Monoclonal antibody for resisting coxsackie B4 virus and nucleic acid molecule application of monoclonal antibody

Abstract

The invention provides a monoclonal antibody for resisting coxsackie B4 virus, a nucleic acid molecule and application of the monoclonal antibody and the nucleic acid molecule. The monoclonal antibody provided by the invention has a remarkable prevention or treatment effect on the coxsackie B4 type virus, and particularly has a remarkable effect on treatment of the infected coxsackie B4 type virus.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and more particularly to a monoclonal antibody against Coxsackie B4 virus, a nucleic acid molecule, and their applications. Background Technology

[0002] Coxsackievirus B4 (CVB4) belongs to the Coxsackievirus B group within the genus Enterovirus of the family Picornaviridae. CVB4 has a typical enterovirus structure, consisting of an icosahedral, non-enveloped viral particle approximately 30 nm in diameter. Its genome is a linear single-stranded RNA of about 7.4 kb enclosed in a viral capsid. The CVB4 capsid is composed of 60 repeating asymmetric units, each consisting of four structural proteins: VP1, VP2, VP3, and VP4. CVB4 infection can cause a variety of clinical symptoms, such as hand-foot-and-mouth disease and respiratory illnesses. Furthermore, multiple studies have confirmed a close association between CVB4 infection and the development of type 1 diabetes. Epidemiological data show that CVB4 continues to circulate globally, posing a threat to global public health. However, effective prevention and treatment methods for CVB4 infection are still lacking.

[0003] In previous studies, the inventors constructed two types of inactivated CVB4 virus particles (F-particles and E-particles). E-particles or F-particles effectively induced mice to produce CVB4 neutralizing antibodies. After mice were inoculated with inactivated CVB4 virus E-particles or F-particles and then infected with a lethal dose of CBV4 virus, the induced neutralizing antibodies protected the mice from lethal CVB4 infection. The immunogenicity and protective efficacy of inactivated CVB4 F-particles and E-particles were determined (Wang T, Wang C, Pang L, et al. Immunogenicity and protective efficacy of inactivated coxsackievirus B4 viral particles[J]. Emerging Microbes & Infections, 2024, 13(1)). The above studies mainly involved preventative vaccines before CVB4 virus infection and failed to provide effective treatments for those already infected with CVB4 virus. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a monoclonal antibody against Coxsackievirus B4 (CVB4), which exhibits significant therapeutic and protective effects against mice infected with CVB4 virus.

[0005] To achieve the aforementioned objective, a first aspect of the present invention provides a monoclonal antibody against Coxsackie B4 virus, the monoclonal antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1 sequence, a heavy chain CDR2 sequence, and a heavy chain CDR3 sequence, and the light chain variable region comprising a light chain CDR1 sequence, a light chain CDR2 sequence, and a light chain CDR3 sequence; wherein: (1) The heavy chain CDR1 sequence is selected from the amino acid sequence shown in SEQ ID NO:1, 2 or 3; The amino acid sequence shown in SEQ ID NO:1 is: GFTFSDFY The amino acid sequence shown in SEQ ID NO:2 is: GFSLTRYG The amino acid sequence shown in SEQ ID NO:3 is: GFTFSRYA (2) The heavy chain CDR2 sequence is selected from the amino acid sequence shown in SEQ ID NO:4, 5 or 6; The amino acid sequence shown in SEQ ID NO:4 is: ISNGGGST The amino acid sequence shown in SEQ ID NO:5 is: WSGGST The amino acid sequence shown in SEQ ID NO:6 is: ISSGGTI (3) The heavy chain CDR3 sequence is selected from the amino acid sequence shown in SEQ ID NO:7, 8 or 9; The amino acid sequence shown in SEQ ID NO:7 is: ARRRSYGYPYYAMDY The amino acid sequence shown in SEQ ID NO:8 is: ARRGYYGSIYYSMDY The amino acid sequence shown in SEQ ID NO:9 is: ARIEEYYGSSYYSMDY (4) The light chain CDR1 sequence is selected from the amino acid sequence shown in SEQ ID NO:10 or 11; The amino acid sequence shown in SEQ ID NO:10 is: SEQGISNY The amino acid sequence shown in SEQ ID NO:11 is: QGISKY (5) The light chain CDR2 sequence is YTS; (6) The light chain CDR3 sequence is selected from the amino acid sequence shown in SEQ ID NO:12, 13 or 14.

[0006] The amino acid sequence shown in SEQ ID NO:12 is: QQYSKLPLT The amino acid sequence shown in SEQ ID NO:13 is: QQYSKLPWT Preferably, the monoclonal antibody is selected from 4E3 monoclonal antibody, 11D4 monoclonal antibody, or 14B3 monoclonal antibody; wherein: the 4E3 monoclonal antibody has heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 sequences that are the amino acid sequences shown in SEQ ID NO:1, 4, and 7, respectively, and its light chain CDR1, light chain CDR2, and light chain CDR3 sequences that are the amino acid sequences shown in SEQ ID NO:10, YTS, and SEQ ID NO:12, respectively (see Table 1 for details); Table 1. CDR sequence of the variable region of the 4E3 monoclonal antibody

[0007] The 11D4 monoclonal antibody has heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 sequences that are the amino acid sequences shown in SEQ ID NO:2, 5, and 8, respectively, and light chain CDR1, light chain CDR2, and light chain CDR3 sequences that are the amino acid sequences shown in SEQ ID NO:10, YTS, and SEQ ID NO:13, respectively (see Table 2 for details). Table 2. CDR sequence of the variable region of 11D4 monoclonal antibody

[0008] The 14B3 monoclonal antibody has heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 sequences that are the amino acid sequences shown in SEQ ID NO:3, 6 and 9, respectively, and light chain CDR1, light chain CDR2 and light chain CDR3 sequences that are the amino acid sequences shown in SEQ ID NO:11, YTS and SEQ ID NO:13, respectively (see Table 3 for details).

[0009] Table 3. CDR sequence of the variable region of the 14B3 monoclonal antibody

[0010] Preferably, the amino acid sequence of the heavy chain variable region of the 4E3 monoclonal antibody is as shown in SEQ ID NO:14, and the amino acid sequence of the light chain variable region of the 4E3 monoclonal antibody is as shown in SEQ ID NO:15; and / or, The amino acid sequence of the heavy chain variable region of the 4E3 monoclonal antibody shown in SEQ ID NO:14 is as follows: EVKLVESGGGLVQPGGSLKLSCATS GFTFSDFY MCWVRQTPEKRLEWVAY ISNGGGST YYPDTVKGRFTISRDSAKNTLYLQMSRLKSEDTAMYYC ARRRSYGYPYYAMDYWGQGTSVTVSS (The bold and underlined regions are the complementary determinant regions CDR1-3, respectively) The amino acid sequence of the light chain variable region of the 4E3 monoclonal antibody shown in SEQ ID NO:15 is as follows: DIQMTQTTSSLSASLGDRVTISCSAS QGISNY LNWYQQKPDGTVKLLIY YTS SLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYC QQYSKLPLT FGGGTKLEIK (The bold and underlined regions are the complementary determinant regions CDR1-3, respectively) The amino acid sequence of the heavy chain variable region of the 11D4 monoclonal antibody is shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region of the 11D4 monoclonal antibody is shown in SEQ ID NO:18; and / or, The amino acid sequence of the heavy chain variable region of the 11D4 monoclonal antibody shown in SEQ ID NO:16 is as follows: QVQLKQSGPGLVQPSQSLSITCTVS GFSLTRYG VDWVRQSPGKGLEWLGV IWSGGST DYNAPFISRLSITKDNSKSQVFFKMNSLQADDSAIYYC ARRGYYGSIYYSMDY WGQGTSVTVSS (The bold and underlined regions are the complementary determinant regions CDR1-3, respectively) The amino acid sequence of the light chain variable region of the 11D4 monoclonal antibody shown in SEQ ID NO:17 is as follows: DIQMTQTTSSLSASLGDRVTITCSAS QGISNY LNWYQQKPDGTVKLLIY YTS SLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYC QQYSKLPWT FGGGTKLEIK (The bold and underlined regions are the complementary determinant regions CDR1-3, respectively) The amino acid sequence of the heavy chain variable region of the 14B3 monoclonal antibody is shown in SEQ ID NO:19, and the amino acid sequence of the light chain variable region of the 14B3 monoclonal antibody is shown in SEQ ID NO:20.

[0011] The amino acid sequence of the heavy chain variable region of the 14B3 monoclonal antibody shown in SEQ ID NO:18 is as follows: EVKLVESGGGLVKPGGSLKLSCAAS GFTFSRYA MSWVRQTPEKRLEWVAS ISSGGTIYYLDSVKGRFTISRDNAGNILYLQMSSLRSEDTAMFYC ARIEYYGSSYYSMDY WGQGTSVTVSS (The bold and underlined regions are the complementary determinant regions CDR1-3, respectively) The amino acid sequence of the variable region of the light chain of the 14B3 monoclonal antibody shown in SEQ ID NO:19 is as follows: DIQMTQTTSSLSASLGDRVTISCSAS QGISKY LNWYQQKPDGTVKLLIY YTS SLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYFC QQYSKLPWT FGGGTKLEIK (The bold and underlined regions are the complementary determinant regions CDR1-3, respectively) Preferably, the monoclonal antibody is a recombinant chimeric antibody, comprising a murine variable region and a human constant region, wherein the heavy chain variable region and the light chain variable region are located in the murine variable region; wherein, The heavy chain amino acid sequence of the 4E3 monoclonal antibody is shown in SEQ ID NO:20, and the light chain amino acid sequence is shown in SEQ ID NO:21; and / or, The heavy chain amino acid sequence of the 4E3 monoclonal antibody shown in SEQ ID NO:20: MHSSALLCCLVLLTGVRAEVKLVESGGGLVQPGGSLKLSCATS GFTFSDFY MCWVRQTPEKRLEWVAY ISNGGGST YYPDTVKGRFTISRDSAKNTLYLQMSRLKSEDTAMYYC ARRRSYGYPYYAMDYWGQGTSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKV DKKIEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVGFSCSVMHEALHNHYTQKSLSLSPGK* (Positions 1-18 are the leader peptide sequence, positions 19-140 are the variable region sequence, positions 141-470 are the constant region sequence, and * is the stop codon. The bold and underlined areas indicate the complementarity-determining region (CDR)) The light chain amino acid sequence of the 4E3 monoclonal antibody shown in SEQ ID NO:21: MHSSALLCCLVLLTGVRADIQMTQTTSSLSASLGDRVTISCSAS QGISNY LNWYQQKPDGTVKLLIY Y TS SLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYC QQYSKLPLT FGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC* (Positions 1-18 are the leader peptide sequence, positions 19-125 are the variable region sequence, positions 126-232 are the constant region sequence, and * is the stop codon. The bold and underlined regions are the complementarity-determining regions (CDRs)) The heavy chain amino acid sequence of the 11D4 monoclonal antibody is shown in SEQ ID NO:22, and the light chain amino acid sequence is shown in SEQ ID NO:23; and / or, The heavy chain amino acid sequence of the 11D4 monoclonal antibody shown in SEQ ID NO:22: MHSSALLCCLVLLTGVRAQVQLKQSGPGLVQPSQSLSITCTVSGFSLTRYG VDWVRQSPGKGLEWLGV IWSGGST DYNAPFISRLSITKDNSKSQVFFKMNSLQADDSAIYYC ARRGYYGSIYYSMDY WGQGTSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTK VDKKIEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* (Positions 1-18 are the leader peptide sequence, positions 19-139 are the variable region sequence, positions 140-468 are the constant region sequence, and * is the stop codon. The bold and underlined areas indicate the complementarity-determining region (CDR)) The light chain amino acid sequence of the 11D4 monoclonal antibody shown in SEQ ID NO:23: MHSSALLCCLVLLTGVRADIQMTQTTSSLSASLGDRVTITCSAS QGISNY LNWYQQKPDGTVKLLIY Y TS SLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYC QQYSKLPWT FGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC* (Positions 1-18 are the leader peptide sequence, positions 19-125 are the variable region sequence, positions 126-232 are the constant region sequence, and * is the stop codon. The bold and underlined regions are the complementarity-determining regions (CDRs)) The heavy chain amino acid sequence of the 14B3 monoclonal antibody is shown in SEQ ID NO:24, and the light chain amino acid sequence is shown in SEQ ID NO:25.

[0012] The heavy chain amino acid sequence of the 14B3 monoclonal antibody, as shown in SEQ ID NO:24, is as follows: MHSSALLCCLVLLTGVRAEVKLVESGGGLVKPGGSLKLSCAAS GFTFSRYA MSWVRQTPEKRLEWVAS ISSGGTI YYLDSVKGRFTISRDNAGNILYLQMSSLRSEDTAMFYC ARIEYYGSSYYSMDY WGQGTSVTVSSATTTAPSVYPLVPGCSDTSGSSVTLGCLVKGYFPEPVTVKWNYGALSSGVRTVSSVLQSGFYSLSSLVTVPSSTWPSQTVICNVAHPASKTEL IKRIEEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* (Positions 1-19 are the leader peptide sequence, positions 20-140 are the variable region sequence, positions 141-470 are the constant region sequence, and * is the stop codon. The bold and underlined areas indicate the complementarity-determining region (CDR)) The light chain amino acid sequence of the 14B3 monoclonal antibody as shown in SEQ ID NO:25: MHSSALLCCLVLLTGVRADIQMTQTTSSLSASLGDRVTISCSAS QGISKY LNWYQQKPDGTVKLLIY Y TS SLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYFC QQYSKLPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC* (Positions 1-18 are the leader peptide sequence, positions 19-125 are the variable region sequence, positions 126-232 are the constant region sequence, and * is the stop codon. The bold and underlined regions are the complementarity-determining regions (CDRs)) A second aspect of the present invention provides a nucleic acid molecule encoding the monoclonal antibody described in the above technical solution, comprising a first nucleotide sequence encoding the heavy chain variable region and a second nucleotide sequence encoding the light chain variable region; The first nucleotide sequence includes the nucleotide sequence encoding heavy chain CDR1, the nucleotide sequence encoding heavy chain CDR2, and the nucleotide sequence encoding heavy chain CDR3; among which, (a) The nucleotide sequence encoding the heavy chain CDR1 is selected from the nucleotide sequence shown in SEQ ID NO:26, 27 or 28; The nucleotide sequence shown in SEQ ID NO:26 is: ggattcactttcagtgacttttac The nucleotide sequence shown in SEQ ID NO:27 is: ggtttctcattaactagatatggt The nucleotide sequence shown in SEQ ID NO:28 is: ggattcactttcagtagatatgcc (b) The nucleotide sequence encoding the heavy chain CDR2 is selected from the nucleotide sequence shown in SEQ ID NO:29, 30 or 31; The nucleotide sequence shown in SEQ ID NO:29 is: ttagtaatggtggtggtagcacc The nucleotide sequence shown in SEQ ID NO:30 is: atatggagtggtggaagcaca The nucleotide sequence shown in SEQ ID NO:31 is: ataagtagtggtggtactatt (c) The nucleotide sequence encoding the heavy chain CDR3 is selected from the nucleotide sequence shown in SEQ ID NO:32, 33 or 34; The nucleotide sequence shown in SEQ ID NO:32: gcaagacggcgtagctatggttacccttactatgctatggactac The nucleotide sequence shown in SEQ ID NO:33: gccagaagaggatactacggtagtatttactattctatggactac The nucleotide sequence shown in SEQ ID NO:34: gcaaggattgagtactacggtagtagctactattctatggactac The second nucleotide sequence includes the nucleotide sequence encoding light chain CDR1, the nucleotide sequence encoding light chain CDR2, and the nucleotide sequence encoding light chain CDR3; wherein, (d) The nucleotide sequence encoding the light chain CDR1 is selected from the nucleotide sequence shown in SEQ ID NO:35 or 36; The nucleotide sequence shown in SEQ ID NO:35 is: cagggcattagcaattat The nucleotide sequence shown in SEQ ID NO:36 is: cagggcattagcaaatat (e) The nucleotide sequence encoding the light chain CDR2 is selected from the nucleotide sequence shown in SEQ ID NO:38; The nucleotide sequence shown in SEQ ID NO:37 is: tacacatca (f) The nucleotide sequence encoding the light chain CDR3 is selected from the nucleotide sequence shown in SEQ ID NO:38 or 39.

[0013] The nucleotide sequence shown in SEQ ID NO:38 is: cagcagtatagtaagcttcctctcacg The nucleotide sequence shown in SEQ ID NO:39 is: cagcagtatagtaagcttccgtggacg Preferably, the nucleic acid is selected from nucleic acid molecules encoding 4E3 monoclonal antibodies, nucleic acid molecules encoding 11D4 monoclonal antibodies, or nucleic acid molecules encoding 14B3 monoclonal antibodies; wherein: The nucleic acid molecule encoding the 4E3 monoclonal antibody has nucleotide sequences encoding heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 as shown in SEQ ID NO:26, 29, and 32, respectively, and nucleotide sequences encoding light chain CDR1, light chain CDR2, and light chain CDR3 as shown in SEQ ID NO:35, 37, and 38, respectively (see Table 1). The nucleic acid molecule encoding the 11D4 monoclonal antibody has nucleotide sequences encoding heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 as shown in SEQ ID NO:27, 30, and 33, respectively, and nucleotide sequences encoding light chain CDR1, light chain CDR2, and light chain CDR3 as shown in SEQ ID NO:35, 37, and 39, respectively (see Table 2). The nucleic acid molecule encoding the 14B3 monoclonal antibody has nucleotide sequences encoding heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 as shown in SEQ ID NO:28, 31, and 34, respectively, and nucleotide sequences encoding light chain CDR1, light chain CDR2, and light chain CDR3 as shown in SEQ ID NO:36, 37, and 39, respectively (see Table 3).

[0014] Preferably, the nucleotide sequence encoding the 4E3 monoclonal antibody has the first nucleotide sequence as shown in SEQ ID NO:40 and the second nucleotide sequence as shown in SEQ ID NO:41; and / or, The first nucleotide sequence (nucleotide sequence of the heavy chain variable region) encoding the 4E3 monoclonal antibody shown in SEQ ID NO:40 is: gaagtgaagctggtggagtctgggggaggcttagtgcagcctggagggtccctgaaactctcctgtgcaacctct ggattcactttcagtgacttttac atgtgttgggttcgccagactccagagaagaggctggagtgggtcgcataca ttagtaatggtggtggtagcacc tattatccagacactgtaaagggccgattcaccatctccagagacagtgccaagaacaccctgtacctgcaaatgagccgtctgaagtctgaggacacagccatgtattactgt gcaagacg gcgtagctatggttacccttactatgctatggactac tggggtcaaggaacctcagtcaccgtctcctca (The bold and underlined regions are the complementary determinant regions CDR1-3, respectively) The second nucleotide sequence (nucleotide sequence of the light chain variable region) encoding the 4E3 monoclonal antibody shown in SEQ ID NO:41 is: gatatccagatgacacagactacatcctccctgtctgcctctctggggagacagtcaccatcagttgcagtgcaagt cagggcattagcaattat ttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctat tacacatca agtttacactcaggagtcccatcaaggttcagtggcagtgggtctgggacagattattctctcaccatcagcaacctggaacctgaagatattgccacttactattgt cagcagtatagtaagcttcctctcacg ttcggaggggggaccaagctggaaataaaa (The bold and underlined regions are the complementary determinant regions CDR1-3, respectively) The nucleotide sequence encoding the 11D4 monoclonal antibody, the first nucleotide sequence is shown in SEQ ID NO:42, the second nucleotide sequence is shown in SEQ ID NO:43; and / or, The first nucleotide sequence (nucleotide sequence of the heavy chain variable region) encoding the 11D4 monoclonal antibody shown in SEQ ID NO:42 is as follows: caggtgcagctgaagcagtcaggacctggcctagtgcagccctcacaggcctgtccatcacctgcacagtctct ggtttctcattaactagatatggt gtagactgggttcgccagtctccaggaaagggtctggagtggctgggcgtg atatggagtggtggaagcaca gactataatgctcctttcatatccagactgagcatcaccaaggacaactccaagagccaagttttctttaaaatgaacagtctgcaagctgatgactcagccatatactactgt gccagaagagg atactacggtagtatttactattctatggactac tggggtcaaggaacctcagtcaccgtctcctca (The bold and underlined regions are the complementary determinant regions CDR1-3, respectively) The second nucleotide sequence (nucleotide sequence of the light chain variable region) encoding the 11D4 monoclonal antibody shown in SEQ ID NO:43 is as follows: gatatccagatgacacagactacatcctccctgtctgcctctctggggagacagtcaccatcacttgcagtgcaagt cagggcattagcaattatttaaactggtatcaacagaaaccagatggaactgttaaactcctgatctat tacacatca agtttacactcaggagtcccatcaaggttcagtggcagtgggtctgggacagattattctctcaccatcagcaacctggaacctgaagatattgccacttactattgt cagcagtatagtaagcttccgtggacg ttcggtggaggcaccaagctggaaatcaaa (The bold and underlined regions are the complementary determinant regions CDR1-3, respectively) The nucleotide sequence encoding the 14B3 monoclonal antibody has the first nucleotide sequence shown in SEQ ID NO:44 and the second nucleotide sequence shown in SEQ ID NO:45.

[0015] The first nucleotide sequence (nucleotide sequence of the heavy chain variable region) encoding the 14B3 monoclonal antibody shown in SEQ ID NO:44 is as follows: gaagtgaaactagtggagtctgggggaggcttagtgaagcctggagggtccctgaaactctcctgtgcagcctct ggattcactttcagtagatatgcc atgtcttgggttcgccagactccagagaagaggctggagtgggtcgcatcc ataagtagtggtggtactatt tactatctagacagtgtgaagggccgattcaccatctccagagataatgccgggaacatcctgtacctgcaaatgagcagtctgaggtctgaggacacggccatgttttactgt gcaaggattga gtactacggtagtagctactattctatggactac tggggtcaaggaacctcagtcaccgtctcctca (The bold and underlined regions are the complementary determinant regions CDR1-3, respectively) The second nucleotide sequence (nucleotide sequence of the light chain variable region) encoding the 14B3 monoclonal antibody shown in SEQ ID NO:45 is as follows: gatatccagatgacacagactacatcctccctctctgcctctctggggagacagtcaccatcagttgcagtgcaagt cagggcattagcaaatat ttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctat tacacatcaagtttacactcaggagtcccatcaaggttcagtggcagtgggtctgggacagattattctctcaccatcagcaacctggaacctgaagatattgccacttacttttgt cagcagtatagtaagcttccgtggacg ttcggtggaggcaccaagctggaaatcaaa (The bold and underlined regions are the complementary determinant regions CDR1-3, respectively) Preferably, the nucleic acid molecule encoding the 4E3 monoclonal antibody has the nucleotide sequence of its heavy chain as shown in SEQ ID NO:46 and the nucleotide sequence of its light chain as shown in SEQ ID NO:47; and / or, The nucleotide sequence encoding the heavy chain of the 4E3 monoclonal antibody shown in SEQ ID NO:47 is as follows: atgcacagctcagcactgctctgttgcctggtcctcctgactggggtgagggccgaagtgaagctggtggagtctgggggaggcttagtgcagcctggagggtccctgaaactctcctgtgcaacctct ggattcactttcagt gacttttac atgtgttgggttcgccagactccagagaagaggctggagtgggtcgcataca ttagtaatggtggtg gtagcacc tattatccagacactgtaaagggccgattcaccatctccagagacagtgccaagaacaccctgtacctgcaaatgagccgtctgaagtctgaggacacagccatgtattactgt gcaagacggcgtagctatggttacccttac tatgctatggactac The nucleotide sequence encoding the light chain of the 4E3 monoclonal antibody shown in SEQ ID NO:47 is as follows: atgcacagctcagcactgctctgttgcctggtcctcctgactggggtgagggccgatatccagatgacacagactacatcctccctgtctgcctctctgggagacagtcaccatcagttgcagtgcaagt cagggcattagc aattat ttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctat tacacatca agtttacactcaggagtcccatcaaggttcagtggcagtgggtctgggacagattattctctcaccatcagcaacctggaacctgaagatattgccacttactattgt cagcagtatagtaagcttcctctcacg ttcggaggggggaccaagctggaaataaaaagagcagatgctgcaccaactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttcttgaacaacttctaccccaaagacatcaatgtcaagtggaagattgatggcagtgaacgacaaaatggcgtcctgaacagttggactgatcaggacagcaaagacagcacctacagcatgagcagcaccctcacgttgaccaaggacgagtatgaacgacataacagctatacctgtgaggccactcacaagacatcaacttcacccattgtcaagagcttcaacaggaatgagtgt (Positions 1-54 are the leader peptide sequence, positions 55-375 are the variable region sequence, positions 376-696 are the constant region sequence, and the bolded and underlined parts are the complementarity-determining regions (CDRs)) A nucleic acid molecule encoding a 11D4 monoclonal antibody, the nucleotide sequence of its heavy chain is shown in SEQ ID NO:48, and the nucleotide sequence of its light chain is shown in SEQ ID NO:49; and / or, The nucleotide sequence encoding the heavy chain of the 11D4 monoclonal antibody shown in SEQ ID NO:48 is as follows: atgcacagctcagcactgctctgttgcctggtcctcctgactggggtgagggcccaggtgcagctgaagcagtcaggacctggcctagtgcagccctcacagagcctgtccatcacctgcacagtctct ggtttctcattaact agatatggt gtagactgggttcgccagtctccaggaaagggtctggagtggctgggcgtg atatggagtggtggaa gcaca gactataatgctcctttcatatccagactgagcatcaccaaggacaactccaagagccaagttttctttaaaatgaacagtctgcaagctgatgactcagccatatactactgt gccagaagaggatactacggtagtatttactat tctatggactac The nucleotide sequence encoding the light chain of the 11D4 monoclonal antibody shown in SEQ ID NO:49 is as follows: atgcacagctcagcactgctctgttgcctggtcctcctgactggggtgagggccgatatccagatgacacagactacatcctccctgtctgcctctctggggagacagtcaccatcacttgcagtgcaagt cagggcattagc aattat ttaaactggtatcaacagaaaccagatggaactgttaaactcctgatctat tacacatca agtttacactcaggagtcccatcaaggttcagtggcagtgggtctgggacagattattctctcaccatcagcaacctggaacctgaagatattgccacttactattgt cagcagtatagtaagcttccgtggacg ttcggtggaggcaccaagctggaaatcaaaagagcagatgctgcaccaactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttcttgaacaacttctaccccaaagacatcaatgtcaagtggaagattgatggcagtgaacgacaaaatggcgtcctgaacagttggactgatcaggacagcaaagacagcacctacagcatgagcagcaccctcacgttgaccaaggacgagtatgaacgacataacagctatacctgtgaggccactcacaagacatcaacttcacccattgtcaagagcttcaacaggaatgagtgt (Positions 1-54 are the leader peptide sequence, positions 55-375 are the variable region sequence, positions 376-696 are the constant region sequence, and the bolded and underlined parts are the complementarity-determining regions (CDRs)) The nucleic acid molecule encoding the 14B3 monoclonal antibody has the heavy chain nucleotide sequence shown in SEQ ID NO:50 and the light chain nucleotide sequence shown in SEQ ID NO:51.

[0016] The nucleotide sequence encoding the heavy chain of the 14B3 monoclonal antibody shown in SEQ ID NO:50 is as follows: atgcacagctcagcactgctctgttgcctggtcctcctgactggggtgagggccgaagtgaaactagtggagtctgggggaggcttagtgaagcctggagggtccctgaaactctcctgtgcagcctct ggattcactttcagt agatatgcc atgtcttgggttcgccagactccagagaagaggctggagtgggtcgcatcc ataagtagtggtggta ctatt tactatctagacagtgtgaagggccgattcaccatctccagagataatgccgggaacatcctgtacctgcaaatgagcagtctgaggtctgaggacacggccatgttttactgt gcaaggattgagtactacggtagtagctactat tctatggactac The nucleotide sequence encoding the light chain of the 14B3 monoclonal antibody shown in SEQ ID NO:51 is as follows: atgcacagctcagcactgctctgttgcctggtcctcctgactggggtgagggccgatatccagatgacacagactacatcctccctctctgcctctctgggagacagtcaccatcagttgcagtgcaagt cagggcattagc aaatat ttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctat tacacatca agtttacactcaggagtcccatcaaggttcagtggcagtgggtctgggacagattattctctcaccatcagcaacctggaacctgaagatattgccacttacttttgt cagcagtatagtaagcttccgtggacg ttcggtggaggcaccaagctggaaatcaaaagagcagatgctgcaccaactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttcttgaacaacttctaccccaaagacatcaatgtcaagtggaagattgatggcagtgaacgacaaaatggcgtcctgaacagttggactgatcaggacagcaaagacagcacctacagcatgagcagcaccctcacgttgaccaaggacgagtatgaacgacataacagctatacctgtgaggccactcacaagacatcaacttcacccattgtcaagagcttcaacaggaatgagtgt (Positions 1-54 are the leader peptide sequence, positions 55-375 are the variable region sequence, positions 375-696 are the constant region sequence, and the bolded and underlined parts are the complementarity-determining regions (CDRs)) A third aspect of the present invention provides a carrier comprising the nucleic acid molecule described in the above-described technical solution.

[0017] The fourth aspect of the present invention provides the use of the monoclonal antibody, nucleic acid molecule or vector described in the foregoing technical solution in the preparation of a medicament for treating Coxsackie B4 virus infection.

[0018] In the application described in this invention, the medicament for treating Coxsackie B4 virus infection comprises at least one active component and one or more pharmaceutically acceptable carriers or excipients; the active component comprises any one or more of the monoclonal antibody or nucleic acid molecule encoding the monoclonal antibody described in the foregoing technical solutions or its carrier. In this invention, the pharmaceutically acceptable carrier or excipient includes, but is not limited to, conventional pharmaceutical diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbent carriers, lubricants, etc., and flavoring agents, sweeteners, etc., may be added if necessary. The medicament described in this invention can be formulated into various forms such as tablets, powders, granules, capsules, oral liquids, and injectable drugs, and all of the above dosage forms can be prepared according to conventional pharmaceutical methods. In this invention, the active component in the medicament may also include other effective ingredients that have therapeutic effects or enhance therapeutic effects, reduce toxic side effects, or prolong metabolic time.

[0019] In some specific embodiments of the present invention, the drug for treating Coxsackie B4 virus infection prepared in the application includes a monoclonal antibody selected from 4E3 monoclonal antibody, 11D4 monoclonal antibody or 14B3 monoclonal antibody. Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] This invention unexpectedly yielded a monoclonal antibody with significant preventive and therapeutic effects against a mouse model infected with Coxsackievirus B4 (CVB4) using hybridoma technology. The complementarity-determining regions (CDRs) of its heavy and light chains are as defined in this invention. In vitro and in vivo experiments confirmed that the monoclonal antibody provided by this invention possesses broad-spectrum neutralizing activity and can specifically bind to CVB4 virus particles, and demonstrates significant preventive ability against lethal CVB4 infection in a mouse model infected with CVB4 virus for 1 day.

[0021] In further infection experiments, the 4E3 monoclonal antibody showed a strong therapeutic effect on mouse models infected with CVB4 virus for 2-3 days, and was able to further improve the survival rate of infected mice. Attached Figure Description

[0022] Figure 1 The graph shows the results of neutralizing activity assays of 4E3, 11D4, and 14B3 monoclonal antibodies against CVB4; the data are expressed as mean ± standard error of the mean from three replicates.

[0023] Figure 2The graph shows the results of the binding ability of monoclonal antibodies to CVB4 solid particles; where A represents the ELISA results of the binding activity of monoclonal antibodies (4E3, 11D4, and 14B3) to CVB4 solid particles; BD represents the binding affinity of monoclonal antibodies (4E3, 11D4, and 14B3) to CVB4 solid particles as determined by BLI. The binding affinity at steady state is expressed as the equilibrium dissociation constant (KD).

[0024] Figure 3 The images show the complete amino acid sequences of the variable regions of monoclonal antibodies (4E3, 11D4, and 14B3); where A is the amino acid sequence comparison of the heavy chain variable region (VH) and B is the amino acid sequence comparison of the light chain variable region (VL). Figure 3 The location of the complementarity-determining region (CDR) was identified and marked using the IgBLAST tool. The dots (.) in the figure represent amino acid residues identical to those in the variable region of the monoclonal antibody 4E3.

[0025] Figure 4 The results show the efficacy of monoclonal antibodies 4E3, 11D4, and 14B3 against CVB4 infection in newborn mice. A is a schematic diagram of the experimental process (Dpi represents the number of days post-infection); B is a survival rate graph of mice in each antibody treatment group; C is a clinical symptom monitoring graph (clinical symptom grading is as follows: 0, healthy; 1, lethargy and slow movement; 2, limb weakness; 3, limb paralysis; 4, death). (Statistical significance is as follows: ns, no significant difference, P≥0.05; **, P <0.01; ***, P <0.001).

[0026] Figure 5This section presents the results of therapeutic efficacy assays for monoclonal antibodies 4E3, 11D4, and 14B3 against CVB4 infection in newborn mice. A shows a schematic diagram of the experimental process involving antibody treatment one day post-infection (Dpi represents the number of days post-infection); B shows the survival rates of mice treated with monoclonal antibodies 4E3 and 11D4 one day post-infection; C shows the clinical symptom monitoring of mice treated with monoclonal antibodies 4E3 and 11D4 one day post-infection; D shows the survival rate of mice treated with monoclonal antibody 14B3 one day post-infection; and E shows the results of treatment with monoclonal antibody 14B3 one day post-infection. Clinical symptom monitoring of mice after treatment; F is a schematic diagram of the experimental process of antibody treatment two days after infection; G is a survival rate graph of mice treated with monoclonal antibodies 4E3, 11D4, and 14B3 two days after infection; H is a clinical symptom monitoring graph of mice treated with monoclonal antibodies 4E3, 11D4, and 14B3 two days after infection; I is a schematic diagram of the experimental process of antibody treatment three days after infection; J is a survival rate graph of mice treated with monoclonal antibody 4E3 three days after infection; K is a clinical symptom monitoring graph of mice treated with monoclonal antibody 4E3 three days after infection. Detailed Implementation

[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Example 1: Preparation of Monoclonal Antibodies

[0028] 1. Materials and Methods 1.1 Cells and Viruses Human rhabdomyosarcoma (RD) cells and HeLa cells were purchased from the American Type Culture Collection (ATCC) and cultured in Dulbecco Modified Eagle Medium (DMEM, Hyclone) containing 10% fetal bovine serum (FBS). Myeloma cell line SP2 / 0 (ATCC-CRL-1581) was cultured in RPMI 1640 medium (Lifetechnologies, USA) supplemented with 10% FBS at 37°C and 5% CO2.

[0029] The CVB4 strains used in the examples include the prototype strain JVB (GenBank No.: X05690.1) and two clinical isolates GZ-R6 (GenBank No.: MZ540958.1) and GZ-HFM01 (GenBank No.: MZ540957.1).

[0030] 1.2 Antigen Inactivated viral particles were produced in RD cells, and the amplification and purification methods for the viral particles were performed according to the literature "Immunogenicity and protective efficacy of inactivated coxsackievirus B4 viral particles" (Wang T, Wang C, Pang L, et al. Immunogenicity and protective efficacy of inactivated coxsackievirus B4 viral particles[J]. Emerging Microbes & Infections, 2024, 13(1).). The three monoclonal antibodies prepared in this patent used F particles as antigens.

[0031] 1.3 Preparation of Monoclonal Antibodies The animal research was approved by the Animal Management and Use Committee of the Shanghai Institute for Major Infectious Diseases and Biosafety, Fudan University. All mice used were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Charles River, China).

[0032] Prior to immunization, inactivated CVB4 virus solid particles (5 μg / dose) were thoroughly mixed with aluminum hydroxide adjuvant (500 μg / dose; Invivogen, USA). The aluminum-adsorbed antigen was intraperitoneally injected into 6-8 week old female BALB / c mice at weeks 0, 2, and 4. A booster immunization was then administered via intraperitoneal injection of 10 μg of CVB4 virus solid particle antigen. Three days after the booster immunization, spleen cells were harvested and fused with SP2 / 0 myeloma cells using polyethylene glycol (PEG) 1450 (Sigma, USA). The fused cells were cultured for 8 days in hypoxanthine, aminopterin, and thymidine (HAT; Sigma) selective growth medium to obtain hybridoma cells. Positive hybridoma cells were cloned 2 to 4 times through limiting dilution to obtain monoclonal cell lines.

[0033] Through screening and research, this invention unexpectedly yielded three hybridoma monoclonal antibodies with significant therapeutic effects that secrete neutralizing antibodies, named 4E3, 11D4, and 14B3, respectively.

[0034] The selected hybridoma monoclonal antibodies were amplified and then injected intraperitoneally into BALB / c mice induced by liquid paraffin. The ascites supernatant was then collected and the antibodies were purified using a Monofinity A Resin affinity chromatography column (GenScript, USA).

[0035] Preparation of control antibodies: The Coxsackievirus A16 (CVA16) specific monoclonal antibody 8C4 was prepared in the applicant's laboratory. The preparation method is detailed in the literature "Molecular mechanism of antibody neutralization of coxsackievirus A16" (Zhang C, Liu C, Shi J, Wang Y, Xu C, Ye X, et al.Molecular mechanism of antibody neutralization of coxsackievirus A16. NatCommun. 2022;13:7854.). Monoclonal antibody 8C4 was used as the control antibody in this invention.

[0036] 1.4 Neutralization Activity Detection The neutralizing activity of hybridoma supernatant and purified 4E3, 11D4, and 14B3 monoclonal antibodies against CVB4 virus was determined by a micro-neutralization assay. CVB4 virus was first diluted to 2 TCID⁻¹. 50 / μl; mix 50 μl of antibody dilution with 100 TCID50. 50 After mixing the virus solution, incubate at 37°C for 1 hour, then add 2×10 4 After culturing HeLa cells at 37 °C for 72 hours, the presence of cytopathic effects was observed to determine the neutralizing concentration of the monoclonal antibody. The neutralizing concentration of the monoclonal antibody was defined as the lowest antibody concentration that could completely inhibit the cytopathic effect.

[0037] Immediately after observing cytopathic effects, cell viability was determined using the CellTiter-Glo 2.0 kit (Promega, USA) according to the manufacturer's instructions. The neutralization percentage was calculated using the following formula: 100 × (fluorescence of antibody-treated cells - fluorescence of virus-only control samples) / (fluorescence of cell-only control samples - fluorescence of virus-only control samples). The half-maximal inhibitory concentration (IC50) of each monoclonal antibody was calculated using a nonlinear regression method with GraphPad Prism software. 50 IC 50 Defined as the antibody concentration required to inhibit 50% of viral infection compared to samples infected with the virus alone. Results are as follows: Figure 1 As shown, the data is expressed as the average value of the three replicates ± the standard error of the mean.

[0038] 1.5 Enzyme-linked immunosorbent assay (ELISA) According to the manufacturer's instructions, antibody types were identified using a sandwich ELISA assay with the SBA Clonotyping System-HRP kit (SouthernBiotech, USA).

[0039] To determine the specificity of monoclonal antibody binding, 100 ng / well of CVB4 solid particles (also known as F-particles) were coated into micro-ELISA plates (Nunc, USA) and incubated overnight at 4°C. After washing, the plates were blocked with PBS-Tween 20 (PBST) buffer containing 5% skim milk. After washing with PBST, 50 µL / well of serially diluted purified monoclonal antibody was added and incubated at 37°C for 2 hours. After washing, horseradish peroxidase (HRP)-conjugated anti-mouse IgG (1:10,000 dilution; Sigma-Aldrich, USA) was added and incubated at 37°C for 1 hour. After color development, the absorbance at 450 nm was monitored using a microplate reader.

[0040] 1.6 Biomembrane Interferometry (BLI) Measurement Prior to the BLI assay, purified CVB4 virus solid particles were labeled with the EZ-Link™ Sulfo-NHS-LC-LC-Biotin Kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions, followed by removal of excess unreacted biotin using a Zeba™ centrifugal desalting column (Thermo Fisher Scientific). To determine the binding affinity of the antibody to CVB4, the BLI assay was performed on an Octet RED96 machine (FortéBio, USA) according to the manufacturer's instructions. In short, a specific concentration (20 µg / ml) of biotinylated CVB4 virus particles was immobilized on a streptavidin-coated biosensor (PallFortéBio) until saturated. The antigen-bound biosensor was then placed in wells containing a series of diluted monoclonal antibody samples for approximately 500 seconds to allow antigen-antibody binding, followed by immersion in dissociation buffer (0.01 M PBS supplemented with 0.1% bovine serum albumin and 0.02% Tween 20) for dissociation. The equilibrium dissociation constant (KD) was calculated using Octet Data Analysis V11.0 software (FortéBio).

[0041] 1.7 Determination and analysis of monoclonal antibody sequences To determine the antibody coding sequence, total RNA was isolated from hybridoma cells using TRIzol reagent (Invitrogen, USA). cDNA was obtained by reverse transcription using a reverse transcription kit (Promega, USA) with antibody heavy chain type-specific primers and Kappa chain type-specific primers. The cDNA was amplified by PCR using Ex Taq enzyme (Takara, Japan) and sequenced to obtain the variable region sequences of the heavy and light chains of the monoclonal antibody. The location of the complementarity-determining region (CDR) was determined using the IgBLAST tool.

[0042] To verify the correctness of the prepared monoclonal antibody sequences 4E3, 11D4, and 14B3, light and heavy chain plasmids containing mouse variable regions and human constant regions were constructed, respectively. Specifically, the variable region genes of the heavy and light chains of the monoclonal antibodies were cloned into pcDNA3.4 vectors containing the interleukin-10 (IL-10) signal sequence and the constant region genes of human immunoglobulins (gamma 1, kappa) using the NEBuilder® High Fidelity DNA Assembly Kit (NEB, USA). The recombinant chimeric antibody was expressed in CHO cell lines, and after purification, the neutralizing concentration of the chimeric antibody was determined using the aforementioned micro-neutralization assay.

[0043] 2. Results In this invention, mice were immunized with inactivated CVB4 solid particles. Subsequently, spleen cells from these immunized mice were fused with SP2 / 0 myeloma cells to obtain a series of hybridoma cell clones. The hybridomas were screened through limiting dilution and in vitro virus neutralization experiments, ultimately yielding three hybridoma monoclonal antibodies that secrete neutralizing antibodies, named 4E3, 11D4, and 14B3, respectively. The heavy and light chain sequences of the three monoclonal antibodies are shown in SEQ ID NO:47-52.

[0044] Antibody subtype identification experiments showed that 4E3 and 11D4 belong to the IgG2a subtype, while monoclonal antibody 14B3 belongs to the IgG3 subtype (Table 4). These three monoclonal antibodies exhibited strong neutralizing activity against the CVB4 prototype strain (JVB strain). Monoclonal antibodies 4E3, 11D4, and 14B3 all showed dose-dependent inhibition of viral infection, with their half-maximal inhibitory concentrations (IC50) reaching [value missing]. 50 The concentrations were determined to be 0.14, 0.39, and 0.06 μg / mL, respectively. Figure 1 ).

[0045] Table 1. Characteristics of anti-CVB4 monoclonal antibodies

[0046] The KD (equilibrium) value of the interaction between monoclonal antibodies and CVB4 solid virus particles was determined by BLI (see [link to BLI]). Figure 2 ).

[0047] b The neutralizing concentration of a monoclonal antibody is defined as the minimum antibody concentration required to completely suppress cytopathic effects.

[0048] 4E3, 11D4, and 14B3 all exhibited good neutralizing activity against the prototype JVB strain and clinical isolates (GZ-R6 and GZ-HFM01). As shown in Table 4, the neutralizing concentrations (minimum antibody concentrations required to completely inhibit cytopathic effects) of monoclonal antibodies 4E3, 11D4, and 14B3 against JVB strain were 0.97, 3.9, and 0.12 μg / mL, respectively; against GZ-R6 strain were 0.57, 1.7, and 0.02 μg / mL, respectively; and against GZ-HFM01 strain were 0.57, 5.2, and 0.02 μg / mL, respectively.

[0049] These results indicate that monoclonal antibodies 4E3, 11D4, and 14B3 are broad-spectrum neutralizing antibodies against CVB4.

[0050] 2.2 Binding characteristics of anti-CVB4 monoclonal antibody This invention first uses ELISA to detect the ability of monoclonal antibodies to recognize CVB4 viral particles. The results show that monoclonal antibodies 4E3, 11D4, and 14B3 can all bind to CVB4 viral particles, and the binding activity is positively correlated with the antibody dose. Figure 2 A). Among them, monoclonal antibody 14B3 showed stronger binding activity to CVB4 viral particles than monoclonal antibodies 4E3 and 11D4. Figure 2 A).

[0051] This invention further determines the binding affinity of anti-CVB4 monoclonal antibodies to CVB4 virus particles using a biomembrane interference (BLI) experiment. In this experiment, biotin-labeled CVB4 virus particles are immobilized on a sensor, and then interacted with different concentrations of anti-CVB4 monoclonal antibodies. The binding and dissociation curves are analyzed to obtain the equilibrium dissociation constant (KD) of the antibodies. The results show that the KD values ​​of the 4E3, 11D4, and 14B3 monoclonal antibodies are 3.82 nM, 9.61 nM, and 5.23 nM, respectively. Figure 2 BD).

[0052] 2.3 Sequencing of anti-CVB4 monoclonal antibody To determine the antibody sequences, we extracted RNA from hybridoma cells and then reverse transcribed it into cDNA using light and heavy chain type-specific primers. Subsequently, we amplified the nucleotide sequences encoding the antibodies using degenerate primers PCR, thereby obtaining the amino acid sequences of the antibody light and heavy chain variable regions. The locations of the complementarity-determining regions (CDRs) were then determined using IgBLAST. The light and heavy chain variable region sequences and CDR boundaries of monoclonal antibodies 4E3, 11D4, and 14B3 are presented below. Figure 3 Sequence alignment revealed that 4E3, 11D4, and 14B3 are different antibodies.

[0053] To verify the correctness of the nucleotide sequences of the three antibodies, the neutralizing concentrations of the chimeric monoclonal antibodies 4E3, 11D4, and 14B3 were determined by a micro-neutralization assay: 0.48, 1.95, and 0.06 μg / mL for JVB strain, 0.48, 0.97, and 0.06 μg / mL for GZ-R6 strain, and 0.48, 3.9, and 0.02 μg / mL for GZ-HFM01 strain (Table 5). These results confirm the neutralizing ability of the chimeric monoclonal antibodies and indicate that the determined nucleotide sequences of the variable regions of the light and heavy chains of the antibodies are correct.

[0054] Table 2 Neutralizing activity of anti-CVB4 chimeric monoclonal antibodies

[0055] a c4E3: Chimeric antibody 4E3. The sequence of this chimeric antibody contains a murine variable region and a human constant region. Meanwhile, both 11D4 and 14B3 monoclonal antibodies use "c" to indicate chimerism. Example 2: Evaluation of the preventive and therapeutic efficacy of the monoclonal antibody against the present invention in a mouse model of CVB4 infection.

[0056] 1. Experimental Methods 1.1 Preventive efficacy testing Following the method described in the literature "Immunogenicity and protective efficacy of inactivated coxsackievirus B4 viral particles" (Wang T, Wang C, Pang L, et al. Immunogenicity and protective efficacy of inactivated coxsackievirus B4 viral particles[J]. Emerging Microbes & Infections, 2024, 13(1).), multiple groups of 1-day-old ICR mice were intraperitoneally injected with PBS, 10 μg / g anti-CVB4 monoclonal antibody (4E3, 11D4, or 14B3), or 10 μg / g control monoclonal antibody (8C4), respectively. One day after immunization, they were intraperitoneally inoculated with 2.0 × 10⁻⁶ saturated PBS. 5 TCID 50 CVB4 / JVB.

[0057] 1.2 Therapeutic efficacy testing This invention further evaluates the therapeutic efficacy of monoclonal antibodies.

[0058] Multiple groups of 2-day-old ICR mice were intraperitoneally injected with 2.0 × 10⁻⁶ mg / L. 5 TCID 50 For CVB4 / JVB infection, treatment involves intraperitoneal injection of PBS, 10 μg / g of 4E3 monoclonal antibody, 10 μg / g of 11D4 monoclonal antibody, or 14B3 monoclonal antibody, 24 hours or 48 hours after infection, respectively. Another treatment strategy involves intraperitoneal injection of PBS or 50 μg / g of 4E3 monoclonal antibody 72 hours after infection.

[0059] All mice were observed daily for two weeks post-infection for survival and clinical signs. Clinical grading criteria were as follows: 0, healthy; 1, lethargy and immobility; 2, limb weakness; 3, limb paralysis; 4, death.

[0060] 2. Results 2.1 Efficacy of monoclonal antibodies in preventing CVB4 infection Multiple groups of 1-day-old ICR mice were injected with PBS, 10 μg / g body weight of anti-CVB4 monoclonal antibody (4E3, 11D4, or 14B3), or isotype control monoclonal antibody (8C4), respectively. One day later, the mice were inoculated with the CVB4 / JVB strain, and their clinical symptoms and mortality were observed for 14 consecutive days. Figure 4As shown, neonatal mice in the PBS group and the control antibody group began to show clinical symptoms 2 days after infection, and all of these mice eventually died; in contrast, the survival rate of newborn mice that received monoclonal antibodies 4E3, 11D4, or 14B3 was 100%. These results indicate that monoclonal antibodies 4E3, 11D4, and 14B3 can effectively prevent lethal CVB4 infection in mice.

[0061] 2.2 Therapeutic efficacy of monoclonal antibodies against CVB4 infection This invention further evaluates the therapeutic efficacy of monoclonal antibodies.

[0062] First, the therapeutic effect of a single dose administered 1 day after infection was evaluated. Two-day-old ICR mice were infected with CVB4 / JVB, and 24 hours later, they were injected with PBS, monoclonal antibody 4E3 (10 μg / g body weight), monoclonal antibody 11D4 (10 μg / g body weight), or monoclonal antibody 14B3 (10 μg / g body weight). Survival rate and clinical symptoms were monitored daily after infection. PBS-treated newborn mice began to show symptoms 3 days after infection, and 100% of the mice eventually died. Figure 5 In contrast, all mice treated with monoclonal antibodies 4E3 or 14B3 had a 100% survival rate, while the survival rate of mice treated with monoclonal antibody 11D4 was 90.9% (10 / 11). Furthermore, mice treated with the 14B3 monoclonal antibody showed milder clinical symptoms compared to mice treated with the 4E3 or 11D4 groups. Figure 5 C and E).

[0063] Secondly, this invention evaluates the therapeutic effect of a single administration two days after infection. This invention found that when antibodies were injected at 10 μg / g body weight two days after infection, only the 4E3 treatment group had a 10% survival rate, while all mice in the 11D4 and 14B3 treatment groups died. Figure 5 (G and H). These results suggest that, at the same dose, the 4E3 monoclonal antibody has a better therapeutic effect.

[0064] This invention further tested the protective effect of a high dose (50 μg / g body weight) of 4E3 monoclonal antibody administered 3 days after viral infection. Figure 5 As shown in J and K, the final mortality rate of newborn mice treated with PBS was 100%; in contrast, the survival rate of mice treated with high-dose monoclonal antibody 4E3 was 77.8%. These results indicate that high-dose monoclonal antibody 4E3 still demonstrates a strong therapeutic effect in mice already exhibiting disease symptoms.

[0065] As can be seen from the above experiments, the 4E3, 11D4 and 14B3 monoclonal antibodies provided by the present invention have significant preventive and therapeutic effects, especially in treating mouse models infected with CVB4 virus, which have significant protective effects and can be used to further construct therapeutic monoclonal antibody drugs against CVB4.

[0066] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A monoclonal antibody against Coxsackie B4 virus, characterized in that, The monoclonal antibody includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 sequences, and the light chain variable region includes light chain CDR1, light chain CDR2, and light chain CDR3 sequences; wherein: (1) The heavy chain CDR1 sequence is selected from the amino acid sequence shown in SEQ ID NO:1, 2 or 3; (2) The heavy chain CDR2 sequence is selected from the amino acid sequence shown in SEQ ID NO:4, 5 or 6; (3) The heavy chain CDR3 sequence is selected from the amino acid sequence shown in SEQ ID NO:7, 8 or 9; (4) The light chain CDR1 sequence is selected from the amino acid sequence shown in SEQ ID NO:10 or 11; (5) The light chain CDR2 sequence is YTS; (6) The light chain CDR3 sequence is selected from the amino acid sequence shown in SEQ ID NO:12 or 13.

2. The monoclonal antibody against Coxsackie B4 virus according to claim 1, characterized in that, The monoclonal antibody is selected from 4E3 monoclonal antibody, 11D4 monoclonal antibody, or 14B3 monoclonal antibody; wherein: The 4E3 monoclonal antibody has heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 sequences that are the amino acid sequences shown in SEQ ID NO:1, 4 and 7, respectively, and light chain CDR1, light chain CDR2 and light chain CDR3 sequences that are the amino acid sequences shown in SEQ ID NO:10, YTS and SEQ ID NO:12, respectively. The 11D4 monoclonal antibody has heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 sequences that are the amino acid sequences shown in SEQ ID NO:2, 5 and 8, respectively, and light chain CDR1, light chain CDR2 and light chain CDR3 sequences that are the amino acid sequences shown in SEQ ID NO:10, YTS and SEQ ID NO:13, respectively. The 14B3 monoclonal antibody has heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 sequences that are the amino acid sequences shown in SEQ ID NO:3, 6, and 9, respectively, and light chain CDR1, light chain CDR2, and light chain CDR3 sequences that are the amino acid sequences shown in SEQ ID NO:11, YTS, and SEQ ID NO:13, respectively.

3. The monoclonal antibody against Coxsackie B4 virus according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the 4E3 monoclonal antibody is shown in SEQ ID NO:14, and the amino acid sequence of the light chain variable region of the 4E3 monoclonal antibody is shown in SEQ ID NO:15; and / or, The amino acid sequence of the heavy chain variable region of the 11D4 monoclonal antibody is shown in SEQ ID NO:16, and the amino acid sequence of the light chain variable region of the 11D4 monoclonal antibody is shown in SEQ ID NO:17; and / or, The amino acid sequence of the heavy chain variable region of the 14B3 monoclonal antibody is shown in SEQ ID NO:18, and the amino acid sequence of the light chain variable region of the 14B3 monoclonal antibody is shown in SEQ ID NO:

19.

4. The monoclonal antibody against Coxsackie B4 virus according to claim 2 or 3, characterized in that, The monoclonal antibody is a recombinant chimeric antibody, comprising a murine variable region and a human constant region, wherein the heavy chain variable region and the light chain variable region are located in the murine variable region; wherein... The heavy chain amino acid sequence of the 4E3 monoclonal antibody is shown in SEQ ID NO:20, and the light chain amino acid sequence is shown in SEQ ID NO:21; and / or, The heavy chain amino acid sequence of the 11D4 monoclonal antibody is shown in SEQ ID NO:22, and the light chain amino acid sequence is shown in SEQ ID NO:23; and / or, The heavy chain amino acid sequence of the 14B3 monoclonal antibody is shown in SEQ ID NO:24, and the light chain amino acid sequence is shown in SEQ ID NO:

25.

5. A nucleic acid molecule encoding the monoclonal antibody according to any one of claims 1-4, characterized in that, It includes the first nucleotide sequence encoding the heavy chain variable region and the second nucleotide sequence encoding the light chain variable region; The first nucleotide sequence includes the nucleotide sequence encoding heavy chain CDR1, the nucleotide sequence encoding heavy chain CDR2, and the nucleotide sequence encoding heavy chain CDR3; among which, (a) The nucleotide sequence encoding the heavy chain CDR1 is selected from the nucleotide sequence shown in SEQ ID NO:26, 27 or 28; (b) The nucleotide sequence encoding the heavy chain CDR2 is selected from the nucleotide sequence shown in SEQ ID NO:29, 30 or 31; (c) The nucleotide sequence encoding the heavy chain CDR3 is selected from the nucleotide sequence shown in SEQ ID NO:32, 33 or 34; The second nucleotide sequence includes the nucleotide sequence encoding light chain CDR1, the nucleotide sequence encoding light chain CDR2, and the nucleotide sequence encoding light chain CDR3; wherein, (d) The nucleotide sequence encoding the light chain CDR1 is selected from the nucleotide sequence shown in SEQ ID NO:35 or 36; (e) The nucleotide sequence encoding the light chain CDR2 is selected from the nucleotide sequence shown in SEQ ID NO:37; (f) The nucleotide sequence encoding the light chain CDR3 is selected from the nucleotide sequence shown in SEQ ID NO:38 or 39.

6. The nucleic acid molecule according to claim 5, characterized in that, The nucleic acid is selected from nucleic acid molecules encoding 4E3 monoclonal antibody, nucleic acid molecules encoding 11D4 monoclonal antibody, or nucleic acid molecules encoding 14B3 monoclonal antibody; wherein: The nucleic acid molecule encoding the 4E3 monoclonal antibody has nucleotide sequences encoding heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 as shown in SEQ ID NO:26, 29, and 32, respectively, and nucleotide sequences encoding light chain CDR1, light chain CDR2, and light chain CDR3 as shown in SEQ ID NO:35, 37, and 38, respectively. The nucleic acid molecule encoding the 11D4 monoclonal antibody has nucleotide sequences encoding heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 as shown in SEQ ID NO:27, 30, and 33, respectively, and nucleotide sequences encoding light chain CDR1, light chain CDR2, and light chain CDR3 as shown in SEQ ID NO:35, 37, and 39, respectively. The nucleic acid molecule encoding the 14B3 monoclonal antibody has nucleotide sequences encoding heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 as shown in SEQ ID NO:28, 31, and 34, respectively, and nucleotide sequences encoding light chain CDR1, light chain CDR2, and light chain CDR3 as shown in SEQ ID NO:36, 37, and 39, respectively.

7. The nucleic acid molecule according to claim 6, characterized in that, The nucleotide sequence encoding the 4E3 monoclonal antibody, wherein the first nucleotide sequence is shown in SEQ ID NO:40, the second nucleotide sequence is shown in SEQ ID NO:41; and / or, The nucleotide sequence encoding the 11D4 monoclonal antibody, the first nucleotide sequence is shown in SEQ ID NO:42, the second nucleotide sequence is shown in SEQ ID NO:43; and / or, The nucleotide sequence encoding the 14B3 monoclonal antibody has the first nucleotide sequence shown in SEQ ID NO:44 and the second nucleotide sequence shown in SEQ ID NO:

45.

8. The nucleic acid molecule according to claim 6 or 7, characterized in that, A nucleic acid molecule encoding a 4E3 monoclonal antibody, wherein the nucleotide sequence of the heavy chain is shown in SEQ ID NO:46 and the nucleotide sequence of the light chain is shown in SEQ ID NO:47; and / or, A nucleic acid molecule encoding a 11D4 monoclonal antibody, the nucleotide sequence of its heavy chain is shown in SEQ ID NO:48, and the nucleotide sequence of its light chain is shown in SEQ ID NO:49; and / or, The nucleic acid molecule encoding the 14B3 monoclonal antibody has the heavy chain nucleotide sequence shown in SEQ ID NO:50 and the light chain nucleotide sequence shown in SEQ ID NO:

51.

9. A vector comprising the nucleic acid molecule as described in any one of claims 5-8.

10. The use of the monoclonal antibody of any one of claims 1-4, the nucleic acid molecule of any one of claims 5-8, or the vector of claim 9 in the preparation of a medicament for treating Coxsackie B4 virus infection.

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