A charge-regulated antigen protein capable of enhancing the synergistic immune effect with adjuvant

Abstract

The application discloses a charge-regulated antigen protein capable of enhancing the synergistic immunization effect with an adjuvant, and the synergistic effect with a charged adjuvant is changed by precisely regulating the surface charge of the antigen protein, so that the vaccine immunization effect is significantly enhanced, and the use dose of the antigen and the adjuvant is greatly reduced. The application also provides a variety of charge-regulated antigen protein variants obtained by using the above method and a synergistic composition of the antigen protein variants and the adjuvant, and application of the antigen protein variants and the synergistic composition in the biological medicine field.

Description

Technical Field

[0001] This invention relates to a charge-regulated antigen protein that can enhance the synergistic immune efficacy with adjuvants, belonging to the field of biomedicine. Background Technology

[0002] In recent years, outbreaks of emerging infectious diseases such as SARS, MERS, avian influenza, Ebola, and the novel coronavirus have occurred globally, seriously threatening public health and social stability. Vaccination is one of the most economical and effective means of combating infectious diseases. Furthermore, developing therapeutic vaccines is an emerging strategy for addressing global challenges such as cancer and Alzheimer's disease. Currently, vaccines are mainly classified into traditional vaccines and novel vaccines. Traditional vaccines include inactivated vaccines and live attenuated vaccines, while novel vaccines mainly include recombinant protein vaccines, viral vector vaccines, and nucleic acid vaccines. Among these, recombinant protein vaccines have many advantages, such as a single, clearly defined component, a clear mechanism of immune protection, simple preparation processes, safe and risk-free operation, and ease of large-scale preparation and production. However, they also have disadvantages, such as weak immunogenicity of the individual protein antigen, often requiring adjuvants to enhance their immunogenicity.

[0003] Aluminum adjuvants, as the most classic vaccine adjuvants, are widely used and administered due to their ease of access and low cost. Commonly used aluminum adjuvants include aluminum hydroxide adjuvant (AH) and aluminum phosphate adjuvant (AP). AH is chemically crystalline aluminum hydroxide; the aluminum on the surface of AH coordinates to amphoteric hydroxyl groups, allowing it to accept or donate protons depending on the pH of the solution. Therefore, commercial AH formulations (isoelectric point 11.4) carry a positive surface charge at physiological neutral pH (approximately 7.4). In contrast, commercial AP formulations typically have an isoelectric point of 4.6-5.6 and carry a negative surface charge at physiological neutral pH (approximately 7.4). In addition, commonly used aluminum adjuvants also include mixed systems of the above-mentioned aluminum adjuvants, as well as adjuvant systems composed of aluminum adjuvants and other adjuvant components, such as aluminum + CpG adjuvant systems and ASO4 adjuvant systems. The exact mechanism of action of aluminum adjuvants is not fully understood. Existing research suggests that they primarily exert their immunomodulatory effects through electrostatic interactions and ligand exchange, adsorbing onto antigen proteins. Aluminum adjuvants micronize soluble antigens, enhancing absorption through phagocytosis by dendritic cells; they also lock antigens onto antigen-presenting cells, simultaneously enhancing antigen presentation; and they retain antigens at the injection site, releasing cytokines and inducing local inflammatory responses, allowing time for antigen-presenting cells to be recruited. Therefore, enhancing the electrostatic adsorption between antigen proteins and aluminum adjuvants may help prolong vaccine bioavailability and promote the co-delivery of vaccine components to lymph nodes, thereby enhancing vaccine immunogenicity. Besides aluminum adjuvants, some novel adjuvants, such as CpG (negatively charged oligonucleotides, pattern recognition receptor agonist adjuvants) and DOTAP (positively charged cationic liposomes), also exhibit electrostatic adsorption with protein antigens, potentially affecting the antigen-adjuvant synergistic immunogenicity.

[0004] Existing reports indicate that adjusting the particle size distribution of the adjuvant or antigen protein itself, the buffer composition, the antigen-adjuvant ratio, and chemically coupling the adjuvant or antigen protein can all influence the degree of adjuvant adsorption to the adjuvant, thereby affecting the adjuvant's synergistic immune enhancement ability. However, existing methods generally suffer from problems such as complex operation, difficulty in precisely regulating antigen-adjuvant interactions, inability to target neutralizing epitopes, and insufficient immune enhancement efficacy, severely limiting the application of existing recombinant protein vaccines.

[0005] The purpose of this invention is to propose a method for enhancing the synergistic immune efficacy of antigen proteins and adjuvants and its application in the biomedical field, which is widely applicable to a variety of antigen proteins and charged adjuvants. Summary of the Invention

[0006] Based on the above objectives, this invention proposes a concept to enhance the synergistic immune efficacy of antigen proteins and adjuvants. This is achieved by introducing charged amino acid fragments at specific sites on vaccine antigen proteins, thereby altering the local charge distribution characteristics of the protein. This enhances the electrostatic adsorption and directional display of neutralizing epitopes between the antigen protein and the charged adjuvant, thus strengthening the synergistic immune efficacy of the antigen protein and adjuvant and significantly reducing the dosage of both antigen and adjuvant. Based on this concept, this invention first provides an antigen protein variant with inserted charged amino acid fragments. These variants are obtained by introducing charged amino acid fragments into the non-neutralizing epitope region of the wild-type antigen protein. The charge can be positive or negative.

[0007] The antigens described in this invention refer to substances that can bind to the TCR / BCR of T or B cells, promoting their proliferation and differentiation to produce antibodies or sensitized lymphocytes, which then bind to these antibodies and exert an immune effect. The ability of an antigen to stimulate the body to produce an immune response, as described in this invention, including inducing the production of antibodies and sensitized T lymphocytes (referred to as immunogenicity), and the ability to specifically bind to antibodies or sensitized lymphocytes and immune response products (referred to as antigenicity), are described in this invention.

[0008] The "antigen protein" mentioned in this invention refers to a preventive or therapeutic vaccine antigen protein obtained through recombination and peptide synthesis using eukaryotic expression systems (including but not limited to mammalian cells, baculovirus-insect cells, yeast cells, and plant cells), prokaryotic expression systems (including but not limited to Escherichia coli and Bacillus), cell-free expression systems, and other methods.

[0009] The non-neutralizing epitope region described in this invention refers to the region where the molecular structure and surface charge of the antigen protein are analyzed using software such as ProtParam and Discovery Studio™ (DS). Known core antigen epitope regions that neutralize antibody binding are excluded. Then, flexible non-neutralizing epitope regions within the antigen protein are manually selected as candidate sites for the introduction of charged amino acids. Structural modeling and molecular dynamics simulations are performed using software. Finally, considering all factors, regions with minimal impact on the overall conformation of the antigen protein and the neutralizing epitope are selected as the charged amino acid introduction sites. These sites can be the N-terminus or C-terminus of the antigen protein, or a flexible intermediate region. Charged amino acid fragments can be introduced directly or through a flexible linker.

[0010] In a preferred embodiment of the present invention, the charged amino acid fragment is a tandemly repeated charged amino acid fragment or a charged natural antigenic protein epitope.

[0011] More preferably, the number of charges carried by the charged amino acid fragment is 3-24. Based on the charge distribution characteristics of the antigen protein itself, this invention sets the number of basic charge units (e or -e) to be 3-24. When less than 3 basic charge units (e or -e) are introduced, the change in the antigen's charge properties is usually small, and it cannot exert a significant regulatory effect on electrostatic adsorption. When more than 24 basic charge units (e or -e) are introduced, the spatial conformation of the added amino acid fragment is larger, which may significantly affect the antigen's own conformation. Specific embodiments of this invention show that when antigen protein variants and adjuvants synergistically enhance immunogenicity, the enhancement of synergistic immunogenicity is correlated with the number of charges, gradually increasing from 3 Asp, 6 Asp to 9 Asp, reaching the highest level at 9 Asp. Further increasing the number of charges to 12e and 24e maintains a basically unchanged antibody level. Therefore, in a preferred embodiment of this invention, a more superior technical effect can be obtained when the number of charges carried by the charged amino acid fragment is 9-24.

[0012] The "tandemly repeated charged amino acid" described in this invention refers to the tandem repetition of multiple charged amino acids. Under normal human physiological conditions (pH approximately 7.4), there are two polar amino acids carrying a negative charge: glutamate (Glu, isoelectric point pI 3.22) and aspartic acid (Asp, isoelectric point pI 2.77); and three amino acids carrying a positive charge: histidine (His, isoelectric point pI 7.59), lysine (Lys, isoelectric point pI 9.74), and arginine (Arg, isoelectric point pI 10.76). The tandemly repeated charged amino acid fragments described in this invention are selected from one of the positively charged histidine, lysine, or arginine, or from the negatively charged glutamate or aspartic acid.

[0013] In one specific technical solution of the present invention, the tandemly repeated charged amino acid fragments are one of the following: 3 aspartic acid (3Asp), 6 aspartic acid (6Asp), 9 aspartic acid (9Asp), 12 aspartic acid (12Asp), 24 aspartic acid (24), 9 glutamic acid (9Glu), or 9 arginine (9Arg) repeated in series. The number of charges carried by the "charged amino acid fragments" varies, ranging from 3e (3Asp), 6e (6Asp), 9e (9Asp), 12e (12Asp), 24e (24Asp), 9e (9Glu), or -9e (9Arg).

[0014] The “naturally charged epitope” mentioned in this invention refers to a naturally occurring antigenic protein epitope with a charge. In order to avoid introducing irrelevant sequences in vaccine antigen design, the “naturally charged epitope” can be derived from the human body (or other test species such as mice, rabbits, livestock and other mammals)’s own protein epitopes or pathogen natural antigenic epitopes. By scanning the pathogen protein sequence for charge, pathogen natural antigenic epitopes with different amounts of charge can be discovered and selected.

[0015] In one specific technical solution of the present invention, the charged natural antigenic protein epitope is selected from a human sequence as shown in SEQ ID NO. 1, which is defined as ARIH2 in the present invention and carries a charge of 23e, or from the SARS-CoV-2 S protein, as shown in SEQ ID NO. 2, which is defined as S1139 in the present invention and carries a charge of 10e.

[0016] Other naturally charged antigenic protein epitopes that can be used in this invention include human extreme isoelectric point proteins listed in the Proteome-pI database, covering 13,614 extremely negatively charged proteins with pI < 5 and 9,959 extremely positively charged proteins with pI > 9, including but not limited to tr|C9JAU2|C9JAU2_HUMAN Isoform of O95376 E3 ubiquitin-protein ligase ARIH2 (Fragment) (pI=2.58, MSVDMNSQGSDSNEEDYDPNCEEEEEEEEDDPGDIEDYYVGVASDVEQQGADAFDP), tr|A0A096LP13|A0A096LP13_HUMAN Uncharacterized protein (pI=13.595, SRRRSKSSRRSSRRSSRRSRSKRSRSRRRSKSSRRSSRRSRRRSRTRRSRSSSRRSSTNRSSSK); and extreme charged epitopes from pathogen proteins themselves, including but not limited to SARS-CoV-2 (NC_045512). 2) Epitopes S281-18AA (pI=2.96, ENGTITDAVDCALDPLSE), S568-19AA (pI=3.26, DIADTTDAVRDPQTLEILD), and S1163-42AA (pI=3.64, DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELG) on the spike S protein (YP_009724390.1).

[0017] In a preferred embodiment of the present invention, the non-neutralizing epitope region of the antigen protein variant refers to a flexible region located at the N-terminus, C-terminus, or between both of the wild-type antigen protein. The flexible region described in this invention is defined as a region with a high B-Factor value for amino acid residues, specifically the region corresponding to the top 20% of the amino acid B-Factor values ​​of all amino acids in the antigen protein, ranked from highest to lowest. This region exhibits significant structural uncertainty and is therefore flexible.

[0018] To verify the feasibility of the technical solution of the present invention, the present invention selects some antigen proteins known to those skilled in the art as specific embodiments of the present invention. However, those skilled in the art will understand that these antigen proteins are merely illustrative technical solutions and should not be regarded as limitations on the claims of the present invention. According to the technical solution defined by the claims of the present invention, other antigen proteins already known in the art, such as viral pathogen vaccine antigens, bacterial pathogen vaccine antigens, tumor antigens, or newly discovered antigen proteins, can also achieve the same immune response effect as these antigen proteins.

[0019] In some specific embodiments of the present invention, the antigen protein variant is selected from one of the following: SARS-CoV-2 RBD antigen, SARS-CoV-2 RBD antigen, MERS-CoV-2 RBD antigen, Neisseria meningitidis fHbp antigen, or ovalbumin OVA antigen. Among these antigen proteins, SARS-CoV-2 RBD... SARS-CoV-2 The antigen has a molecular weight of 25.5 kDa and an isoelectric point (pI) of 8.96. (SARS virus RBD) SARS The antigen has a molecular weight of 25.4 kDa and an isoelectric point (pI) of 8.56. (MERS virus RBD) MERS The molecular weight of the antigen is 26.6 kDa, and the isoelectric point (pI) is 5.95. The molecular weight of the Neisseria meningitidis fHbp antigen is 27.8 kDa, and the isoelectric point (pI) is 7.25. The ovalbumin (OVA) has a molecular weight of 43.7 kDa and an isoelectric point (pI) of 5.58. These proteins cover viral pathogen vaccine antigens, bacterial pathogen vaccine antigens, and tumor model antigens. The above proteins cover a wide range of isoelectric point (pI) distributions (5.58 to 8.96).

[0020] This invention relates to the RBD of the novel coronavirus. SARS-CoV-2 The antigen's flexible, non-neutralizing epitope, for example, the introduction of a "charged amino acid fragment" or a charged natural antigen protein epitope into the C-terminus or the flexible region at position 519, is exemplified by any of the SARS-CoV-2 RBD antigens shown in SEQ ID NO. 3-8, wherein:

[0021] This invention relates to the RBD of the novel coronavirus. SARS-CoV-2The C-terminus of the antigen's flexible non-neutralizing epitope was modified by introducing a "tandem repeat charged amino acid," specifically a tandem repeat of the negatively charged aspartic acid fragment 9Asp (Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp), to construct the SARS-CoV-2 RBD. SARS-CoV-2 -9Asp antigen (SEQ ID NO: 3).

[0022] This invention relates to the RBD of the novel coronavirus. SARS-CoV-2 The flexible non-neutralizing epitope H519 introduced a "tandemly repeated charged amino acid," namely the tandemly repeated negatively charged aspartic acid fragment 9Asp (Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp), to construct the SARS-CoV-2 RBD. SARS-CoV-2 -519-9Asp antigen (SEQ ID NO: 4).

[0023] This invention relates to the RBD of the novel coronavirus. SARS-CoV-2 The flexible, non-neutralizing epitope C-terminus introduced a "tandem repeat charged amino acid," namely the tandem repeat negatively charged glutamate fragment 9Glu (Glu-Glu-Glu-Glu-Glu-Glu-Glu-Glu-Glu), to construct the SARS-CoV-2 RBD. SARS-CoV-2 -9Glu antigen (SEQ ID NO: 5).

[0024] This invention relates to the RBD of the novel coronavirus. SARS-CoV-2 A "naturally charged epitope" was introduced at the C-terminus of the flexible non-neutralizing epitope, namely a tandem repeat (DPLQPELDSFKEELD-DPLQPELDSFKEELD) of the naturally negatively charged epitope S1139 on the SARS-CoV-2 S protein (YP_009724390.1), to construct the SARS-CoV-2 RBD. SARS-CoV-2 -S1139 antigen (SEQ ID NO: 6).

[0025] This invention relates to the RBD of the novel coronavirus. SARS-CoV-2 The C-terminus of the flexible non-neutralizing epitope was introduced with a "naturally charged epitope," namely the naturally negatively charged epitope ARIH2 (MSVDMNSQGSDSNEEDYDPNCEEEEEEEEDDPGDIEDYYVGVASDVEQQGADAFDP, tr|C9JAU2|C9JAU2_HUMAN Isoform of O95376 E3 ubiquitin-protein ligase ARIH2 (Fragment)), which was used to construct the SARS-CoV-2 RBD. SARS-CoV-2 -ARIH2 antigen (SEQ ID NO: 7).

[0026] This invention relates to the RBD of the novel coronavirus. SARS-CoV-2 The flexible, non-neutralizing epitope C-terminus introduced a "tandemly repeated charged amino acid," namely the tandemly repeated positively charged arginine fragment -9Arg (Arg-Arg-Arg-Arg-Arg-Arg-Arg), to construct the SARS-CoV-2 RBD. SARS-CoV-2 -9Arg antigen (SEQ ID NO: 8).

[0027] This invention relates to SARS virus RBD SARS The C-terminus of the antigen's flexible non-neutralizing epitope was modified by introducing a "charged amino acid fragment," namely, a tandem repeat of negatively charged aspartic acid 9Asp (Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp), to construct the SARS virus RBD. SARS -9Asp antigen (SEQ ID NO: 9).

[0028] This invention introduces a "charged amino acid fragment," namely a tandem repeat of negatively charged aspartic acid-9Asp (Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp), at the C-terminus of the flexible non-neutralizing epitope of the MERS virus RBD antigen, to construct the MERS virus RBD. MERS -9Asp antigen (SEQ ID NO: 10).

[0029] This invention introduces a “tandem repeat charged amino acid”, namely a tandem repeat negatively charged aspartic acid fragment 9Asp (Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp), into the C-terminus of the flexible non-neutralizing epitope of Neisseria meningitidis fHbp, to construct the fHbp-9Asp antigen (SEQ ID NO: 11).

[0030] This invention introduces a “tandem repeat charged amino acid”, namely a tandem repeat negatively charged aspartic acid fragment 9Asp (Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp), into the C-terminus of the flexible non-neutralizing epitope of the model antigen chicken ovalbumin (OVA), thus constructing the model antigen chicken ovalbumin OVA-9Asp (SEQ ID NO: 12).

[0031] Secondly, the present invention provides a vaccine composition containing the above-mentioned antigen protein variant, the vaccine composition further comprising a charged adjuvant carrying a charge opposite to the charge property of the charged amino acid fragment introduced in the antigen protein variant.

[0032] In a preferred embodiment, the charged adjuvant is selected from one or more of charged metal salt adjuvants, pattern recognition receptor agonist adjuvants, liposome adjuvants, oil emulsion adjuvants, cytokine adjuvants, and chemokine adjuvants.

[0033] In a more preferred embodiment, the charged metal salt adjuvant is selected from one of aluminum hydroxide adjuvant, aluminum-containing composite adjuvant, manganese adjuvant, or CpG adjuvant.

[0034] The "charged adjuvant" mentioned in this invention refers to an adjuvant that can exert synergistic immune efficacy by electrostatic adsorption with antigen proteins. The technical effects of this invention can also be achieved by using other metal salt adjuvants and novel adjuvant systems with the help of the inventive concept of this invention.

[0035] In one specific technical solution of the present invention, the "charged adjuvant" uses aluminum hydroxide adjuvant (Denmark, Denmark), manganese adjuvant (Qimeng Biotechnology, China), CpG adjuvant (Takara, Japan), and a composite adjuvant system such as an aluminum + CpG adjuvant system. The above adjuvants are used only as examples in the present invention and should not be regarded as limiting the claims of the present invention.

[0036] When the antigen protein and charged adjuvant described in this invention are used in combination, wherein when the vaccine antigen protein is used in combination with an adjuvant that is positively charged on the surface under physiological conditions, negatively charged amino acid fragments are introduced into specific regions of the antigen protein to precisely regulate the surface charge of the antigen protein; and when the vaccine antigen protein is used in combination with an adjuvant that is negatively charged on the surface under physiological conditions, positively charged amino acid fragments are introduced into specific regions of the antigen protein to precisely regulate the surface charge of the antigen protein; thereby enhancing the electrostatic adsorption and neutralizing epitope orientation between the antigen protein and the adjuvant, and thus improving the synergistic immune efficacy of the antigen protein-adjuvant combination.

[0037] In one specific technical solution of the present invention, when used in combination with aluminum hydroxide adjuvant and manganese adjuvant, which are positively charged under physiological conditions, the effects on the RBD of the novel coronavirus are respectively... SARS-CoV-2 Antigen, SARS virus RBD SARS Antigen, MERS virus RBD MERS The antigen, Neisseria meningitidis fHbp antigen, and ovalbumin OVA antigen were modified by introducing negatively charged amino acids to precisely regulate the surface charge of the antigen proteins, thereby enhancing the synergistic immunogenicity of the antigen protein-adjuvant combination. When used in combination with a CpG adjuvant that carries a negative surface charge under physiological conditions, it showed efficacy against SARS-CoV-2 RBD. SARS-CoV-2 The antigen incorporates positively charged amino acids to precisely regulate the surface charge of the antigen protein, thereby enhancing the synergistic immune efficacy of the antigen protein and adjuvant.

[0038] The COVID-19 RBD obtained by this invention SARS-CoV-2-9Asp antigen (SEQ ID NO: 3) compared to wild-type RBD SARS-CoV-2 The antigen, when combined with aluminum hydroxide adjuvant (Denmark, Denmark) for immunization of mice, rapidly elicited an immune response, with antibody levels reaching 10^5 mmol / L 14 days after the initial immunization; it significantly increased the level of specific antibodies against SARS-CoV-2 (>1000-fold increase) and neutralizing antibody levels (>50-fold increase); and it greatly reduced the dosage of the antigen, requiring only 1 μg of charge-regulated RBD. SARS-CoV-2 -9Asp antigen can achieve 5μg wild-type RBD SARS-CoV-2 It possesses the same immunogenic efficacy as the antigen; simultaneously, it can significantly reduce the dosage of adjuvants required, in conjunction with charge-regulated RBD. SARS-CoV-2 When used in combination with -9Asp antigen, 2μg of aluminum hydroxide adjuvant is sufficient to achieve the original wild-type RBD. SARS-CoV-2 The antigen, when used in combination with 50 μg of aluminum hydroxide adjuvant, has the same immunogenicity.

[0039] The COVID-19 RBD obtained by this invention SARS-CoV-2 -519-9Asp antigen (SEQ ID NO: 4) compared to wild-type RBD SARS-CoV-2 The antigen, when combined with aluminum hydroxide adjuvant to immunize mice, significantly increased the level of specific antibodies against the novel coronavirus (>3-fold increase).

[0040] The COVID-19 RBD obtained by this invention SARS-CoV-2 -9Glu antigen (SEQ ID NO: 5) compared to wild-type RBD SARS-CoV-2 The antigen, when combined with aluminum hydroxide adjuvant to immunize mice, significantly increased the level of specific antibodies against the novel coronavirus (>40-fold increase).

[0041] The COVID-19 RBD obtained by this invention SARS-CoV-2 -S1139 antigen (SEQ ID NO: 6) compared to wild-type RBD SARS-CoV-2 The antigen, when combined with aluminum hydroxide adjuvant (Denmark, Denmark) to immunize mice, significantly increased the level of specific antibodies against the novel coronavirus (>20-fold increase).

[0042] The COVID-19 RBD obtained by this invention SARS-CoV-2 -ARIH2 antigen (SEQ ID NO: 7) compared to wild-type RBD SARS-CoV-2 The antigen, when combined with aluminum hydroxide adjuvant (Denmark, Denmark) to immunize mice, significantly increased the level of specific antibodies against the novel coronavirus (>4-fold increase).

[0043] The COVID-19 RBD obtained by this invention SARS-CoV-2-9Asp antigen (SEQ ID NO: 3) compared to wild-type RBD SARS-CoV-2 The antigen, when combined with manganese adjuvant (China Qimeng Biotechnology) to immunize mice, can significantly increase the level of specific antibodies against the novel coronavirus (>100-fold increase).

[0044] The COVID-19 RBD obtained by this invention SARS-CoV-2 -9Arg antigen (SEQ ID NO: 8), in RBD SARS-CoV-2 The flexible, non-neutralizing epitope C-terminus introduces a "tandem repeat charged amino acid," namely the tandem repeat positively charged arginine fragment 9Arg (Arg-Arg-Arg-Arg-Arg-Arg-Arg), compared to wild-type RBD. SARS-CoV-2 When combined with CpG adjuvant (ODN 2006, Takara, Japan) to immunize mice, it can significantly increase the level of specific antibodies against the novel coronavirus (>3-fold increase).

[0045] The COVID-19 RBD obtained by this invention SARS-CoV-2 -9Asp antigen (SEQ ID NO: 3) compared to wild-type RBD SARS-CoV-2 The antigen, when combined with a compound adjuvant consisting of aluminum hydroxide adjuvant (Denmark, Denmark) and CpG, significantly increased the level of specific antibodies against the novel coronavirus in mice (>40-fold increase).

[0046] The SARS virus RBD obtained in this invention SARS -9Asp antigen (SEQ ID NO: 9) compared to wild-type RBD SARS The antigen, when combined with aluminum hydroxide adjuvant (Denmark, Denmark) to immunize mice, significantly increased the level of specific antibodies against the SARS virus (>80-fold increase).

[0047] The MERS virus RBD obtained by this invention MERS -9Asp antigen (SEQ ID NO: 10) compared to wild-type RBD MERS The antigen, when combined with aluminum hydroxide adjuvant (Denmark, Denmark) to immunize mice, significantly increased the level of specific antibodies against MERS virus (>10-fold increase).

[0048] The Neisseria meningitidis fHbp-9Asp antigen (SEQ ID NO: 11) obtained in this invention significantly enhances the level of specific antibodies against Neisseria meningitidis (>60-fold increase) when mice are immunized with aluminum hydroxide adjuvant (Denmark, Denmark).

[0049] The model antigen chicken ovalbumin OVA-9Asp (SEQ ID NO: 12) obtained in this invention significantly increases the level of specific antibodies against OVA itself (>3-fold increase) when mice are immunized with aluminum hydroxide adjuvant (Denmark, Denmark).

[0050] The various types of vaccine antigen proteins selected in this invention are widely representative and can all be used to implement the methods and applications described above, achieving excellent technical results. Therefore, those skilled in the art, based on the application of this invention and common knowledge in the field, can understand and reasonably anticipate that other vaccine antigen proteins can also be used to implement this invention.

[0051] The various charged amino acid fragments selected in this invention are widely representative and can all be used to implement the methods and applications described above, achieving excellent technical results. Therefore, those skilled in the art, based on the application of this invention and common technical knowledge, can understand and reasonably anticipate that other charged amino acid fragments can also be used to implement this invention.

[0052] The aforementioned "non-neutralizing epitope regions" selected in this invention are widely representative and can all be used to implement the methods and applications described above, achieving excellent technical results. Therefore, those skilled in the art, based on the application of this invention and common technical knowledge in the field, can understand and reasonably anticipate that introducing charged amino acid sequences using other specific sites in the antigen can also implement this invention.

[0053] The various types of "charged adjuvants" selected in this invention are widely representative and can all be used to implement the methods and applications described above, achieving excellent technical results. Therefore, those skilled in the art, based on the application of this invention and common knowledge in the field, can understand and reasonably anticipate that other charged adjuvants can also be used to implement this invention.

[0054] This invention, through artificial design, introduces naturally charged amino acid fragments with specific charges at specific sites on the antigen protein, thereby altering the local charge distribution characteristics of the protein. This achieves electrostatic adsorption and neutralization epitope display between the antigen protein and the adjuvant, enhancing the synergistic immune effect of the antigen protein and the adjuvant, and thus increasing the immunogenicity of the vaccine.

[0055] The novel vaccine antigens obtained in this invention, including OVA pattern antigens for novel coronavirus, SARS virus, MERS virus, and Neisseria meningitidis, significantly enhance the levels of specific antibodies or neutralizing antibodies compared to wild-type antigens when combined with various types of adjuvants. This greatly reduces the dosage of antigens and adjuvants used, overcoming the efficacy bottleneck of existing recombinant protein vaccines. The method has a clear mechanism and simple antigen modification process, requiring no additional chemical modifications. It can achieve more efficient synergistic immunogenicity with charged adjuvants while maintaining the original characteristics of the antigen protein. It is widely applicable to recombinant protein vaccines containing charged adjuvants and has promising application prospects. Attached Figure Description

[0056] Figure 1 Schematic diagram of a method to enhance the synergistic immunogenicity of antigen proteins and adjuvants through charge regulation;

[0057] Figure 2 COVID-19 RBD SARS-CoV-2 Schematic diagram of variant antigen design;

[0058] Figure 3 COVID-19 RBD SARS-CoV-2 Diagram showing the purification and identification of variant antigen proteins;

[0059] Figure 4 COVID-19 RBD SARS-CoV-2 Figure 1. Immunological evaluation of RBD-specific antibodies in mice with different charge number (3e, 6e, 9e) variant antigens combined with aluminum adjuvant.

[0060] Figure 5 COVID-19 RBD SARS-CoV-2 Figure 1. Evaluation of neutralizing antibody results in mice with different charge numbers (3e, 6e, 9e) variant antigens combined with aluminum adjuvant;

[0061] Figure 6 COVID-19 RBD SARS-CoV-2 Figure 1. Evaluation of RBD-specific antibodies in mice with different charge numbers (9e, 12e, 24e) variant antigens combined with aluminum adjuvant;

[0062] Figure 7 COVID-19 RBD SARS-CoV-2 Figure 1: Results of mouse immune evaluation of RBD-specific antibodies using a reduced dose of variant antigen (RBD-9Asp) combined with aluminum adjuvant;

[0063] Figure 8 COVID-19 RBD SARS-CoV-2 Figure 1: Immunological evaluation of RBD-specific antibodies in mice with reduced aluminum adjuvant dosage of variant antigen (RBD-9Asp);

[0064] Figure 9COVID-19 RBD SARS-CoV-2 Immunological evaluation results of mice with different introduction site variant antigens (RBD-519-9Asp) combined with aluminum adjuvant;

[0065] Figure 10 COVID-19 RBD SARS-CoV-2 Figure showing the results of immune evaluation in mice using different charged amino acid (Asp, Arg, Glu, Ser) variant antigens combined with aluminum adjuvant;

[0066] Figure 11 COVID-19 RBD SARS-CoV-2 Immunological evaluation results in mice using the viral naturally charged amino acid fragment variant antigen (RBD-S1139) combined with aluminum adjuvant;

[0067] Figure 12 COVID-19 RBD SARS-CoV-2 Immunological evaluation results in mice using the human naturally charged amino acid fragment variant antigen (RBD-ARIH2) combined with aluminum adjuvant;

[0068] Figure 13 COVID-19 RBD SARS-CoV-2 Immunological evaluation results of mice with variant antigen (RBD-9Asp) combined with manganese adjuvant;

[0069] Figure 14 COVID-19 RBD SARS-CoV-2 Immunological evaluation results of mice with variant antigen (RBD-9Asp) combined with AL+CpG adjuvant;

[0070] Figure 15 COVID-19 RBD SARS-CoV-2 Immunological evaluation results of mice with variant antigen (RBD-9Arg) combined with CpG adjuvant;

[0071] Figure 16 SARS coronavirus variant antigen RBD SARS -9Asp design diagram and protein purification and identification diagram;

[0072] Figure 17 SARS coronavirus variant antigen RBD SARS Figure 1; Immunological evaluation results of mice with -9Asp combined with aluminum adjuvant;

[0073] Figure 18 MERS coronavirus variant antigen RBD MERS -9Asp design diagram and protein purification and identification diagram;

[0074] Figure 19 MERS coronavirus variant antigen RBD MERS Figure 1; Immunological evaluation results of mice with -9Asp combined with aluminum adjuvant;

[0075] Figure 20 Schematic diagram of the design of the Neisseria meningitidis variant antigen fHbp-9Asp and diagram of protein purification and identification;

[0076] Figure 21 Figure 1. Immunological evaluation results of mice with Neisseria meningitidis variant antigen fHbp-9Asp combined with aluminum adjuvant;

[0077] Figure 22 Schematic diagram of the design of the model antigen chicken ovalbumin variant OVA-9Asp and diagram of protein purification and identification;

[0078] Figure 23 Figure showing the results of the immune evaluation of mice with the model antigen chicken ovalbumin variant OVA-9Asp combined with aluminum adjuvant. Detailed Implementation

[0079] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0080] Example 1: COVID-19 RBD SARS-CoV-2 Variant antigen design, expression preparation and immunological evaluation

[0081] 1.1 Spatial Structure and Surface Charge Analysis of SARS-CoV-2 RBD

[0082] The amino acid sequence (YP_009724390.1) of the RBD of the wild-type SARS-CoV-2 strain (Genebank accession number: NC_045512.2) was obtained from the Genebank database. The molecular weight, theoretical isoelectric point (pI), amino acid composition, and other physicochemical properties of the given protein were calculated using ProtParam. Based on the theoretical isoelectric point results, the overall charge characteristics of the protein were analyzed, revealing that the SARS-CoV-2 RBD exhibits a strong positive charge (pI=8.95). Structural data of the SARS-CoV-2 RBD were obtained from the PDB database. The surface charge of the protein was analyzed using Discovery Studio™ (DS) simulation software, revealing a relatively concentrated positive charge distribution at the N-terminus and C-terminus of the RBD, far from the RBM core neutralizing epitope region N437-P507 (71 amino acids in total).

[0083] 1.2 Introducing charged amino acids to regulate RBD charge

[0084] Currently, most RBD-based recombinant protein vaccines use aluminum hydroxide adjuvants to modify the charge characteristics of natural RBD proteins, allowing for better electrostatic adsorption of the positively charged aluminum adjuvant and targeted display of neutralizing epitopes. Figure 1 First, we attempted to design and introduce repeating negatively charged amino acids to regulate the charge of the RBD. The negatively charged amino acid was aspartic acid (D-Asp) (or other negatively charged amino acids such as glutamic acid (E-Glu), etc.), and the number of introduced amino acids was designed as a gradient distribution of 3Asp, 6Asp, 9Asp, 12Asp, and 24Asp. Combining the reported distribution information of neutralizing epitopes in RBD, flexible and non-neutralizing epitope regions were used as candidate sites for the introduction of negatively charged amino acids. Structural modeling and molecular dynamics simulations were performed, and finally, the C-terminal region and the flexible non-neutralizing epitope H519 position, which have less impact on the overall conformation, were selected as the introduction sites. RBD-nAsp and RBD-nAsp were designed and obtained. SARS-CoV-2 -519-9Asp, RBD-9Glu and other charge-regulated variant antigens ( Figure 2 ).

[0085] Considering that the introduced repetitive negatively charged amino acids (nAsp or nGlu) are non-natural exogenous sequences, potentially posing risks such as strong autoimmunogenicity and safety concerns, a design was devised to directly scan negatively charged regions from SARS-CoV-2 proteins, such as the S protein, to identify negatively charged epitopes to replace the repetitive negatively charged amino acid fragments. Structural data of the SARS-CoV-2 S protein were obtained from the PDB database, and the surface charge of the S protein was analyzed using Discovery Studio™ (DS) simulation software to identify concentrated negatively charged epitopes. Structural and functional analyses were performed on several naturally occurring negatively charged epitopes of the S protein identified during the scan. Epitopes with relatively stable conformation and high charge density were selected for application, and the S1139 epitope was ultimately chosen as the "naturally charged epitope," resulting in the design and acquisition of the RBD. SARS-CoV-2 -S1139 charge-regulated variant antigen ( Figure 2 ).

[0086] Alternatively, one could consider selecting naturally charged amino acid fragments from the human body as "natural charged epitopes" to reduce the immunogenicity of charged fragments. Human proteins with extreme isoelectric points listed in the Proteome-pI database were selected, and the naturally negatively charged epitope ARIH2 (tr|C9JAU2|C9JAU2_HUMANIsoform of O95376 E3 ubiquitin-protein ligase ARIH2 (Fragment, MSVDMNSQGSDSNEEDYDPNCEEEEEEEEDDPGDIEDYYVGVASDVEQQGADAFDP) with the sequence shown in SEQ ID NO.1 was designed as a "natural charged epitope," and RBD was obtained.SARS-CoV-2 -ARIH2 charge-regulated variant antigen ( Figure 2 ).

[0087] Finally, in addition to enhancing the synergistic immunogenicity of RBD with aluminum hydroxide adjuvant through negative charge modulation, when the RBD antigen protein is used in combination with an adjuvant that carries a negative surface charge under physiological conditions, introducing positively charged amino acid fragments into specific regions of the antigen protein to further enhance the positive charge of the RBD antigen protein can also enhance electrostatic adsorption and targeted display of neutralizing epitopes with the negatively charged adjuvant, thereby improving the synergistic immunogenicity of the antigen protein-adjuvant combination. By introducing repeating positively charged amino acids to regulate the RBD charge, with arginine R-Arg (or other positively charged amino acids such as lysine K-Lys), a novel coronavirus RBD was designed and obtained. SARS-CoV-2 -9Arg antigen ( Figure 2 Simultaneously, the neutral-charged amino acid serine S-Ser was introduced as a control, and the SARS-CoV-2 RBD was designed and obtained. SARS-CoV-2 -9Ser antigen ( Figure 2 ).

[0088] 1.3 Construction of eukaryotic expression vectors and protein expression, purification, and identification

[0089] A secretory signal peptide tPA and a His purification tag were added to the N-terminus of the RBD charge-regulated variant antigen. The amino acid sequence was artificially optimized using mammalian cell codons. The prebase sequence of the start codon ATG was designed and adjusted to conform to the Kozak rule. The translation stop codon TGA was added to the 3' end. The gene was ligated into the pcDNA3.1 eukaryotic expression vector via EcoRI (GAATTC) and HindIII (AAGCTT) restriction sites.

[0090] Expi293F mammalian suspension cells were cultured and expanded, and the cell density was adjusted to 3 × 10⁻⁶ cells using Expi293 expression medium. 6 Cells were transfected with the constructed expression plasmid at a concentration of 120 rpm / mL. Cells were then transfected using a transfection kit and cultured on a shaker at 37°C, ≥80% relative humidity, and 8% carbon dioxide. After 72 hours of transfection, the cell culture medium was centrifuged, and the supernatant was filtered through a 0.45 μm syringe filter to remove cell debris. A GE His-trap affinity chromatography column was installed on an AKTA protein purification system, and the protein was purified using His-tagged affinity chromatography. SDS-PAGE analysis confirmed that the target protein with the expected molecular weight was obtained (…). Figure 3 ).

[0091] 1.4 COVID-19 RBD SARS-CoV-2 Immunological evaluation of mouse antigens with different charge number variants

[0092] Female BALB / c mice aged 6-8 weeks were selected, with 6-8 mice in each group. Wild-type and RBD variants with different charge numbers, obtained through recombinant expression and purification, were administered intramuscularly to the mice in combination with aluminum hydroxide adjuvant. A booster immunization was given 14 days later, with each dose containing 100 μL of vaccine (containing 5 μg antigen and 50 μg adjuvant). Post-immunization, blood was collected from the tail vein of the mice, and the levels of RBD-specific IgG antibodies, pseudovirus, and live virus neutralizing antibodies in the immune serum were detected using an enzyme-linked immunosorbent assay (ELISA).

[0093] Method for detecting pseudovirus neutralizing antibodies: Based on the wild-type novel coronavirus sequence published by NCBI (Genebank accession number: NC_045512.2), the nucleotide coding sequence of the S protein was synthesized and inserted into the pCAGGS expression vector; the S protein expression vector and the pNL4.3-Luc-RE-backbone plasmid were co-transfected into 293T cells. Six hours after transfection, the supernatant was replaced with MEM medium containing 10% FBS. After 48 hours, the supernatant was collected, filtered through a 0.45μm sterile filter, and the packaged pseudovirus was obtained. Mouse immunized serum was serially diluted and added to 96-well cell culture plates for further serial dilution. 50 μL of sham virus solution was added to each well and incubated at 37°C for 1 h. 100 μL of ACE2-293T cells were added to each well of the 96-well plate and cultured at 37°C with 5% CO2 for 48 h. The plates were then vortexed at 500 rpm for 15 min using 5× lysis buffer. 20 μL of the lysate from each 96-well plate was transferred to the corresponding 96-well chemiluminescence assay plate and the luminescence value was read using a chemiluminescence analyzer. The 50% inhibitory dilution (ID50) was defined as the serum dilution with a 50% reduction in relative light units (RLU) compared to the virus control well (virus + cells) after subtracting the background relative fluorescence readings (RLU) from the control group.

[0094] Method for detecting SARS-CoV-2 true virus neutralizing antibodies: SARS-CoV-2 live virus neutralization assay was performed using mouse immunized serum (original strain: SARS-CoV-2 / human / CHN / Beijing_IME-BJ01 / 2020). Mouse serum inactivated at 56°C was serially diluted and incubated with 100 TCID50 of the virus strain at 37°C for 1 hour. The serum-virus complex was added to 96-well plates pre-coated with Vero E6 cells and incubated for 48–72 hours. Cells were stained with 0.05% crystal violet for 40 minutes, and after adding destaining solution, OD was measured at 570 nm / 630 nm.

[0095] The COVID-19 RBD obtained by this invention SARS-CoV-2 -nAsp antigen, in RBD SARS-CoV-2 The flexible, non-neutralizing epitope C-terminus introduces "tandemly repeating charged amino acids," compared to wild-type RBD. SARS-CoV-2 The antigen, when combined with aluminum hydroxide adjuvant (Denmark, Denmark) to immunize mice, significantly increased the level of specific antibodies against the novel coronavirus. The enhanced immunogenicity was correlated with the number of antibodies, reaching its highest level at 9 Asp. It rapidly elicited an immune response, with antibody levels reaching 10^5 mmol / L 14 days after the first immunization, more than 1000 times higher than wild-type antibodies (P < 0.01). Figure 4 Neutralizing antibodies also showed a similar significant upward trend, increasing by more than 50 times (P < 0.0001). Figure 5 Further increasing the charge quantity to 12e and keeping antibody levels essentially unchanged at 24e. Figure 6 Therefore, RBD-9Asp is the optimal design for introducing the smallest fragment while achieving the most significant enhancement in immunogenicity.

[0096] Further optimization of antigen and adjuvant dosages revealed that the charge-modulation strategy significantly reduced the antigen dosage, using as little as 1 μg of charge-modulated RBD. SARS-CoV-2 -9Asp antigen can achieve 5μg wild-type RBD SARS-CoV-2 The same antigen has the same immunogenicity ( Figure 7 ); at the same time, it can greatly reduce the dosage of adjuvants used, in conjunction with charge-regulated RBD SARS-CoV-2 When used in combination with -9Asp antigen, 2μg of aluminum hydroxide adjuvant is sufficient to achieve the original wild-type RBD. SARS-CoV-2 The antigen, when used in combination with 50 μg of aluminum hydroxide adjuvant, has the same immunogenicity. Figure 8 ).

[0097] 1.5 COVID-19 RBD SARS-CoV-2 Immunological evaluation of mouse variant antigens at different introduction sites

[0098] SARS-CoV-2 RBD obtained by recombinant expression and purification SARS-CoV-2 -519-9Asp antigen (SEQ ID NO: 4), i.e., in RBD SARS-CoV-2 A tandemly repeated negatively charged aspartic acid fragment (9Asp) was introduced at the H519 position of the flexible non-neutralizing epitope for mouse immunoassay. The evaluation and detection protocols were the same as above. The results showed that, compared with wild-type RBD... SARS-CoV-2 Antigen, RBD SARS-CoV-2 The -519-9Asp antigen, when combined with aluminum hydroxide adjuvant for immunization of mice, significantly increased the level of specific antibodies against the novel coronavirus (>3-fold increase). Figure 9).

[0099] 1.6 COVID-19 RBD SARS-CoV-2 Immunological evaluation of mice with different charged amino acid fragment variant antigens

[0100] SARS-CoV-2 RBD obtained by recombinant expression and purification SARS-CoV-2 -9Glu antigen (SEQ ID NO: 5), i.e., in RBD SARS-CoV-2 A tandemly repeated negatively charged glutamate fragment 9Glu was introduced at the C-terminus of a flexible, non-neutralizing epitope, and immunological evaluation was conducted. Results showed that, after immunization of mice with aluminum hydroxide adjuvant (Denmark, Denmark), compared to wild-type RBD... SARS-CoV-2 Antigen, RBD SARS-CoV-2 -9Glu significantly increased the level of specific antibodies against the novel coronavirus (>40-fold, P<0.0001). Figure 10 This indicates that different types of tandemly repeated negatively charged amino acids can be used to regulate the surface charge of antigen proteins, thereby altering their synergistic effect with charged adjuvants and significantly enhancing vaccine efficacy. RBD... SARS-CoV-2 -9Ser、RBD SARS-CoV-2 -9Arg did not increase antibody levels relative to wild-type when combined with aluminum hydroxide adjuvant, indicating that the modification of neutral serine (Ser) and positively charged arginine (Arg) cannot enhance the synergistic immune efficacy of antigen and aluminum hydroxide adjuvant.

[0101] SARS-CoV-2 RBD obtained by recombinant expression and purification SARS-CoV-2 -S1139 antigen (SEQ ID NO: 6), i.e., in RBD SARS-CoV-2 A tandem repeat of the naturally occurring negatively charged epitope S1139 from the SARS-CoV-2 spike protein was introduced at the C-terminus of the flexible non-neutralizing epitope for immunological evaluation. Results showed that, in combination with aluminum hydroxide adjuvant (Denmark, Denmark), mice immunized with this formula showed significantly better results compared to wild-type RBD. SARS-CoV-2 Antigen, RBD SARS-CoV-2 -S1139 can significantly increase the level of specific antibodies against the novel coronavirus (>20-fold, P<0.0001). Figure 11 Furthermore, it maintains high immunogenicity even when the immunization dose is reduced. This indicates that the "naturally charged epitopes" derived from the virus itself can also be used to regulate the surface charge of antigen proteins, thereby altering the synergistic effect with charged adjuvants and significantly enhancing vaccine efficacy.

[0102] RBD obtained from recombinant expression purification SARS-CoV-2 -ARIH2 antigen (SEQ ID NO: 7), i.e., in RBD SARS-CoV-2The C-terminus of the flexible, non-neutralizing epitope ARIH2, derived from a natural negatively charged epitope on a human protein, was introduced for immunological evaluation. Results showed that, after immunization of mice with aluminum hydroxide adjuvant (Denmark, Denmark), compared to wild-type RBD... SARS-CoV-2 Antigen, RBD SARS-CoV-2 -ARIH2 significantly increased the level of specific antibodies against the novel coronavirus (>4-fold, P<0.05). Figure 12 This indicates that the body's own "natural charged surface sites" can also be used to regulate the surface charge of antigen proteins, thereby altering the synergistic effect with charged adjuvants and significantly enhancing vaccine efficacy.

[0103] 1.7 COVID-19 RBD SARS-CoV-2 Immunological evaluation of mice with variant antigens combined with different charged adjuvants

[0104] SARS-CoV-2 RBD obtained by recombinant expression and purification SARS-CoV-2 -9Asp antigen (SEQ ID NO: 3), i.e., in RBD SARS-CoV-2 A tandemly repeated negatively charged aspartic acid fragment 9Asp was introduced into the C-terminus of the flexible non-neutralizing epitope for mouse immunoassay, and the evaluation and detection protocols were the same as above. Results showed that, compared to wild-type RBD... SARS-CoV-2 Antigen, RBD SARS-CoV-2 -9Asp, when combined with manganese adjuvant (China Qimeng Biotechnology) for immunization of mice, significantly increased the level of specific antibodies against SARS-CoV-2 (>100-fold increase, P<0.0001). Figure 13 This indicates that the charge control strategy is applicable to other charged metal salt adjuvants besides aluminum adjuvants, such as manganese adjuvants.

[0105] SARS-CoV-2 RBD obtained by recombinant expression and purification SARS-CoV-2 -9Asp antigen (SEQ ID NO: 3), i.e., in RBD SARS-CoV-2 A tandemly repeated negatively charged aspartic acid fragment 9Asp was introduced into the C-terminus of the flexible non-neutralizing epitope for mouse immunoassay, and the evaluation and detection protocols were the same as above. Results showed that, compared to wild-type RBD... SARS-CoV-2 Antigen, RBD SARS-CoV-2 -9Asp, when combined with an aluminum hydroxide adjuvant (Denmark, Denmark) and CpG in a compound adjuvant, significantly increased the level of specific antibodies against SARS-CoV-2 in mice (>40-fold increase, P<0.01). Figure 14 This indicates that the charge control strategy is applicable to aluminum-containing compound adjuvants other than aluminum adjuvants alone.

[0106] SARS-CoV-2 RBD obtained by recombinant expression and purification SARS-CoV-2-9Arg antigen (SEQ ID NO: 8), i.e., in RBD SARS-CoV-2 The flexible, non-neutralizing epitope C-terminus introduces a "tandemly repeated charged amino acid," namely the tandemly repeated positively charged arginine fragment 9Arg, compared to wild-type RBD. SARS-CoV-2 When combined with CpG adjuvant (ODN 2006, Takara, Japan) for immunization of mice, it significantly increased the level of specific antibodies against SARS-CoV-2 (>3-fold increase, P<0.05). Figure 15 This indicates that the charge control strategy is applicable to other charged adjuvants besides charged metal salt adjuvants.

[0107] Example 2: SARS coronavirus RBD SARS Variant antigen design, expression preparation and immunological evaluation

[0108] 2.1 SARS Coronavirus RBD SARS Design and expression preparation of -9Asp variant antigen

[0109] The amino acid sequence of the SARS coronavirus RBD (YP_009825051.1) was obtained from the Genebank database. Referring to the RBD antigen charge regulation method in SARS-CoV-2, a repeating negatively charged amino acid was designed and introduced to regulate the RBD charge. The negatively charged amino acid was aspartic acid D-Asp, and the number of introduced amino acids was designed to be 9 Aspartic acid molecules. The C-terminal region was used as the introduction site, and the RBD was successfully designed. SARS -9Asp charge-regulated variant antigen (SEQ ID NO: 9) Figure 16 ).

[0110] Following the methods described above for constructing eukaryotic expression vectors and purifying proteins of the RBD antigen from SARS-CoV-2, RBD was prepared. SARS RBD SARS -9Asp charge-regulated variant antigen, identified by SDS-PAGE, showed that its molecular weight was as expected. Figure 16 ).

[0111] 2.2 Coronavirus RBD SARS Immunological evaluation of mice with -9Asp variant antigen combined with aluminum adjuvant

[0112] Female BALB / c mice aged 6-8 weeks were selected, with 6-8 mice in each group. Wild-type RBD obtained through recombinant expression and purification was... SARS and RBD SARSMice were immunized intramuscularly with the -9Asp charge-regulated variant antigen and aluminum hydroxide adjuvant, with a booster dose 14 days later. Each dose consisted of 100 μL of vaccine (containing 5 μg antigen and 50 μg adjuvant). Blood was collected from the tail vein of the mice after immunization, and the level of RBD-specific IgG antibodies in the immune serum was detected by ELISA.

[0113] The results showed that the SARS virus RBD obtained in this invention SARS -9Asp antigen (SEQ ID NO: 9), in RBD SARS The flexible, non-neutralizing epitope C-terminus introduces a "tandemly repeated charged amino acid," namely the tandemly repeated negatively charged aspartic acid fragment 9Asp, compared to wild-type RBD. SARS The antigen, when combined with aluminum hydroxide adjuvant (Denmark, Denmark) for immunization of mice, significantly increased the level of specific antibodies against SARS virus (>80-fold increase, P<0.0001). Figure 17 This indicates that the charge regulation strategy is applicable to antigens other than the SARS-CoV-2 RBD antigen.

[0114] Example 3: MERS coronavirus RBD MERS Variant antigen design, expression preparation and immunological evaluation

[0115] 3.1 MERS Coronavirus RBD MERS Design and expression preparation of -9Asp variant antigen

[0116] The amino acid sequence of the MERS coronavirus RBD (YP_009047204.1) was obtained from the Genebank database. Referring to the RBD antigen charge regulation method in SARS-CoV-2, a repeating negatively charged amino acid was designed and introduced to regulate the RBD charge. The negatively charged amino acid was aspartic acid D-Asp, and the number of introduced amino acids was designed to be 9 Aspartic acid molecules. The C-terminal region was used as the introduction site. The resulting RBD sequence was obtained. MERS -9Asp charge-regulated variant antigen (SEQ ID NO: 10) Figure 18 ).

[0117] Following the methods described above for constructing eukaryotic expression vectors and purifying proteins of the RBD antigen from SARS-CoV-2, RBD was prepared. MERS RBD MERS -9Asp charge-regulated variant antigen, identified by SDS-PAGE, showed that its molecular weight was as expected. Figure 18 ).

[0118] 3.2 Coronavirus RBD SARS Immunological evaluation of mice with -9Asp variant antigen combined with aluminum adjuvant

[0119] Female BALB / c mice aged 6-8 weeks were selected, with 6-8 mice in each group. Wild-type RBD obtained through recombinant expression and purification was... MERS and RBD MERS Mice were immunized intramuscularly with the -9Asp charge-regulated variant antigen and aluminum hydroxide adjuvant, with a booster dose 14 days later. Each dose consisted of 100 μL of vaccine (containing 5 μg antigen and 50 μg adjuvant). Blood was collected from the tail vein of the mice after immunization, and the level of RBD-specific IgG antibodies in the immune serum was detected by ELISA.

[0120] The results show that the MERS virus RBD obtained in this invention MERS -9Asp antigen (SEQ ID NO: 10), in RBD MERS The flexible, non-neutralizing epitope C-terminus introduces a "tandemly repeated charged amino acid," namely the tandemly repeated negatively charged aspartic acid fragment 9Asp, compared to wild-type RBD. MERS The antigen, when combined with aluminum hydroxide adjuvant (Denmark, Denmark) for immunization of mice, significantly increased the level of specific antibodies against MERS virus (>10-fold increase, P<0.01). Figure 19 This indicates that the charge regulation strategy is applicable to viral antigens other than the SARS-CoV-2 RBD antigen.

[0121] Example 4: Design, expression, preparation, and immunological evaluation of Neisseria meningitidis variant antigen fHbp-9Asp

[0122] 4.1 Design and Expression Preparation of Neisseria meningitidis Variant Antigen fHbp-9Asp

[0123] The amino acid sequence of *Neisseria meningitidis* antigen fHbp (Q6QCC2) was obtained from the Uniprot database. Referring to the charge regulation method for the RBD antigen in SARS-CoV-2, a repeating negatively charged amino acid was designed to regulate the charge of fHbp. The negatively charged amino acid was aspartic acid D-Asp, and the number of introduced amino acids was designed to be 9 Asp. The C-terminal region was used as the introduction site to design and obtain the fHbp-9Asp charge-regulated variant antigen (SEQ ID NO: 11). Figure 20 ).

[0124] The target protein was prepared by expression using a conventional E. coli prokaryotic expression system. Codons were optimized and gene sequences were synthesized. The pTIG E. coli expression vector was constructed, and after selection and verification of correct clones, expression was induced using IPTG in the OD range of 0.6-0.8. The bacterial cells were lysed, the supernatant was collected, and the target protein was purified using affinity chromatography. The fHbp and fHbp-9Asp charge-regulated variant antigens were obtained. SDS-PAGE analysis confirmed that the molecular weights met expectations. Figure 20 ).

[0125] 4.2 Immunological evaluation of mice with Neisseria meningitidis variant antigen fHbp-9Asp combined with aluminum adjuvant

[0126] Female BALB / c mice aged 6-8 weeks were selected, with 6-8 mice in each group. Mice were immunized intramuscularly with the recombinantly expressed and purified wild-type fHbp and fHbp-9Asp charge-regulated variant antigens, combined with aluminum hydroxide adjuvant. A booster immunization was administered 14 days later, with each dose containing 100 μL of vaccine (containing 5 μg antigen and 50 μg adjuvant). Post-immunization, blood was collected from the tail vein of the mice, and the level of fHbp-specific IgG antibodies in the immune serum was detected by ELISA.

[0127] The results showed that the fHbp-9Asp antigen (SEQ ID NO: 11) obtained in this invention introduced a "tandem repeat charged amino acid," namely the tandem repeat negatively charged aspartic acid fragment 9Asp, at the C-terminus of the flexible non-neutralizing epitope of fHbp. Compared with the wild-type fHbp antigen, after immunizing mice with aluminum hydroxide adjuvant (Denmark, Denmark), it significantly increased the level of specific antibodies against fHbp (>60-fold increase, P<0.001). Figure 21 This indicates that the charge control strategy is applicable to bacterial antigens other than viral antigens.

[0128] Example 5: Design, expression, preparation, and immunological evaluation of the model antigen chicken ovalbumin variant OVA-9Asp

[0129] 5.1 Design and Expression Preparation of the Model Antigen Chicken Ovalbumin Variant OVA-9Asp

[0130] The amino acid sequence of the model antigen chicken ovalbumin OVA (P01012) was obtained from the Uniprot database. Referring to the charge regulation method for the RBD antigen in SARS-CoV-2, a repeating negatively charged amino acid was designed to regulate the OVA charge. The negatively charged amino acid was aspartic acid D-Asp, and the number of introduced amino acids was designed to be 9 Aspartic acid. The C-terminal region was used as the introduction site to design and obtain the OVA-9Asp charge-regulated variant antigen (SEQ ID NO: 12). Figure 22 ).

[0131] Following the methods described above for constructing eukaryotic expression vectors and purifying proteins of the RBD antigen from SARS-CoV-2, OVA and OVA-9Asp charge-regulated variant antigens were prepared. SDS-PAGE analysis confirmed that their molecular weights met expectations. Figure 22 ).

[0132] 5.2 Immunological evaluation of mice using the model antigen chicken ovalbumin variant OVA-9Asp combined with aluminum adjuvant

[0133] Female BALB / c mice aged 6-8 weeks were selected, with 6-8 mice in each group. Mice were immunized intramuscularly with the recombinantly expressed and purified wild-type OVA and OVA-9Asp charge-regulated variant antigen, combined with aluminum hydroxide adjuvant. A booster immunization was administered 14 days later, with each dose containing 100 μL of vaccine (containing 5 μg antigen and 50 μg adjuvant). Post-immunization, blood was collected from the tail vein of the mice, and the level of OVA-specific IgG antibodies in the immune serum was detected by ELISA.

[0134] The results showed that the OVA-9Asp antigen (SEQ ID NO: 12) obtained in this invention introduced a "tandem repeat charged amino acid," namely the tandem repeat negatively charged aspartic acid fragment 9Asp, at the C-terminus of the flexible non-neutralizing epitope of OVA. Compared with the wild-type OVA antigen, after immunizing mice with aluminum hydroxide adjuvant (Denmark, Denmark), it could significantly increase the level of specific antibodies against OVA (>3-fold increase, P<0.01). Figure 23 This indicates that the charge regulation strategy is applicable to other model antigens besides viral and bacterial antigens.

[0135] The above embodiments are preferred embodiments of the present invention, but are not intended to limit the present invention. Any modifications and alterations made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. An antigen protein variant with an insertion of a charged amino acid fragment, characterized in that, The antigen protein variant is selected from the SARS-CoV-2 RBD antigen as shown in SEQ ID NO.

8.

2. A vaccine composition containing the antigen protein variant of claim 1, characterized in that, The vaccine composition further comprises a charged adjuvant carrying a charge opposite to that of the charged amino acid fragments introduced into the antigen protein variant.

3. The vaccine composition according to claim 2, characterized in that, The charged adjuvant is selected from one or more of the following: charged metal salt adjuvants, pattern recognition receptor agonist adjuvants, liposome adjuvants, oil emulsion adjuvants, cytokine adjuvants, and chemokine adjuvants.

4. The vaccine composition according to claim 3, characterized in that, The charged metal salt adjuvant is selected from one of aluminum hydroxide adjuvant, aluminum-containing composite adjuvant, manganese adjuvant, or CpG adjuvant.

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