Recombinant protein for use as a mycoplasma pneumonia vaccine and preparation method and application thereof

Abstract

The application discloses a fusion protein and a preparation method and application thereof. The fusion protein comprises a P1 protein C-terminal fragment and an adjuvant protein, and can be used for preparing a Mycoplasma pneumoniae recombinant protein vaccine. The P1 antigen fragment is fused and expressed with a mucosal adjuvant, so that high expression quantity, strong immunogenicity and induction of respiratory mucosal immune response are realized.

Description

Technical Field

[0001] This invention relates to the field of immunobiology, particularly to the prevention and treatment of Mycoplasma pneumoniae infection in humans. Specifically, this invention relates to a recombinant protein that can be used for the prevention or treatment of Mycoplasma pneumoniae infection in humans, its preparation method, and its uses. Background Technology

[0002] Mycoplasma pneumoniae is one of the important pathogens causing community-acquired pneumonia. However, due to the lack of safe and effective protein vaccine candidate antigens, there are currently no mature and commercially available Mycoplasma pneumoniae vaccines. The P1 adhesion protein of Mycoplasma pneumoniae is involved in host cell adhesion and is one of the most widely studied candidate targets, especially its C-terminal region (P1C), which is considered to contain important protective epitopes and is therefore often used as a candidate protein component for exploration.

[0003] However, the research based on P1C components still has the following shortcomings: (1) The range of the C-terminal fragments of P1 protein verified in different studies varies greatly, and there is a lack of a unified and clear preferred antigen fragment. There may be significant differences in expression level, stability and immune protection effect between different fragments; (2) The expression efficiency and solubility of many candidate proteins in prokaryotic expression systems are not ideal, which brings difficulties to the preparation, purification and subsequent industrialization of recombinant proteins; (3) The immunogenicity of single protein antigens is usually limited, and often requires additional adjuvants or more complex delivery strategies to induce a more ideal protective immune response; (4) Mycoplasma pneumoniae is a respiratory mucosal pathogen, and its initial infection site is mainly located on the surface of the respiratory epithelium. However, existing studies still mainly use conventional immunization methods such as subcutaneous or intramuscular immunization, and research on mucosal delivery, especially lung delivery, is still relatively limited.

[0004] No research has yet proposed how to design vaccine antigens with higher stability, immunogenicity, and expression efficiency based on the conserved C-terminal domain of the P1 protein. Therefore, there is still an urgent need in this field to develop a Mycoplasma pneumoniae vaccine that can effectively induce mucosal immune responses, has high expression levels, and provides excellent protective effects. Summary of the Invention

[0005] The purpose of this invention is to develop a recombinant protein vaccine against Mycoplasma pneumoniae. This invention identifies and validates a P1C fragment with higher expression levels, higher stability, and better immunoprotective efficacy. In particular, when this P1C fragment is fused with a specific adjuvant protein, its immunoprotective efficacy against Mycoplasma pneumoniae is further enhanced. Therefore, this invention provides a P1C fragment capable of achieving better immunoprotective efficacy, a fusion protein comprising said fragment, and its applications, particularly in the preparation of a recombinant protein vaccine against Mycoplasma pneumoniae.

[0006] In one aspect, the present invention provides a fusion protein comprising: (a) an antigenic polypeptide derived from Mycoplasma pneumoniae P1 adhesion protein; and (b) an adjuvant protein operatively linked to said antigenic polypeptide.

[0007] In one aspect, the present invention provides a fusion protein comprising: (a) an antigenic polypeptide derived from Mycoplasma pneumoniae P1 adhesion protein, the antigenic polypeptide comprising a sequence represented by amino acid residues 1376-1521 of the Mycoplasma pneumoniae M129 strain P1 adhesion protein or an amino acid sequence having at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or higher) sequence identity with it; and (b) an adjuvant protein operatively linked to the antigenic polypeptide.

[0008] In some embodiments, the antigenic polypeptide comprises an amino acid sequence as shown in SEQ ID NO:2, or an amino acid sequence having at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or higher) sequence identity with SEQ ID NO:2.

[0009] In some embodiments, the adjuvant protein is CTB or a variant thereof. In some embodiments, the adjuvant protein comprises an amino acid sequence as shown in SEQ ID NO:4, or an amino acid sequence having at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or higher) sequence identity with SEQ ID NO:4.

[0010] In some embodiments, the operative linking is either a direct linking or a linking via a peptide linker, for example, a peptide linker comprising one or more glycine residues and / or one or more serine residues. In some embodiments, the peptide linker is (GGGGS)n, where n = 1-3. In some embodiments, the antigenic polypeptide is operatively linked to the C-terminus of the adjuvant protein.

[0011] In some embodiments, the fusion protein comprises an amino acid sequence as shown in SEQ ID NO:3, or an amino acid sequence having at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or higher) sequence identity with SEQ ID NO:3.

[0012] In one aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a fusion protein as disclosed herein.

[0013] In one aspect, the present invention provides a carrier comprising nucleic acid molecules as disclosed herein.

[0014] In one aspect, the present invention provides a host cell comprising a nucleic acid molecule as disclosed herein or a vector as disclosed herein. Such a host cell may be a prokaryotic cell, such as a bacterial cell (e.g., *Escherichia coli* cells). For example, the host cell is an *Escherichia coli* BL21 (DE3) cell.

[0015] In one aspect, the present invention provides a method for preparing a fusion protein as disclosed herein, comprising: culturing a host cell containing a nucleic acid molecule or a vector as disclosed herein under suitable conditions, and recovering the fusion protein from the cell culture. Those skilled in the art will understand that the fusion protein can be prepared by the above method, or by artificial chemical synthesis, or by other biochemical or molecular biological methods known to those skilled in the art.

[0016] In one aspect, the present invention provides a composition comprising a fusion protein as disclosed herein, a nucleic acid molecule as disclosed herein, a vector comprising said nucleic acid molecule, or a host cell as disclosed herein. In some embodiments, the composition is a pharmaceutical composition or an immunogenic composition (e.g., a vaccine composition). In some embodiments, the composition further comprises a pharmaceutically acceptable carrier and / or excipient. In some embodiments, the pharmaceutically acceptable carrier and / or excipient comprises an adjuvant. The carrier and / or excipient (e.g., an adjuvant) used for co-administration or included in the immunogenic composition according to the invention should preferably be potentially safe, well-tolerated, and effective in humans. Such carriers and / or excipients are well known to those skilled in the art.

[0017] On the other hand, this application provides a method for inducing an immune response against Mycoplasma pneumoniae in a subject and / or for preventing and / or treating Mycoplasma pneumoniae infection or disease associated with Mycoplasma pneumoniae infection (e.g., respiratory infection) in a subject, comprising: administering an effective amount of the fusion protein or composition as described above to a subject (e.g., a human) in need.

[0018] In one aspect, the present invention relates to the use of fusion proteins as disclosed herein, nucleic acid molecules as disclosed herein, vectors comprising said nucleic acid molecules, or host cells as disclosed herein in the preparation of formulations for inducing an immune response against Mycoplasma pneumoniae in a subject and / or for preventing and / or treating Mycoplasma pneumoniae infection or disease associated with Mycoplasma pneumoniae infection in a subject.

[0019] In one aspect, the present invention relates to the use of an antigenic polypeptide in the preparation of formulations for inducing an immune response against Mycoplasma pneumoniae in a subject and / or for the prevention and / or treatment of Mycoplasma pneumoniae infection or disease associated with Mycoplasma pneumoniae infection in a subject, wherein the antigenic polypeptide is derived from Mycoplasma pneumoniae P1 adhesion protein, preferably, the antigenic polypeptide comprises the sequence shown for amino acid residues 1376-1521 of the Mycoplasma pneumoniae M129 strain P1 adhesion protein. In some embodiments, the antigenic polypeptide comprises the amino acid sequence shown in SEQ ID NO:2.

[0020] In some embodiments, the disease associated with Mycoplasma pneumoniae infection is a respiratory infection, such as mycoplasma pneumoniae. In some embodiments, the preparation is a vaccine.

[0021] In some embodiments, the formulation is administered to the subject via the nose or subcutaneously. In some embodiments, the subject is a mammal, such as a human.

[0022] In some respects, the present invention provides the use of the fusion proteins disclosed herein in the production of antibodies against Mycoplasma pneumoniae (e.g., polyclonal or monoclonal antibodies). Attached Figure Description

[0023] Figure 1 The P1 protein fragment P1C was shown. 1376-1521 With P1C 1287-1521 The expression output.

[0024] Figure 2 The P1 protein fragment P1C was shown. 1376-1521 P1C 1287-1521 Its immune protective effect.

[0025] Figure 3 The soluble expression of different forms of fusion proteins is shown.

[0026] Figure 4 The UV spectra of the purified products of three fusion proteins after passing through a molecular sieve are shown. A. P1C 1376-1521 - PFFer with Superose 6 UV value; B. LTB-P1C 1376-1521 Exceeds Superdex 200 UV value; C. CTB-P1C 1376-1521 Exceeds Superdex 200 UV value.

[0027] Figure 5 GST-P1C was displayed. 1376-1521 Protein purification diagram.

[0028] Figure 6The levels of antibody production via pulmonary delivery and subcutaneous immunization pathways are shown. A. Serum IgA antibody levels in mice after immunization via different routes; B. Serum IgG antibody levels in mice after immunization via different routes.

[0029] Figure 7 The study evaluated the bacterial clearance efficacy of the pulmonary delivery immune route versus the subcutaneous immune route.

[0030] Figure 8 The following figures show the antibody concentrations in post-immunization serum as detected by ELISA. A. IgA antibody concentration in mouse serum; B. IgG antibody concentration in mouse serum.

[0031] Figure 9 The results show the changes in body weight and the measurement of pathogenic load in the lungs of mice after challenge. A. Changes in body weight of mice after challenge; B. Measurement of pathogenic load in the lungs of mice after challenge. Detailed Implementation

[0032] In this invention, unless otherwise stated, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art. All patents, patent applications, and other publications cited herein are incorporated herein by reference in their entirety. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are widely used terms and routine procedures in their respective fields. If any definition presented herein conflicts with a definition presented in a patent, patent application, or other publication incorporated herein by reference, the definition presented herein shall prevail.

[0033] Unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. More specifically, as used in this specification and the appended claims, unless the context explicitly indicates otherwise, the singular forms “a,” “an,” and “the” include plural indicators. Thus, for example, reference to “a protein” includes multiple proteins; reference to “a cell” includes a mixture of cells, etc. In this application, unless otherwise stated, the use of “or” means “and / or.” Furthermore, the use of the term “comprising” and other forms such as “including” and “containing” is not restrictive. Moreover, the scope provided in the specification and the appended claims includes all values ​​between endpoints.

[0034] Numerical ranges include numbers within defined ranges. Taking into account significant figures and measurement-related errors, measured and measurable values ​​should be understood as approximate values. As used in this application, the terms "about" and "approximately" have their meanings as understood in the art. Unless otherwise specified, figures used in this application, with or without modifiers such as "about" or "approximately," should be understood to cover normal divergence and / or fluctuations, as will be understood by one of ordinary skill in the art. In some embodiments, the terms "about" or "approximately" refer to a range of values ​​(greater than or less than) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in any direction of the reference value, unless otherwise stated or otherwise apparent from the context (except where such values ​​would exceed 100% of possible values).

[0035] As used herein, when referring to the amino acid positions of the Mycoplasma pneumoniae P1 adhesion protein, the amino acid sequences of the P1 adhesion protein of known Mycoplasma pneumoniae subtypes or strains can be used for description. The Mycoplasma pneumoniae strains mentioned herein may include Mycoplasma pneumoniae M129 strain (ATCC 29342) and Mycoplasma pneumoniae FH strain (ATCC 15531 / NCTC 10119). Exemplary P1 protein amino acid sequences are shown in UniProt_P11311 or UniProt_Q548R2. For example, the expression "amino acid residues 1376-1521 of the P1 adhesion protein (aa 1376-1521)" refers to amino acid residues 1376-1521 of the amino acid sequence shown in UniProt_P11311, which, because they are located at the C-terminus of the P1 adhesion protein, can also be abbreviated as "P1C" in this document. 1376-1521However, those skilled in the art will understand that the amino acid sequence of the P1 adhesion protein described herein may also encompass corresponding sequence fragments in its natural or artificial variants, as well as corresponding fragments in the P1 adhesion protein of different Mycoplasma pneumoniae subtypes or strains. For example, the expression "amino acid residues 1376-1521 of the P1 adhesion protein" may include amino acid residues 1376-1521 of the amino acid sequence shown as UniProt_P11311, and corresponding fragments in its variants (natural or artificial). According to the present invention, the expression "corresponding sequence fragment" or "corresponding fragment" refers to a fragment located at an equivalent position in the compared sequences when the sequences are optimally aligned, i.e., when the sequences are aligned to obtain the highest percentage of identity. For example, the corresponding fragment of amino acid residues 1376-1521 of the P1 adhesion protein of Mycoplasma pneumoniae strain M129 may include amino acid residues 1383-1528 of the P1 adhesion protein of Mycoplasma pneumoniae strain FH.

[0036] As used herein, the term "fusion protein" refers to a polypeptide having two (or more) parts operatively linked together, each part being a polypeptide with different properties. The two parts may be directly linked by a single peptide bond or by a peptide linker containing one or more amino acid residues. In some embodiments, the fusion protein is a fusion of a fragment of Mycoplasma pneumoniae P1 protein and an adjuvant protein.

[0037] As used herein, the term "adjuvant protein" refers to a protein molecule capable of fusing with an antigen protein or fragment thereof and enhancing or modulating a specific immune response (especially a mucosal immune response) to the antigen. Adjuvant proteins are typically derived from mutants of pathogenic microorganisms or their toxins, bacterial flagellar components, or other proteins with immunostimulatory activity. Adjuvant proteins can enhance the strength, persistence, and directionality of the immune response by activating pattern recognition receptors (such as TLR5, TLR4, NLR, etc.) or binding to cell surface molecules to promote the uptake and presentation of antigen-presenting cells. Preferred adjuvant proteins are those capable of serving as mucosal immune adjuvants. Non-limiting examples of adjuvant proteins include CTB, LTB, Flagellin, and CRM197 and their variants.

[0038] As used herein, the term "variant," in the context of polypeptides (including polypeptides), also refers to a polypeptide or peptide that contains an amino acid sequence altered by the introduction of amino acid residue substitutions, deletions, or additions. As used herein, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to the polypeptide or peptide). For example, but not limited to, polypeptides can be modified, such as by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linking to cellular ligands or other proteins, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, and the metabolic synthesis of tunicamycin. Furthermore, variants have functions similar to, identical to, or improved upon those of the polypeptide or peptide from which they are derived.

[0039] As used herein, the term "fragment" refers to a peptide or polypeptide that contains several (e.g., 50-100, 100-150) consecutive amino acid residues of the amino acid sequence of another polypeptide, and that the fragment retains the desired function or property (e.g., immunogenicity) of the polypeptide.

[0040] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., a gap may be introduced in the first amino acid sequence or nucleic acid sequence to best align with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecule is identical at that position. The percentage identity between two sequences is a function of the number of identity positions shared by the sequences (i.e., percentage identity = number of identical overlapping positions / total number of positions × 100%). In some embodiments, the two sequences are of the same length.

[0041] The determination of percentage identity between two sequences can also be achieved using mathematical algorithms. A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, as an improvement upon that in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such algorithms are integrated into the NBLAST and XBLAST procedures of Altschul et al., 1990, J. Mol. Biol. 215:403.

[0042] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0043] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector. Common host cells include, but are not limited to, prokaryotic cells such as *Escherichia coli* or *Bacillus subtilis*, fungal cells such as yeast cells or *Aspergillus*, insect cells such as S2 *Drosophila* cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells. In some embodiments, the host cell described herein is a prokaryotic cell, such as *E. coli* cells. In some embodiments, the host cell described herein is *E. coli* BL21(DE3) cells.

[0044] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. In some exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution).

[0045] As used herein, the terms “antigenicity” or “immunogenicity” refer to the property of a peptide or protein to induce, trigger, increase, or enhance a cellular and / or humoral immune response and to be recognized by the products of that response (T cells, antibodies).

[0046] As used herein, the term "adjuvant" refers to a nonspecific immune enhancer that, when administered to the body along with or before an antigen, can enhance the body's immune response to the antigen or alter the type of immune response. There are many types of adjuvants, including but not limited to aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete and incomplete Freund's adjuvants), Corynebacterium breve, lipopolysaccharides, and cytokines. Freund's adjuvant is currently the most commonly used adjuvant in animal studies. Aluminum hydroxide adjuvant is more frequently used in clinical trials.

[0047] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom (e.g., Mycoplasma pneumoniae infection) in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include (but are not limited to) alleviating symptoms, reducing the extent of disease, stabilizing (i.e., no longer worsening) the state of disease, delaying or slowing the progression of disease, improving or alleviating the state of disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to the expected survival (if no treatment was received).

[0048] As used herein, the term "subject" refers to a mammal, such as a human. In some embodiments, the subject (e.g., a human) has a Mycoplasma pneumoniae infection or a disease associated with Mycoplasma pneumoniae infection, or is at risk of having such a disease.

[0049] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing disease (e.g., Mycoplasma pneumoniae infection) means an amount sufficient to prevent, stop, or delay the onset of disease (e.g., Mycoplasma pneumoniae infection); an effective amount for treating disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the method of administration of the drug, and other concurrent treatments, etc.

[0050] Antigenic polypeptide derived from Mycoplasma pneumoniae P1 protein

[0051] Mycoplasma pneumoniae is a prokaryotic microorganism intermediate between bacteria and viruses, belonging to the class Mollicutes, order Mycoplasma, and family Mycoplasmatidae. It is a significant pathogen causing respiratory infections in humans, primarily leading to primary atypical pneumonia (PAP) and is also a common cause of community-acquired pneumonia (CAP), especially prevalent in children, adolescents, and immunocompromised individuals. The motility and infectivity of Mycoplasma pneumoniae depend on the expression of its immunodominant proteins P1 and P40 / P90. These two proteins together form a transmembrane adhesion complex, mediating the binding of Mycoplasma pneumoniae to host respiratory epithelial cells, thereby causing cell damage and an immune inflammatory response.

[0052] P1 adhesin (also known as P1 protein) is a major adhesin expressed on the surface of Mycoplasma pneumoniae, composed of a polypeptide chain of approximately 170 kDa. P1 protein is a key molecule for the adhesion and colonization of Mycoplasma pneumoniae in the host respiratory tract, and is also one of the main target antigens recognized by the host immune system, exhibiting strong immunogenicity. Structurally, P1 protein includes a signal peptide region at the N-terminus, a large N-terminal domain (adhesion functional region), and a smaller conserved C-terminal domain. Specific antigenic peptides can induce the production of specific neutralizing antibodies against Mycoplasma pneumoniae, blocking its binding to host cells, thereby exerting preventive or therapeutic effects.

[0053] Through design, screening, and experimental verification, the inventors have for the first time discovered that antigenic peptides containing specific fragments of the Mycoplasma pneumoniae P1 protein exhibit high expression levels, good stability, and excellent immunoprotective effects. Therefore, these antigenic peptides can be used to induce an immune response against Mycoplasma pneumoniae, or to prepare vaccines against Mycoplasma pneumoniae (e.g., recombinant protein vaccines), vaccine compositions, etc. Preferably, the antigenic peptide contains 140-160 consecutive amino acid residues at the C-terminus of the P1 protein. In some embodiments, the P1 protein may be derived from Mycoplasma pneumoniae strain M129 (ATCC29342) or Mycoplasma pneumoniae strain FH (ATCC 15531 / NCTC 10119). In some embodiments, the amino acid sequence of the P1 protein is as shown in UniProt_P11311 or UniProt_Q548R2. In some embodiments, the antigenic peptide contains the amino acid sequence shown in amino acid residues 1376-1521 of the P1 protein derived from Mycoplasma pneumoniae strain M129. In some embodiments, the antigenic polypeptide comprises the amino acid sequence represented by amino acid residues 1383-1528 of the P1 protein derived from Mycoplasma pneumoniae FH strain.

[0054] It should be understood that the specific fragment of the P1 protein contained in the antigenic polypeptide may also encompass corresponding sequence fragments in different natural or artificial variants of the P1 protein, as well as corresponding fragments in the P1 protein of different Mycoplasma pneumoniae subtypes or strains. In some embodiments, the antigenic polypeptide may contain amino acid residues 1376-1521 of the P1 protein of strain M129 and corresponding fragments in its variants (natural or artificial), the length of which varies but has similar functions or properties (e.g., immunogenicity). In some embodiments, the antigenic polypeptide may contain amino acid residues 1383-1528 of the P1 protein derived from Mycoplasma pneumoniae FH strain and corresponding fragments in its variants (natural or artificial).

[0055] In some embodiments, the antigenic polypeptide comprises an amino acid sequence as shown in SEQ ID NO:2, or an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:2.

[0056] Fusion proteins containing antigenic peptides

[0057] In one aspect, the present invention provides a fusion protein comprising an antigenic polypeptide derived from Mycoplasma pneumoniae P1 protein as described above, and an adjuvant protein operatively linked to said antigenic polypeptide. In some embodiments, the fusion protein comprises: (a) an antigenic polypeptide derived from Mycoplasma pneumoniae P1 protein, said antigenic polypeptide comprising a sequence represented by amino acid residues 1376-1521 of the Mycoplasma pneumoniae M129 strain P1 protein, or an amino acid sequence having at least 95% sequence identity with it; and (b) an adjuvant protein operatively linked to said antigenic polypeptide.

[0058] In some embodiments, the antigenic polypeptide comprises the amino acid sequence shown at amino acid residues 1376-1521 of UniProt_P11311 or UniProt_Q548R2. In some embodiments, the antigenic polypeptide comprises the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence having at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or higher) sequence identity with SEQ ID NO:2. In some embodiments, the antigenic polypeptide comprises a specific fragment of the P1 protein derived from Mycoplasma pneumoniae strain M129 (ATCC 29342), Mycoplasma pneumoniae strain PI 1428 (ATCC 29085), or Mycoplasma pneumoniae strain FH (ATCC 15531 / NCTC 10119) corresponding to the sequence shown in SEQ ID NO:2.

[0059] Preferably, the adjuvant protein, after being fused to the antigenic polypeptide, retains its desired function, such as self-assembling into a multimer and allowing the antigenic polypeptide to be presented on the surface of the adjuvant protein multimer. Examples of such adjuvant proteins include LTB and CTB. In some embodiments, the adjuvant protein is CTB or a variant thereof. In some embodiments, the adjuvant protein comprises the amino acid sequence shown in SEQ ID NO:4, or an amino acid sequence having at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or higher) sequence identity with SEQ ID NO:4.

[0060] In some embodiments, the operatively linked structure is either a direct link or a link via a peptide linker. The peptide linker can be a rigid or flexible linker, preferably a flexible linker. A variety of known peptide linkers can be used in the fusion protein of the present invention and are well known to those skilled in the art. For example, the peptide linker is a linker comprising one or more glycines and / or one or more serines. In some embodiments, the peptide linker is (GGGGS)n, where n = 1-3. In some embodiments, the antigenic polypeptide is operatively linked to the N-terminus or C-terminus of the adjuvant protein. In some embodiments, the antigenic polypeptide is operatively linked to the C-terminus of the adjuvant protein.

[0061] In some embodiments, the fusion protein comprises an amino acid sequence as shown in SEQ ID NO:3, or an amino acid sequence having at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or higher) sequence identity with SEQ ID NO:3. Preferably, the fusion protein is in the form of a pentamer.

[0062] Adjuvant protein

[0063] The adjuvant proteins included in the fusion proteins disclosed herein enable the fusion proteins to achieve enhanced immunoprotective effects against Mycoplasma pneumoniae. Common adjuvant proteins include, but are not limited to, CTB, LTB, Flagellin, CRM197, or variants thereof. Typically, adjuvant proteins themselves have the ability to bind to specific receptors, and when fused to a target antigen, they can directly deliver the antigen to the surface of cells expressing that specific receptor (e.g., antigen-presenting cells). Adjuvant proteins can act as innate immune agonists, activating immune-related signaling pathways by binding to receptors to enhance the immune response. Adjuvant proteins capable of spontaneously assembling into multimers can also serve as antigen delivery carriers, increasing the uptake of the fusion protein by antigen-presenting cells. The adjuvant proteins described herein encompass any adjuvant protein capable of being used to construct fusion proteins with the antigenic peptides described herein and allowing the antigenic peptides to exhibit immunogenicity.

[0064] CTB is the non-toxic B subunit of cholera toxin (CT), consisting of a ring structure composed of five identical subunits. It has the ability to specifically bind to the GM1 ganglioside receptor on the surface of host cells. The mechanisms by which CTB contributes to mucosal immunity include promoting antigen crossing of the mucosal barrier, enhancing antigen presentation by dendritic cells (DCs) and other antigen-presenting cells, and increasing the secretion of TGF-β by suppressor T cells. When CTB is conjugated with or used in conjunction with antigens, it can significantly enhance the production of IgA antibodies.

[0065] LTB is the non-toxic B subunit of Escherichia coli heat-labile enterotoxin (LT). It can also bind to cell surface gangliosides (GM1), thereby exerting an adjuvant effect. Flagellin is a major structural protein of bacterial flagella, derived from various flagellated bacteria (such as Salmonella typhimurium and Pseudomonas aeruginosa). It exerts its immune-activating effect by binding to Toll-like receptor 5 (TLR5) and NOD-like receptor family C4 (NLRC4) on the surface of host cells.

[0066] Preferably, the adjuvant protein is CTB or a variant thereof. The antigenic peptide described herein can be fused to the C-terminus of the CTB adjuvant protein via a peptide linker to construct the fusion protein described herein. Upon expression, the CTB of the fusion protein can spontaneously assemble into a pentamer, wherein the antigenic peptide is presented on the surface of the pentamer. The CTB variants described herein include molecules that are non-toxic but retain CT adjuvant and immunogenic properties after site-directed mutagenesis screening, such as CTS106. In some embodiments, the adjuvant protein comprises the amino acid sequence shown in SEQ ID NO:4, or an amino acid sequence having at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or higher) sequence identity with SEQ ID NO:4.

[0067] Nucleic acid molecules, vectors, and host cells

[0068] In one aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a fusion protein as disclosed herein. The nucleic acid molecule disclosed herein can be obtained using standard molecular biology techniques. The nucleic acid encoding an antigenic polypeptide can be operatively linked to another DNA molecule encoding an adjuvant protein. DNA fragments containing these regions can be obtained by standard PCR amplification.

[0069] Once DNA fragments encoding antigenic peptides and adjuvant proteins are obtained, these DNA fragments can be further manipulated using standard recombinant DNA techniques, such as incorporation into expression vectors known in the art. In some embodiments, the nucleic acids encoding these DNA fragments are each contained in a single expression vector, typically under the control of different or the same promoter. In some other embodiments, the nucleic acids encoding these DNA fragments are operably ligated and contained in a single expression vector under the control of the same promoter. In some embodiments, the vector is a pET-28a plasmid. The term "operably ligated" as used herein is intended to mean that the ligation of two DNA fragments results in the amino acid sequences encoded by the two DNA fragments remaining within their reading frames. In one aspect, the present invention provides a vector comprising nucleic acid molecules as disclosed herein.

[0070] In one aspect, the present invention provides a host cell comprising a nucleic acid molecule as disclosed herein or a vector as disclosed herein. Such a host cell can be any cell suitable for expressing the fusion protein of the present invention, preferably a prokaryotic cell such as a bacterial cell (e.g., *Escherichia coli* cells). In some embodiments, the host cell is *Escherichia coli* BL21 (DE3) cells.

[0071] In one aspect, the present invention provides a method for preparing a fusion protein as disclosed herein, comprising: culturing a host cell containing a nucleic acid molecule or a vector as disclosed herein under suitable conditions, and recovering the fusion protein from the cell culture. When an expression vector encoding a fusion protein is introduced into a mammalian host cell, the fusion protein is produced by culturing the host cell for a sufficient period to allow expression of the fusion protein in the host cell or by secreting the fusion protein into the culture medium in which the host cell grows. The fusion protein can be recovered from the culture medium using standard protein purification methods. Those skilled in the art will understand that the fusion protein can be prepared by the above method, or by artificial chemical synthesis, or by other biochemical or molecular biological methods known to those skilled in the art.

[0072] Composition

[0073] In one aspect, the present invention provides a composition comprising a fusion protein as disclosed herein, a nucleic acid molecule as disclosed herein, a vector comprising said nucleic acid molecule, or a host cell as disclosed herein. In some embodiments, the composition is a pharmaceutical composition or an immunogenic composition (e.g., a vaccine composition). In some embodiments, the composition further comprises a pharmaceutically acceptable carrier and / or excipient. In some embodiments, the pharmaceutically acceptable carrier and / or excipient comprises an adjuvant. The carrier and / or excipient (e.g., an adjuvant) used for co-administration or included in the immunogenic composition according to the invention should preferably be potentially safe, well-tolerated, and effective in humans. Such carriers and / or excipients are well known to those skilled in the art.

[0074] The compositions of the present invention can be administered to subjects in need via various routes, including but not limited to mucosal (e.g., nasal), oral, intramuscular, intradermal, and subcutaneous administration, or otherwise via implantation or inhalation. The compositions can be formulated as preparations in liquid or gaseous form; including but not limited to solutions, injections, inhalers, and aerosols. Appropriate formulations and routes of administration can be selected according to the intended application and treatment regimen. Preferably, the compositions of the present invention are administered nasally or subcutaneously.

[0075] In some embodiments, the composition is a vaccine composition. The vaccine composition can be prepared by mixing the fusion protein disclosed herein with various adjuvants known in the art in a specific ratio. These adjuvants include, but are not limited to, TLR (Toll-like receptor) agonists, NOD agonists, CLR (C-type lectin receptor) agonists, RLR agonists, aluminum adjuvants (e.g., aluminum hydroxide, aluminum phosphate), squalene-containing oil-in-water emulsions (e.g., MF59), AS adjuvant systems (e.g., AS01, AS03, AS04), saponin adjuvants, α-galactosylceramide-derived adjuvants, and polysaccharide adjuvants. The inventors have unexpectedly discovered that vaccine compositions comprising the fusion protein of the present invention, when delivered subcutaneously or via the lungs (nasal delivery), can induce a specific immune response against pneumonia pathogens, particularly when delivered via the lungs, where a potent immune response is efficiently induced.

[0076] The vaccine composition of the present invention can be used in single-dose or multi-dose administration regimens, such as single-dose, two-dose, or three-dose administration regimens. In the multi-dose administration regimen, the doses administered at different times can be the same or different. The interval between two or three administrations can be from two weeks to six months, for example, three weeks to three months. Regular long-term booster doses can also be provided, for example, every 2 to 10 years.

[0077] Suitably, the vaccine composition of the present invention can be prepared in a vaccine dose volume of 0.1 mL to 1 mL, preferably 0.2 mL, 0.25 mL, or 0.5 mL. The vaccine dose volume can be adjusted to less than 0.1 mL or more than 1 mL as needed. A single dose of the vaccine composition of the present invention may contain 1-10 μg of the fusion protein of the present invention, for example, it may contain 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, or 10 μg of the fusion protein.

[0078] Application of the present invention

[0079] Mycoplasma pneumoniae invades respiratory epithelial cells through a specific adhesion mechanism, triggering a wide range of clinical manifestations, from upper respiratory tract infections to severe lower respiratory tract diseases (atypical pneumonia), and may even involve various extrapulmonary complications. The compositions of the present invention (e.g., immunogenic compositions) can be readily used in various therapeutic or preventative applications to treat Mycoplasma pneumoniae infection in subjects or to elicit an immune response against Mycoplasma pneumoniae. For example, the compositions can be administered to subjects to induce an immune response against Mycoplasma pneumoniae, such as inducing the production of broadly neutralizing antibodies against Mycoplasma pneumoniae. For subjects at risk of developing Mycoplasma pneumoniae infection, the immunogenic compositions of the present invention can be administered to provide prophylactic protection against viral infection.

[0080] Therefore, in one aspect, this application provides a method for inducing an immune response against Mycoplasma pneumoniae in a subject and / or for preventing and / or treating Mycoplasma pneumoniae infection or disease associated with Mycoplasma pneumoniae infection (e.g., respiratory infection) in a subject, comprising: administering an effective amount of the fusion protein or composition disclosed herein to a subject (e.g., a human) in need.

[0081] Those skilled in the art will understand that appropriate dosages can vary from patient to patient. Determining the optimal dosage typically involves balancing the level of therapeutic benefit against any risks or adverse side effects. The immunogenic compositions of the present invention should be administered in an amount sufficient to induce an immune response against Mycoplasma pneumoniae. The level of the immune response can be determined by methods known in the art. The appropriate amount of the immunogen (i.e., the fusion protein or antigenic peptide in the fusion protein disclosed herein) can be determined based on the specific disease or condition to be treated or prevented, its severity, the age of the subject, and other individual attributes of the subject (e.g., the general health status of the subject and the robustness of the subject's immune system). The determination of an effective dosage is also guided by animal model studies, followed by human clinical trials, and by administration regimens that significantly reduce the occurrence or severity of the target disease symptoms or condition in the subject.

[0082] For prophylactic application, the fusion protein, nucleic acid molecule, vector, host cell, or composition (e.g., immunogenic composition) of the present invention is provided prior to any symptoms, such as prior to infection. Prophylactic application of the immunogenic composition is intended to prevent or mitigate any subsequent infection, to reduce the expected severity, duration, or extent of symptoms of infection and / or related disease after exposure to or suspected exposure to a virus, or after actual onset of infection. Therefore, in some embodiments, the subject to be treated is a subject with or at risk of developing Mycoplasma pneumoniae infection, for example, due to exposure to or potential exposure to Mycoplasma pneumoniae. Following administration of a therapeutically effective amount of the disclosed therapeutic composition, the subject may be monitored for Mycoplasma pneumoniae infection, or symptoms associated with Mycoplasma pneumoniae infection.

[0083] For therapeutic applications, the fusion proteins, nucleic acid molecules, vectors, host cells, or compositions (e.g., immunogenic compositions) of the present invention are provided during or after the onset of symptoms of a disease or infection, such as after the onset of symptoms of Mycoplasma pneumoniae infection or after a diagnosis of Mycoplasma pneumoniae infection.

[0084] In one aspect, the present invention relates to the use of fusion proteins as disclosed herein, nucleic acid molecules as disclosed herein, vectors comprising said nucleic acid molecules, or host cells as disclosed herein in the preparation of formulations for inducing an immune response against Mycoplasma pneumoniae in a subject and / or for preventing and / or treating Mycoplasma pneumoniae infection or disease associated with Mycoplasma pneumoniae infection in a subject.

[0085] In one aspect, the present invention relates to the use of an antigenic polypeptide in the preparation of formulations for inducing an immune response against Mycoplasma pneumoniae in a subject and / or for the prevention and / or treatment of Mycoplasma pneumoniae infection or disease associated with Mycoplasma pneumoniae infection in a subject, wherein the antigenic polypeptide is derived from Mycoplasma pneumoniae P1 adhesion protein, preferably, the antigenic polypeptide comprises the sequence shown for amino acid residues 1376-1521 of the Mycoplasma pneumoniae M129 strain P1 adhesion protein. In some embodiments, the antigenic polypeptide comprises the amino acid sequence shown in SEQ ID NO:2.

[0086] In some embodiments, the illness associated with Mycoplasma pneumoniae infection is a respiratory infection, such as mycoplasma pneumoniae pneumonia or atypical pneumonia. In some embodiments, the preparation is a vaccine, such as a protein vaccine. In some embodiments, the preparation is administered to the subject nasally or subcutaneously. In some embodiments, the subject is a mammal, such as a human. Beneficial effects of the present invention

[0087] Compared with the prior art, the present invention has the following beneficial effects:

[0088] Compared to other P1 protein fragments (such as P1C) 1287-1521 The P1 protein fragment P1C of this invention 1376-1521 The soluble expression level was significantly higher in the *E. coli* expression system, and it produced a better immune response. This was achieved through P1C... 1376-1521 By fusing the protein with a mucosal adjuvant protein (such as CTB) to construct a recombinant fusion protein, immunization of mice via subcutaneous or pulmonary delivery induced high titers of antibodies against P1C. In particular, immunization of mice via the nasal route (pulmonary delivery) with the fusion protein induced significantly higher titers of specific IgA and IgG antibodies compared to the groups immunized with the P1 fragment without adjuvant and those immunized subcutaneously, demonstrating that the fusion protein of this invention is particularly suitable for inducing respiratory mucosal immune responses.

[0089] In summary, this invention provides a recombinant protein vaccine that is highly immunogenic, can induce broad-spectrum mucosal immune protection, has high safety, and is easy to produce, which has significant technological advancements and clinical value compared to existing technologies.

[0090] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0091] Example

[0092] Example 1. Detection of P1C protein fragment expression level and immunoprotective effect

[0093] P1 adhesion protein is an important candidate antigen for Mycoplasma pneumoniae protein vaccines, especially its C-terminal region, which is considered to contain important protective epitopes and is often used as a candidate protein component. However, different C-terminal fragments of the P1 protein may show significant differences in expression levels, stability, and immunoprotective efficacy. This invention selects two P1C fragments, namely P1C... 1376-1521 (SEQ ID NO: 2) and P1C 1287-1521 (SEQ ID NO: 1) was recombinantly expressed using the Escherichia coli prokaryotic expression system, and its expression level and immunoprotective effect were compared and evaluated.

[0094] Two protein fragments were reverse-translated into nucleotide sequences, optimized for E. coli codons, and inserted between the Nco I and Xho I restriction sites on the pET-28a expression plasmid. These sequences were then transformed into the BL21 (DE3) expression strain for subsequent protein expression. This work was commissioned to Beijing Qingke Biotechnology Co., Ltd.

[0095] Protein expression assay:

[0096] 1. Inoculate the two protein expression strains into 5 mL LB Kana (50 μg / mL) medium and incubate overnight at 37°C and 180 rpm;

[0097] 2. Transfer the overnight cultured strain to 500 mL LB Kana (50 μg / mL) medium and incubate at 37°C and 180 rpm until OD500. 600 =0.5;

[0098] 3. Add IPTG to a final concentration of 1 mM, and induce expression at 25°C and 180 rpm for 8 h;

[0099] 4. Centrifuge the expressed bacterial culture at 8000 rpm for 10 min to collect the bacterial cells and discard the supernatant;

[0100] 5. Resuspend the bacterial cells in 150 mL buffer A (20 mM PB, 0.5 M NaCl, 20 mM imidazole, pH 7.4), homogenize using an ultra-high pressure homogenizer (Ningbo Xinzhi Biotechnology Co., Ltd., SCIENTZ-207A), centrifuge at 8000 rpm to collect the supernatant, and filter the supernatant through a 0.22 μm filter membrane to remove impurities.

[0101] 6. Purify the target protein using the AKTA Pure protein purifier. First, equilibrate the HisTrapHP protein purification column with buffer A. Load the supernatant at 3 mL / min. After loading, wash away unbound protein with buffer A until the A280 UV absorbance is close to 0 mAU. Perform linear elution with buffer B (20 mM PB, 0.5 M NaCl, 0.5 M imidazole, pH 7.4), increasing to 100% buffer B after 50 min. Collect the protein based on the UV absorbance.

[0102] 7. The collected samples were analyzed and identified using a 4-20% protein precast gel to determine the protein expression level. Results are as follows: Figure 1 As shown, the same volume of bacterial culture was used for purification, P1C 1376-1521 The expression level of P1C was significantly higher than that of P1C. 1287-1521 .

[0103] Example 2. Evaluation of the immunoprotective effect of two P1C protein fragments

[0104] The purified protein concentration was determined using a BCA protein quantification kit. Fifteen 8-week-old SPF-grade BALB / c female mice were ordered from Beijing SPAF Biotechnology Co., Ltd., and randomly divided into three groups: a PBS control group, a P1C control group, and a control group. 1287-1521 Group, P1C 1376-1521 Each group consisted of 5 mice. 20 μg of protein was mixed with 50 μg of Al(OH)3 adjuvant and 20 μg of CpG2006 adjuvant, and then supplemented with physiological saline to a final volume of 100 μL. Mice were immunized subcutaneously in the nape of the neck for a total of three immunizations. Each immunization was performed 14 days after the first immunization. Fourteen days after the third immunization, mice were infected with Mycoplasma pneumoniae NCTC10119 via lung delivery, with each mouse infected with 1 × 10⁻⁶ NCTC10119. 8Four days after infection, mice were sacrificed in the CCU (Coronary Care Unit). Lung tissue was collected and mixed with PBS in equal weight ratios. After grinding, 10 mg of lung tissue was taken, and nucleic acid was extracted using the DNeasy blood & tissue kit (QIAGEN, 69504). The content of Mycoplasma pneumoniae was determined by digital PCR. The digital PCR detection reagent was the QIAcuity Probe PCR Kit (QIAGEN, 250102), and the digital PCR consumables were QIAcuity Nanoplate 26k 24-well (QIAGEN, 250001). The digital PCR primer and probe sequences were: upstream primer: 5'-GCAGTTGCTGGCGCTAAGTT-3' (SEQ ID NO: 7), downstream primer: 5'-AGCGAGGTACGGTAGCGGTAT-3' (SEQ ID NO: 8), and probe sequence: 5'-FAM-CTCGTTTTAGCGGGTACC-MGB-3' (SEQ ID NO: 9). The digital PCR detection system consisted of: 10 μL of 4×Probe PCR Master Mix, 3.2 μL of upstream primer (10 μM), 3.2 μL of downstream primer (10 μM), 1.6 μL of probe (10 μM), 17 μL of nuclease-free water, and 5 μL of genomic template. Results were as follows: Figure 2 As shown, P1C 1376-1521 The immune protection effect is better than P1C 1287-1521 via P1C 1376-1521 The levels of Mycoplasma pneumoniae were significantly lower in immunized mice.

[0105] Example 3. P1C 1376-1521 Expression effects of fusing different delivery carrier proteins

[0106] To further enhance P1C 1376-1521 To enhance the immunoprotective effect, we fused the protein to the N-terminus of *Pyrococcus furiosus* ferritin nanoparticles (PFFer), and to the C-terminus of cholera toxin B subunit (CTB) adjuvant protein and *Escherichia coli* heat-labile enterotoxin B subunit (LTB) adjuvant protein, linked by a (GGGGS)3 linker. The amino acid sequences of the three fusion proteins are shown in SEQ ID NO: 5, SEQ ID NO: 3, and SEQ ID NO: 6, respectively.

[0107] After reverse translating the amino acids into nucleotides, codon optimization was performed on *E. coli*, and the nucleotides were inserted between the Nco I and Xho I restriction sites on the pET-28a expression plasmid. The plasmid was then transformed into the BL21 (DE3) expression strain for subsequent protein expression. This work was commissioned to Beijing Qingke Biotechnology Co., Ltd.

[0108] P1C 1376-1521 The purification method for PFFer is as follows:

[0109] 1. Containing P1C 1376-1521 The BL21(DE3) strain expressing the PFFer expression plasmid was inoculated into 5 mL of LB kanamycin-resistant medium and cultured at 37°C and 180 rpm until the mid-to-late logarithmic growth phase. It was then transferred to 500 mL of LB kanamycin-resistant medium and cultured at 37°C and 180 rpm until the OD phase. 600 =0.6, add IPTG to a final concentration of 0.5mM, incubate overnight at 20℃ and 150rpm for 14h, and collect the expression cells by centrifugation at 8000rpm for 10min;

[0110] 2. Resuspend the bacterial cells in 80 mL of solution A (20 mM Tris-HCl (pH=8)), sonicate to disrupt the cells, centrifuge at 8000 rpm for 10 min, and collect the supernatant.

[0111] 3. Incubate the supernatant after centrifugation in a 70℃ water bath for 10 min to remove most of the heat-sensitive proteins. Centrifuge at 8000 rpm for 10 min and collect the supernatant. Dilute the supernatant to 200 mL with solution A.

[0112] 4. Ferritin nanoparticles were purified using a protein purifier. First, the HiTrap Q HP anion exchange chromatography column was equilibrated with solution A. After loading the diluted supernatant, it was leveled again with solution A. The elution flow rate was set to 1.5 mL / min, and linear elution was performed for 50 min with 50% solution B (20 mM Tris-HCl (pH=8), 0.5 M NaCl). Collection began after UV exposure increased and continued until 50% solution B was reached. Then, the column was washed with 100% solution B. No further collection was required at this point.

[0113] 5. Perform SDS-PAGE analysis on the collected liquid to identify the target protein.

[0114] CTB-P1C 1376-1521 With LTB-P1C 1376-1521 The expression and purification methods are as follows:

[0115] 1. Inoculate the two protein expression strains into 5 mL LB Kana (50 μg / mL) medium and incubate overnight at 37°C and 180 rpm;

[0116] 2. Transfer the overnight cultured strain to 500 mL LB Kana (50 μg / mL) medium and incubate at 37°C and 180 rpm until OD500. 600 =0.6;

[0117] 3. Add IPTG to a final concentration of 0.5 mM, incubate overnight at 18°C ​​and 150 rpm for 14 h, and collect the expression cells by centrifugation at 8000 rpm for 10 min;

[0118] 4. Centrifuge the expressed bacterial culture at 8000 rpm for 10 min to collect the bacterial cells and discard the supernatant;

[0119] 5. Resuspend the bacterial cells in 150 mL buffer A (20 mM PB, 0.5 M NaCl, 20 mM imidazole, pH 7.4), homogenize using an ultra-high pressure homogenizer (Ningbo Xinzhi Biotechnology Co., Ltd., SCIENTZ-207A), centrifuge at 8000 rpm to collect the supernatant, and filter the supernatant through a 0.22 μm filter membrane to remove impurities.

[0120] 6. Purify the target protein using the AKTA Pure protein purifier. First, equilibrate the HisTrapHP protein purification column with buffer A. Load the supernatant at 3 mL / min. After loading, wash away unbound protein with buffer A until the A280 UV absorbance is close to 0 mAU. Perform linear elution with buffer B (20 mM PB, 0.5 M NaCl, 0.5 M imidazole, pH 7.4), increasing to 100% buffer B after 50 min. Collect the protein based on the UV absorbance.

[0121] 7. Perform SDS-PAGE analysis on the collected liquid to identify the target protein.

[0122] Purification results are as follows Figure 3 As shown, P1C 1376-1521 -PFFer expression was highest, CTB-P1C 1376-1521 Expression levels higher than LTB-P1C 1376-1521 .

[0123] The key to ferritin nanoparticles' carrier delivery effect lies in their ability to self-assemble into 24-mer nanoparticles, thereby enhancing antigen presentation efficiency. Similarly, the key to CTB and LTB's adjuvant protein effects lies in the formation of pentamer proteins. We further used molecular sieve methods to determine whether the three proteins achieved correct assembly, and the results showed (e.g.) Figure 4 ), P1C 1376-1521 -PFFer are all in monomeric form and have not assembled into nanoparticles, while CTB-P1C 1376-1521 and LTB-P1C1376-1521 It can be assembled into a pentamer.

[0124] Based on the above results, it can be concluded that P1C 1376-1521 Although PFFer is expressed at high levels, it cannot self-assemble into nanoparticles, thus failing to exert its carrier delivery effect. (CTB-P1C) 1376-1521 and LTB-P1C 1376-1521 Soluble protein in pentamer form, CTB-P1C, can be obtained. 1376-1521 The expression level of the protein was higher than that of LTB-P1C. 1376-1521 Therefore, CTB-P1C was selected. 1376-1521 The form of vaccine to be evaluated later.

[0125] Example 4. Delivery Method Evaluation

[0126] Recombinantly expressed CTB-P1C 1376-1521 Mice were immunized with the protein via subcutaneous and pulmonary delivery, respectively, in three groups: PBS pulmonary delivery group, CTB-P1C group, and CTB-P1C group. 1376-1521 Subcutaneous immunization group, CTB-P1C 1376-1521 The lung delivery immunization group consisted of 8 animals per group, with a protein immunization dose of 5 μg. A total of three immunizations were administered, with each immunization two weeks apart. One week after the third immunization, blood was collected from the tail tip using ELISA to measure serum P1C levels. 1376-1521 Antibody titer.

[0127] To eliminate the influence of the His tag, GST-P1C was first completed. 1376-1521 The fusion protein was purified and prepared to evaluate serum P1C antibody levels. P1C... 1376-1521 The protein's nucleotide sequence was inserted between the BamHI and Xho I restriction sites of the pGEX-4T-1 expression plasmid and transformed into the BL21(DE3) expression strain for GST-P1C. 1376-1521 Protein expression was performed by Beijing Qingke Biotechnology Co., Ltd. (GST-P1C) 1376-1521 The protein expression and purification methods are as follows:

[0128] 1. GST-P1C 1376-1521 The protein-expressing strain was inoculated into 5 mL of LB Amp (100 μg / mL) medium and cultured overnight at 37°C and 180 rpm.

[0129] 2. Transfer the overnight cultured strain to 500 mL LB Amp (100 μg / mL) medium and incubate at 37°C and 180 rpm until OD500. 600 =0.6;

[0130] 3. Add IPTG to a final concentration of 1 mM, induce at 25°C and 150 rpm for 6 h, and collect the expression cells by centrifugation at 8000 rpm for 10 min;

[0131] 4. Centrifuge the expressed bacterial culture at 8000 rpm for 10 min to collect the bacterial cells and discard the supernatant;

[0132] 5. Resuspend the bacterial cells in 150 mL PBS, homogenize them using an ultra-high pressure homogenizer (Ningbo Xinzhi Biotechnology Co., Ltd., SCIENTZ-207A), centrifuge at 8000 rpm to collect the supernatant, and filter the supernatant through a 0.22 μm filter membrane to remove impurities.

[0133] 6. Purify the target protein using the AKTA Pure protein purification system. First, equilibrate the GSTrapFF protein purification column with PBS solution. Load the supernatant at a rate of 2 mL / min. After loading, wash away unbound protein with PBS solution until the A280 UV absorbance is close to 0 mAU. Elute with buffer B (50 mM Tris-HCl, 10 mM reduced glutathione, pH=8.0) and collect the elution based on the UV absorbance.

[0134] 7. Perform SDS-PAGE analysis on the collected liquid. Figure 5 ), to identify the target protein.

[0135] ELISA was used to detect P1C in serum after immunization. 1376-1521 The antibody levels were determined using the following ELISA method: purified GST-P1C... 1376-1521 Recombinant protein was used as the coating antigen, diluted to 1 μg / mL with coating buffer. 100 μL was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. After discarding the coating buffer, the plate was washed three times with TBST buffer and patted dry. 100 μL / well of blocking buffer (TBST buffer containing 3% BSA) was added, and the plate was blocked overnight at 4°C. The blocking buffer was then discarded and the plate was patted dry. Post-immunization mouse serum was serially diluted 10-fold with TBST, with double replicates for each dilution. TBST was used as a negative control, and the plate was incubated at 37°C for 1 h. After washing the plate three times with TBST, 100 μL / well of HRP-labeled goat anti-mouse IgG secondary antibody (1:10000 dilution) was added, and the plate was incubated at 37°C in the dark for 30 min. After washing, 100 μL of single-component TMB chromogenic solution was added, and the plate was incubated at room temperature in the dark for 10 min. Finally, 50 μL of stop solution was added to terminate the reaction. The OD of each well was measured using an ELISA reader. 450The antibody titer was determined by adding three times the mean square deviation to the average optical density value of the negative control. The dilution factor of serum corresponding to optical density values ​​exceeding this threshold was used as the antibody titer. From the perspective of antibody titer, both pulmonary delivery and subcutaneous immunization could induce specific antibodies against P1C, with pulmonary delivery showing better immunization efficacy than subcutaneous immunization. Figure 6 ).

[0136] Fourteen days after the third immunization, mice were infected with Mycoplasma pneumoniae NCTC10119 via lung delivery, with each mouse infected with 1×10⁻⁶ NCTC10119. 8 In the CCU, mice were sacrificed 4 days after infection, and lung tissue was collected. PBS was added in equal weight proportions, and the tissue was ground and homogenized. 10 mg of lung tissue was collected, and nucleic acid was extracted using the DNeasy blood & tissue kit (QIAGEN, 69504). The content of Mycoplasma pneumoniae was determined using digital PCR. The digital PCR detection reagent was the QIAcuity Probe PCR Kit (QIAGEN, 250102), and the digital PCR consumables were QIAcuity Nanoplate 26k 24-well (QIAGEN, 250001). The digital PCR primer and probe sequences were: upstream primer: 5'-GCAGTTGCTGGCGCTAAGTT-3', downstream primer: 5'-AGCGAGGTACGGTAGCGGTAT-3', and probe sequence: 5'-FAM-CTCGTTTTAGCGGGTACC-MGB-3'. The digital PCR detection system consisted of: 10 μL of 4×Probe PCR Master Mix, 3.2 μL of upstream primer (10 μM), 3.2 μL of downstream primer (10 μM), 1.6 μL of probe (10 μM), 17 μL of nuclease-free water, and 5 μL of genomic template. Results were as follows: Figure 7 As shown, both pulmonary delivery and subcutaneous immunization can produce immune protection, with pulmonary delivery showing better results than subcutaneous immunization.

[0137] The above results indicate that, through CTB-P1C 1376-1521 Both subcutaneous and pulmonary delivery of the protein can produce immunoprotective effects. Pulmonary delivery can induce higher antibody titers against P1C and significantly reduce the pathogen load in immunized mice.

[0138] Example 5. Evaluation of the Immunogenic Effect of Different Adjuvants

[0139] To further confirm CTB-P1C 1376-1521 The immunoprotective effect of recombinant proteins was investigated in four experimental groups, including P1C. 1376-1521 Protein (5 μg) + CpG adjuvant group, CTB-P1C 1376-1521(1μg) group, CTB-P1C 1376-1521 (2.5μg) group, CTB-P1C 1376-1521 The (5μg) group and the PBS control group were both immunized via lung delivery. Seven days after immunization, blood was collected from the tail tip to measure serum P1C levels. 1376-1521 Antibody titers of the protein. Results showed that all CTB-P1C... 1376-1521 IgA in the vaccine group ( Figure 8 A) and IgG ( Figure 8 B) Antibody titers were significantly better than P1C. 1376-1521 The protein + CpG adjuvant group indicates that P1C 1376-1521 After the protein is fused with the CTB component, its immunogenicity can be effectively enhanced, stimulating a more balanced and effective Th1 and Th2 immune response, which is superior to CpG adjuvant compatibility.

[0140] Fourteen days after the third immunization, mice were infected with Mycoplasma pneumoniae, and their weight was monitored daily. Results showed that all vaccine groups effectively alleviated post-infection weight loss, with CTB-P1C showing the best results. 1376-1521 The group experienced a smaller weight loss. Figure 9 A) Mice were sacrificed 4 days after infection, and lung tissue was collected, ground, and nucleic acid was extracted. The concentration of the pathogen in the lung tissue was determined using digital PCR. The results showed that P1C 1376-1521 After the protein is fused with the CTB component, the immunoprotective effect of the protein can be effectively enhanced. Even at an immunization dose as low as 1 μg, it can still effectively clear Mycoplasma pneumoniae, and is significantly superior to P1C. 1376-1521 Protein (5 μg) + CpG adjuvant group ( Figure 9 B).

[0141] The present application has been described in detail above with reference to specific embodiments and exemplary examples; however, these descriptions should not be construed as limiting the present application. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and implementation methods of the present application without departing from the spirit and scope of the present application, and all such modifications and improvements fall within the scope of the present application. The scope of protection of the present application is determined by the appended claims.

[0142] Sequence Summary

[0143] Information on some sequences involved in this invention is provided in the table below.

Claims

1. A fusion protein, said fusion protein comprising: (a) An antigenic polypeptide derived from the P1 adhesion protein of Mycoplasma pneumoniae, said antigenic polypeptide comprising a sequence represented by amino acid residues 1376-1521 of the P1 adhesion protein of Mycoplasma pneumoniae M129 strain, or an amino acid sequence having at least 95% sequence identity with it; and (b) An adjuvant protein operatively linked to the antigenic polypeptide.

2. The fusion protein of claim 1, wherein the antigenic polypeptide comprises an amino acid sequence as shown in SEQ ID NO:2, or an amino acid sequence having at least 95% sequence identity with SEQ ID NO:

2.

3. The fusion protein of claim 1 or 2, wherein the adjuvant protein is CTB or a variant thereof.

4. The fusion protein of claim 1 or 2, wherein the adjuvant protein comprises an amino acid sequence as shown in SEQ ID NO:4, or an amino acid sequence having at least 95% sequence identity with SEQ ID NO:

4.

5. The fusion protein of any one of claims 1-4, wherein the operably linked connection is a direct connection or a connection via a peptide linker, for example, the peptide linker being a peptide linker comprising one or more glycines and / or one or more serines.

6. The fusion protein of any one of claims 1-5, wherein the antigenic polypeptide is operatively linked to the C-terminus of the adjuvant protein.

7. The fusion protein of any one of claims 1-6, comprising the amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence having at least 95% sequence identity with SEQ ID NO:

3.

8. A nucleic acid molecule comprising a nucleotide sequence encoding a fusion protein as described in any one of claims 1-7.

9. A vector comprising the nucleic acid molecule as described in claim 8.

10. A host cell comprising the nucleic acid molecule of claim 8 or the vector of claim 9, preferably, the host cell being a prokaryotic cell.

11. A method for preparing the fusion protein according to any one of claims 1-7, comprising: The host cell of claim 10 is cultured under suitable conditions, and the fusion protein is recovered from the cell culture.

12. A composition comprising the fusion protein of any one of claims 1-7, the nucleic acid molecule of claim 8, the vector of claim 9, or the host cell of claim 10; Optionally, the composition is a pharmaceutical composition or an immunogenic composition (e.g., a vaccine composition); Optionally, the composition may further comprise a pharmaceutically acceptable carrier and / or excipient.

13. Use of the fusion protein of any one of claims 1-7, the nucleic acid molecule of claim 8, the vector of claim 9, or the host cell of claim 10 in the preparation of formulations for inducing an immune response against Mycoplasma pneumoniae in a subject and / or for preventing and / or treating Mycoplasma pneumoniae infection or disease associated with Mycoplasma pneumoniae infection in a subject.

14. Use of the antigenic polypeptide in the preparation of formulations for inducing an immune response against Mycoplasma pneumoniae in a subject and / or for the prevention and / or treatment of Mycoplasma pneumoniae infection or disease associated with Mycoplasma pneumoniae infection in a subject, wherein the antigenic polypeptide comprises the sequence shown as amino acid residues 1376-1521 of the P1 adhesion protein of Mycoplasma pneumoniae strain M129. Optionally, the antigenic polypeptide comprises an amino acid sequence as shown in SEQ ID NO:

2.

15. The use as described in claim 13 or 14, wherein the disease associated with Mycoplasma pneumoniae infection is a respiratory infection, such as mycoplasma pneumoniae.

16. The use according to any one of claims 13-15, wherein the formulation is a vaccine.

17. The use according to any one of claims 13-16, wherein the formulation is administered to the subject via the nose or subcutaneous route.

18. The use according to any one of claims 13-17, wherein the subject is a mammal, such as a human.

19. Use of the fusion protein of any one of claims 1-7 in the production of an antibody against Mycoplasma pneumoniae, for example, said antibody being a polyclonal antibody or a monoclonal antibody.

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