Optimized influenza A H3N2 mRNA vaccine

Abstract

The invention provides an optimized influenza A (H3N2) mRNA (messenger Ribonucleic Acid) vaccine. Specifically, two lysine sites of the hemagglutinin antigen HA protein of the H3N2 influenza virus are mutated, so that the expression level of the HA protein is remarkably improved; meanwhile, the intracellular region of the HA protein is truncated, so that the sequence of a coding region is simplified, and the protein expression is not influenced. Through sequence simplification and mutation of only two conservative amino acid sites, the HA expression level is obviously improved, and the method can be applied to development of most mRNA vaccines aiming at the H3N2 subtype, so that the protective force of the vaccines is finally improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to an optimized mRNA vaccine against influenza A H3N2 virus. Background Art

[0002] Seasonal influenza is an acute respiratory infection caused by influenza viruses. Influenza viruses can be classified into four types, namely A, B, C, and D, according to their core proteins (Influenza A / B / C / D). Among them, influenza A viruses can be further divided into various subtypes according to different combinations of hemagglutinin antigen (HA, H) and neuraminidase antigen (NA, N) on the surface of virus particles. Currently, the influenza A viruses that are prone to seasonal epidemics are H1N1 and H3N2 subtypes.

[0003] According to the data released by the US Centers for Disease Control and Prevention over the years, the effectiveness of influenza vaccines generally does not exceed 60%. Compared with traditional inactivated vaccines, the research and production of mRNA vaccines are more suitable for seasonal influenza viruses that require rapid response, and are easy to be mixed and prepared into multivalent vaccines against multiple virus subtypes, having the potential to exert higher protective efficacy. Currently, several mRNA influenza vaccines have entered phase III clinical trials.

[0004] As the main binding site of influenza virus neutralizing antibodies, HA is a key research object of influenza vaccines. Most of the existing mRNA vaccines against H3N2-HA adopt full-length HA sequences in the coding region, and the protective efficacy of these mRNA vaccines is not yet satisfactory.

[0005] Therefore, there is an urgent need in this field to develop mRNA vaccines against HA antigen with higher expression levels and higher immunological efficacy. Summary of the Invention

[0006] The object of the present invention is to provide an optimized mutant HA antigen and mRNA encoding the same.

[0007] In the first aspect of the present invention, there is provided a hemagglutinin antigen HA protein mutant of influenza A H3N2 virus, wherein the protein mutant has amino acid mutations of K43Q and / or K484Q, and the numbering of the mutation sites is based on the full-length sequence (SEQ ID NO:1) or its truncated sequence of the hemagglutinin antigen HA protein of influenza A H3N2 virus; and the protein mutant has the immunogenicity of the hemagglutinin antigen HA protein.

[0008] In another preferred example, the truncated sequence has the sequence shown from the 1st position to the Zth position in SEQ ID NO:1, where Z is a positive integer selected from 554-566.

[0009] In another preferred example, Z is 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565 or 566.

[0010] In another preferred example, Z is 556 or 566.

[0011] In another preferred example, the truncated sequence is as shown in SEQ ID NO:2.

[0012] In another preferred example, the protein mutant is a sequence having K43Q and K484Q mutations based on the sequence shown in SEQ ID NO:1, and the amino acid sequence of the protein mutant is as shown in SEQ ID NO:3.

[0013] In another preferred example, the protein mutant is a sequence having K43Q and K484Q mutations based on the sequence shown in SEQ ID NO:2, and the amino acid sequence of the protein mutant is as shown in SEQ ID NO:4.

[0014] In another preferred example, the protein mutant further includes its active fragment, variant form or derivative protein, and the active fragment, variant form or derivative protein of the protein mutant has amino acid mutations of K43Q and K484Q, has a sequence identity of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% compared with the protein mutant, and has the immunogenicity of the hemagglutinin antigen HA protein.

[0015] In another preferred example, the derivative protein includes: an amino acid sequence formed by having K43Q and K484Q mutations on the sequence shown in SEQ ID NO:1 or SEQ ID NO:2, and further having a deletion, insertion and / or substitution of one or several (for example, usually 1-30, preferably 1-10, more preferably 1-6, still more preferably 1-3, most preferably 1) amino acid residues, and still having the immunogenicity of the protein mutant.

[0016] In the second aspect of the present invention, there is provided a polynucleotide which encodes the protein mutant described in the first aspect of the present invention.

[0017] In another preferred example, the polynucleotide is selected from the group consisting of: DNA, RNA, cDNA or a combination thereof.

[0018] In another preferred example, the polynucleotide is RNA.

[0019] In the third aspect of the present invention, there is provided a nucleic acid construct which contains the polynucleotide described in the second aspect of the present invention.

[0020] In another preferred example, the nucleic acid construct is mRNA.

[0021] In another preferred example, the mRNA has a structure as shown in Formula I:

[0022] Z1-Z2-Z3-Z4-Z5-Z6 (I)

[0023] In the formula,

[0024] Z1 is a cap (5'-Cap) element;

[0025] Z2 is a 5' untranslated region (5'-UTR) element;

[0026] Z3 is an absent or signal peptide sequence;

[0027] Z4 is the coding sequence of the protein mutant described in the first aspect of the present invention;

[0028] Z5 is a 3' untranslated region (3'-UTR) element;

[0029] Z6 is a polyadenylation tail (polyA) element.

[0030] In another preferred example, the sequence of Z4 is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0031] In another preferred example, the sequence of Z4 is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 3. Preferably, the sequence of Z4 is as shown in SEQ ID NO: 5.

[0032] In another preferred example, the sequence of Z4 is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 4. Preferably, the sequence of Z4 is as shown in SEQ ID NO: 6.

[0033] In the fourth aspect of the present invention, a vector is provided, which contains the polynucleotide described in the second aspect of the present invention or the nucleic acid construct described in the third aspect of the present invention.

[0034] In another preferred example, the vector includes: bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0035] In another preferred example, the vector is an expression vector.

[0036] In another preferred example, the vector is a plasmid.

[0037] In a fifth aspect of the present invention, there is provided a host cell containing the vector described in the fourth aspect of the present invention, or a polynucleotide described in the second aspect of the present invention is integrated into the genome of the host cell.

[0038] In another preferred embodiment, the host cell is a prokaryotic cell or a eukaryotic cell.

[0039] In another preferred embodiment, the prokaryotic cell is Escherichia coli.

[0040] In another preferred embodiment, the host cell further contains a vector for expressing a fusion protein or a protein mutant is integrated into the chromosome.

[0041] In another preferred embodiment, the host cell expresses a protein mutant.

[0042] In a sixth aspect of the present invention, there is provided a pharmaceutical composition comprising: (i) the protein mutant described in the first aspect of the present invention, the polynucleotide described in the second aspect of the present invention, or the nucleic acid construct described in the third aspect of the present invention, and (ii) a pharmaceutically acceptable carrier.

[0043] In another preferred embodiment, the pharmaceutical composition is a vaccine composition.

[0044] In another preferred embodiment, the pharmaceutical composition is an mRNA vaccine composition.

[0045] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from liquid, solid, or gel state.

[0046] In another preferred embodiment, the pharmaceutical composition is administered by a method selected from the group consisting of subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, or inhalation.

[0047] In another preferred embodiment, the vaccine composition is monovalent or multivalent.

[0048] In a seventh aspect of the present invention, there is provided the use of the protein mutant described in the first aspect of the present invention, the polynucleotide described in the second aspect of the present invention, the nucleic acid construct described in the third aspect of the present invention, or the pharmaceutical composition described in the sixth aspect of the present invention for preparing a pharmaceutical composition for preventing or treating influenza A H3N2.

[0049] In another preferred embodiment, the pharmaceutical composition is an mRNA vaccine composition.

[0050] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be repeated here one by one. Brief Description of the Drawings

[0051] Figure 1 Shows the molecular design of the mRNA of the present invention. From the 5'-end to the 3'-end are in sequence the cap, 5'-untranslated region (5'UTR), coding region (including signal peptide, extracellular region, transmembrane region and intracellular region), 3'-untranslated region (3'UTR) and polyadenylation tail (polyA). Among them, WT-FL is the full-length sequence; WT is the sequence after truncating the intracellular region of the full-length sequence; K43 / 484Q is the sequence after mutating lysine (K) at positions 43 and 484 in the coding region of the WT sequence to glutamine (Q).

[0052] Figure 2 Shows the results of detecting the effect of single amino acid mutations at different sites on HA expression levels by western blot. WT: the truncated intracellular region sequence without mutation; K43Q: lysine at position 43 in the WT coding region is replaced by glutamine; K254Q: lysine at position 254 in the WT coding region is replaced by glutamine; I276M: isoleucine at position 276 in the WT coding region is replaced by methionine; K275Q: lysine at position 275 in the WT coding region is replaced by glutamine; K484Q: lysine at position 484 in the WT coding region is replaced by glutamine. Tubulin is the internal reference protein, and HA / Tubulin is the semi-quantitative result.

[0053] Figure 3 Shows the detection results of detecting the HA expression levels of different mRNA sequences by western blot. Mock: blank control; WT-FL: wild-type full-length HA sequence; WT: wild-type truncated intracellular region HA sequence; K43 / 484Q: truncated intracellular region and lysine at positions 43 / 484 in the coding region is replaced by glutamine; Q17G: truncated intracellular region and glutamine at position 17 in the coding region is replaced by glycine. Tubulin is the internal reference protein, and HA / Tubulin is the semi-quantitative result.

[0054] Figure 4 Shows the alignment results of the HA amino acid sequences of 8 H3N2 strains in the influenza vaccine components recommended by the World Health Organization from 2016 to 2024. The K43 and K484 sites are marked with red boxes. Detailed Description of the Invention

[0055] Through extensive and in - depth research, the inventor of the present invention optimized the expression level of the hemagglutinin antigen (HA) of the H3N2 virus strain A / Darwin / 9 / 2021, which is one of the components of the 2023 - 2024 influenza vaccine recommended by the World Health Organization, through a large number of screenings. It was unexpectedly found that mutations in the amino acids at specific sites (K43Q and / or K484Q) of HA could lead to a significant increase in the expression level of the HA antigen in human cells. Based on this, the present invention was completed.

[0056] Experiments showed that the mutations of K43Q and / or K484Q not only led to a significant increase in the expression level of HA, but also this significant increase in expression was not related to codon optimization, nor was it related to whether the intracellular region was truncated, and it could be used for full - length or truncated HA coding sequences. In addition, in view of the fact that these two lysines are conserved sites of the HA of H3N2 subtype viruses, the present invention can be applied to the development of most mRNA vaccines against the H3N2 subtype, improving the expression level of mRNA and the protective efficacy of the vaccine.

[0057] Influenza A virus H3N2

[0058] Influenza A viruses are subdivided into various subtypes according to different combinations of the hemagglutinin antigen (hemagglutinin, HA, H) and neuraminidase antigen (neuraminidase, NA, N) on the surface of the virus particles. Among them, the H3N2 subtype is one of the influenza A viruses that currently cause winter seasonal epidemics.

[0059] Wild - type HA protein and its coding sequence

[0060] The hemagglutinin antigen (hemagglutinin, HA, H) is the main binding site of influenza virus neutralizing antibodies and is a key research object for influenza vaccines. Among the mRNA vaccine information for H3N2 - HA disclosed so far, the coding region mostly adopts the full - length HA sequence. The wild - type HA protein of the present invention comes from the influenza virus strain A / Darwin / 9 / 2021, and its full - length wild - type HA protein sequence (WT - FL) is shown as SEQ ID NO:1.

[0061] The intracellular region of the full - length wild - type HA protein sequence was truncated to obtain a streamlined wild - type truncated sequence (WT), and its amino acid sequence is shown as SEQ ID NO:2. The truncated sequence only streamlined the length of the intracellular region, deleted the redundant sequence, and did not affect antigen expression.

[0062] In the present invention, the amino acid sequence of the full - length HA protein (WT - FL) is as follows:

[0063] MKTIIALSNILCLVFAQKIPGNDNSTATLCLGHHAVPNGTIVKTITNDRIEVTNATELVQNSSIGEICDSPHQILDGGNCTLIDALLGDPQCDGFQNKEWDLFVERSRANSNCYPYDVPDYASLRSLVASSGTLEFKNESFNWTGVKQNGTSSACIRGSSSSFFSRLNWLTSLNNIYPAQNVTMPNKEQFDKLYIWGVHHPNTDKNQISLFAQSSGRITVSTKRSQQAVIPNIGSRPRIRGIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCKSECITPNGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRVQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNETYDHNVYRDEALNNRFQIKGVELKSGYKDWILWISFAMSCFLLCIALLGFIMWACQKGNIRCNICI(SEQ ID NO:1);

[0064] The full-length nucleotide sequence of the codon-optimized HA protein is shown below:

[0065]

[0066] Preferably, the amino acid sequence of the truncated protein (WT) is as follows:

[0067] MKTIIALSNILCLVFAQKIPGNDNSTATLCLGHHAVPNGTIVKTITNDRIEVTNATELVQNSSIGEICDSPHQILDGGNCTLIDALLGDPQCDGFQNKEWDLFVERSRANSNCYPYDVPDYASLRSLVASSGTLEFKNESFNWTGVKQNGTSSACIRGSSSSFFSRLNWLTSLNNIYPAQNVTMPNKEQFDKLYIWGVHHPNTDKNQISLFAQSSGRITVSTKRSQQAVIPNIGSRPRIRGIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCKSECITPNGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRVQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNETYDHNVYRDEALNNRFQIKGVELKSGYKDWILWISFAMSCFLLCIALLGFIMWACQ (SEQ ID NO:2);

[0068] The nucleotide sequence of the codon-optimized HA truncated protein is as follows:

[0069]

[0070] Mutant HA protein and its coding sequence

[0071] As used herein, the terms "mutant protein of the present invention", "mutant protein of the present invention", "protein mutant of the present invention", "mutant HA protein of the present invention", and "HA protein mutant of the present invention" are used interchangeably and refer to the mutant protein described in the first aspect of the present invention.

[0072] In the present invention, site-specific mutations were made to the full-length sequence of the wild-type HA protein (WT-FL) or its truncated sequence (WT) as shown in SEQ ID NO: 1, resulting in mutants with significantly improved expression levels.

[0073] Specifically, the mutant protein of the present invention generally refers to a sequence having K43Q and K484Q mutations based on the sequence shown in SEQ ID NO: 1 (wild-type full-length sequence) or SEQ ID NO: 2 (wild-type truncated sequence), wherein the numbering of the mutation sites is based on the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; and the HA mutant protein has the immunogenicity of the wild-type HA protein.

[0074] Preferably, the amino acid sequence of the HA mutant protein having K43Q and K484Q mutations based on the full-length sequence of the wild-type HA protein (WT-FL, SEQ ID NO: 1) is as follows:

[0075] MKTIIALSNILCLVFAQKIPGNDNSTATLCLGHHAVPNGTIVQTITNDRIEVTNATELVQNSSIGEICDSPHQILDGGNCTLIDALLGDPQCDGFQNKEWDLFVERSRANSNCYPYDVPDYASLRSLVASSGTLEFKNESFNWTGVKQNGTSSACIRGSSSSFFSRLNWLTSLNNIYPAQNVTMPNKEQFDKLYIWGVHHPNTDKNQISLFAQSSGRITVSTKRSQQAVIPNIGSRPRIRGIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCKSECITPNGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRVQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFQIYHKCDNACIGSIRNETYDHNVYRDEALNNRFQIKGVELKSGYKDWILWISFAMSCFLLCIALLGFIMWACQKGNIRCNICI(SEQ ID NO:3);

[0076] Preferably, the amino acid sequence of the HA mutant protein with K43Q and K484Q mutations based on the truncated wild-type HA protein sequence (WT, SEQ ID NO: 2) is shown below:

[0077] MKTIIALSNILCLVFAQKIPGNDNSTATLCLGHHAVPNGTIVQTITNDRIEVTNATELVQNSSIGEICDSPHQILDGGNCTLIDALLGDPQCDGFQNKEWDLFVERSRANSNCYPYDVPDYASLRSLVASSGTLEFKNESFNWTGVKQNGTSSACIRGSSSSFFSRLNWLTSLNNIYPAQNVTMPNKEQFDKLYIWGVHHPNTDKNQISLFAQSSGRITVSTKRSQQAVIPNIGSRPRIRGIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCKSECITPNGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRVQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFQIYHKCDNACIGSIRNETYDHNVYRDEALNNRFQIKGVELKSGYKDWILWISFAMSCFLLCIALLGFIMWACQ(SEQ ID NO:4).

[0078] The mutant protein of the present invention also includes fragments, derivatives and analogs of the mutants shown in SEQ ID NO:3 or SEQ ID NO:4, and the said fragments, derivatives and analogs substantially retain the antigenic activity of the HA protein mutant of the present invention. They may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, or (ii) polypeptides having substitution groups in one or more amino acid residues, or (iii) polypeptides formed by fusing the polypeptide of the present invention with another compound (such as a compound that prolongs the half-life of the polypeptide, for example polyethylene glycol), or (iv) polypeptides formed by fusing additional amino acid sequences to this polypeptide sequence (fusion proteins formed by fusing with a leader sequence, a secretion sequence or a tag sequence such as 6His). These fragments, derivatives and analogs are within the scope well known to those skilled in the art.

[0079] In view of the teachings of the present invention and the prior art, those skilled in the art should understand that the mutant protein of the present invention should also include active fragments of the said mutant protein, modified or unmodified variant forms or their derivative proteins.

[0080] In a preferred example, the protein mutant further includes its active fragment, variant form or derivative protein, and the active fragment, variant form or derivative protein of the protein mutant has amino acid mutations of K43Q and K484Q, has a sequence identity of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% compared with the protein mutant, and has the immunogenicity of the hemagglutinin antigen HA protein.

[0081] In a preferred example, the derivative protein includes: the amino acid sequence formed by the mutations of K43Q and K484Q on the sequence shown in SEQ ID NO:1 or SEQ ID NO:2, and still has the immunogenicity of the protein mutant after having a deletion, insertion and / or substitution of one or several (for example, usually 1-30, preferably 1-10, more preferably 1-6, still more preferably 1-3, most preferably 1) amino acid residues.

[0082] Those skilled in the art can produce mutants with conservative variations by making conservative amino acid substitutions according to, for example, the following table.

[0083] Table A

[0084]

[0085]

[0086] Nucleic acid construct

[0087] As used herein, the terms "the nucleic acid construct of the present invention", "the mRNA of the present invention", "the mRNA molecule of the present invention", "the mRNA sequence of the present invention" can be used interchangeably, and refer to the mRNA construct having the structure of formula I:

[0088] Z1-Z2-Z3-Z4-Z5-Z6 (I)

[0089] In the formula,

[0090] Z1 is a cap (5'-Cap) element;

[0091] Z2 is a 5' untranslated region (5'-UTR) element;

[0092] Z3 is no or a signal peptide sequence;

[0093] Z4 is the coding sequence of the protein mutant of the present invention;

[0094] Z5 is a 3' untranslated region (3'-UTR) element;

[0095] Z6 is a polyadenylation tail (polyA) element.

[0096] In a preferred example, the sequence of Z4 is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0097] In a preferred example, the sequence of Z4 is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 3, and preferably, the sequence of Z4 is as follows:

[0098]

[0099] In a preferred example, the sequence of Z4 is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 4. Preferably, the sequence of Z4 is as follows:

[0100]

[0101] Drug composition

[0102] The present invention also provides a drug composition, which comprises (i) the protein mutant described in the first aspect of the present invention, or the nucleic acid construct described in the third aspect of the present invention, and (ii) a pharmaceutically acceptable carrier.

[0103] The drug composition can be in any suitable form, depending on the administration method required by the patient. It can be provided in the form of unit dosage forms, usually placed in a sealed container, and can be provided as part of a kit. Such kits usually (but not necessarily) contain instructions for use. It can contain multiple said unit dosage forms.

[0104] The drug composition is suitable for any appropriate administration route, such as injection (including subcutaneous, intradermal, intramuscular, intraperitoneal, microneedle or intravenous injection), inhalation or oral administration, or nasal or anal administration, etc. The composition can be prepared by any method known in the pharmaceutical field, for example, by mixing the active ingredient with a carrier or excipient under sterile conditions.

[0105] In a preferred embodiment, the drug composition is a vaccine composition. In another preferred embodiment, the vaccine composition is monovalent or multivalent.

[0106] Application

[0107] The present invention provides the use of the protein mutant of the present invention and the nucleic acid construct encoding the protein mutant of the present invention, for example, for preparing a drug composition for preventing or treating influenza A H3N2.

[0108] Preferably, the drug composition is a vaccine composition. More preferably, the drug composition is an mRNA vaccine composition.

[0109] The main advantages of the present invention include:

[0110] 1. By only modifying the amino acid sites of K43Q or K484Q, the protein expression level of the mRNA vaccine is increased in the present invention; and when both sites of K43Q and K484Q are mutated simultaneously, the degree of increase in the protein expression level is more significant, showing a synergistic effect. This modification can enhance the vaccine effectiveness or reduce the vaccine dosage under the same effect.

[0111] 2. The obvious increase in the HA expression level of the present invention has nothing to do with codon optimization and also has nothing to do with whether the intracellular region is truncated, so it can be used for full-length or truncated HA coding sequences.

[0112] 3. Since these two lysines are conserved sites of the H3N2 subtype virus, the present invention can be applied to the development of most mRNA vaccines against the H3N2 subtype, improving the expression level of mRNA and the protective efficacy of the vaccine.

[0113] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and fractions are weight percentages and weight fractions.

[0114] Example 1: Design of Mutant Proteins and Their Encoding mRNA Sequences

[0115] Multiple single or double point mutations were made based on the intracellular domain truncated amino acid sequence (WT, SEQ ID NO: 2), and the mutation sites included K43Q, K254Q, I276M, K275Q, K484Q, K43 / 484Q, Q17G. The corresponding encoding sequences were obtained by artificial synthesis, and the coding regions of all sequences were codon-optimized. For all nucleic acid coding sequences, except for the codons corresponding to the point mutation sites, the amino acid codons at the same locus of the nucleic acid sequences were exactly the same, and the codons before and after mutation are shown in Table 1.

[0116] Table 1 Codon Changes at Mutation Sites

[0117]

[0118]

[0119] Schematic diagrams of the mRNA constructs encoding full-length HA (WT-FL), the intracellular domain truncated mRNA construct (WT), and the mRNA construct with double point mutations based on the truncated sequence (K43 / 484Q) are as Figure 1 shown.

[0120] The amino acid sequence corresponding to the coding region of the full-length sequence WT-FL is the full-length sequence of the hemagglutinin protein HA of influenza virus A / Darwin / 9 / 2021 (H3N2), and its amino acid sequence is shown in SEQ ID NO: 1, and the nucleotide sequence after codon optimization is shown in SEQ ID NO: 7.

[0121] The truncated sequence WT retains only the first two amino acids (CQ) in the intracellular region of the full-length sequence WT-FL, and the sequences of the remaining regions are exactly the same as those of WT-FL. Its amino acid sequence is shown in SEQ ID NO:2, and the codon-optimized nucleotide sequence is shown in SEQ ID NO:8.

[0122] The amino acid mutant sequence K43 / 484Q is based on the truncated sequence, and the lysine (K) at positions 43 and 484 in the coding region is mutated to glutamine (Q). The nucleotide sequence encoding the K43 / 484Q mutant truncated sequence is shown in SEQ ID NO:6.

[0123] Example 2: mRNA Preparation

[0124] (1) Preparation of Transcription Template

[0125] The circular plasmid loaded with the target RNA sequence was synthesized by GenScript. 40 ng was transformed into stable competent cells and spread on an LB agar plate containing kanamycin resistance, and cultured overnight at 30 °C. Single colonies were picked and amplified in 20 mL of LB liquid medium, cultured overnight at 30 °C and 220 rpm, and then transferred to 37 °C and 220 rpm for continuous culture for 2 h. According to the kit instructions, the plasmid was extracted using the Tiangen Mini Plasmid Kit, and the concentration was measured using Nanodrop and detected by 1% agarose gel electrophoresis.

[0126] The plasmid used as the transcription template needs to be linearized. 8 μL of BsaI and 40 μL of reaction buffer were added to 40 μg of circular plasmid, and nuclease-free water was added to make up to 400 μL, and digested overnight at 37 °C and 150 rpm. According to the SV Gel and PCR Clean-Up System instructions (clean-up step), the linearized plasmid was purified. An equal volume of membrane-binding solution was added to the digested product and mixed well, then loaded onto the column, incubated at room temperature for 1 minute, centrifuged at 14000 rpm for 1 minute, and the filtrate was discarded. 700 μL of washing solution diluted with absolute ethanol was added, centrifuged at 14000 rpm for 1 minute, and the filtrate was discarded. 500 μL of washing solution diluted with absolute ethanol was added, centrifuged at 14000 rpm for 5 minutes, and the filtrate was discarded. The column was transferred to a 1.5 mL EP tube, 40 μL of nuclease-free water was added to elute the DNA, incubated at room temperature for 1 minute, centrifuged at 14000 rpm for 1 minute, the filtrate was collected, and the concentration was measured using Nanodrop and detected by 1% agarose gel electrophoresis.

[0127] (2) In Vitro Capped Co-Transcription

[0128] Add each reagent to the EP tube in the order shown in Table 2. After mixing, incubate at 37 °C for 3 hours. After the reaction, add DNase I to the reaction system to digest the remaining plasmid template. After mixing, incubate at 37 °C for 30 minutes. After the reaction, add 1.5 volumes of 7.5 M LiCl precipitation solution to the reaction system. After mixing, place at -20 °C for 30 minutes and centrifuge to obtain a precipitate (12,000 rpm, 4 °C, 15 min). Wash the RNA precipitate three times with 70% ethanol (12,000 rpm, 4 °C, 5 min), resuspend with 50 μL of enzyme-free water, and measure the concentration using Nanodrop. Store at -80 °C in the refrigerator.

[0129] Table 2. In vitro co-transcription capping system

[0130]

[0131] (3) Detection of mRNA integrity by capillary electrophoresis

[0132] The percentage of the target mRNA product in the total transcription products was detected by capillary electrophoresis. The RNA was diluted to 50 - 60 ng / μL with 1× Dilution Buffer, heated in a metal bath at 70 °C for 2 min, quickly transferred to ice and placed for 5 min, and detected using a Qsep1 fully automatic nucleic acid and protein analysis system with an R1 cassette. The molecular weight of the sample was determined based on the electrophoresis results of the RNA 6000 molecular weight standard. The proportion of the main peak corresponding to the expected mRNA molecular weight was determined by Smear analysis to obtain the mRNA integrity data.

[0133] Example 3: Protein expression and detection

[0134] According to the methods of Examples 1 and 2, mRNA sequences with full length (WT-FL), truncation (WT), and multiple amino acid mutations were prepared, and it was confirmed that the mRNA integrity was greater than 80%. The mRNA was transfected into cells, and protein expression was detected by western blot. The experimental method is as follows:

[0135] (1) Cell transfection

[0136] Take adherent human embryonic kidney cells HEK-293T cells grown into a confluent monolayer, obtain a cell suspension after trypsin digestion, and seed in a 6-well plate at a cell density of 3×10 5 . The cell culture medium is 2 mL / well of high-glucose DMEM liquid medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, 1% non-essential amino acids, and 1% sodium pyruvate. The next day, add 400 ng of the mRNA prepared in Example 2 and 1.2 μL MessengerMAX TM to two tubes of 50 μL Opti- In Reduced Serum Medium, let it stand for 10 min. After mixing the two, 100 μL of transfection solution was obtained and incubated at room temperature for 5 min. Replace the old medium in the well plate with fresh complete medium, and then slowly add the transfection solution containing the required amount of mRNA (60 ng) along the well wall and mix well. The cell culture conditions were 37 °C and 5% CO2.

[0137] (2) Detect protein expression by western blot

[0138] Twenty-four hours after cell transfection, rinse the cells in the 6-well plate with PBS, add 1 mL of PBS, and collect the cells with a cell scraper into a 1.5 mL EP tube. Centrifuge at 4000 rpm at 4 °C for 5 minutes, collect the precipitate, add 100 μL of lysis buffer (RIPA added with 1x protease inhibitor and 1% nuclease), and lyse on ice for 15 min. Centrifuge at 13200 rpm at 4 °C for 5 min and collect the supernatant. Add 25 μL of 5x SDS protein loading buffer to 100 μL of cell lysate in each tube and heat in a metal bath at 100 °C for 5 min. Take a 4-12% SurePAGE TM protein precast gel, load 10 μL for electrophoresis, at 160 V for 40 min, and then transfer the membrane at 400 mA for 30 min. Place the membrane in blocking solution (5% milk-TBST) and block at room temperature for 1 h. Discard the blocking solution, incubate with anti-HA Antibody primary antibody (Sino Biological, diluted 1:2000 in blocking solution) at room temperature for 2 h or overnight at 4 °C. Discard the primary antibody, wash the membrane 3 times with TBST, 5 min each time, and incubate with HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) (Proteintech, diluted 1:5000 in blocking solution) at room temperature for 1 h. Discard the secondary antibody, wash the membrane 5 times with TBST, 5 min each time. Mix the enhanced chemiluminescence substrate ECL 1:1 and drop it on the membrane for 1 min. The internal reference protein uses α-tubulin (1:10000) antibody and the secondary antibody HRP-conjugated Affinipure Goat Anti-mouse IgG(H+L) (Proteintech, diluted 1:5000 in blocking solution). Place the membrane in a 4600SF chemiluminescence imaging system for imaging.

[0139] (3) Results

[0140] The western blot results of single amino acid mutations are as Figure 2 shown.

[0141] The results showed that some point mutations had no effect on the expression level and even led to a decrease in protein expression level (such as K254Q, I276M, K275Q, etc., and the semi-quantitative results were less than 1). However, unexpectedly, compared with the wild type, after the single point mutations of K43Q and K484Q, the expression level of HA protein actually increased slightly (the semi-quantitative results were slightly greater than 1).

[0142] On this basis, double point mutations of K43Q and K484Q were made to HA to test their effects on the expression level and perform semi-quantitative calculations. The results were as Figure 3 shown. The level of HA expressed in cells with the double point substitution sequence of K43Q and K484Q was increased by more than 1.5 times compared to WT (see Figure 3 A, 1.61 times; or Figure 3 B, 1.78 times), which was significantly higher than the increase in the single point mutation, indicating that the double point mutation of K43Q / K484Q could produce a superimposed effect.

[0143] In addition, the expression levels of the HA truncated sequence (SEQ ID NO:6) of the double point mutation of K43Q and K484Q, the wild type HA truncated sequence (WT, SEQ ID NO:8), and the wild type full-length sequence (WT-FL, SEQ ID NO:7) were further compared under the same conditions. The results were as Figure 3 shown in B. The expression level of the sequence-optimized full-length sequence (WT-FL) (relative expression level was 1.05) was relatively similar to that of the HA truncated sequence (WT) (the relative expression level was set as 1.0). After the double mutation of K43Q and K484Q, the expression level of the HA truncated sequence was significantly increased (the relative expression level was as high as 1.78). In addition, the expression level of the mutant control Q17G decreased slightly (relative expression level 0.85). The results showed that the expression level of the wild type truncated sequence was comparable to that of the wild type full-length sequence, and truncating the intracellular region would not affect the expression level. Therefore, it was considered that the amino acid mutations at the K43Q and K484Q sites could not only significantly increase the expression of the truncated HA, but also be used to significantly increase the expression of the full-length HA or other truncated HA proteins containing these two sites.

[0144] Example 4: Study on the conservation of mutation sites

[0145] To confirm whether the mutation sites were universal, the HA amino acid sequences of 8 H3N2 virus strains, which were the influenza vaccine components recommended by the World Health Organization from 2016 to 2024, were aligned. The results were as Figure 4 shown. It could be seen that both positions 43 and 484 were lysine, indicating that these two sites were conserved in most epidemic strains. Therefore, the mutations of the present invention could be applied to vaccines against other H3N2 strains.

[0146] Discussion:

[0147] For existing mRNA vaccines against H3N2-HA, the coding regions mostly adopt the full-length HA sequence and are not optimized for antigen expression. For example, patent application US2023 / 0000970A1 discloses an mRNA vaccine sequence against H3N2-HA. The coding region of this sequence is from the HA protein sequence of influenza virus strain A / California / 55 / 2020, and the only mutation is that valine at the 21st position is replaced by glycine in the vaccine. The reason for this replacement may be that this site is glycine in most virus strains, so a repair mutation is made from an evolutionary perspective.

[0148] In the present invention, through structural biology analysis, several amino acid sites that may cause protein degradation or affect protein translation efficiency are determined, and they are replaced with relatively similar amino acids, and the effects brought about by these mutations are detected. Surprisingly, it is found that when lysine (K) at positions 43 and 484 in the coding region is replaced by glutamine (Q) respectively, the HA expression level increases. When lysine (K) at these two sites is replaced by glutamine (Q) simultaneously, the increase in HA expression level is more than 1.5 times. The above findings are confirmed in the truncated sequence of the HA intracellular region. Since the expression levels of the full-length HA protein and the truncated protein are similar, it is considered that this mutation can be applied to both the truncated sequence and the full-length sequence.

[0149] The HA protein on the surface of the influenza virus envelope exists in the form of a trimer and is structurally divided into a head and a stem. The head is located at the distal end of the membrane and contains receptor-binding sites; the stem is located at the proximal end of the membrane and contains a membrane fusion peptide and a transmembrane region. The amino acid sequence of the head varies among different virus strains, while the stem tends to be conserved. When the virus invades host cells, HA changes from the pre-fusion conformation to the post-fusion conformation as the pH decreases, and mediates the fusion of the virus envelope and the host cell membrane. From the crystal structure of the HA protein (PDB ID: 6PDX), it can be seen that the two mutated sites K43 and K484 in the present invention are located in the stem and are both in relatively concave regions. Therefore, this modification basically does not affect the HA antigen epitope or the immunogenicity of HA.

[0150] In addition, considering that the sequence of HA has been codon-optimized, the codon adaptation indices (CAIs) before and after mutation are 0.9215 and 0.9206 respectively, and the codons of lysine at the two positions before mutation are AAA, and the mutated glutamine is CAA and CAG, which are not rare codons in the human body. Therefore, it can be basically excluded that the mutation affects protein expression at the codon level, but rather the amino acid substitution in the present invention unexpectedly has a positive impact on translation efficiency or protein stability.

[0151] All documents mentioned in this invention are cited herein by reference as if each individual document was cited by reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A hemagglutinin antigen HA protein mutant of influenza A H3N2 virus, characterized in that, The protein mutant has amino acid mutations of K43Q and / or K484Q, wherein the numbering of the mutation sites is based on the full-length sequence (SEQ ID NO:1) or its truncated sequence of the hemagglutinin antigen HA protein of influenza A virus subtype H3N2; and the protein mutant has the immunogenicity of the hemagglutinin antigen HA protein.

2. The protein mutant according to claim 1, wherein The protein mutant is a sequence with K43Q and K484Q mutations based on the sequence shown in SEQ ID NO:2, and the amino acid sequence of the protein mutant is as shown in SEQ ID NO:

4.

3. A polynucleotide, characterized in that, The polynucleotide encodes the protein mutant as claimed in claim 1.

4. A nucleic acid construct, characterized in that, The nucleic acid construct contains the polynucleotide as claimed in claim 3.

5. The nucleic acid construct according to claim 4, characterized in that, The nucleic acid construct is mRNA, and the mRNA has a structure as shown in formula I: Z1-Z2-Z3-Z4-Z5-Z6 (I) In the formula, Z1 is a cap (5'-Cap) element; Z2 is a 5' untranslated region (5'-UTR) element; Z3 is an absent or signal peptide sequence; Z4 is the coding sequence of the protein mutant as claimed in claim 1; Z5 is a 3' untranslated region (3'-UTR) element; Z6 is a polyadenylation tail (polyA) element.

6. A carrier, characterized in that, The vector contains the polynucleotide as claimed in claim 3 or the nucleic acid construct as claimed in claim 4.

7. A host cell, characterized in that, The host cell contains the vector as claimed in claim 6, or the polynucleotide as claimed in claim 3 is integrated into the genome of the host cell.

8. A pharmaceutical composition, characterized in that, The composition contains: (i) the protein mutant as claimed in claim 1, the polynucleotide as claimed in claim 3, or the nucleic acid construct as claimed in claim 4, and (ii) a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, wherein The pharmaceutical composition is an mRNA vaccine composition.

10. Use of the protein mutant according to claim 1, the nucleic acid construct according to claim 4, or the pharmaceutical composition according to claim 8, characterized in that, For preparing a pharmaceutical composition for preventing or treating influenza A virus subtype H3N2.

Images