, or SEQ ID NO:

206.

3. The composition of claim 1 , wherein the RSV RNA further comprises a 5′ untranslated region (UTR).

4. The composition of claim 3, wherein the sequence of the 5' untranslated region is SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220 or SEQ ID NO:

221.

5. The composition of claim 1 , wherein the RSV RNA further comprises a 3′ untranslated region (UTR).

6. The composition of claim 5, wherein the sequence of the 3' untranslated region is SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224 or SEQ ID NO:

225.

7. The composition of claim 1 , wherein the RSV RNA further comprises a poly(A) tail.

8. The composition of claim 1 , wherein the RSV RNA further comprises a 5′ end cap structure.

9. 9. The composition of claim 8, wherein the 5' end cap structure is 7mG(5')ppp(5')NlmpNp.

10. 2. The composition of claim 1, wherein the sequence of the open reading frame (ORF) encoding the RSV F protein or variant thereof in the RSV RNA is codon-optimized.

11. The composition of claim 10, wherein the sequence of the ORF comprises at least one base modification.

12. 12. The composition of claim 11, wherein the base modifications comprise any one or more selected from pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2'-O-methyluridine.

13. 10. The composition of claim 1, which is a vaccine and further comprises a pharmaceutically acceptable carrier.

14. 14. The composition of claim 13, wherein the pharmaceutically acceptable carrier comprises a lipid mixture, and the lipid mixture is a lipid nanoparticle (LNP).

15. the vaccine is an mRNA vaccine, or the effective amount of RSV RNA is 25 μg to 200 μg; or 14. The composition of claim 13, wherein the effective amount of RSV RNA is 50 μg to 100 μg.

16. 15. The composition of claim 14, wherein the lipid nanoparticles comprise cationic lipids, neutral lipids, structured lipids, and polymer-conjugated lipids.

17. The cationic lipid is a compound of the structure of Formula I, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein G １ is C １～６ alkylene, and G ２ is C ２～８ alkylene, and G ３ is C １～３ alkylene, and L １ is C ６～１５ is a linear alkyl; L ２ is C １２～２５ is a branched alkyl; 【Chemistry 1】 Alternatively, the cationic lipid is a compound of the structure of Formula II, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein G １ is C ２～８ alkylene, and G ２ is C ２～８ alkylene, and L １ is —C(O)O— or —OC(O)—, and L ２ is —C(O)O— or —OC(O)—, and R １ is C ６～２５ is a straight or branched chain alkyl; R ２ is C ６～２５ is a straight or branched chain alkyl; G ３ is HO(CH ２ ) ２ - or HO(CH ２ ) ３ - and G ４ is HO(CH ２ ) ２ - or HO(CH ２ ) ３ - and L is (CH ２ ) ２ - or - (CH ２ ) ３ - or - (CH ２ ) ４ - and 【Chemistry 2】 Alternatively, the cationic lipid is a compound of the structure of Formula III, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein G １ is C １～６ alkylene, and G ２ is C ２～８ alkylene, and R １ is C ６～２０ is a straight or branched chain alkyl; R ２ is C １２～２５ is a branched alkyl; G ３ is HO(CH ２ ) ２ N (CH ３ ) (CH ２ ) ２ -, HO(CH ２ ) ２ N (CH ２ CH ３ ) (CH ２ ) ２ -, (HO(CH ２ ) ２ ) ２ N (CH ２ ) ２ -, CH ３ O (CH ２ ) ２ N (CH ３ ) (CH ２ ) ２ -, (CH ３ ) ２ N (CH ２ ) ３ SC(O)O(CH ２ ) ２ -, (CH ３ ) ２ N (CH ２ ) ３ SC(O)-, CH ３ NH (CH ２ ) ２ N (CH ３ ) (CH ２ ) ２ - or CH ３ CH ２ NH (CH ２ ) ２ - and 【Transformation 3】 Alternatively, the cationic lipid is a compound of the structure of Formula IV, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein G １ is C １～８ alkylene, and G ２ is C ２～８ alkylene, and R １ is C ６～２５ is a straight or branched chain alkyl; R ２ is C １２～２５ is a straight or branched chain alkyl; G ３ is HO(CH ２ ) ２ N (R ３ ) CH ２ CH(OH)CH ２ -, where R ３ is -CH ３ or -CH ２ CH ３ or -CH ２ CH ２ OH, 【Chemistry 4】 Alternatively, the cationic lipid is a compound of the structure of Formula V, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein G １ and G ２ are each independently an unsubstituted C ６ ~C １０ alkylene, and G ３ is unsubstituted C １ ~C １２ alkylene, and R １ and R ２ are each independently C ６ ~C ２４ Alkyl or C ６ ~C ２４ alkenyl, and R ３ is OR ５ , N, -C(=O)OR ４ , —OC(═O)R ４ or -NR ５ C(=O)R ４ and R ４ is C １ ~C １２ alkyl, and R ５ is H or C １ ~C ６ is alkyl, 【Transformation 5】 Alternatively, the cationic lipid is a compound of the structure of Formula VI, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein R ４ is -(CH ２ ) ｎ Q and -(CH ２ ) ｎ CHQR, where Q is —OR, —OH, —O(CH ２ ) ｎ N (R) ２ , -OC(O)R, -CX ３ , -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O) ２ R, -N(H)S(O) ２ R, -N(R)C(O)N(R) ２ , -N(H)C(O)N(R) ２ , -N(H)C(O)N(H)(R), -N(R)C(S)N(R) ２ , -N(H)C(S)N(R) ２ , -N(H)C(S)N(H)(R), -N(R)S(O) ２ R ８ and heterocyclyl, n is 1, 2 or 3, where R is hydrogen, C １～３ Alkyl, C ２～３ alkenyl or (C H ２ ) ｑ OR ＊ where q is 1, 2 or 3, and R ＊ is C １～１２ Alkyl or C ２～１２ alkenyl, X is fluorine, chlorine, bromine or iodine, R ８ is C ３～６ is cycloalkyl or heterocyclyl, 【Transformation 6】 Alternatively, the cationic lipid is a compound of the structure of Formula VII, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof: 【Transformation 7】 Alternatively, the composition of claim 16, wherein the cationic lipid is selected from the compounds YK-009, YK-401, YK-305, ALC0315, SM102, DLIN-MC3. 【Transformation 8】

18. the molar ratio of the cationic lipid to the neutral lipid is (1-10):1; or 17. The composition of claim 16, wherein the molar ratio of the cationic lipid to the structural lipid is (1-5):

1.

19. The molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (25-75):(5-25):(15-65):(0.5-10), Or, The composition of claim 16, wherein the molar ratio of the cationic lipid, the neutral lipid, the structured lipid, and the polymer-conjugated lipid is (35-49):(7.5-15):(35-55):(1-5).

20. the neutral lipid is selected from one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol and derivatives thereof; or The neutral lipids include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether). PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,or selected from one or more of 2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine (LPE); the neutral lipid is DOPE and / or DSPC, or 17. The composition of claim 16, wherein the structured lipid is selected from one or more of cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, corticosteroids.

21. 17. The composition of claim 16, wherein the polymer-conjugated lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol.

22. The polymer-conjugated lipid is a distearoylphosphatidylethanolamine-polyethylenediamine.

22. The composition of claim 21, wherein the hydroxybenzoate is selected from one or more of dimethicone glycol 2000 (DSPE-PEG2000), 1,2-dimyristoyl-rac-glycero-methoxypolyethylene glycol-2000 (DMG-PEG2000), and methoxypoly(ethylene glycol) ditetradecylacetamide (ALC-0159).

23. The composition according to any one of claims 13 to 22, wherein the vaccine is in the form of an injectable dosage form.

24. A method for producing the composition of any one of claims 13 to 23, comprising the step of mixing the RSV RNA with the pharmaceutically acceptable carrier.

25. A vaccine for inducing a protective immune response against RSV in a subject, comprising the composition of any one of claims 1 to 23.

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