Vaccine against respiratory syncytial virus infection

Abstract

This invention relates to a vaccine for respiratory syncytial virus infection and pertains to the field of medicine. To address the unmet need for effective prophylactic and therapeutic agents for respiratory syncytial virus (RSV)-induced respiratory infections in the elderly, infants, and immunocompromised individuals, this invention provides a vaccine for respiratory syncytial virus infection, the vaccine using adenovirus as a vector to express an engineered antigen gene specifically designed and optimized based on the fusion (F) protein sequence of RSV. The adenovirus vector vaccine can help the host resist respiratory syncytial virus infection and has good prophylactic and therapeutic effects.

Description

[Technical Field]

[0001] This invention relates to a vaccine for respiratory syncytial virus infection and pertains to the field of medicine. [Background technology]

[0002] Respiratory syncytial virus (RSV) is the most important pathogen causing lower respiratory tract infections in children under 5 years of age and the elderly worldwide. RSV belongs to the genus Pneumovirus of the family Paramyxoviridae and is characterized as an antisense single-stranded RNA virus with only one serotype. It is transmitted primarily by contact with virus-containing secretions or contaminants on the nasopharyngeal or ocular mucosa. Scientists began researching RSV vaccines in 1960. However, RSV vaccines that were inactivated at that time not only failed to prevent RSV infection but also worsened the severity of illness in infants newly infected with RSV.

[0003] The virus is spherical and has an envelope with a diameter of 120 nm to 300 nm. The genome is characterized as unsegmented single-strand negative RNA and mainly encodes 10 proteins: three transmembrane proteins including a fusion protein (F), a small hydrophobic protein (SH), and an adhesion protein (G), two matrix proteins M1 and M2, three proteins (L, N, and P) that bind to viral RNA to form a nucleocapsid, and two non-structural proteins (NS1 and NS2). The viral envelope has spikes composed of glycoproteins that do not contain HA, NA, and HL.

[0004] The fusion protein (F) is the surface-exposed viral envelope protein of RSV, primarily mediating viral entry into host cells. Due to its high sequence conservation, it is a primary target protein in vaccine development. Protein F exists in two conformational states: pre-fusion (pre-F) and post-fusion (post-F). Infectious RSV exhibits both pre-fusion and post-fusion conformations of the F protein on its surface. After a cell is infected with the virus, the pre-F conformation is converted to the post-F conformation. Since the virus was first discovered in 1956, its complex molecular structure and safety concerns have hindered vaccine development. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] To address the unmet need for effective prophylactic and therapeutic agents for respiratory syncytial virus (RSV)-induced respiratory infections in the elderly, infants, and immunocompromised individuals, the present invention provides an adenovirus vector vaccine for respiratory syncytial virus infection, the vaccine using adenovirus as a vector, the RSV F protein sequence being optimized and then designed as a series of pre-F protein sequences, the pre-F protein sequences being used as an antigen gene and recombined into the adenovirus vector. In particular, since the antigen gene is designed based on the F protein of subtype A RSV, the adenovirus vector can express the RSV antigen protein and help the host resist respiratory syncytial virus infection, which has good prophylactic and therapeutic effects. [Means for solving the problem]

[0006] In a first aspect, the present invention provides an adenovirus vector vaccine for preventing and / or treating respiratory syncytial virus infection, the adenovirus vector vaccine being obtained by constructing a recombinant adenovirus vector comprising a respiratory syncytial virus expression gene, the polynucleotide sequence of the respiratory syncytial virus expression gene being selected from at least one of SEQ ID NOs: 12, SEQ ID NOs: 14, SEQ ID NOs: 16, SEQ ID NOs: 18, SEQ ID NOs: 20, SEQ ID NOs: 22, SEQ ID NOs: 24, SEQ ID NOs: 26, SEQ ID NOs: 28, SEQ ID NOs: 30, SEQ ID NOs: 32, and SEQ ID NOs: 34.

[0007] Furthermore, the amino acid sequence of the protein encoded by the polynucleotide sequence is selected from at least one of SEQ ID NOs: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33.

[0008] Furthermore, the adenovirus vector vaccine further comprises a pharmaceutically acceptable adjuvant, vector, diluent, or excipient.

[0009] Furthermore, the recombinant adenovirus vector is selected from at least one of the following: adenovirus, ankara waxinia virus, and adeno-associated virus.

[0010] Preferably, the adenovirus vector is selected from human type 5, 35, or 26 and / or chimpanzee AdC68 or AdC7 replication-deficient adenoviruses.

[0011] More preferably, the adenovirus vector is selected from human type 5 replication-deficient adenovirus with combined E1 and E3 deletions.

[0012] Adenovirus vector vaccines are also formulated as intradermal or subcutaneous injections, intramuscular injections, intravenous injections, oral formulations, or nasal spray formulations.

[0013] Preferably, the vaccine is formulated as a nasal spray or an intramuscular injection.

[0014] The present invention provides a method for preparing adenovirus in an adenovirus vector vaccine, the method comprising the following steps: constructing a shuttle plasmid vector containing polynucleotides; transfecting the constructed shuttle plasmid vector and backbone plasmid into host cells and culturing the host cells; obtaining replication-deficient recombinant adenovirus; and performing large-scale culture and purification.

[0015] Sequence ID 11 RSV-A-PreF-1(R1)(Del104-145aa)

[0016] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTSAIASGVAVSKVLHLEGEVNKI KSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIK EEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYG KTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL

[0017] Sequence ID 12 encodes the polypeptide of Sequence ID 11.

[0018]

[0019] SEQ ID NO: 13 RSV-A-PreF-1F(R2)(Del104-145aa)

[0020] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0021] SEQ ID NO: 14 encoding the polypeptide of SEQ ID NO: 13

[0022]

[0023] Sequence ID 15 RSV-A-PreF-2(R3) (two disulfide bond mutations)

[0024] MELLILKANAITTILTAVTFCFASGQNCTEEFYQSTCSAVSKGYLSALRTGWYTCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRR FLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKK LMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVIT SLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQECKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL

[0025] Sequence ID 16 encodes the polypeptide of Sequence ID 15.

[0026]

[0027] Sequence ID 17 RSV-A-PreF-2F(R4) (two disulfide bond mutations)

[0028] MELLILKANAITTILTAVTFCFASGQNCTEEFYQSTCSAVSKGYLSALRTGWYTCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYS IMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRG IIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQECKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0029] Sequence ID 18 encodes the polynucleotide of Sequence ID 17.

[0030]

[0031] Sequence ID 19 RSV-A-PreF-3(R5) (Del104-145aa+1 disulfide bond mutation)

[0032] MELLILKANAITTILTAVTFCFASGQNCTEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTSAIASGVAVSKVLHLEGEVNKI KSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIK EEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYG KTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQECKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL

[0033] Sequence ID 20 encodes the polynucleotide of Sequence ID 19.

[0034]

[0035] Sequence ID 21 RSV-A-PreF-3F(R6) (Del104-145aa+1 disulfide bond mutation)

[0036] MELLILKANAITTILTAVTFCFASGQNCTEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTSAIASGVAVSKVLHLEGEVNKIKSALLSTN KAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGV IDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDY VSNKGMDTVSVGNTLYYVNKQECKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0037] Sequence ID 22 encodes the polynucleotide of Sequence ID 21.

[0038]

[0039] Sequence ID 23 RSV-B-PreF-1(R7)(Del104-145aa)

[0040] MELLIHRSSAIFLTLAVNALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAASAIASGIAVSKVLHLEGEVNKI KNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNRLLPIVNQQSCRISNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIK EEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYG KTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL

[0041] Sequence ID 24 encodes the polynucleotide of Sequence ID 23.

[0042]

[0043] Sequence ID 25 RSV-B-PreF-1F(R8)(Del104-145aa)

[0044] MELLIHRSSAIFLTLAVNALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAASAIASGIAVSKVLHLEGEVNKIKNALLSTN KAVVSLSNGVSVLTSKVLDLKNYINNRLLPIVNQQSCRISNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGV IDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDY VSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIMITTIIIVIIVVLLSLIAIGLLLYCKAKNTPVTLSKDQLSGINNIAFSK

[0045] Sequence ID 26 encodes the polynucleotide of Sequence ID 25.

[0046]

[0047] Sequence ID 27 RSV-B-PreF-2(R9) (two disulfide bond mutations)

[0048] MELLIHRSSAIFLTLAVNALYLTSSQNCTEEFYQSTCSAVSRGYFSALRTGWYTCVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRR FLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVCTSKVLDLKNYINNRLLPIVNQQSCRISNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKK LMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVIT SLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLECKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL

[0049] Sequence ID 28 encodes the polynucleotide of Sequence ID 27.

[0050]

[0051] Sequence ID 29 RSV-B-PreF-2F(R10) (two disulfide bond mutations)

[0052] MELLIHRSSAIFLTLAVNALYLTSSQNCTEEFYQSTCSAVSRGYFSALRTGWYTCVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLG VGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVCTSKVLDLKNYINNRLLPIVNQQSCRISNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYS IMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRG IIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLECKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIMITTIIIVIIVVLLSLIAIGLLLYCKAKNTPVTLSKDQLSGINNIAFSK

[0053] Sequence ID 30 encodes the polynucleotide of Sequence ID 29.

[0054]

[0055] Sequence ID 31 RSV-B-PreF-3(R11)(Del104-145aa+1 disulfide bond mutation)

[0056] MELLIHRSSAIFLTLAVNALYLTSSQNCTEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAASAIASGIAVSKVLHLEGEVNKI KNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNRLLPIVNQQSCRISNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIK EEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYG KTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLECKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL

[0057] Sequence ID 32 encodes the polynucleotide of Sequence ID 31.

[0058]

[0059] Sequence ID 33 RSV-B-PreF-3F(R12) (Del104-145aa+1 disulfide bond mutation)

[0060] MELLIHRSSAIFLTLAVNALYLTSSQNCTEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAASAIASGIAVSKVLHLEGEVNKIKNALLSTN KAVVSLSNGVSVLTSKVLDLKNYINNRLLPIVNQQSCRISNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGV IDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDY VSNKGVDTVSVGNTLYYVNKLECKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIMITTIIIVIIVVLLSLIAIGLLLYCKAKNTPVTLSKDQLSGINNIAFSK

[0061] Sequence ID 34 encodes the polynucleotide of Sequence ID 33.

[0062]

[0063] In a second aspect, the present invention provides a composition for preventing and / or treating respiratory syncytial virus infection, the composition being a compound formulation comprising a recombinant protein vaccine and an adenovirus vector vaccine as active ingredients for preventing and / or treating respiratory syncytial virus infection.

[0064] The present invention provides a combination drug for the prevention and / or treatment of respiratory syncytial virus infection, the combination drug comprising a recombinant protein vaccine and an adenovirus vector vaccine for the prevention and / or treatment of respiratory syncytial virus infection, the vaccines of which are administered separately or simultaneously.

[0065] Furthermore, the recombinant protein vaccine contains a recombinant protein for the prevention and / or treatment of respiratory syncytial virus infection, and the amino acid sequence of the recombinant protein has more than 70% sequence identity and identical or substantially equivalent biological activity to SEQ ID NO: 1 or SEQ ID NO: 2.

[0066] Preferably, the amino acid sequence of the recombinant protein is selected from at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0067] Furthermore, recombinant protein vaccines are obtained by constructing protein precursors having an amino acid sequence selected from SEQ ID NO: 7 or SEQ ID NO: 8.

[0068] Furthermore, the nucleotide sequence encoding the protein precursor is selected from at least one of SEQ ID NO: 9 and SEQ ID NO: 10.

[0069] Furthermore, the method for preparing recombinant proteins in recombinant protein vaccines includes the following steps: culturing host cells to express a protein or protein precursor, and then recovering the protein.

[0070] Furthermore, insect cells or mammalian cells are used as host cells. Preferably, the insect cells are selected from at least one of sf9 cells, sf21 cells, and Hi5 cells. Preferably, the mammalian cells are CHO cells. More preferably, the CHO cell expression vector is pTT5 or FTP-002.

[0071] The host cell contains a recombinant vector, and the recombinant vector contains nucleotides encoding a protein or a protein precursor. The recombinant vector is either an insect baculovirus expression vector or a mammalian cell expression vector. Preferably, the insect baculovirus expression vector is pFastBac1. Preferably, the mammalian cell expression vector is a CHO cell expression vector.

[0072] Furthermore, recombinant protein vaccines contain recombinant proteins and / or protein precursors, as well as pharmaceutically acceptable excipients or adjuvant components.

[0073] Furthermore, the adjuvant component is an immunoadjuvant. Preferably, the immunoadjuvant is selected from at least one of the following: aluminum salts, calcium salts, phytosaponins, phytopolysaccharides, monophosphatide A, muramyl dipeptides, muramyl tripeptides, squalene-based oil-in-water emulsions, recombinant cholera toxin, GM-CSF cytokines, lipids and cationic liposome materials, and CpG ODN.

[0074] Furthermore, the squalene-based oil-in-water emulsion is MF59. Furthermore, the aluminum salt is selected from at least one of aluminum hydroxide and alum. Furthermore, the calcium salt is tricalcium phosphate. Furthermore, the phytosaponin is either QS-21 or ISCOM. Furthermore, the phytopolysaccharide is astragalus polysaccharide. Furthermore, the lipid is selected from at least one of the following: phosphatidylethanolamine (PE), phosphatidylcholine (PC), cholesterol (Chol), and dioleoylphosphatidylethanolamine (DOPE). Furthermore, the cationic liposome material is selected from at least one of the following: (2,3-dioleoyloxypropyl)trimethylammonium chloride (DOTAP), N-[1-(2,3-dioleoylchloride)propyl]-N,N,N-trimethylamine chloride (DOTMA), cationic cholesterol (DC-Chol), dimethyl-2,3-dioleyloxypropyl-2-(2-spermidinecarboxamide)ethylammonium trifluoroacetate (DOSPA), trimethyldodecylammonium bromide (DTAB), trimethyltetradecylammonium bromide (TTAB), trimethylhexadecylammonium bromide (CTAB), and dimethyldioctadecylammonium bromide (DDAB).

[0075] Furthermore, the composition or concomitant drug may be formulated as an intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral formulation, or nasal spray formulation, and preferably, the vaccine may be formulated as an intramuscular injection formulation or a nasal spray formulation.

[0076] In a third aspect, the present invention provides the use of an adenovirus vector vaccine, composition, or concomitant drug in preparing a drug for the prevention and / or treatment of respiratory syncytial virus infection.

[0077] Sequence ID 1 is the RSV subtype AF sequence:

[0078] QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEG EVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAY VVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFS NGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0079] Sequence ID 2 is the RSV subtype BF sequence:

[0080] QNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEG EVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNRLLPIVNQQSCRISNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAY VVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFS NGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIMITTIIIVIIVVLLSLIAIGLLLYCKAKNTPVTLSKDQLSGINNIAFSK

[0081] Sequence ID 3: Optimized RSV subtype AF protein truncated sequence

[0082] QNCTEEFYQSTCSAVSKGYLSALRTGWYTCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAV VSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMS IIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTD VSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQECKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL

[0083] Sequence ID 4: Optimized RSV subtype BF protein truncated sequence

[0084] QNCTEEFYQSTCSAVSRGYFSALRTGWYTCVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAASAIASGIAVSKVLHLEGEVNKIKNALLSTNKAV VSLSNGVSVCTSKVLDLKNYINNRLLPIVNQQSCRISNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMS IIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTD ISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLECKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELL

[0085] Sequence ID 5: SAIG ​​and Foldon sequences attached to the optimized RSV subtype AF shortened sequence.

[0086] QNCTEEFYQSTCSAVSKGYLSALRTGWYTCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVS VCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPL YGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTA SNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQECKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL

[0087] Sequence ID 6: SAIG ​​and Foldon sequences attached to the optimized RSV subtype BF shortened sequence.

[0088] QNCTEEFYQSTCSAVSRGYFSALRTGWYTCVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAASAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVS VCTSKVLDLKNYINNRLLPIVNQQSCRISNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPI YGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTA SNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLECKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL

[0089] Sequence ID 7: Signal peptide + optimized RSV subtype AF protein truncated sequence + (SAIG) + Foldon sequence + thrombin cleavage site + His 6 tag + strep II tag

[0090] MELLILKANAITTILTAVTFCFASGQNCTEEFYQSTCSAVSKGYLSALRTGWYTCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTSAIASGVAVSKVLHLEGEVNKIKSALL STNKAVVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQL PLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKT FSNGCDYVSNKGMDTVSVGNTLYYVNKQECKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLVPRGSHHHHHHGSWSHPQFEK

[0091] Sequence ID 8: Signal peptide + constructed RSV subtype BF protein truncated sequence + (SAIG) + Foldon sequence + thrombin cleavage site + His 6 tag + Strep II tag

[0092] MELLILKANAITTILTAVTFCFASGQNCTEEFYQSTCSAVSRGYFSALRTGWYTCVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAASAIASGIAVSKVLHLEGEVNKIKNALL STNKAVVSLSNGVSVCTSKVLDLKNYINNRLLPIVNQQSCRISNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQL PIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKT FSNGCDYVSNKGVDTVSVGNTLYYVNKLECKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLVPRGSHHHHHHGSWSHPQFEK

[0093] Sequence ID 9 encodes the polynucleotide sequence shown in Sequence ID 7, which is codon-optimized based on the optimized RSV subtype AF protein.

[0094]

[0095] Sequence ID 10, which encodes the polynucleotide sequence shown in Sequence ID 8, is codon-optimized based on the optimized RSV subtype BF protein.

[0096] [Effects of the Invention]

[0097] Beneficial Effects: This invention designs a series of adenovirus vector vaccines against respiratory syncytial virus infections based on an optimized F protein sequence of respiratory syncytial virus. Animal studies have demonstrated that recombinant adenovirus vector vaccines carrying the codon-optimized subtype A RSV-PreF (RSV-A-PreF) gene can simultaneously induce the production of serum antibodies against pre-F and post-F glycoproteins after immunization of animals. Furthermore, such vaccines can induce the production of highly effective antibodies against pre-F and post-F glycoproteins in the bronchi. Notably, adenovirus vector vaccines exhibit potent humoral immunoprotective efficacy against RSV.

[0098] A recombinant adenovirus vaccine containing the optimized subtype A RSV-PreF (RSV-A-PreF)R2 gene (amino acid sequence shown in SEQ ID NO: 13) exhibits potent humoral immunoprotective efficacy and safety after immunizing animals and can be prepared on a large scale to help hosts resist respiratory syncytial virus infections, which has potential for broad applications. [Brief explanation of the drawing]

[0099] [Figure 1] This figure shows a plaque diagram of recombinant RSV adenovirus in Embodiment 1. [Figure 2] Embodiment 1: This figure shows the PCR identification diagram of recombinant RSV adenovirus in 1 negative control and 2 Ad-RSV-PreF. [Figure 3] This figure shows the results of anti-preF IgG levels in the serum of mice immunized intranasally with different doses of the adenovirus vector RSV vaccine R1 in Embodiment 3. [Figure 4]This figure shows the results of anti-preF IgG levels in the serum of mice immunized intranasally with different doses of the adenovirus vector RSV vaccine R2 in Embodiment 3. [Figure 5] This figure shows the results of anti-preF IgG levels in the serum of mice immunized intranasally with different doses of the adenovirus vector RSV vaccine R3 in Embodiment 3. [Figure 6] This figure shows the results of anti-preF IgG levels in the serum of mice immunized intranasally with different doses of the adenovirus vector RSV vaccine R4 in Embodiment 3. [Figure 7] This figure shows the results of anti-preF IgG levels in the serum of mice immunized intranasally with different doses of the adenovirus vector RSV vaccine R5 in Embodiment 3. [Figure 8] This figure shows the results of anti-postF IgG levels in the serum of mice immunized intranasally with different doses of the adenovirus vector RSV vaccine R2 in Embodiment 3. [Figure 9] This figure shows the results of anti-preF IgG levels in the serum of mice immunized intramuscularly with different doses of the adenovirus vector RSV vaccine R2 in Embodiment 3. [Figure 10] This figure shows the results of anti-postF IgG levels in the serum of mice immunized intramuscularly with different doses of the adenovirus vector RSV vaccine R2 in Embodiment 3. [Figure 11] This figure shows the results of anti-postF IgG levels in the bronchi of mice immunized intranasally with different doses of the adenovirus vector RSV vaccine R2 in Embodiment 3. [Figure 12] This figure shows the results of anti-preF IgG levels in the bronchi of mice immunized intranasally with different doses of the adenovirus vector RSV vaccine R2 in Embodiment 3. [Figure 13] This figure shows the results of anti-postF IgA in the bronchi of mice immunized intranasally with different doses of the adenovirus vector RSV vaccine R2 in Embodiment 3. [Figure 14]This figure shows the results of anti-preF in the bronchi of mice immunized intranasally with different doses of the adenovirus vector RSV vaccine R2 in Embodiment 3. [Figure 15] This figure shows the results of anti-postF IgG levels in the serum of rats immunized intranasally with different doses of the adenovirus vector RSV vaccine R2 in Embodiment 3. [Figure 16] This figure shows the results of anti-preF IgG levels in the serum of rats immunized intranasally with different doses of the adenovirus vector RSV vaccine R2 in Embodiment 3. [Figure 17] This figure shows the results of RSV neutralizing antibodies in the serum of mice immunized intranasally with the adenovirus vector RSV vaccine R2 in Embodiment 4. [Figure 18] This figure shows the results of viral loading in the lung tissue of cotton rats immunized intranasally with the adenovirus vector RSV vaccine R2 in Embodiment 5. [Figure 19] This is a pathological diagram of lung tissue from a challenge experiment of Cotton rats immunized intranasally with the adenovirus vector RSV vaccine R2 in Embodiment 5. [Modes for carrying out the invention]

[0100] Detailed description of the invention The solutions of the present invention will be described with reference to specific embodiments. Those skilled in the art will understand that the following embodiments are not intended to limit the scope of the invention, but merely to illustrate it. Where specific techniques or conditions are not explicitly stated in the embodiments, the techniques and conditions described in the literature or product specifications of the art should be followed. Where the manufacturer of the reagents or equipment used is not indicated, they are all conventional products available on the market. [Examples]

[0101] Embodiment 1: Preparation of an adenovirus vector RSV vaccine (1) Construction of RSV recombinant adenovirus

[0102] 1. Based on the F protein sequence of subtype A RSV, a series of PreF candidate sequences were obtained by performing amino acid shortening or amino acid mutation. Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to optimize human-derived codons and synthesize the gene to obtain the optimized subtype A RSV-PreF (RSV-A-PreF) gene (having the amino acid sequences shown in SEQ ID NOs. 11, 13, 15, 17, and 19 respectively; and having the corresponding nucleotide sequences encoding the amino acid sequences shown in SEQ ID NOs. 12, 14, 16, 18, and 20 respectively).

[0103] 2. The optimized subtype A RSV-PreF (RSV-A-PreF) gene was inserted between the XmaI restriction enzyme cleavage site and the SacI restriction enzyme cleavage site of the pDC516 vector by seamless cloning (homologous recombination), and the resulting shuttle plasmid was named pDC516-RSV-A-PreF. Following the instructions for the Lipofectamine 3000 transfection kit, the shuttle plasmid pDC516-RSV-A-PreF (2ug) containing the RSV-A-PreF gene and the AdMax adenovirus system skeleton plasmid pBHGfrt_E1,3FLP (4ug) were simultaneously transfected into HEK293A cells to package recombinant adenovirus. The specific process was as follows:

[0104] 1) 8 × 10 5 HEK293A cells were inoculated into a 6-well plate at a cell / well ratio, cultured in high-glucose DMEM + 10% FBS culture medium, and placed overnight in a 5% CO2 cell incubator at 37°C.

[0105] 2) The following day, high-glucose DMEM + 2% FBS was used for the culture medium change, and HEK293A cells were co-transfected with lipofectamine 3000 using the backbone plasmid (pBHGfrt_E1, 3FLP) and the shuttle plasmid (pDC516-RSV-A-PreF). The specific procedure was as follows: 4 μg of backbone plasmid and 2 μg of shuttle plasmid were taken into each transfection well, diluted in 125 μL of Opti-MEM culture medium, then 12 μL of P3000 reagent was added to the diluent, another 1.5 mL EP tube was taken, 7.5 μL of lipofectamine 3000 was diluted in 125 μL of Opti-MEM culture medium, and the diluted plasmid and diluted lipofectamine 3000 were mixed in a 1:1 ratio. The resulting mixture was incubated at room temperature for 10-15 minutes, then added to the cells, and the cells were cultured until they had fully grown, then incubated for 25 cm. 2 Transfer the cells to a cell culture bottle and observe for signs of cytotoxicity daily. Then, once the cells have fully grown at the bottom of the bottle, continue until they appear as a distinct plaque, as shown in Figure 1, for 75 cm. 2 The cells were transferred to cell culture bottles, and the virus stock was collected when most of the cells had lysed and detached from the growth surface.

[0106] 3) The cytotoxic cell culture was collected, centrifuged at 1200 rpm for 3 minutes, the virus-containing supernatant was aspirated, the cell pellet was resuspended in 1 / 10 of the culture volume of virus-containing supernatant, and subjected to three cycles of freezing and thawing in a -80°C refrigerator and a 37°C water bath. The prepared mixture was then centrifuged at 3000 rpm for 20 minutes, the virus-containing supernatant was collected, and mixed with the above virus-containing supernatant. The resulting mixture is the virus strain for the adenovirus vaccine.

[0107] 3. Identification of the virus strain A 50 μL stock of vaccine candidate virus strains was taken, 2 μL of protease K was added, and the resulting mixture was digested at 50°C for 30 minutes to release the viral genome. The resulting digest was used as a template for PCR amplification of the gene sequence, and the PCR product would be sequenced and identified after the electrophoresis gel was recovered. As shown in Figure 2, 1 represents the negative control, and 2 represents the Ad5 vector encoding the preF RSV F protein (Ad-RSV-PreF) with a stable conformation. The conditions, parameters, and primers for PCR amplification are as follows.

[0108] The PCR amplification conditions are as follows: denaturation: 98°C, 2 minutes, denaturation: 98°C, 10 seconds, annealing: 60°C, 15 seconds, extension: 72°C, 1 minute, extension: 72°C, 5 minutes, number of cycles: 35.

[0109] The primers for PCR amplification are as follows: Ad2304-F:ACGTGGGTATAAGAGGCGCGAC(Sequence ID 35) Ad2304-R:CTTCGGATCTTCGATGCTAGACGATCC (Sequence ID 36)

[0110] (2) Large-scale amplification of RSV recombinant adenovirus Qualified RSV recombinant adenovirus strains were stepwise amplified in 293 cells. The specific process was as follows: 1.0–4.0 × 10⁻⁶ 6293H cells were added at a rate of 1 / mL according to an MOI of 1-30, and the virus culture was collected when the cell viability reached 50%-80% to prepare master virus strain stocks and working virus strain stocks. Recombinant adenovirus vaccine was amplified in a cell factory or bioreactor, and the virus culture was collected after the majority of the cells had lysed. Cells and viruses were processed using the following steps: adding PBS to the bioreactor, sterilizing the bioreactor, discarding the PBS from the bioreactor after sterilization, adding the cell culture medium to the bioreactor, and, while maintaining operating conditions of 37°C, pH 7.0, DO 50%, and 50 rpm, collecting 293H cells in vials and in the logarithmic growth phase, and inoculating these cells into the bioreactor, where the inoculated cells were 0.5-1.0 × 10⁶. 6 A step of having a cell density of cells / mL, a step of replenishing the cell culture medium to 5L, wherein the cell culture conditions of the bioreactor are as follows: temperature 37°C, rotation speed 30-50 rpm, pH 7.15-7.25, and DO 30%-50%, a step of taking a culture sample daily to detect glucose concentration on microcarriers, cell density and cell morphology, and a step of the cell density in the bioreactor being 1.0-4.0 × 10⁻⁶. 6 The adenovirus strain was amplified in the bioreactor by the following steps: when the cell / mL reached a certain level, a recombinant adenovirus strain was inoculated into the bioreactor at an MOI of 1-30; after inoculation, the inoculated sample was collected daily to detect the glucose concentration and the viral titer in the culture supernatant and cell pellet, and the morphology of the cells on the microcarrier was observed; when the majority of the cells had detached from the microcarrier, the culture was stopped, and a viral lysate solution was added to the bioreactor at 37°C for 2-4 hours using Tween80 at a final concentration of 0.05%-1%, followed by the collection of the viral solution.

[0111] (3) Purification of RSV recombinant adenovirus

[0112] The recovered virus was purified by cesium chloride ultracentrifugation or ion-exchange chromatography, and the purified adenovirus was directly aliquoted and stored in the dark at -20°C. The specific process was as follows:

[0113] (1) Purification of adenovirus by cesium chloride ultracentrifugation The collected virus culture was centrifuged at 1200g for 10 minutes, and the virus-containing culture supernatant was aspirated. The cell pellet was resuspended in 1 / 10 the culture volume of virus-containing supernatant, and freeze-thawed for 3 cycles in a -80°C refrigerator and a 37°C water bath. The cell pellet was then centrifuged at 3000 rpm for 10-20 minutes, and the supernatant was aspirated. The virus-containing culture supernatant was concentrated 10-fold using an ultrafiltration membrane at 100K-300K to prepare 1.4 g / mL cesium chloride solution (53 g cesium chloride + 87 mL of 10 mM Tris-HCl, pH 7.9) and 1.2 g / mL cesium chloride solution (26.8 g cesium chloride + 92 mL of 10 mM Tris-HCl, pH 7.9). 8 mL of the 1.4 g / mL cesium chloride solution and 6 mL of the 1.2 g / mL cesium chloride solution were slowly added sequentially to an overspeed tube. Finally, 20 mL of the virus-containing culture supernatant was added at the peak of the discontinuous gradient. The equilibrium tube was centrifuged at 100,000 x g for 90 minutes at 4°C. After centrifugation, the blue virus band was aspirated with a syringe, followed by dialysis to remove cesium chloride, and the purified virus was stored at -80°C.

[0114] (2) Purification of adenovirus by ion exchange chromatography The virus culture was collected and lysed in 0.05%~1% Tween80 at 37°C for 2~4 hours. The lysed culture was filtered through 1.2 μm and 0.45 μm cartridge filters. The culture sample was concentrated 10-fold on a tangential flow membrane with a molecular weight of 100~300 kD, washed with 10 volumes of washing buffer (50 mM Tris-HCl, 2 mM MgCl2, 0~500 mM NaCl, pH 8.0), then collected. Nuclease was added to the washed sample at a final concentration of 10~50 U / mL, and the mixture was digested at 37°C for 1~3 hours. The washed sample was then treated with Q Sepharose XL, Source 30Q, Source The samples were subjected to anion exchange chromatography using 15Q or other packing materials, and the specific process was as follows: the buffer was equilibrated at 20 mL / min and 5 column volumes; after equilibration, the sample was loaded at 10 mL / min; after loading, the buffer was equilibrated to the conductivity level; linear gradient elution was performed under elution conditions of 100% low-salt buffer and 100% high-salt buffer, elution column volume of 10 V, and flow rate of 10 mL / min; each eluted peak was collected; and after elution, the column was regenerated with 2 M NaCl buffer at a rate of 20 mL / min in 5-10 column volumes. The viral peak was collected, and then the eluted viral sample was dialyzed or filtered by tangential flow for buffer replacement.

[0115] The RSV recombinant adenovirus prepared using the method described above was used as a vector vaccine for subsequent research, such as animal immunization studies.

[0116] Embodiment 2: Animal preparation, animal immunization, and sample collection

[0117] 1. Immunization procedure for intramuscular adenovirus vector vaccines: Female BALB / c mice aged 6-8 weeks were purchased from Charles River Laboratories and housed at the National Key Laboratory of Biotherapy, Sichuan University, under specific pathogen-free (SPF) conditions. Mice were intramuscularly immunized twice, on day 0 and day 21. The immunization dose of the adenovirus vector RSV vaccines R1, R2, R3, R4, and R5 (each having the amino acid sequences shown in SEQ ID NOs. 11, 13, 15, 17, and 19, respectively; and the corresponding nucleotide sequences encoding the amino acid sequences shown in SEQ ID NOs. 12, 14, 16, 18, and 20, respectively) was 2.5 × 10⁻⁶. 9 VP / mouse / time or 5 x 10 9 The vaccine was administered via VP / mouse / dose. Blood samples were collected via the orbital vein on day 35 (14 days after secondary immunization) and centrifuged at 6000 rpm for 10 minutes at 4°C. Serum samples were stored at -20°C before use, and the immunosuppressant effect of the vaccine was evaluated by detecting conjugated and neutralizing antibodies in the serum. Mice were sacrificed on day 49, and bronchoalveolar lavage fluid was collected by rinsing twice with 1 mL of physiological saline. Subsequently, the immunosuppressant effect of the vaccine was evaluated by detecting conjugated and neutralizing antibodies in the bronchoalveolar lavage fluid.

[0118] 2. Immunization procedure of intranasal immunoadenovirus vector vaccine: Female BALB / c mice at 6 - 8 weeks old were purchased from Charles River Laboratories and housed in the National Key Laboratory of Biotherapy, Sichuan University under specific pathogen - free (SPF) conditions. The mice were immunized intranasally twice, on day 0 and day 21. The immunization doses of the adenovirus vector RSV vaccines R1, R2, R3, R4, and R5 used (each having the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19 respectively; and having the corresponding nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20 respectively) were 2.5×10 9 VP / mouse / time or 5×10 9 VP / mouse / time. Blood samples were collected through the orbital vein on day 35 (14 days after the secondary immunization) and centrifuged at 6000 rpm for 10 minutes at 4°C. Serum samples were stored at - 20°C before use, and then the binding antibodies and neutralizing antibodies in the serum were detected to evaluate the immunization effect of the vaccine. The mice were sacrificed on day 49, and bronchoalveolar lavage fluid was collected by repeatedly bubbling twice with 1 mL of physiological saline. Then, the binding antibodies and neutralizing antibodies in the bronchoalveolar lavage fluid were detected to evaluate the immunization effect of the vaccine.

[0119] 3. Immunization procedure of rats: Female rats at 6 - 8 weeks old were purchased from Charles River Laboratories and housed in the National Key Laboratory of Biotherapy, Sichuan University under specific pathogen - free (SPF) conditions. The rats were immunized intranasally twice, on day 0 and day 21. The immunization doses of the adenovirus vector RSV vaccines R1, R2, R3, R4, and R5 used (each having the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19 respectively; and having the corresponding nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20 respectively) were 2×1010 VP / rat / session or 4 x 10 10 The vaccine was administered via VP / rat / dose. Blood samples were collected via the orbital vein on day 35 (14 days after secondary immunization) and centrifuged at 6000 rpm for 10 minutes at 4°C. Serum samples were stored at -20°C before use, and the immunosuppressive effect of the vaccine was evaluated by detecting conjugated and neutralizing antibodies in the serum.

[0120] 4. Immunization procedure for challenged cotton rats: Female cotton rats aged 6-8 weeks were purchased from SPF Biotechnology Co., Ltd. and housed in the National Key Laboratory of Biotherapy, Sichuan University under specific pathogen-free (SPF) conditions. The cotton rats were immunized intranasally twice on days 0 and 21, with an immunization dose of 2.5 × 10⁻¹⁴ of the adenovirus vector RSV vaccine R2 (having the amino acid sequence shown in SEQ ID NO: 13; and the corresponding nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 14). 9 The vaccine was administered as VP / rat / dose or 5 x 10⁹VP / rat / dose, and the immunoprotective effect was evaluated by challenging the rat on day 35 (14 days after secondary immunization).

[0121] Embodiment 3 Detection of antibodies by enzyme-linked immunosorbent assay (ELISA)

[0122] To detect specific IgG and IgA for RSV preF (preF) and postF (postF), 1 μg / mL RSV preF (Sino Biological Inc.) or RSV postF (Sino Biological Inc.) solutions were prepared using 50 mM carbonate coated buffer (pH 9.6). The prepared solutions were added to 96-well coated plates at a rate of 100 μL / well (Thermo Scientific Company, NUNC-MaxiSorp), and the plates were incubated overnight at 4°C. Preparation of 50 mM carbonate coated buffer (pH 9.6): 0.15 g Na2CO3 and 0.293 g NaHCO3 were weighed, dissolved in redistilled water, and the prepared buffer was adjusted to pH 9.6, with a final volume of 100 mL, and stored at 4°C for later use.

[0123] The following day, the covered plates were washed three times with PBS solution containing 0.1% Tween20 (PBST), then sealed and insulated at room temperature for 1 hour with blocking buffer (prepared with PBST) containing 1% BSA or 5% skim milk, followed by one wash with PBST. Mouse serum or bronchoalveolar lavage fluid was diluted to various ratios with blocking buffer, then the diluent was added to the covered plates at a rate of 100 μL / well, the plates were incubated at 37°C for 1-2 hours, and then washed three times with PBST.

[0124] Subsequently, 100 μL / well of HRP-goat anti-mouse IgG or HRP-anti-mouse IgA antibody (both diluted 1:10000 with blocking buffer) was added to the coated plate, the plate was incubated at 37°C for 1 hour, and then washed five times with PBST. Finally, 100 μL / well of 3,3',5,5'-tetramethylbenzidine (TMB) was added to the coated plate, the plate was allowed to develop color in the dark for 10-15 minutes, and then 50 μL / well of 1 M H2SO4 stop solution (preparation of 1H2SO4 stop solution: 2.7 mL of concentrated sulfuric acid (98%) was added dropwise to 47.3 mL of re-distilled water) was added to the coated plate, mixed thoroughly, and then read at a wavelength of 450 nm with a microplate reader.

[0125] The results are shown in Figures 3 to 7. After administering two doses of intranasal immunization to mice, different doses of adenovirus vector RSV vaccines (R1, R2, R3, R4, R5) were able to induce high levels of anti-preF IgG in the serum. In particular, the level of anti-preF IgG induced by adenovirus vector RSV vaccine R2 (amino sequence shown in SEQ ID NO: 13, nucleotide sequence shown in SEQ ID NO: 14, RSV-A-PreF-1F(R2)(Del104-145aa)) was significantly higher than that of the other intranasal adenovirus vector RSV vaccines, indicating that R2 may have potent humoral immunoprotective efficacy against RSV.

[0126] The results are shown in Figure 8. After administering two doses of intranasal immunization to mice, different doses of the adenovirus vector RSV vaccine R2 were able to induce anti-PostF IgG in the serum, indicating that R2 may have potent humoral immunoprotective efficacy against RSV.

[0127] The results are shown in Figures 9 and 10. After subjecting mice to two intramuscular immunization doses, different doses of the adenovirus vector RSV vaccine R2 were able to induce anti-preF and anti-postF IgG in the serum, indicating that R2 may have potent humoral immunoprotective efficacy against RSV.

[0128] The results are shown in Figures 11 and 12. After administering two doses of intranasal immunization to mice, the adenovirus vector RSV vaccine R2 was able to induce anti-preF and anti-postF IgG in bronchoalveolar lavage fluid, indicating that R2 may have potent in situ humoral immunoprotective efficacy against RSV.

[0129] The results are shown in Figures 13 and 14. After mice were subjected to two doses of intranasal immunization, the adenovirus vector RSV vaccine R2 was able to induce anti-preF and anti-postF IgA in bronchoalveolar lavage fluid, indicating that R2 may have potent in situ humoral immunoprotective efficacy against RSV.

[0130] The results are shown in Figures 15 and 16. After subjecting rats to two doses of intranasal immunization, the adenovirus vector RSV vaccine R2 was able to induce anti-preF and anti-postF IgG in the serum, indicating that R2 may have potent humoral immunoprotective efficacy against RSV.

[0131] Embodiment 4: RSV neutralizing antibody detection experiment

[0132] RSV neutralizing antibody detection experiments were performed to detect the titer of neutralizing antibodies in serum and bronchoalveolar lavage (BALF) samples. RSV strains A2, Long, B18357, and B9320 were purchased from ATCC Company in the United States.

[0133] Cell culture: Place A549 cells in complete DMEM medium (DMEM culture medium containing 10% fetal bovine serum and 1000 IU penicillin / streptomycin) in 4 × 10⁶ cells. 5 The cells were resuspended at a cell density of / mL. 100 μL of the cell suspension was added to a 96-well flat-bottom sterile plate and incubated overnight in a 37°C incubator (5% CO2).

[0134] Positive control: Before use, the positive serum was inactivated in a 56°C water bath for 30 minutes, diluted 100-fold in DMEM medium without fetal bovine serum, and 110 μL of the 100-fold diluted positive serum was added to well A12. 55 μL of DMEM culture medium without fetal bovine serum was added to wells B12-H12, and 55 μL of the liquid was transferred from well A12 to well B12, mixed thoroughly by gently pipetting up and down 6-8 times. This transfer process was repeated until the liquid was transferred to well H12, and finally 55 μL of the liquid was aspirated and discarded from well H12.

[0135] Sample Dilution: The sample was first diluted 30-fold in a triple dilution system, and then the sample with seven concentration gradients was diluted using a 2-fold dilution method. The initially diluted sample was added to wells A1-A9, with the sample well volume before gradient dilution being 110 μL / well. 55 μL of DMEM culture medium without fetal bovine serum was added to wells A2-H11. A multichannel pipette was adjusted to 55 μL, and the liquid in wells A1-A9 was gently pipetted up and down 6-8 times to mix thoroughly. 55 μL of liquid was then transferred to the corresponding wells B1-B9, gently pipetted up and down 6-8 times, and then transferred to wells D1-D9. This transfer process was repeated, and finally 50 μL of liquid was aspirated and discarded from wells H1-H9.

[0136] Incubation of samples with viruses: 55 μL of RSV virus was added to each well of columns 1-10 and 12 so that the pseudovirus load in each well was 200 PFU / well. Column 10 represented the positive control group. 55 μL of DMEM culture medium without fetal bovine serum was added to column 11, and column 11 represented the negative control group. The 96-well plate was incubated in a cell incubator (37°C, 5% CO2) for 1 hour.

[0137] Cell and virus inoculation: A 96-well culture plate containing A549 cells was placed upside down on sterile absorbent paper, the supernatant was aspirated, PBS was added to prevent cell washout, aspirated twice, the final PBS was aspirated and discarded, 100 μL of a virus and serum mixture was added to the cell well plate, and the plate was incubated in a 37°C incubator for 1 hour. Subsequently, 90 μL of liquid was aspirated from each well using a multichannel pipette, 100 μL of complete culture medium was added to each well, and the plate was incubated in a 37°C incubator for 3 days.

[0138] Fixation: The plate was gently rotated to remove the supernatant, and 200 μL of fixation buffer (80% acetone, 20% PBS) was added to each well to fix the cells. The plate was incubated at -20°C for 20 minutes. The fixation buffer was discarded, and the plate was dried upside down for 10 minutes. 200 μL of blocking buffer (5% milk diluent prepared with PBST) was added to each well, and the plate was incubated at room temperature for 30 minutes. The blocking buffer was discarded by gently inverting the plate, and goat anti-RSV antibody (primary antibody, 1:500) diluted in 50 μL / well of blocking buffer was added, incubated at 37°C for 1 hour, and washed three times with PBST. Subsequently, Alexa-Fluor donkey anti-goat IgG antibody (secondary antibody, 1:50000) diluted in 50 μL / well of blocking buffer was added, incubated at 37°C for 1 hour, and washed five times with PBST.

[0139] Reading: Read the plate using the fluorescein isothiocyanate (FITC) channel with the count setting on the automatic spot reader.

[0140] Determination of 50% neutralization titer: Inhibition rate = [1 - (average luminescence intensity of sample group - average of negative control group) / (average luminescence intensity of positive group - average of negative control group CC)] x 100%. EC50 (IC50) was calculated from the neutralization inhibition rate results using the Reed-Mune method.

[0141] The results are shown in Figure 17. Mice were immunized twice with an intranasal dose (5 × 10⁻¹). 9 After exposure to VP / mouse / dose, the adenovirus vector RSV vaccine R2 was able to induce neutralizing antibodies against A2, B18537, B9320, and Long, indicating that R2 intranasal immunization can induce broad and potent immunoprotection.

[0142] Embodiment 5: RSV Challenge Experiment

[0143] On days 0 and 14, a low dose (2.5 × 10) was administered. 9VP / rat) and high dose (5 × 10 9 Cotton rats were intranasally immunized with the adenovirus vector vaccine R2 (VP / rat). Cotton rats immunized with the adenovirus empty vector vaccine served as the control group, with n=5 rats per group. Intranasal immunization was performed at a volume of 50 μL / mouse, administered in two divided doses at 2-hour intervals. On day 35, a challenge experiment was conducted. Mice were given a volume of 100 μL / mouse and 3 × 10⁶ doses of either RSV-A2 strain or RSV-B18537 strain. 6 Intranasal challenge was performed with a dose of PFU. Cotton rats were euthanized 5 days after infection, the left lung lobe was fixed, sliced, and stained with HE for pathological examination, and the right lung lobe was collected for viral load detection. The tissue was cut into small pieces, homogenized, and approximately 100 mg of tissue was weighed and homogenized in 800 μL of TRIzol reagent. 400 μL of tissue homogenate was collected and RNA was extracted. Finally, the RNA was dissolved in 50 μL of nuclease-free solution and stored at -80°C for later use. The RNA was used for subsequent detection of viral load by qRT-PCR (one-step method). Viral genomic RNA (gRNA) and subgenomic RNA (sgRNA) were determined by real-time quantitative reverse transcription PCR (qRT-PCR). The primer and probe sequences used for the gRNA are derived from the N gene, primer sequences: 5'-CTCCTAATTATGATGTGCAGAAACACA-3' (SEQ ID NO: 37 forward), 5'-CCAGTGAATTTATGATTAGCATCTTCT-3' (SEQ ID NO: 38 reverse); probe sequence: 5'-FAM-ATAACATGCCACATAACTTA-BHQ1-3' (SEQ ID NO: 39).

[0144] Post-respiratory syncytial virus (RSV) infection lung viral load and pathological changes are important indicators for evaluating the protective efficacy of candidate respiratory syncytial virus vaccines. Cotton rats were vaccinated with two doses of vaccine, followed by live RSV-A2 and RSV-B18537 strains (3 × 10⁻¹⁰). 6PFU / cotton rats were challenged. On the fifth day after infection, the animals were euthanized, and the right lung lobe was collected for viral load detection (Figure 18). The left lung lobe was fixed, sliced, and stained with HE for pathological examination (Figure 19). The results showed that after challenging with two virus strains, the viral load in the lung tissue of experimental animals in the empty load control group was approximately 10%. 6 The data is in copies / mg tissue, and the viral load in the lung tissue of animals in the vaccine-immunized group was 10 3 The study showed a reduction to less than copies / mg of tissue (Figure 18), demonstrating that vaccine immunization effectively neutralizes and eliminates the virus in the lung tissue of Cotton rats. Pathological results showed that the lung tissue of animals in the empty-loaded group exhibited clear alveolar wall thickening with local hemorrhage and infiltration of numerous inflammatory cells. The pathological state of the lung tissue of animals in the vaccine-immunized group was significantly improved. Alveolar contours were clear, and infiltration was limited to small amounts of inflammatory cell infiltration (Figure 19). These results demonstrate that vaccine immunization provides substantial protection against RSV-induced disease in Cotton rats.

Claims

1. An adenovirus vector vaccine for preventing and / or treating respiratory syncytial virus infection, wherein the adenovirus vector vaccine is obtained by constructing a recombinant adenovirus vector containing a respiratory syncytial virus expression gene, the polynucleotide sequence of the respiratory syncytial virus expression gene is selected from at least one of SEQ ID NOs: 12, SEQ ID NOs: 14, SEQ ID NOs: 16, SEQ ID NOs: 18, SEQ ID NOs: 20, SEQ ID NOs: 22, SEQ ID NOs: 24, SEQ ID NOs: 26, SEQ ID NOs: 28, SEQ ID NOs: 30, SEQ ID NOs: 32, and SEQ ID NOs:

34.

2. The adenovirus vector vaccine according to claim 1, wherein the amino acid sequence of the protein encoded by the polynucleotide sequence is selected from at least one of SEQ ID NOs: 11, SEQ ID NOs: 13, SEQ ID NOs: 15, SEQ ID NOs: 17, SEQ ID NOs: 19, SEQ ID NOs: 21, SEQ ID NOs: 23, SEQ ID NOs: 25, SEQ ID NOs: 27, SEQ ID NOs: 29, SEQ ID NOs: 31, and SEQ ID NOs:

33.

3. The adenovirus vector vaccine according to claim 1 or 2, further comprising a pharmaceutically acceptable adjuvant, vector, diluent, or excipient.

4. The adenovirus vector vaccine according to claim 1 or 2, wherein the adenovirus vector is selected from at least one of adenoviruses, Ankarawaxinia virus and adeno-associated viruses; preferably, human type 5, 35 or 26 and / or chimpanzee AdC68 or AdC7 replication-deficient adenovirus; and more preferably, human type 5 replication-deficient adenovirus having a combined E1 and E3 deletion.

5. The adenovirus vector vaccine according to any one of claims 1 to 4, wherein the vaccine is formulated as an intradermal injection preparation, a subcutaneous injection preparation, an intramuscular injection preparation, an intravenous injection preparation, an oral preparation, or a nasal spray preparation, preferably the vaccine is formulated as the nasal spray preparation or the intramuscular injection preparation.

6. A method for preparing an adenovirus in an adenovirus vector vaccine according to any one of claims 1 to 5, comprising the steps of: constructing a shuttle plasmid vector containing polynucleotides; transfecting the constructed shuttle plasmid vector and a backbone plasmid into host cells and culturing the host cells; obtaining a replication-deficient recombinant adenovirus; and performing large-scale culture and purification.

7. A composition for preventing and / or treating respiratory syncytial virus infection, wherein the composition is a compound formulation comprising a recombinant protein vaccine for preventing and / or treating respiratory syncytial virus infection and an adenovirus vector vaccine according to any one of claims 1 to 5 as active ingredients.

8. A combination drug for preventing and / or treating respiratory syncytial virus infection, comprising a recombinant protein vaccine for preventing and / or treating respiratory syncytial virus infection and an adenovirus vector vaccine according to any one of claims 1 to 5, wherein the vaccines are administered separately or simultaneously.

9. The composition according to claim 7 or the combination drug according to claim 8, wherein the recombinant protein vaccine comprises a recombinant protein for preventing and / or treating respiratory syncytial virus infection, the amino acid sequence of the recombinant protein having more than 70% sequence identity and the same or substantially equivalent biological activity as SEQ ID NO: 1 or SEQ ID NO: 2, preferably the amino acid sequence of the recombinant protein is selected from at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO:

6.

10. The composition or combination drug according to claim 9, wherein the recombinant protein vaccine comprises a protein precursor, the protein precursor is constructed of at least one amino acid sequence selected from SEQ ID NO: 7 and SEQ ID NO: 8, and preferably the nucleotide sequence encoding the protein precursor is selected from SEQ ID NO: 9 or SEQ ID NO:

10.

11. The composition or the concomitant drug according to any one of claims 7, 9, and 10, wherein the composition or the concomitant drug is formulated as an intradermal injection preparation, a subcutaneous injection preparation, an intramuscular injection preparation, an intravenous injection preparation, an oral preparation, or a nasal spray preparation, and preferably the vaccine is formulated as a nasal spray preparation.

12. Use of an adenovirus vector vaccine according to any one of claims 1 to 5, a composition according to claims 7, 9 to 11, or a combination drug according to any one of claims 8 to 11, when preparing a drug for the prevention and / or treatment of respiratory syncytial virus infection.

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