A pharmaceutical composition for preventing and / or treating clostridium difficile infection and use thereof

Abstract

This invention provides a pharmaceutical composition for the prevention and / or treatment of Clostridium difficile infection and its use therein. The pharmaceutical composition of this invention comprises a fusion protein and an immune adjuvant, wherein the fusion protein comprises an epitope fusion peptide having an HTL epitope cluster, a CTL epitope cluster, and a B-cell epitope cluster, wherein the HTL epitope cluster comprises one or more HTL epitopes selected from those shown in SEQ ID NO. 1 to SEQ ID NO. 7; the CTL epitope cluster comprises one or more CTL epitopes selected from those shown in SEQ ID NO. 8 to SEQ ID NO. 17; and the B-cell epitope cluster comprises one or more B-cell epitopes selected from those shown in SEQ ID NO. 18 to SEQ ID NO. 26. The pharmaceutical composition of this invention is effective in preventing and treating Clostridium difficile infection and exhibits high safety and broad-spectrum activity.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. Specifically, this invention relates to a pharmaceutical composition for the prevention and / or treatment of Clostridium difficile infection and its use. Background Technology

[0002] Clostridium difficile (C. difficile) is one of the leading pathogens of hospital-acquired infections and antibiotic-associated diarrhea (CDI) worldwide. Infection can cause a variety of intestinal-related diseases, ranging from mild diarrhea to pseudomembranous colitis, and in severe cases, toxic megacolon and even death. With an aging population and the increasing use of antibiotics, the incidence and recurrence rate of CDI have risen significantly, becoming a major challenge for global healthcare systems. In 2019, the U.S. Centers for Disease Control and Prevention (CDC) listed Clostridium difficile as an "Urgent Threat" in its "Antibiotic Resistance Threat Report," urging proactive prevention and control measures. In 2023, data from 10 Emerging Infections Program (EIP) surveillance stations in the United States showed that the crude incidence rate of CDI in the U.S. in 2021 was approximately 110.2 per 100,000. Data from the European Centre for Disease Prevention and Control (ECDC) shows that from 2018 to 2020, EU / EEA acute care hospitals reported a cumulative total of 86,034 healthcare-associated clinical injury diseases (CDIs) (HA-CDIs), with an average incidence density of 2.58 cases per 10,000 inpatient days (95% CI: 2.42–2.75). Related studies in my country indicate that the overall prevalence of CDIs is approximately 11.4% (95% CI: 9.1%–13.9%), suggesting a high disease burden of CDIs in my country as well.

[0003] Currently, CDI treatment still primarily relies on antibiotics. However, existing drugs are insufficient to eliminate spores and may exacerbate gut microbiota imbalance, thereby increasing the risk of relapse. While adjuvant or alternative therapies such as febrile malignant tumors (FMT), monoclonal antibodies, and probiotics show promise, their effectiveness is limited by factors such as efficacy variations, safety, accessibility, and long-term stability, making them insufficient to meet the long-term prevention and control needs of CDI. Therefore, developing safe, effective, and widely applicable proactive immunization strategies is of great significance.

[0004] Current research has identified toxin A (TcdA), toxin B (TcdB), and some surface-associated proteins (such as FliC, FliD, and Cwp84) as immune targets, designing various candidate vaccines. Some of these have entered animal or clinical trials, demonstrating certain immunogenicity and protective effects. However, existing CDI candidate vaccines still have limitations: toxin-targeted vaccines mostly rely on TcdA / TcdB neutralization, offering limited intervention against bacterial colonization, spore transmission, and recurrence; non-toxin antigen vaccines are mostly based on single or a few surface antigens, resulting in insufficient broad-spectrum coverage and protective efficacy; and research on the combined application of toxin antigens with flagellar, adhesion, cell wall, and spore-associated antigens is still insufficient. Currently, there are no approved CDI prophylactic vaccines on the market, and multi-antigen, multi-epitope combined strategies still require further systematic validation.

[0005] Therefore, there is an urgent need to provide feasible technical solutions that meet the pressing clinical needs for the prevention and control of CDI and can effectively reduce the incidence and recurrence rate of the disease. Summary of the Invention

[0006] Therefore, the object of the present invention is to address the above-mentioned deficiencies by providing a pharmaceutical composition for the prevention and / or treatment of Clostridium difficile infection and its use therein.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a pharmaceutical composition comprising a fusion protein and an immune adjuvant, wherein the fusion protein comprises an epitope fusion peptide having an HTL epitope cluster, a CTL epitope cluster, and a B cell epitope cluster; wherein:

[0009] The HTL epitope cluster includes one or more HTL epitopes selected from those shown in SEQ ID NO. 1 to SEQ ID NO. 7;

[0010] The CTL epitope cluster includes one or more CTL epitopes selected from those shown in SEQ ID NO. 8 to SEQ ID NO. 17;

[0011] The B-cell epitope cluster comprises one or more B-cell epitopes selected from those shown in SEQ ID NO. 18 to SEQ ID NO. 26.

[0012] Specifically, the amino acid sequences shown in SEQ ID NO. 1 to SEQ ID NO. 26 are as follows:

[0013] SEQ ID NO. 1:YYFNTNTAEAATGWQ

[0014] SEQ ID NO. 2: IETGWIYDMENESDK

[0015] SEQ ID NO. 3:GEMAVKILNGESVSD

[0016] SEQ ID NO. 4:GCTEFVALAFPAVRA

[0017] SEQ ID NO. 5:MEKLSSGVRIKRAAD

[0018] SEQ ID NO. 6:AEKINYQFAVSQMAE

[0019] SEQ ID NO. 7:GAKYEVYYAPVVNGV

[0020] SEQ ID NO. 8:NIEGQAIVY

[0021] SEQ ID NO. 9:NIEGQAIRY

[0022] SEQ ID NO. 10:QELVERWNL

[0023] SEQ ID NO. 11:HQNTLDENF

[0024] SEQ ID NO. 12:KALEKNGFK

[0025] SEQ ID NO. 13:RVDYVEFTF

[0026] SEQ ID NO. 14:IQPRLVDTF

[0027] SEQ ID NO. 15:RVNTNVSAL

[0028] SEQ ID NO. 16:KEKRLYAKY

[0029] SEQ ID NO. 17:DLSEEHLRW

[0030] SEQ ID NO. 18:EYFAPANTDANNIEG

[0031] SEQ ID NO. 19:FEYFAPANTHNNNIE

[0032] SEQ ID NO. 20:HNMSENTVVEKYLKLK

[0033] SEQ ID NO. 21:TLDENLEGEAIDFTG

[0034] SEQ ID NO. 22: CSQGGDSGNSKQESN

[0035] SEQ ID NO. 23: VGRNCETTFEFFAVCGE

[0036] SEQ ID NO. 24:RAKLGAQQNRLESTQ

[0037] SEQ ID NO. 25:AEKINYQFAVSQMAEP

[0038] SEQ ID NO. 26:EKDIPYNLKSEAQGAT

[0039] According to some embodiments of the present invention, the B-cell epitope cluster is located at the C-terminus of the epitope fusion peptide. Accordingly, in the epitope fusion peptide of the present invention, the three epitope clusters can be connected in series in the order of HTL epitope cluster, CTL epitope cluster, and B-cell epitope cluster from N-terminus to C-terminus, or they can be connected in series in the order of CTL epitope cluster, HTL epitope cluster, and B-cell epitope cluster from N-terminus to C-terminus. The three epitope clusters can be directly connected or connected through a adapter. The adapter can be a commonly used adapter in the art, such as KK, AAY, GGPPG, GGGS, GGGGS, GGSSG, GS, GPG, or EAAAK, etc.

[0040] According to some embodiments of the present invention, the HTL epitope cluster includes HTL epitopes as shown in SEQ ID NO. 1 to SEQ ID NO. 7.

[0041] Preferably, in the HTL epitope cluster, the HTL epitopes, such as those shown in SEQ ID NO. 1 to SEQ ID NO. 7, are connected in series sequentially. These HTL epitopes can be directly connected or connected via connectors. The connectors can be commonly used connectors in the art, such as KK, AAY, or GGPPG connectors.

[0042] More preferably, the amino acid sequence of the HTL epitope cluster comprises or is composed of the amino acid sequence shown in SEQ ID NO. 27.

[0043] SEQ ID NO. 27:

[0044] YYFNTNTAEAATGWQKKIETGWIYDMENESDKKKGEMAVKILNGESVSDKKGCTEFVALAFPAVRAKKMEKLSSGVRIKRAADKKAEKINYQFAVSQMAEKKGAKYEVYYAPVVNGV

[0045] According to some embodiments of the present invention, the CTL epitope cluster includes CTL epitopes as shown in SEQ ID NO. 8 to SEQ ID NO. 17.

[0046] Preferably, in the CTL epitope cluster, the CTL epitopes, such as those shown in SEQ ID NO. 8 to SEQ ID NO. 17, are connected in series sequentially. These CTL epitopes can be directly connected or connected via connectors. The connectors can be commonly used connectors in the art, such as KK, AAY, or GGPPG connectors.

[0047] More preferably, the amino acid sequence of the CTL epitope cluster comprises or is composed of the amino acid sequence shown in SEQ ID NO. 28.

[0048] SEQ ID NO. 28:

[0049] NIEGQAIVYAAYNIEGQAIRYAAYQELVERWNLAAYHQNTLDENFAAYKALEKNGFKAAYRVDYVEFTFAAYIQPRLVDTFAAYRVNTNVSALAAYKEKRLYAKYAAYDLSEEHLRW

[0050] According to some embodiments of the present invention, the B-cell epitope cluster includes B-cell epitopes as shown in SEQ ID NO. 18 to SEQ ID NO. 26.

[0051] Preferably, in the B-cell epitope cluster, the B-cell epitopes, such as those shown in SEQ ID NO. 18 to SEQ ID NO. 26, are sequentially connected in series. These B-cell epitopes can be directly connected or connected via adapters. The adapters can be adapters commonly used in the art, such as KK, AAY, or GGPPG.

[0052] More preferably, the amino acid sequence of the B cell epitope cluster comprises or is composed of the amino acid sequence shown in SEQ ID NO. 29.

[0053] SEQ ID NO. 29:

[0054] EYFAPANTDANNIEGKKFEYFAPANTHNNNIEKKHNMSENTVVEKYLKLKKKTLDENLEGEAIDFTGKKCSQGGDSGNSKQESNKKVGRNCETTFEFFAVCGEKKRAKLGAQQNRLESTQKKAEKINYQFAVSQMAEPKKEKDIPYNLKSEAQGAT

[0055] According to some preferred embodiments of the present invention, the amino acid sequence of the epitope fusion peptide comprises the amino acid sequence shown in SEQ ID NO. 30, or the amino acid sequence of the epitope fusion peptide is shown in SEQ ID NO. 30.

[0056] SEQ ID NO. 30:

[0057] YYFNTNTAEAATGWQKKIETGWIYDMENESDKKKGEMAVKILNGESVSDKKGCTEFVALAFPAVRAKKMEKLSSGVRIKRAADKKAEKINYQFAVSQMAEKKGAKYEVYYAPVVNGVAAYNIEGQAIVYAAYNIEGQAIRYAAYQELVERWNLAAYHQNTLDENFAAYKALEKNGFKAAYRVDYVEFTFAAYIQPRL VDTFAAYRVNTNVSALAAYKEKRLYAKYAAYDLSEEHLRWKKEYFAPANTDANNIEGKKFEYFAPANTHNNNIEKKHNMSENTVVEKYLKLKTLDENLEGEAIDFTGKKCSQGGDSGNSKQESNKKVGRNCETTFEFFAVCGEKKRAKLGAQQNRLESTQKKAEKINYQFAVSQMAEPKKEKDIPYNLKSEAQGAT

[0058] According to other preferred embodiments of the present invention, the amino acid sequence of the epitope fusion peptide comprises the amino acid sequence shown in SEQ ID NO. 31, or the amino acid sequence of the epitope fusion peptide is shown in SEQ ID NO. 31.

[0059] SEQ ID NO. 31:

[0060] NIEGQAIVYAAYNIEGQAIRYAAYQELVERWNLAAYHQNTLDENFAAYKALEKNGFKAAYRVDYVEFTFAAYIQPRLVDTFAAYRVNTNVSALAAYKEKRLYAKYAAYDLSEEHLRWKKYYFNTNTAEAATGWQKKIETGWIYDMENESDKKKGEMAVKILNGESVSDKKGCTEFVALAFPAVRAKKMEKLSSGVR IKRAADKKAEKINYQFAVSQMAEKKGAKYEVYYAPVVNGVKKEYFAPANTDANNIEGKKFEYFAPANTHNNNIEKKHNMSENTVVEKYLKKKTLDENLEGEAIDFTGKKCSQGGDSGNSKQESNKKVGRNCETTFEFAVCGEKKRAKLGAQQNRLESTQKKAEKINYQFAVSQMAEPKKEKDIPYNLKSEAQGAT

[0061] According to some embodiments of the present invention, the fusion protein further comprises a panepitope region and an adjuvant region sequentially connected to the N-terminus of the epitope fusion peptide.

[0062] Preferably, the generic region includes PADRE.

[0063] Preferably, the adjuvant region comprises one or more selected from the following: cholera enterotoxin B subunit (CTXB), type II heat-sensitive enterotoxin C subunit (LT-IIC), type II heat-sensitive enterotoxin B subunit (LT-IIB), 50S ribosome L7 / L12 (i.e., 50S ribosome large subunit protein L7 / L12), β-defensin, RS09, and Pam2Cys.

[0064] In this invention, RS09 is a synthetic heptapeptide with the amino acid sequence APPHALS (SEQ ID NO.36), which belongs to the TLR4 specific agonist and its core function is to mimic LPS to activate innate immunity.

[0065] In this invention, Pam2Cys, or dipalmitoylated cysteine, is a synthetic diacylated lipopeptide and a classic TLR2 / 6 agonist.

[0066] Furthermore, the N-terminus of the fusion protein also includes an initiation methionine (M) to initiate protein synthesis.

[0067] According to some embodiments of the present invention, the fusion protein further comprises a 6×His tag linked to the C-terminus of the epitope fusion peptide.

[0068] The fusion protein of this invention comprises segments that can be directly connected or connected via adapters. The adapters can be those commonly used in the art, such as KK, AAY, or GGPPG.

[0069] According to some embodiments of the present invention, the fusion protein comprises or is composed of the following amino acid sequence:

[0070] (1) The amino acid sequence shown in any one of SEQ ID NO. 32 to SEQ ID NO. 35;

[0071] (2) An amino acid sequence obtained by deleting, substituting, or adding one or more amino acids, such as 1, 2, 3, 4, or 5, from any of the amino acid sequences shown in any of SEQ ID NO. 32 to SEQ ID NO. 35; or

[0072] (3) An amino acid sequence that has more than 90% homology with any of the amino acid sequences shown in SEQ ID NO. 32 to SEQ ID NO. 35.

[0073] Specifically, the amino acid sequences described in SEQ ID NO. 32 to SEQ ID NO. 35 are as follows:

[0074] SEQ ID NO. 32:

[0075] MTPQNITDLCAEYHNTQIYTLNDKIFSYTESLAGKREMAIITFKNGAIFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNKTPHAIAAISMANEAAAKAKFVAAWTLKAAAKKYYFNTNT AEAATGWQKKIETGWIYDMENESDKKKGEMAVKILNGESVSDKKGCTEFVALAFPAVRAKMEKLSSGVRIKRAADKKAEKINYQFAVSQMAEKKGAKYEVYYAPVVNGVAAYNIEGQAIVYAAYNIEGQA IRYAAYQELVERWNLAAYHQNTLDENFAAYKALEKNGFKAAYRVDYVEFTFAAYIQPRLVDTFAAYRWNTNVSALAAYKEKRLYAKYAAYDLSEEHLRWKKEYFAPANTDANNIEGKKFEYFAPANTHNNN IEKKHNMSENTVVEKYLKLKKTDLDENLEGEAIDFTGKKCSQGGDSGNSKQESNKKVGRNCETTFEFAVCGEKKRAKLGAQQNRLESTQKKAEKINYQFAVSQMAEPKKEKDIPYNLKSEAQGATHHHHH

[0076] SEQ ID NO. 33:

[0077] MVSKTFKDKCASTTAKLVQSVQLVNISSDVNKDSKGIYISSSAGKTWFIPGGQYYPDNYLSNEMRKIAMAAVLSNVRVNLCASEAYTPNHVWAIELAPEAAAKAKFVAAWTLKAAAKKYYFNTNTAEAAA TGWQKKIETGWIYDMENESDKKKGEMAVKILNGESVSDKKGCTEFVALAFPAVRAKKMEKLSSGVRIKRAADKKAEKINYQFAVSQMAEKKGAKYEVYYAPVVNGVAAYNIEGQAIVYAAYNIEGQAIRY AAYQELVERWNLAAYHQNTLDENFAAYKALEKNGFKAAYRVDYVEFTFAAYIQPRLVDTFAAYRWNTNVSALAAYKEKRLYAKYAAYDLSEEHLRWKKEYFAPANTDANNIEGKKFEYFAPANTHNNNI EKKHNMSENTVVEKYLKKKTLDENLEGEAIDFTGKKCSQGGDSGNSKQESNKKVGRNCETTFEFAVCGEKKRAKLGAQQNRLESTQKKAEKINYQFAVSQMAEPKKEKDIPYNLKSEAQGATHHHHH

[0078] SEQ ID NO. 34:

[0079] MGASQFFKDNCNRTTASLVEGVELTKYSDINNNTDGMYVVSSTGGVWRISRAKDYPDNVMTAEMRKIAMAAVLSGMRVNMCASPASSPNVIWAIELEAAEEAAAKAKFVAAWTLKAAAKKYYFNTNTAEA ATGWQKKIETGWIYDMENESDKKKGEMAVKILNGESVSDKKGCTEFVALAFPAVRAKKMEKLSSGVRIKRAADKKAEKINYQFAVSQMAEKKGAKYEVYYAPVVNGVAAYNIEGQAIVYAAYNIEGQAIR YAAYQELVERWNLAAYHQNTLDENFAAYKALEKNGFKAAYRVDYVEFTFAAYIQPRLVDTFAAYRWNTNVSALAAYKEKRLYAKYAAYDLSEEHLRWKKEYFAPANTDANNIEGKKFEYFAPANTHNNNI EKKHNMSENTVVEKYLKKKTLDENLEGEAIDFTGKKCSQGGDSGNSKQESNKKVGRNCETTFEFAVCGEKKRAKLGAQQNRLESTQKKAEKINYQFAVSQMAEPKKEKDIPYNLKSEAQGATHHHHH

[0080] SEQ ID NO. 35:

[0081] MTPQNITDLCAEYHNTQIYTLNDKIFSYTESLAGKREMAIITFKNGAIFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANEAAAKAKFVAAWTLKAAAAAYNIEGQA IVYAAYNIEGQAIRYAAYQELVERWNLAAYHQNTLDENFAAYKALEKNGFKAAYRVDYVEFTFAAYIQPRLVDTFAAYRVNTNVSALAAYKEKRLYAKYAAYDLSEEHLRWKKYYFNTNTAEAATGWQKKI ETGWIYDMENESDKKKGEMAVKILNGESVSDKKGCTEFVALAFPAVRAKKMEKLSSGVRIKRAADKKAEKINYQFAVSQMAEKKGAKYEVYYAPVVNGVKKEYFAPANTDANNIEGKKFEYFAPANTHNNN IEKKHNMSENTVVEKYLKLKKKTLDENLEGEAIDFTGKKCSQGGDSGNSKQESNKKVGRNCETTFEFACGEKKRAKLGAQQNRLESTQKKAEKINYQFAVSQMAEPKKEKDIPYNLKSEAQGATHHHHHHH

[0082] According to some embodiments of the present invention, the immune adjuvant is a pharmaceutically acceptable immune adjuvant suitable for protein vaccines or multi-epitope vaccines, including but not limited to aluminum salt adjuvants, emulsion adjuvants, TLR receptor agonist adjuvants, saponin adjuvants, or combinations thereof.

[0083] Preferably, the immune adjuvant is selected from aluminum hydroxide (Al(OH)3), aluminum phosphate, MF59, MPLA, CpG oligodeoxynucleotide, Poly(I:C), QS-21 or a combination thereof; more preferably, the immune adjuvant is aluminum hydroxide (Al(OH)3) or MF59.

[0084] According to some embodiments of the present invention, the fusion protein is mixed with an exogenous immune adjuvant in a proportion suitable for inducing an immune response.

[0085] Preferably, when the exogenous immune adjuvant is aluminum hydroxide (Al(OH)3), the mass ratio of the fusion protein to aluminum hydroxide is 1:(1-10); when the exogenous immune adjuvant is MF59, the volume ratio of the fusion protein solution to MF59 is 1:(0.5-2), more preferably 1:1.

[0086] In a second aspect, the present invention provides the use of the pharmaceutical composition according to the first aspect of the present invention in the preparation of a medicament, wherein the medicament is used for the prevention and / or treatment of Clostridium difficile infection or diseases caused by Clostridium difficile infection.

[0087] Preferably, the disease caused by Clostridium difficile infection is selected from one or more of Clostridium difficile-associated diarrhea, Clostridium difficile-associated colitis, pseudomembranous colitis, toxic megacolon, intestinal perforation, sepsis, and septic shock.

[0088] Thirdly, the present invention provides a vaccine for the prevention and / or treatment of Clostridium difficile infection or diseases caused by Clostridium difficile infection, comprising an effective amount of the pharmaceutical composition according to the first aspect of the present invention, and optionally a pharmaceutically acceptable carrier or excipient.

[0089] Preferably, the disease caused by Clostridium difficile infection is selected from one or more of Clostridium difficile-associated diarrhea, Clostridium difficile-associated colitis, pseudomembranous colitis, toxic megacolon, intestinal perforation, sepsis, and septic shock.

[0090] In this invention, "effective amount" refers to the dosage that, after the pharmaceutical composition or vaccine of this invention is administered to the body, is sufficient to prevent or treat Clostridium difficile infection, reduce the incidence of infection, alleviate infection symptoms, inhibit the proliferation of pathogens, induce the body to produce a specific immune response, and achieve the expected preventive or therapeutic effect. This effective amount can be routinely adjusted according to the target population, route of administration, frequency of administration, age, weight, immune status, and severity of the disease, and can be routinely determined by those skilled in the art without excessive experimentation.

[0091] In this invention, "pharmaceutically acceptable carriers or excipients" refer to pharmaceutically suitable formulation adjuvants that are suitable for human or animal administration within a reasonable dosage range and do not affect the stability, immunogenicity, or safety of the fusion protein of this invention. These carriers or excipients include, but are not limited to, diluents, solvents, buffer solutions, stabilizers, lyophilization protectants, isotonic adjusters, suspending agents, emulsifiers, preservatives, excipients, or dispersants. Those skilled in the art can select appropriate carriers or excipients for formulation preparation based on the physicochemical properties of the fusion protein, the vaccine dosage form, and the route of administration.

[0092] In this invention, the specific preparation methods of the pharmaceutical composition, the drug, and the vaccine can refer to relevant schemes in the prior art, therefore this invention does not make specific limitations.

[0093] In this invention, the vaccine composition can be prepared into a pharmaceutically acceptable injectable dosage form according to conventional pharmaceutical processes in the art, including but not limited to injection solutions, sterile lyophilized powders for injection, suspensions, or pre-filled injectables. The vaccine composition is preferably administered via intramuscular injection, but subcutaneous or intradermal injection may also be used as needed. Those skilled in the art can select appropriate pharmaceutical carriers, excipients, or adjuvants for formulation based on the physicochemical properties of the vaccine antigen, the immunization schedule, and the administration requirements.

[0094] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0095] The epitope fusion peptides provided by this invention were obtained through screening seven key target proteins of Clostridium difficile: TcdA, TcdB, CD0873, CdeC, FliC, FliD, and Cwp84, and include CTL, HTL, and LBL epitopes. Immuninformatics analysis shows that these epitopes theoretically possess high antigenicity, high sequence conservation, no sensitization or potential toxicity, can bind efficiently to multiple HLA molecules, are fully exposed in the protein's three-dimensional structure, and have strong immunogenicity, thus providing stable, efficient, and broad-spectrum core antigenic elements for the construction of multi-epitope vaccines.

[0096] This invention also provides a fusion protein, WLcd2, which is designed and assembled based on the epitope fusion peptide of this invention. Bioinformatics prediction results show that WLcd2 has superior physicochemical properties, structural stability, and solubility; molecular docking and molecular dynamics analysis suggest that WLcd2 can form relatively stable interactions with TLR2, TLR4, and TLR5, possessing the potential to be recognized by innate immune receptors and induce immune responses. HLA population coverage analysis shows that it has high theoretical coverage potential in major countries and regions worldwide, and immune simulation results suggest that it can simultaneously activate humoral and cellular immune responses. This fusion protein can be efficiently expressed and purified using a prokaryotic system. Safety evaluation results indicate that it has no hemolytic toxicity, does not damage liver or kidney function, does not destroy the structure of major organs and tissues, and has no significant impact on the intestinal flora, demonstrating excellent biosafety and good drug development and industrialization prospects.

[0097] Subsequently, in vivo immunoassays showed that WLcd2 can induce the production of specific IgG, exhibiting a certain dose-dependent and programmed immune accumulation, while also activating Th1 / Th2-related immune responses and CD8⁺T cell-related responses.

[0098] Using the fusion protein WLcd2 of this invention as the active ingredient, and in combination with an adjuvant, compositions and vaccines for the prevention or adjuvant treatment of Clostridium difficile infection can be provided. Experimental results show that the adjuvant significantly enhances the antigen-specific immune response induced by WLcd2, increasing specific antibody levels by approximately 8–32 times, and enhances cellular immune-related responses. In a mouse model of Clostridium difficile R20291 infection, the survival rate of the WLcd2 combined with Al(OH)3 group reached 90%, and the survival rate of the WLcd2 combined with MF59 group reached 80%, while all animals in the model group and the adjuvant-only group died. Compared with the control group, WLcd2 combined with adjuvant immunization significantly alleviated infection-induced weight loss and clinical symptoms, reduced colonic tissue pathological damage, and downregulated the mRNA expression levels of colonic pro-inflammatory factors CXCL1, TNF-α, IL-1β, and IL-6, demonstrating good potential for immunoprotective applications. Attached Figure Description

[0099] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0100] Figure 1 The spatial distribution of CTL epitopes screened in the embodiments of the present invention on the surface of target proteins is shown. The epitopes are labeled in the form of "C+start amino acid position", for example, C1914 represents a CTL epitope starting at amino acid 1914.

[0101] Figure 2 The spatial distribution of HTL epitopes screened in the embodiments of the present invention on the surface of target proteins is shown. The epitopes are labeled in the form of "H+ starting amino acid position", for example, H2109 indicates an HTL epitope starting at amino acid 2109.

[0102] Figure 3 The spatial distribution of linear B-cell (LBL) epitopes screened in this embodiment of the invention on the surface of the target protein is shown. The epitopes are labeled in the form of "B+starting amino acid position", for example, B2284 represents an LBL epitope starting at amino acid 2284.

[0103] Figure 4 The molecular design and structural features of WLcd2 in embodiments of the present invention are shown. Figure 4 A shows the construction strategy and full-length amino acid sequence of WLcd2; Figure 4 B shows the results of the transmembrane region prediction analysis for WLcd2; Figure 4 C shows the results of the signal peptide prediction analysis for WLcd2.

[0104] Figure 5 The structure of WLcd2 and its structural quality verification in an embodiment of the present invention are shown. Figure 5A is a schematic diagram of the secondary structure distribution of WLcd2: gray represents random coils, pink represents α-helices, and yellow represents β-sheets. Numbers 1-524 in the diagram represent the positions of amino acid residues, and different colored areas visually demonstrate the distribution of the corresponding secondary structures within the sequence. Figure 5 B shows the optimized three-level structure model of WLcd2; Figure 5 C is the Ramachandran plot of the optimized WLcd2 structure, based on conformational analysis of the dihedral angle (φ / ψ) distribution of the main chain. The red area is the most favorable conformation region, the dark yellow area is the allowed conformation region, the light yellow area is the widely allowed conformation region, and the white area is the disallowed conformation region (where there is spatial conflict between main chain atoms). Figure 5 D is the Z-score distribution diagram of the optimized WLcd2 structure.

[0105] Figure 6 This illustrates the interaction pattern analysis between WLcd2 and TLR receptor molecules in embodiments of the present invention. Figure 6 A is a cartoon diagram of the binding conformation of WLcd2 and TLR2 (left) and the interacting amino acid residues (right). The light purple color represents WLcd2, the grass green color represents TLR2, and the red dashed line represents the intermolecular hydrogen bond. Figure 6 B is a cartoon illustration of the interaction between WLcd2 and TLR4 (left) and the interacting amino acid residues (right). Light purple represents WLcd2, light blue represents TLR4, and red dashed lines represent intermolecular hydrogen bonds. Figure 6 C represents the Cartoon diagram of the interaction between WLcd2 and TLR5 (left) and the interacting amino acid residues (right). Light purple represents WLcd2, light gray represents TLR5, and red dashed lines represent intermolecular hydrogen bonds.

[0106] Figure 7 The figure shows the molecular simulation kinetics of the WLcd2-TLR complex in embodiments of the present invention. The red curve represents the WLcd2-TLR2 complex, the green curve represents the WLcd2-TLR4 complex, and the purple curve represents the WLcd2-TLR5 complex. Figure 7 A is the RMSD curve of the WLcd2-TLR complex over time; Figure 7 B represents the time-series curve of the radius of gyration (Rg) of the complex; Figure 7 C represents the dynamic change in the number of hydrogen bonds between complexes; Figure 7 D represents the RMSF distribution of the WLcd2 peptide chain residues in the WLcd2-TLR2, WLcd2-TLR4, and WLcd2-TLR5 complexes.

[0107] Figure 8 The results of immune simulation of WLcd2 in an embodiment of the present invention are shown. Figure 8 A shows the phase changes in antigen and antibody (IgM, IgG, IgG1, IgG2) titers after immunization; Figure 8 B shows the dynamic evolution of the abundance of each B lymphocyte subset; Figure 8 C shows changes in the levels of helper T cells (TH cells) and memory helper T cells; Figure 8 D shows the dynamics of TH subsets in different activation states; Figure 8 E shows the changing trend of the number of cytotoxic T cells (TC cells); Figure 8 F shows the dynamics of TC cell subsets in different activation states.

[0108] Figure 9 The results of recombinant plasmid construction, prokaryotic expression, and purification of WLcd2 in embodiments of the present invention are shown. Figure 9 A is the pET-22b(+)-WLcd2 plasmid map; Figure 9 Lane B shows the results of double digestion of the recombinant plasmid with NdeI and XhoI; lane M is the DNA Marker; lane 1 shows the double digestion product of the pET-22b(+)-WLcd2 recombinant plasmid, which shows a vector backbone band of about 5000 bp and a WLcd2 target gene fragment of about 1572 bp; lane 2 is the control of the empty pET-22b(+) plasmid. Figure 9 C represents the results of SDS-PAGE analysis of the fusion protein expression trial. Lane M represents the protein markers. Lane 1 represents the total protein before induction. Lanes 2-6 represent the samples after IPTG induction (induction conditions: cell OD). 600 When the concentration is 0.6-0.8, add IPTG to a final concentration of 0.5 mM, incubate at 37°C with shaking for 4 hours, and then collect the bacterial cells. Figure 9 D represents the results of Western Blot analysis of fusion protein expression; lane M represents the protein marker; and lane 1 represents the target protein. Figure 9E represents the expression and solubility analysis results of WLcd2 under different induction conditions, and lane M represents the protein marker; Lane 1: 15℃, 0.2mM IPTG, induced for 16h; Lane 2: 15℃, 1.0mM IPTG, induced for 16h; Lane 3: 37℃, 0.2mM IPTG, induced for 4h; Lane 4: 37℃, 1.0mM IPTG, induced for 4h; Lane 5: Uninduced sample; Lane 6: 37℃, 1.0mM IPTG, induced for 4h followed by precipitation; Lane 7: 37℃, 1.0mM IPTG, induced for 4h followed by supernatant; Lane 8: 37℃, 0.2mM IPTG, induced for 4h followed by precipitation; Lane 9: 37℃, 0.2mM IPTG, induced for 4h followed by supernatant; Lane 10: 15℃, 1.0mM IPTG, induced for 16h followed by precipitation; Lane 11: 15℃, 1.0mM IPTG, induced for 16h followed by precipitation; IPTG, supernatant after 16 h induction; Lane 12: 0.2 mM IPTG at 15℃, precipitation after 16 h induction; Lane 13: 0.2 mM IPTG at 15℃, supernatant after 16 h induction. Figure 9 F represents the results of WLcd2 affinity purification SDS-PAGE assay. Lane M represents protein markers. Lane 1: precipitate after disruption. Lane 2: supernatant after disruption. Lane 3: eluent. Lane 4: washing sample. Lane 5: elution sample. Figure 9 G represents the final protein sample detected by SDS-PAGE, lane M represents the protein marker, and lane 1 represents the final renatured protein; Figure 9 H represents the final sample protein verified by Western blotting, lane M represents the protein marker, and lane 1 represents the target protein.

[0109] Figure 10 The security evaluation results of WLcd2 in the embodiments of the present invention are shown. Figure 10 A shows the results of the WLcd2 hemolytic activity assay; Figure 10 B shows the statistical results of hemolysis rate for each concentration of WLcd2; Figure 10 C to Figure 10 F shows the results of liver function biochemical indicators: alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), and albumin (ALB). Figure 10 G to Figure 10 H shows the results of renal function biochemical indicators: blood urea nitrogen (BUN) and creatinine (Cr); Figure 10 I shows the histopathological results of major organs (HE staining, heart, liver, lung, kidney) of mice immunized with different doses of WLcd2, scale bar = 100 μm; statistical marker: ns indicates no significant difference.

[0110] Figure 11The effects of WLcd2 on the gut microbiota of mice in embodiments of the present invention are shown. Figure 11 A is a schematic diagram of the experimental design, including the immunization protocol, antibiotic intervention, and stool collection time points; Figure 11 B shows the alpha diversity analysis, specifically the changes in the Shannon index at three time points (before immunization, after immunization, and after pretreatment). Different colors represent different experimental groups (Ctrl or mice receiving WLcd2), and "ns" indicates no significant difference between groups. Figure 11 C shows the β diversity analysis, based on principal coordinate analysis (PCoA) of ASV. Figure 11 D is a bar chart showing the relative abundance distribution at the phylum level, illustrating the changes in the composition of the main bacterial communities at different groups and time points; Figure 11 E is a bar chart showing the relative abundance distribution of genera at the genus level, illustrating the distribution of major genera at different groups and time points.

[0111] Figure 12 The results of WLcd2-induced humoral immune responses in embodiments of the present invention are shown. Figure 12 A shows the immunization process; Figure 12 B shows the binding activity of WLcd2 in the serum of mice immunized with different doses (10, 20, 30 μg / mouse); Figure 12 C shows the level of anti-WLcd2 IgG in the serum of mice immunized with different doses of WLcd2; Figure 12 D shows the level of anti-WLcd2 IgG in mouse serum at different time points after immunization with 30 μg WLcd2; Figure 12 E showed the binding activity of 30 μg WLcd2-immunized mouse serum to WLcd2; Figure 12 F shows the anti-WLcd2 IgG levels in the serum of mice immunized with 30 μg WLcd2 in combination with different adjuvants. The control group (Ctrl) received 100 μL of physiological saline; the Al(OH)3 group received an equal volume of Al(OH)3 + NS; the MF59 group received an equal volume of MF59 + NS; the WLcd2 + Al(OH)3 group received an equal volume of Al(OH)3 + WLcd2; and the WLcd2 + MF59 group received an equal volume of MF59 + WLcd2. Statistical markers: ns indicates no significant difference, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0112] Figure 13 The results of the WLcd2-induced cellular immune response analysis in this embodiment of the invention are shown. Figure 13 A is a schematic diagram of the experimental groups and the vaccine immunization process; Figure 13B shows the results of lymphocyte proliferation response in each group of mice. Lymphocytes were isolated 14 days after the last immunization, stimulated in vitro with 10 μg / mL WLcd2 for 48 h, and cell proliferation was detected by CCK-8 assay. Figure 13 C and Figure 13 D shows the secretion levels of IFN-γ and IL-4 in mouse lymphocytes after in vitro restimulation. Lymphocytes were isolated 14 days after the last immunization, stimulated in vitro with 10 μg / mL WLcd2 for 48 h, and the supernatant was collected for ELISA to detect cytokine levels. Figure 13 E shows the CD8 levels of mice in each group. + IFN-γ + Analysis of T cell (Th1-type cytotoxic T cell) proportion. Splenic lymphocytes were isolated 14 days after the last immunization and stimulated in vitro with 10 μg / mL WLcd2 for 1 h, followed by 5 h of induction with 2 μL / ml eBioscience™ Cell Stimulation Cocktail. CD8 count was then detected by flow cytometry. + IFN-γ + T / CD4 + IL-4 + The proportion of T cells. The left figure shows representative flow cytometry scatter plots for each group, and the right figure shows the statistical analysis results. Figure 13 F shows the CD4 count of mice in each group. + IL-4 + Analysis of the proportion of T cells (Th2 helper T cells). Using the same... Figure 13 Samples were processed using the same method as E, and CD4 was detected by flow cytometry. + IL-4 + T cell percentage. The left figure shows representative flow cytometry scatter plots for each group, and the right figure shows the statistical analysis results. Gate strategy: CD8 + IFN-γ + T cells: 2 × 10⁶ cells were collected from each sample. 6 Cells were sequentially selected by FSC-H / FSC-A (R1), live cells were selected by marking with live / dead dyes (R2), and CD3... + Set R3 gate, CD4 - CD8 + Quadrant gate (R4 / Q1), ultimately at CD8 + Analysis of IFN-γ in T cells + Cell frequency (R5, Freq. of Parent). CD4 + IL-4 + The T-cell gatening strategy is the same as above, with R4 being CD4. + CD8 - Quadrant gate (R4 / Q3), ultimately at CD4+ IL-4 analysis in T cells + Cell frequency (R5, Freq. of Parent).

[0113] Figure 14 The results of the immunomodulatory effect analysis of WLcd2 combined with different adjuvants in embodiments of the present invention are shown. Figure 14 A. Experimental Design and Intervention Procedure: The experiment was divided into a sham-infected group, a Clostridium difficile infection group (CDI, i.e., a sham-immunized group), and groups with different immunization conditions. Mice in the immunized group were injected with three doses of immunization mixture (14 days apart), while the sham-infected and sham-immunized groups were injected with normal saline (NS). Seven days after the last immunization, mice were given sterile water containing a mixture of antibiotics (kanamycin (40 mg / kg), gentamicin (3.5 mg / kg), colistin (4.2 mg / kg), metronidazole (21.5 mg / kg), and vancomycin (4.5 mg / kg)) for five consecutive days, followed by two days of feeding with sterile water. One day before Clostridium difficile challenge, mice were pretreated by intraperitoneal injection of clindamycin (10 mg / kg) to clear intestinal flora. Finally, mice were orally inoculated with Clostridium difficile strain R20291. For seven consecutive days after challenge, changes in mouse body weight, the degree of diarrhea, related clinical symptoms, and survival status were monitored, and mice were dissected and harvested at the experimental endpoint (day 7 after challenge). Figure 14 B represents the survival curves of mice in each group; Figure 14 C shows the comparison of the overall survival rates of mice in each group.

[0114] Figure 15 The effects of WLcd2 combined with adjuvant on clinical symptoms in mice, as illustrated in this embodiment of the invention, are shown. Figure 15 A shows the curves of weight change in mice in each group after challenge; Figure 15 B shows the percentage change in body weight of mice in each group on day 2 after challenge; Figure 15 C shows the percentage change in body weight of mice in each group on day 3 after challenge; Figure 15 D represents the overall clinical symptom score of mice in each group after challenge. Figure 15 E represents the clinical symptom score of mice in each group on day 2 after challenge. Figure 15 F represents the clinical symptom score of mice in each group on day 3 after challenge.

[0115] Figure 16 This invention demonstrates the effects of WLcd2 in combination with different adjuvants on colonic inflammatory damage in mice infected with Clostridium difficile. Figure 16A shows a schematic diagram of HE staining of colon tissue from mice in each group on day 2 after challenge. The figure shows the histopathological changes in each group, including the mock infected group, CDI group, CDI+MF59 group, CDI+Al(OH)3 group, CDI+[WLcd2+MF59] group (live observation group), CDI+[WLcd2+MF59] group (death group), CDI+[WLcd2+Al(OH)3] group (live observation group), and CDI+[WLcd2+Al(OH)3] group (pathological dissection group). Figure 16 B represents the histopathological scores of the colon tissue of mice in each group on day 2 after challenge, quantified based on epithelial damage, mucosal edema, and inflammatory cell infiltration. Data are expressed as mean ± standard error; Figure 16 C is a schematic diagram of HE staining of colon tissue from mice in each group on day 3 after challenge, consistent with the grouping order on day 2; Figure 16 D represents the pathological score of the colon tissue of mice in each group on day 3 after challenge; Figure 16 E is a schematic diagram of HE staining of colon tissue of mice in each group on day 7 after challenge. Only three groups of surviving mice are shown: the Mock infected group, the CDI+[WLcd2+Al(OH)3] group (survival observation group), and the CDI+[WLcd2+MF59] group (survival observation group) to highlight the effect of vaccine combined with adjuvant on tissue recovery. Figure 16 F represents the histopathological score of the colon tissue of mice in each group on day 7 after challenge (only three groups of surviving mice).

[0116] Figure 17 The results of WLcd2 combined with adjuvant inhibiting the expression of pro-inflammatory factors in the colon of CDI mice are shown in the embodiments of the present invention. Figure 17 A to Figure 17 D shows the relative mRNA expression levels of CXCL1, TNF-α, IL-1β, and IL-6 in the colon tissue of mice in each group on day 2 after challenge. Figure 17 E to Figure 17 H shows the relative mRNA expression levels of CXCL1, TNF-α, IL-1β, and IL-6 in the colon tissue of mice in each group on day 3 after challenge. Detailed Implementation

[0117] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and do not limit the scope of the present invention.

[0118] Unless otherwise specified, the experimental methods and conditions used in the following examples are conventional methods and conditions. Unless otherwise specified, the reagents used in the following examples are commercially available products.

[0119] (1) The experimental strains and experimental animals used in the examples are as follows:

[0120] Strain number: Clostridium difficile standard strain R20291 (RT027); purchased from the UK Healthy Security Agency (UKHSA) strain bank;

[0121] Mouse strain and age: 6-week-old BALB / C female mice free of specific pathogens; purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.

[0122] (2) The statistical analysis methods used in the embodiments are as follows:

[0123] All statistical analyses and plots were performed using R (v4.4.3) and GraphPad Prism 10.0 (GraphPad Software, San Diego, CA, USA). Experimental data are expressed as mean ± standard deviation (x̄ ± s). For time-repeated measures indicators (such as weight change), two-way repeated measures ANOVA was used, with experimental group and time point as two factors, to test the significance of group effects, time effects, and their interaction effects. If the overall difference was significant, Sidak's multiple comparisons test was used to control for family-wise error. For univariate experimental indicators, if three or more groups were involved, one-way ANOVA was used to test the overall differences between groups, combined with Tukey's multiple comparisons test for pairwise comparisons; if only two groups were involved, independent samples t-test or Mann-Whitney U test was used to analyze differences between groups. Mouse survival rates were plotted using Kaplan-Meier survival curves, and the Log-rank (Mantel-Cox) test was used to compare differences between groups. A two-sided P-value <0.05 was considered statistically significant. Significance markers were uniformly expressed as follows: ns indicates no significant difference, * indicates P <0.05, ** indicates P <0.01, *** indicates P <0.001, and **** indicates P <0.0001.

[0124] (3) The bioinformatics prediction tools used in the embodiments are shown in Table 1.

[0125]

[0126]

[0127] (4) The main reagents for prokaryotic expression and purification of proteins in the examples are shown in Table 2.

[0128]

[0129] (5) The main reagents for SDS-PAGE analysis and Western Blot in the examples are shown in Table 3.

[0130]

[0131] (6) The main reagents for the culture, identification and preservation of Clostridium difficile in the examples are shown in Table 4.

[0132]

[0133] (7) The main reagents for the tissue morphology experiment in the examples are shown in Table 5.

[0134]

[0135] (8) The main reagents for the ELISA experiment in the examples are shown in Table 6.

[0136]

[0137] (9) The main reagents for the qPCR experiment in the examples are shown in Table 7.

[0138]

[0139] (10) The main reagents for flow cytometry in the examples are shown in Table 8.

[0140]

[0141] (11) Other reagents used in the examples are shown in Table 9.

[0142]

[0143] (12) The preparation methods of the relevant reagents used in the examples are as follows:

[0144] (12.1) Weigh 2.42g Tris-HCl and 17.52g NaCl, add an appropriate amount of Milli-Q water to dissolve them completely, add 174 mg PMSF, mix thoroughly, adjust the pH to 8.0, and bring the volume to 1L. Place on ice for later use or use immediately.

[0145] (12.2) Inclusion body washing buffer (containing Triton X-100): Weigh 2.42g Tris-HCl, 17.52g NaCl and 0.372g EDTA, add an appropriate amount of Milli-Q water to dissolve them completely, add 20mL Triton X-100, mix well, adjust the pH to 8.0, and bring the volume to 1L. Stir at room temperature and set aside.

[0146] (12.3) Inclusion body washing buffer (without Triton): Weigh 2.42g Tris-HCl and 17.52g NaCl, add an appropriate amount of Milli-Q water to dissolve them completely, adjust the pH to 8.0, and bring the volume to 1L. Mix thoroughly and set aside.

[0147] (12.4) Denaturation and dissolution buffer: Weigh 2.42g Tris-HCl, 17.52g NaCl, 480g urea and 0.68g imidazole, add an appropriate amount of Milli-Q water to dissolve completely, add 174mg PMSF (freshly dissolved), mix thoroughly, adjust the pH to 8.0, and bring the volume to 1L. Stir at room temperature for 2–4 hours until completely dissolved, and use immediately.

[0148] (12.5) Ni-NTA binding buffer: Weigh 2.42g Tris-HCl, 17.52g NaCl, 480g urea and 0.68g imidazole, add an appropriate amount of Milli-Q water to dissolve them completely, adjust the pH to 8.0, and bring the volume to 1L. Mix thoroughly and store at room temperature for use in resin equilibration.

[0149] (12.6) Ni-NTA washing buffer: Weigh 2.42g Tris-HCl, 17.52g NaCl, 480g urea and 1.36–3.40g imidazole (corresponding to 20–50 mM), add an appropriate amount of Milli-Q water to dissolve completely, adjust the pH to 8.0, and bring the volume to 1L. Mix thoroughly and store at room temperature.

[0150] (12.7) Ni-NTA elution buffer: Weigh 2.42g Tris-HCl, 17.52g NaCl, 480g urea and 17.02–34.04g imidazole (corresponding to 250–500 mM), add an appropriate amount of Milli-Q water to dissolve completely, adjust the pH to 8.0, and bring the volume to 1L. Mix thoroughly and store at room temperature.

[0151] (12.8) Refolding dialysis buffer: Weigh 2.42g Tris-HCl and 8.76–17.52g NaCl (150–300 mM), add an appropriate amount of Milli-Q water to dissolve, adjust the pH to 8.0 and bring the volume to 1L, mix well and store at 4℃ for later use.

[0152] (12.9) Brain and heart extract broth agar plates: Accurately weigh 37g of brain and heart extract dry powder, 5g of yeast extract and 15g of agar powder, add an appropriate amount of Milli-Q water to dissolve and make up to 1L; sterilize at 121℃ and 0.1MPa high pressure steam for 20min, cool to about 60℃, dispense into petri dishes in a clean bench, and store upside down at 4℃ after solidification.

[0153] (12.10) Nutrient broth agar plates: Weigh 21g of nutrient broth powder and 15g of agar powder, add an appropriate amount of Milli-Q water, heat to dissolve and make up to 1L; sterilize at 121℃ and 0.1MPa for 20min, cool and dispense into petri dishes, and store upside down at 4℃ after solidification.

[0154] Example 1

[0155] 1.1 Experimental Methods

[0156] 1.1.1 Acquisition of target protein sequence and structural data

[0157] The target protein sequences in this embodiment were all obtained from the UniProt database, specifically as follows: TcdA (ID: Q189K5), TcdB (ID: Q189K3), CD0873 (ID: Q18A65), CdeC (ID: Q18AS2), FliC (ID: Q18CX7), FliD (ID: Q18CX9), and Cwp84 (ID: Q183M1). The TLR structures required for molecular docking were obtained as follows: the crystal structures of TLR2 (PDB ID: 2Z81) and TLR4 (PDB ID: 2Z63) were directly obtained from the RCSB PDB database; since the RCSB PDB database lacked crystal structures of the human TLR5 extracellular domain that could be directly used in this study, the human TLR5 protein sequence (O60602) was obtained from the UniProt database. Homology modeling was performed using SWISS-MODEL, and the transmembrane region in the TLR5 structure was deleted using PyMOL, retaining only the extracellular domain for subsequent analysis.

[0158] 1.1.2 CTL Epitope Prediction

[0159] The CTL tabletop is CD8. +The core recognition fragments of T cell-mediated cytotoxic immune responses play a crucial role in the construction of multi-epitope vaccines, significantly enhancing vaccine specificity and immunogenicity. This embodiment uses the NetCTL 1.2 server to predict CTL epitopes of candidate antigen proteins. During prediction, a threshold of 0.75 and an epitope length limited to 9 amino acids were used. The predicted epitopes underwent further multi-dimensional screening: antigenicity was assessed using VaxiJen 2.0 (threshold 0.4), toxicity was predicted using ToxinPred, and potential sensitization was predicted using AllerTOP v2.0, eliminating non-potential antigens, toxic or sensitizing epitopes. To further evaluate the conservation of candidate epitopes across different strains, the amino acid sequences of seven target proteins were retrieved and downloaded from the NCBI database, and the conservation of candidate epitopes was analyzed using the IEDB Epitope Conservancy Analysis module.

[0160] 1.1.3 HTL Epitope Prediction

[0161] HTL epitopes activate CD4 by binding to MHC class II molecules. + T cell responses regulate humoral and cellular immune responses, promote B cell differentiation and antibody production, and enhance the cytotoxic response of CTLs. This embodiment utilizes the NetMHCIIpan-4.3 server to predict HTL epitopes of candidate antigen proteins. Considering the global distribution of HLA alleles and the broad applicability of the vaccine, this embodiment selects 27 high-frequency HLA class II alleles for prediction. Epitope length is limited to 15 amino acids, and the screening criterion is Rank% <2%, indicating high binding affinity between the peptide and MHC II molecules. The predicted HTL epitopes are further analyzed for antigenicity, toxicity, sensitization, and conservation, following the same screening procedure as in 1.1.2. To enhance vaccine immunogenicity, the ability of epitopes to induce the secretion of IFN-γ and IL-4 by immune cells was also predicted using the IFNepitope and IL-4pred servers, retaining only epitopes capable of inducing the secretion of one of these key cytokines.

[0162] 1.1.4 LBL Epitope Prediction

[0163] LBL epitopes are composed of consecutive amino acids, can be recognized by the BCR, and stimulate humoral immune responses to induce antibody production. This embodiment uses two tools—ABCpred (based on an Artificial Neural Network, ANN) and LBtope (based on a Support Vector Machine, SVM)—for joint prediction, retaining candidate epitopes predicted by both to reduce the false positive rate. Subsequently, antigenicity, toxicity, sensitization, and conservation analyses are performed on the candidate epitopes to screen for epitopes with high antigenicity, no toxicity, and no sensitization for subsequent multi-epitope vaccine construction, ensuring that the multi-epitope vaccine can effectively induce humoral immunity. The screening process is the same as in 1.1.2.

[0164] 1.1.5 Localization of epitopes on the surface of target proteins

[0165] To determine the spatial exposure of candidate epitopes on target proteins, this embodiment used AlphaFold v2.0 to predict the three-dimensional structures of seven target proteins. Subsequently, ERRAT, PROCHECK, and ProSA-Web were used to validate the model's quality: ERRAT assessed structural plausibility by analyzing the distribution of non-bonded interactions between different atomic types; PROCHECK quantitatively analyzed the model's stereochemical quality using Ramachandran plots, with higher proportions of residues in the optimal region and lower proportions in the disallowed region indicating higher model quality; ProSA-Web calculated the overall Z-score to assess the model's consistency with known experimental structures, with lower negative values ​​indicating more stable and plausible structures. Finally, ChimeraX 1.11 was used to visualize the tertiary structures of the candidate target proteins, clarifying the epitope exposure on the protein surface.

[0166] 1.1.6 Design and Construction of Fusion Proteins

[0167] The final selected CTL, HTL, and B-cell epitopes were tandemly constructed into multi-epitope fusion proteins using specific linkers. In the design of epitope fusion peptides, linkers play a crucial role in maintaining epitope independence and structural integrity. In this embodiment, AAY, KK, and KK linkers were used to connect CTL, HTL, and B-cell epitopes, respectively. The AAY linker serves as a natural proteasome cleavage site in mammalian cells, facilitating the generation of natural epitopes and inhibiting the formation of unnecessary linker epitopes, thereby improving epitope presentation efficiency and enhancing immunogenicity. Using KK linkers between HTL epitopes shortens the epitope spacing, optimizing antigen processing and presentation efficiency while maintaining epitope exposure and immune recognition. The KK linker, composed of lysine residues, provides flexible spacers and spatial separation for different functional domains or epitopes in the protein construct, reducing steric hindrance, maintaining structural independence, and promoting protease cleavage for targeted release of each component. To further enhance immunogenicity, this embodiment introduces the cholera enterotoxin B subunit (CTXB; UniProt: P01556) as a molecular adjuvant at the N-terminus of the fusion peptide via an EAAAK linker. Furthermore, to enhance T-cell and humoral immune responses, a pan-HLA-DR binding epitope (PADRE, sequence: AKFVAAWTLKAAA (SEQ ID NO. 37)) is introduced into the construct. PADRE is a universal 13-amino acid synthetic peptide capable of binding with high affinity to most common human HLA-DR alleles, thereby activating CD4+. + T cells overcome HLA polymorphism limitations, improving vaccine coverage in different populations. Finally, a 6×histidine tag (6×His tag) is added to the C-terminus of the fusion peptide to facilitate subsequent purification and detection of the recombinant protein. This design ensures that the fusion protein structurally balances epitope exposure and stability.

[0168] 1.1.7 Evaluation of the physicochemical properties and biological characteristics of the fusion protein

[0169] To confirm the safety and excellent physicochemical properties of the designed construct, this embodiment systematically evaluated the antigenicity, toxicity, sensitization, and basic physicochemical properties of the final construct. First, VaxiJen 2.0 and ANTIGENpro were used to predict vaccine antigenicity, AllerTOP v2.0 to predict sensitization, and ToxinPred to predict toxicity. Subsequently, the ExPASy ProtParam tool was used to analyze the basic physicochemical properties of the construct, including molecular weight, theoretical isoelectric point (pI), amino acid composition, instability index, aliphatic index, and grand average ofhydropathy (GRAVY), providing a theoretical basis for the subsequent expression and purification of the fusion protein. To ensure that the vaccine can be effectively recognized and function by the immune system, this embodiment further utilized a series of bioinformatics tools to optimize and validate the construct. SignalP-6.0 was used to predict whether the vaccine construct contained a signal peptide sequence to assess its secretion-related characteristics and potential impact on subsequent expression. DeepTMHMM is used to predict and remove transmembrane helical regions that may hinder epitope exposure and antigen presentation. The SOLpro server is used to evaluate the solubility of vaccine constructs when overexpressed in E. coli, providing a reference for the selection of subsequent expression strategies.

[0170] 1.1.8 Prediction of secondary structure, modeling of tertiary structure, and assessment of properties of fusion proteins

[0171] The secondary structure of the candidate fusion protein was predicted using the PSIPRED 4.0 server to assess the proportions of structures such as α-helices, β-sheets, and random coils, providing a reference for subsequent 3D modeling and structural analysis. For the tertiary structure of the fusion protein, this embodiment used AlphaFold v2 and Chai-1 software for 3D modeling, and selected the optimal conformation for further optimization based on model quality to obtain a reasonable protein spatial structure. To verify and obtain structural stability, this embodiment used GROMACS v2023.3 software to perform a 100 ns MD simulation on the optimal model, with temperature and pressure similar to the physiological environment under simulation conditions. After the simulation, the last frame of the protein structure was taken as the stable conformation of the fusion protein, and the GalaxyRefine module in the GalaxyWEB server was used to further optimize the protein side chains, generating five optimized models. Using the MolProbity score as a reference, the model with the lowest score was selected as the optimal 3D model. Subsequently, the structural quality of the model was verified using ERRAT, PROCHECK, and ProSA-Web servers. ERRAT evaluates model quality by analyzing the distribution of non-bonded interactions between different atomic types; a higher score indicates that the model structure is closer to the high-resolution crystal structure. PROCHECK analyzes the stereochemical rationality of the model using Ramachandran plots; a higher proportion of residues in the optimal region and a lower proportion in the disallowed region indicate better structural quality. ProSA-Web assesses overall structural rationality by calculating the Z-score; a Z-score closer to the negative range of the experimentally resolved high-resolution protein indicates a more stable and reliable model. Finally, ChimeraX 1.11.1 is used to visualize the tertiary structure of the candidate fusion protein, providing a structural basis for subsequent molecular docking and immunogenicity analysis.

[0172] 1.1.9 Molecular docking analysis

[0173] To evaluate the interaction between the fusion protein and the immune receptor, this embodiment used the HawkDOCK server to perform molecular docking of the candidate fusion protein with TLR2, TLR4, and TLR5. The binding energy of the protein-receptor complex was calculated using the Molecular Mechanics Generalized Born Surface Area (MM-GBSA) method to quantify the binding stability of the complex. The docking complex with the strongest binding energy was further optimized using the HawkDOCK 2.4 server to improve the conformation and interaction characterization of the binding interface. Key interactions between the complexes, including hydrogen bonds, hydrophobic interactions, and salt bridges, were analyzed using PDBsum and visualized using PyMOL software to identify key amino acid residues and their spatial arrangement at the binding interface.

[0174] 1.1.10 Molecular Dynamics Simulation

[0175] To further evaluate the dynamic stability, binding affinity, and intermolecular interactions of the vaccine-TLR complex, a 100 ns molecular dynamics simulation was performed on the complex using GROMACS v2023.3. All proteins were parameterized using the Amber14SB_parmbsc1 force field, and the complex was dissolved in a cubic TIP3P water tank with the tank boundary at least 10 Å from the outermost atoms to eliminate boundary effects. Simultaneously, 0.145 mol / L NaCl was added to simulate physiological ionic strength, and counterions were added to neutralize the system charge. The simulation first performs energy minimization, using the steepest descent method for 5000 steps, with the convergence criterion being a maximum force less than 1000 kJ·mol⁻¹·nm⁻¹. Next, a 400 ps NVT equilibrium simulation is performed at 310 K, with temperature controlled by a V-rescale thermostat (τ_T = 0.1 ps). Following this, a 400 ps NPT equilibrium simulation is performed at 1 atm, using a Parrinello–Rahman pressure coupler (τ_P = 2.0 ps) to stabilize the pressure and adjust the simulation chamber volume to achieve thermodynamic equilibrium. After equilibrium is achieved, a 100 ns MD simulation is performed with a time step of 2 fs. Hydrogen bond lengths are constrained using the LINCS algorithm to improve computational efficiency, and trajectory coordinates are recorded every 10 ps for subsequent analysis. The simulation analysis metrics include the root mean square deviation (RMSD) of the complex backbone atoms, used to assess overall conformational stability; the radius of gyration (Rg), used to assess structural compactness; the number of hydrogen bonds (H-bonds), defined as donor-acceptor distance ≤ 3.5 Å and donor-hydrogen-acceptor angle ≥ 120°, used to characterize the stability of intermolecular interactions; and the root mean square fluctuation (RMSF) of the vaccine protein residue backbone, used to assess flexibility at the residue level.

[0176] 1.1.11 Calculation of MM-PBSA binding free energy

[0177] This embodiment uses the gmx_MMPBSA v1.62 tool to calculate the binding free energy of the fusion protein-TLR complex based on the molecular mechanics / Poisson-Boltzmann surface area (MM-PBSA) method. The binding free energy is calculated according to the following formula:

[0178]

[0179] Wherein, ΔG bind,solv The binding free energy represents the change in net free energy resulting from the formation of a complex between the receptor and ligand in solution, taking into account the solvation effect; ΔG bind,vacuum Represents the vacuum binding free energy, which is the binding free energy contributed by the direct interaction between the acceptor and ligand without considering the effect of solvent; ΔG solv,complex The solvation free energy of the complex represents the change in free energy as the acceptor-ligand complex transitions from a vacuum environment to a solvent environment; ΔG solv,receptor and ΔG solv,ligand These represent the solvation free energies of the free acceptor and free ligand, respectively, which are the changes in free energy that occur when they are transferred from a vacuum environment to a solvent environment.

[0180] This method estimates the binding affinity between complexes under near-physiological conditions by estimating the gas binding energy and solvation free energy separately, providing a computational basis for the rational design of candidate compounds and the analysis of their mechanisms of action.

[0181] 1.1.12 HLA Population Coverage Analysis

[0182] T-cell epitopes can only elicit an immune response in individuals expressing HLA molecules capable of binding to specific epitopes. However, the distribution of different HLA alleles varies significantly across the globe and among different populations. To assess the potential beneficiaries of a vaccine, this embodiment uses the Population Coverage module of IEDB to predict the population coverage of candidate fusion proteins in different regions of the world. This analysis integrates HLA genotype prevalence data from different geographic regions, combined with epitope HLA binding capacity and T-cell restriction information, to provide a reference for the broad applicability of the vaccine.

[0183] 1.1.13 Immune Simulation

[0184] This embodiment utilizes the C-ImmSim server to predict the immunogenicity of a designed multi-epitope vaccine. The server assesses the vaccine's potential to induce humoral and cellular immune responses by simulating epitope binding to HLA molecules, immune cell activation, and intercellular interactions. The simulation step size was set to 1050 (each time step equivalent to 8 hours), and injection time points were set at steps 0, 42, and 84 (equivalent to day 0, day 14, and day 28) to evaluate changes in the immune system over time. All other simulation parameters remained at their initial default values.

[0185] 1.1.14 Reverse translation, sequence optimization, and recombinant plasmid construction of fusion protein sequences

[0186] In this embodiment, the amino acid sequence of the multi-epitope fusion protein was optimized for E. coli codon bias using the JCat server, and reverse translation was performed to obtain the optimized cDNA sequence. Subsequently, the insertion scheme of the target gene was designed using Vectorbee software, selecting NdeI and XhoI as multiple cloning sites for the pET-22b(+) vector to ensure that the inserted fragment was located downstream of the T7 promoter, while retaining the C-terminal 6×His tag coding sequence for subsequent purification of the recombinant protein. The construction of the recombinant plasmid was completed by Shanghai Sangon Biotech Co., Ltd., and the accuracy and integrity of the inserted sequence were verified by Sanger sequencing.

[0187] 1.1.15 Prokaryotic expression of fusion proteins

[0188] (1) Recombinant plasmid transformation: Take 100 μL of frozen E. coli BL21(DE3) chemocompetent cells from a -80℃ freezer, thaw them slowly on ice for 5-10 min, add 1-2 μL of recombinant plasmid DNA (about 100 ng), mix gently, and incubate on ice for 30 min. Then place the centrifuge tube in a 42℃ water bath for 30-45 s heat shock, and immediately cool on ice for 3 min. Add 900 μL of antibiotic-free LB liquid medium, and revive at 37℃ and 180 rpm for 60 min with shaking. After revival, centrifuge at 4000 rpm for 2 min, discard part of the supernatant, resuspend the bacterial culture, and spread it evenly on LB solid medium containing 30 μg / mL ampicillin. Incubate upside down at 37℃ for 12-16 h.

[0189] (2) Activation and preservation of positive clones: Single colonies were picked and inoculated into 5 mL of LB liquid medium containing 30 μg / mL ampicillin, and cultured at 37℃ and 220 rpm with shaking until OD600 = 0.6-0.8. 0.5 mL of bacterial culture was mixed with an equal volume of sterile glycerol and frozen at −80℃ as a seed bank. The remaining bacterial culture was used for subsequent expression or small-scale induction verification.

[0190] (3) Small-scale induction and condition screening: Seed culture was inoculated at a ratio of 1:100 into LB medium containing 30 μg / mL ampicillin and cultured at 37℃ and 220 rpm until OD600 = 0.6-0.8. Samples were taken as uninduced control. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM and cultured at 37℃ and 220 rpm for 4 h. The bacterial culture was then collected to determine OD600, and the bacterial cells were collected by centrifugation at 4℃ and 4000 rpm for 20 min. The bacterial cells were washed twice with PBS and then added to SDS-PAGE loading buffer. The mixture was boiled at 95℃ for 10 min and stored at −20℃ for SDS-PAGE and Western Blot (WB) analysis.

[0191] (4) Optimization of induction conditions: The activated bacterial solution was inoculated into fresh LB medium (containing 30 μg / mL ampicillin) at a ratio of 1:100 and cultured until OD600=0.6-0.8. Two temperature / time conditions were set: 37℃ for 4 h and 15℃ for 16 h. IPTG was added to a final concentration of 0.2 mM or 1.0 mM under each condition. The protein expression form was analyzed by SDS-PAGE to screen the optimal induction conditions.

[0192] (5) Identification of expression form and determination of conditions: Bacterial cells cultured under optimal induction conditions were resuspended in PBS and then sonicated (power 200W, 2s on / 3s off, total duration 15–20min). After sonication, the cells were centrifuged at 12000×g, 4℃ for 20min to separate the supernatant and precipitate. SDS-PAGE loading buffer was added and the samples were boiled to prepare the samples for analysis of the target protein distribution. If the protein was mainly present in the supernatant, a soluble expression protocol was used; if it was present in the form of inclusion bodies, subsequent renaturation treatment was performed.

[0193] (6) Scale-up expression: Based on the optimal conditions determined in the above steps, scale-up culture was carried out at 3 L. The seed culture was inoculated at a ratio of 1:100 into 3 L of LB medium containing 30 μg / mL ampicillin and cultured at 37℃ and 220 rpm until OD600 = 0.6-0.8. IPTG was added to a final concentration of 1 mM and induced at 37℃ for 4 h. After induction, the bacterial culture was centrifuged (4℃, 4000 rpm, 20 min) to collect the bacterial cells. The cells were washed twice with PBS to remove residual culture medium. The bacterial precipitate was aliquoted and stored at -80℃ for later use or purified immediately.

[0194] 1.1.16 Purification and refolding of inclusion bodies of fusion proteins

[0195] (1) Collection and disruption of bacterial cells: Resuspend frozen or fresh bacterial cells in 10 mL of pre-cooled lysis buffer per gram of wet cells, and sonicate in an ice bath (power 200W, 2 s on, 3 s off, total duration 15-20 min, avoid overheating of the sample). Centrifuge the disruption solution at 12000×g, 4℃ for 20 min, discard the supernatant, and the precipitate is the coarse inclusion bodies.

[0196] (2) Inclusion body washing and impurity removal: Resuspend the precipitate in inclusion body washing buffer (containing Triton X-100), wash with stirring at room temperature for 30 min, centrifuge at 12000×g, 4℃ for 15 min, and discard the supernatant. Repeat the washing 2-3 times until the precipitate is milky white and loose. Finally, wash once with washing buffer without Triton to remove detergent residue, centrifuge at 12000×g, 4℃ for 15 min, and collect the washed inclusion body precipitate.

[0197] (3) Denaturation and dissolution of inclusion bodies: Add 5-10 mL of denaturation and dissolution buffer per gram of wet weight to the washed inclusion body precipitate and stir at room temperature for 2-4 h (or stir gently overnight at 4 °C). Centrifuge at 12000 × g at room temperature for 30 min, collect the supernatant as the denatured protein sample, filter through a 0.45 μm filter membrane to remove insoluble matter, and store temporarily at room temperature or load immediately.

[0198] (4) Ni-NTA affinity chromatography

[0199] ① Resin equilibration: Take 5 mL of Ni-NTA agarose gel (column bed volume), pack it into the chromatography column, and equilibrate it with 10 times the column bed volume of denaturing binding buffer at a flow rate of 1 mL / min until the pH and conductivity of the effluent are stable and consistent with the buffer.

[0200] ② Sample loading: Slowly add the denatured protein sample to the column to ensure sufficient binding of the protein to the Ni-NTA resin. Loading can be done in stages or cyclically to ensure the target protein binds to the resin as much as possible. Collect the flow-through for later use and detect the amount of unbound protein.

[0201] ③ Washing: Wash with 5-10 column bed volumes of denaturing wash buffer at a flow rate of 1 mL / min, while monitoring the A280 value until the baseline stabilizes.

[0202] ④ Elution: Gradient elution was performed using denaturing elution buffer (0→50→100→250→500mM imidazole, 5 column volumes per gradient, flow rate 0.5mL / min), and the eluent was collected in 5mL tubes. The fraction containing the target protein was identified by A280 monitoring and SDS-PAGE, and the high-purity eluents were combined.

[0203] (5) Purification effect analysis: SDS-PAGE analysis was performed on the bacterial cell rupture precipitate, denaturation and dissolution supernatant, Ni-NTA flow-through solution, washing solution and elution sample to evaluate the protein distribution, purity and purification effect of each step.

[0204] (6) Protein refolding: The high-purity denatured protein sample was placed in a dialysis bag with a molecular weight cutoff of 10 kDa and refolded by gradient dialysis at 4°C. The buffer solution was changed 2–3 times during the process to completely remove urea. After refolding, the sample was filtered through a 0.22 μm filter membrane, analyzed by SDS-PAGE, and the protein concentration was determined by the BCA method.

[0205] (7) Concentration and preservation of refolded protein: The refolded protein solution is concentrated by ultrafiltration and sterilized by filtration using a 0.22 μm filter membrane. It is then dispensed into sterile EP tubes (0.5-1 mL per tube) and stored at -80℃ for a long time.

[0206] 1.1.17 SDS-PAGE analysis and WB identification steps for vaccine fusion proteins

[0207] (1) Protein denaturation: Take the protein sample frozen at -80℃ and thaw it slowly on ice to avoid repeated freeze-thaw cycles. After thawing, mix the protein sample with 5×SDS loading buffer at a ratio of 4:1 (v / v) and vortex for 10-15 seconds. Then heat the mixture in a 95℃ metal bath for 10 minutes to completely denature the protein. After denaturation, centrifuge at 10000×g for 1 minute, collect the supernatant for subsequent sample loading and analysis, and rapidly cool it on ice for later use to ensure protein stability.

[0208] (2) Electrophoresis: Place a 10% precast gel into the electrophoresis tank, add fresh electrophoresis buffer to the inner tank, and flush the sample wells with a syringe to remove residual gel fragments. Add protein marker (3 μL / well) and sample (8 μL / well) sequentially, and add an appropriate amount of electrophoresis buffer to the outer tank. Set the electrophoresis parameters: first electrophoresis at a constant voltage of 80 V for 30 min, then adjust to a constant voltage of 120 V until bromophenol blue migrates to the bottom of the gel, ensuring complete protein separation.

[0209] (3) Gel staining: After SDS-PAGE electrophoresis, carefully remove the gel and place it in an incubator. Add sufficient staining solution to cover the gel and place it on a shaker at room temperature for about 2 hours. After staining, pour out the staining solution, add destaining solution to cover the gel, and place it on a shaker for about 2 hours. Change the destaining solution twice until the background is completely removed and the protein bands are clearly visible. After destaining, immerse the gel in water for preservation, so that it can be photographed and recorded.

[0210] (4) Transfer: Cut a 0.22 μm PVDF membrane to an appropriate size (approximately 9 × 6 cm), immerse it in methanol for 5 min to activate, and then equilibrate it in transfer buffer for 5 min. Prepare filter paper and equilibrate it in transfer buffer for 10 min to ensure no air bubbles. After electrophoresis, remove the glass plate and cut a corner mark on the upper right corner of the separating gel to indicate the direction. Stack the membranes in the following order: “graphite electrode → sponge → filter paper × 2 → separating gel → PVDF membrane → filter paper × 2 → sponge”. Gently press the membrane with a roller to remove air bubbles, then place it in the transfer tank. Add ice packs and pre-cooled transfer buffer, and transfer the membrane at a constant current of 400 mA for 80 min. After the transfer is complete, rinse the PVDF membrane three times in 1 × TBST at room temperature for 1 min each time.

[0211] (5) Blocking: Place the rinsed PVDF membrane in 5% skim milk powder (prepared with 1×TBST) and block it in a shaker at room temperature for 1 hour to block non-specific binding sites.

[0212] (6) Primary antibody incubation: Dilute the anti-His tag primary antibody at a ratio of 1:3000 in the antibody dilution solution to cover the entire PVDF membrane surface, with the protein side facing up. Place the membrane in an incubation box and incubate at 4°C on a slow shaker for 12-16 hours to ensure specific binding.

[0213] (7) Primary antibody washing: After the primary antibody incubation is completed, the primary antibody working solution is retrieved and the PVDF membrane is placed in 1×TBST and washed three times at room temperature with a rapid shaker. The first wash is 10 min and the next two washes are 5 min each, in order to remove unbound antibodies.

[0214] (8) Secondary antibody incubation: Dilute the secondary antibody at a ratio of 1:10000 in 5% skim milk powder, completely cover the surface of the PVDF membrane, and incubate slowly on a shaker at room temperature for 1 hour.

[0215] (9) Secondary antibody washing: After the secondary antibody incubation is completed, the PVDF membrane is placed in 1×TBST and washed three times at room temperature with a rapid shaker. The first time is 10 min and the next two times are 5 min each to remove excess secondary antibody.

[0216] (10) Development: Prepare the developing working solution (a 1:1 mixture of solution A and solution B), blot off excess TBST on the surface of the PVDF membrane with filter paper, place the membrane flat in a culture dish with the protein side facing up, and add developing solution to completely cover the membrane surface. Expose the membrane using a Bio-Rad chemiluminescent gel imaging system to obtain clear target bands and save the images.

[0217] 1.2 Experimental Results

[0218] 1.2.1 Filtering Tabletops

[0219] For seven target proteins (TcdA, TcdB, CD0873, CdeC, FliC, FliD, and Cwp84), this embodiment first predicted CTL, HTL, and B-cell epitopes. Subsequently, a systematic evaluation of multidimensional indicators of the candidate epitopes was conducted, including antigenicity, safety (sensitization and toxicity), sequence conservation, and binding potential to HLA molecules. Based on these comprehensive evaluation indicators, 10 CTL epitopes (Table 10), 7 HTL epitopes (Table 11), and 9 LBL epitopes (Table 12) were finally screened. The specific distribution is as follows: 5 epitopes were screened for TcdA, 5 for TcdB, 3 for CD0873, 4 for CdeC, 3 for FliC, 3 for FliD, and 3 for Cwp84. These epitopes all possess high antigenicity, no potential sensitization or toxicity, and exhibit good binding affinity to different HLA molecule types, providing a reliable basis for the design of multi-epitope fusion peptides and fusion proteins. The spatial distribution of the screened epitopes in various target proteins is shown below. Figure 1 , Figure 2 and Figure 3 As shown, this can intuitively reflect the exposure and potential immune accessibility of epitopes in the three-dimensional structure of proteins, thus providing a reference for the construction and structural optimization of subsequent vaccine fusion proteins.

[0220]

[0221]

[0222]

[0223] 1.2.2 Construction and physicochemical properties of fusion proteins

[0224] Based on the final selected CTL, HTL, and B cell epitopes, combined with the PADRE sequence, CTXB adjuvant, and C-terminal 6×His tag, this embodiment constructed a multi-epitope fusion protein named WLcd2, whose amino acid sequence is shown in SEQ ID NO. 32 ( Figure 4A). VaxiJen and ANTIGENpro predictions showed antigenicity of 0.85 and 0.91, respectively, suggesting high immune recognition potential for WLcd2. Both toxicity and sensitization predictions were negative, indicating theoretically good safety. The WLcd2 protein consists of 524 amino acid residues with a theoretical molecular weight of 59.31 kDa, below the generally accepted threshold of 110 kDa for expression. The theoretical isoelectric point (pI) is 9.14, suggesting a possible net positive charge at physiological pH, which may contribute to intermolecular repulsion and thus improve the protein's solubility and stability in solution. Predicted half-lives were greater than 10 h in mammalian reticulocytes, 20 h in yeast, and 30 h in E. coli in vitro, suggesting theoretically good in vivo stability for WLcd2. The instability index (II) was 36.43, within the range for stable proteins (II < 40), and the aliphatic index was 67.77, indicating potentially high thermal stability. The GRAVY score was -0.659, indicating that WLcd2 is generally hydrophilic. The SOLpro predicted solubility probability was 0.94, and the Protein-sol score was 0.482 (higher than the protein average of 0.45), suggesting that WLcd2 may theoretically have good solution solubility. No obvious features were detected in the transmembrane helix and signal peptide predictions. Figure 4 B and Figure 4 (C) This further supports its applicability as a multi-epitope vaccine candidate. In summary, WLcd2 demonstrates superior performance in terms of theoretical antigenicity, immune recognition potential, and in vivo stability, providing a reliable theoretical basis for subsequent in vitro expression and immunological experiments.

[0225] In addition, three sequences were designed and named CD1, CD2 and CD3 respectively. The properties of each sequence are shown in Table 13.

[0226] WLcd2 (SEQ ID NO. 32):

[0227] MTPQNITDLCAEYHNTQIYTLNDKIFSYTESLAGKREMAIITFKNGAIFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNKTPHAIAAISMANEAAAKAKFVAAWTLKAAAKKYYFNTNT AEAATGWQKKIETGWIYDMENESDKKKGEMAVKILNGESVSDKKGCTEFVALAFPAVRAKMEKLSSGVRIKRAADKKAEKINYQFAVSQMAEKKGAKYEVYYAPVVNGVAAYNIEGQAIVYAAYNIEGQA IRYAAYQELVERWNLAAYHQNTLDENFAAYKALEKNGFKAAYRVDYVEFTFAAYIQPRLVDTFAAYRWNTNVSALAAYKEKRLYAKYAAYDLSEEHLRWKKEYFAPANTDANNIEGKKFEYFAPANTHNNN IEKKHNMSENTVVEKYLKLKKTDLDENLEGEAIDFTGKKCSQGGDSGNSKQESNKKVGRNCETTFEFAVCGEKKRAKLGAQQNRLESTQKKAEKINYQFAVSQMAEPKKEKDIPYNLKSEAQGATHHHHH

[0228] CD1 (SEQ ID NO. 33):

[0229] MVSKTFKDKCASTTAKLVQSVQLVNISSDVNKDSKGIYISSSAGKTWFIPGGQYYPDNYLSNEMRKIAMAAVLSNVRVNLCASEAYTPNHVWAIELAPEAAAKAKFVAAWTLKAAAKKYYFNTNTAEAAA TGWQKKIETGWIYDMENESDKKKGEMAVKILNGESVSDKKGCTEFVALAFPAVRAKKMEKLSSGVRIKRAADKKAEKINYQFAVSQMAEKKGAKYEVYYAPVVNGVAAYNIEGQAIVYAAYNIEGQAIRY AAYQELVERWNLAAYHQNTLDENFAAYKALEKNGFKAAYRVDYVEFTFAAYIQPRLVDTFAAYRWNTNVSALAAYKEKRLYAKYAAYDLSEEHLRWKKEYFAPANTDANNIEGKKFEYFAPANTHNNNI EKKHNMSENTVVEKYLKKKTLDENLEGEAIDFTGKKCSQGGDSGNSKQESNKKVGRNCETTFEFAVCGEKKRAKLGAQQNRLESTQKKAEKINYQFAVSQMAEPKKEKDIPYNLKSEAQGATHHHHH

[0230] CD2 (SEQ ID NO. 34):

[0231] MGASQFFKDNCNRTTASLVEGVELTKYSDINNNTDGMYVVSSTGGVWRISRAKDYPDNVMTAEMRKIAMAAVLSGMRVNMCASPASSPNVIWAIELEAAEEAAAKAKFVAAWTLKAAAKKYYFNTNTAEA ATGWQKKIETGWIYDMENESDKKKGEMAVKILNGESVSDKKGCTEFVALAFPAVRAKKMEKLSSGVRIKRAADKKAEKINYQFAVSQMAEKKGAKYEVYYAPVVNGVAAYNIEGQAIVYAAYNIEGQAIR YAAYQELVERWNLAAYHQNTLDENFAAYKALEKNGFKAAYRVDYVEFTFAAYIQPRLVDTFAAYRWNTNVSALAAYKEKRLYAKYAAYDLSEEHLRWKKEYFAPANTDANNIEGKKFEYFAPANTHNNNI EKKHNMSENTVVEKYLKKKTLDENLEGEAIDFTGKKCSQGGDSGNSKQESNKKVGRNCETTFEFAVCGEKKRAKLGAQQNRLESTQKKAEKINYQFAVSQMAEPKKEKDIPYNLKSEAQGATHHHHH

[0232] CD3 (SEQ ID NO. 35):

[0233] MTPQNITDLCAEYHNTQIYTLNDKIFSYTESLAGKREMAIITFKNGAIFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANEAAAKAKFVAAWTLKAAAAAYNIEGQA IVYAAYNIEGQAIRYAAYQELVERWNLAAYHQNTLDENFAAYKALEKNGFKAAYRVDYVEFTFAAYIQPRLVDTFAAYRVNTNVSALAAYKEKRLYAKYAAYDLSEEHLRWKKYYFNTNTAEAATGWQKKI ETGWIYDMENESDKKKGEMAVKILNGESVSDKKGCTEFVALAFPAVRAKKMEKLSSGVRIKRAADKKAEKINYQFAVSQMAEKKGAKYEVYYAPVVNGVKKEYFAPANTDANNIEGKKFEYFAPANTHNNN IEKKHNMSENTVVEKYLKLKKKTLDENLEGEAIDFTGKKCSQGGDSGNSKQESNKKVGRNCETTFEFACGEKKRAKLGAQQNRLESTQKKAEKINYQFAVSQMAEPKKEKDIPYNLKSEAQGATHHHHHHH

[0234]

[0235] 1.2.3 Second-order and tertiary structures of WLcd2

[0236] PSIPRED server predictions show that the secondary structure of WLcd2 consists of 38.17% random coils, 43.7% α-helices, and 18.13% β-sheets. Figure 5 A). The high proportion of random coils provides flexibility for multi-epitope connections, while α-helices and β-sheets help maintain overall protein structural stability. The initial tertiary structure of WLcd2 was significantly improved in quality after MD simulation and refinement on the GalaxyRefine server. Figure 5 B). ERRAT analysis showed that the optimized overall quality factor was 90.533, indicating that the overall non-bonded interactions of the model were reasonable and the structural quality was good. Ramachandran plot analysis showed that 85.7% of the residues were located in the most favorable conformational region, 14.1% were located in the allowed region, and 0.2% were located in the disallowed region. Figure 5C) indicates that the protein backbone has a reasonable dihedral conformation and stable spatial folding. The Z-value predicted by the ProSA-Web server is -6.51, which is within a reasonable range for known protein structures, further validating the overall quality of the model. Figure 5 D). In summary, the secondary structure of WLcd2 supports the flexible display of multiple epitopes, and its optimized tertiary structure exhibits good folding rationality and spatial stability, providing a reliable structural basis for multi-epitope vaccine design and immunogenicity research.

[0237] 1.2.4 Molecular docking of WLcd2 with TLR receptor

[0238] To investigate the interaction characteristics between the WLcd2 vaccine and Toll-like receptors, this study used the HawkDOCK server for initial blind docking and optimized the optimal conformation using the HawkDOCK 2.4 server. The docking results suggest that the WLcd2 vaccine has the potential to form binding complexes with TLR2, TLR4, and TLR5 (Table 14). Figure 6 The HADDOCK scores were all negative, indicating that the three complexes had favorable docking conformations and potential binding tendencies. WLcd2-TLR4 had the best score (-176.2±2.2), followed by WLcd2-TLR5 (-161.9±3.3), while WLcd2-TLR2 had a relatively low score (-146.0±2.0). The RMSD values ​​of all three complexes were less than 0.7 Å, and the Z-Score was 0, indicating good consistency among the conformational clusters. Energy decomposition analysis (Table 4-5) showed that van der Waals energy and electrostatic energy were both negative, representing the main energy contributors besides desolvation energy. The electrostatic energy of WLcd2-TLR4 (-539.5±51.7 kcal / mol) is the largest in absolute value, followed by WLcd2-TLR5 (-520.9±39.6 kcal / mol) and WLcd2-TLR2 (-406.6±12.9 kcal / mol). Van der Waals energies show little difference among the three complexes (-67.0 to -80.9 kcal / mol). Desolvation energies are all positive (9.2–16.2 ​​kcal / mol). These energy components suggest that the complexes have a certain favorable binding tendency. Interface analysis shows that the buried surface area (BSA) of all three complexes is greater than 2600 Å. 2 (2643.7–2730.6 Å) 2PDBsum analysis showed that WLcd2-TLR2 formed 3 salt bridges and 15 hydrogen bonds, WLcd2-TLR4 formed 7 salt bridges and 13 hydrogen bonds, and WLcd2-TLR5 formed 5 salt bridges and 20 hydrogen bonds. These results provide structural clues suggesting that WLcd2 may interact with TLR receptors and indicate its potential innate immune receptor recognition capability, but further experimental verification is needed.

[0239]

[0240] 1.2.5 Molecular dynamics simulation of WLcd2-TLR docking complex

[0241] 1.2.5.1 Molecular Dynamics Simulation Trajectory Analysis

[0242] To systematically evaluate the binding stability and molecular recognition characteristics of the WLcd2 vaccine with immune receptors TLR2, TLR4, and TLR5, this study performed 100 ns molecular dynamics (MD) simulations on each complex. MD simulations can reproduce molecular thermal motion under near-physiological conditions at the atomic level, not only verifying the stability of the docking complex over time but also revealing the dynamic characteristics of non-covalent interactions such as hydrogen bonds and salt bridges. Furthermore, MD simulations can help identify key residues and binding hotspots and reduce the uncertainty of single static docking results. This study used RMSD, Rg, RMSF, and the number of hydrogen bonds at the complex interface to perform multi-dimensional analysis of the simulated trajectories.

[0243] RMSD measures the deviation of the overall structure of a complex from its initial conformation and is one of the core indicators for evaluating the equilibrium and dynamic stability of a system. A smaller RMSD value with less fluctuation generally indicates a more stable overall structure, while a higher or persistently fluctuating value suggests potential conformational adjustments, overall displacement, or changes in flexible regions. The results show ( Figure 7 A) The WLcd2-TLR4 and WLcd2-TLR5 complexes rapidly stabilized within the first 6 ns after the simulation began and maintained relatively stable fluctuations in subsequent simulations. In contrast, the WLcd2-TLR2 complex exhibited more pronounced RMSD fluctuations in the early stages of the simulation, subsequently stabilizing. The average RMSDs for the three complexes were 0.959±0.232 nm for WLcd2-TLR2, 0.356±0.046 nm for WLcd2-TLR4, and 0.424±0.097 nm for WLcd2-TLR5. These results indicate that the WLcd2-TLR4 and WLcd2-TLR5 complexes showed relatively small overall conformational deviations and high dynamic stability; while the WLcd2-TLR2 complex had a higher average RMSD, suggesting that it may have undergone significant conformational adaptation, overall translation, or rotation during the simulation, but this does not necessarily represent local structural unfolding.

[0244] Rg is used to reflect the compactness and spatial extension of the overall structure of a complex. Generally, a lower Rg value with less fluctuation suggests a more compact structure, while an increased Rg value or greater fluctuation may indicate some conformational extension, enhanced local flexibility, or conformational breathing. Simulation results show ( Figure 7 (B) The average Rg values ​​of the WLcd2-TLR2, WLcd2-TLR4, and WLcd2-TLR5 complexes were 3.253±0.505 nm, 3.225±0.224 nm, and 2.954±0.197 nm, respectively. Among them, the WLcd2-TLR5 complex had the lowest Rg value and the smallest fluctuation range, suggesting that it maintained a more compact and stable overall conformation during the simulation. The Rg value of the WLcd2-TLR4 complex was slightly higher than that of TLR5, but the fluctuation range was relatively limited, indicating that its overall structure was slightly more extended than TLR5, but still maintained a relatively stable spatial conformation. In contrast, the WLcd2-TLR2 complex had the highest average Rg value and the largest fluctuation range, consistent with its higher RMSD result, suggesting that this complex may have undergone a more significant overall conformational adjustment or spatial stretching during the simulation.

[0245] Hydrogen bond quantity analysis was used to assess the abundance and stability of non-covalent interactions at the complex interface. Simulation results show that ( Figure 7 C) The average number of hydrogen bonds in the WLcd2-TLR2, WLcd2-TLR4, and WLcd2-TLR5 complexes are 8±3, 15±3, and 17±4, respectively. The TLR4 and TLR5 complexes form significantly more hydrogen bonds than TLR2, suggesting richer polar interactions at their bonding interface. The TLR5 complex exhibits the lowest RMSD, the lowest Rg, and the highest number of hydrogen bonds, indicating good overall conformational stability, structural compactness, and interfacial interactions. While the Rg of the TLR4 complex is slightly higher than that of TLR5, suggesting a slightly more open conformation, its lowest RMSD and high number of hydrogen bonds indicate good overall dynamic stability and interfacial bonding stability. In contrast, the TLR2 complex has fewer hydrogen bonds and higher RMSD and Rg, suggesting relatively weaker interfacial interactions accompanied by more pronounced overall conformational adjustments.

[0246] RMSF analysis was used to assess the residue-level flexibility and local fluctuation characteristics of the vaccine chain in the complex. The mean RMSF values ​​showed that the flexibility of WLcd2 in the TLR2, TLR4, and TLR5 complexes was 0.272±0.083 nm, 0.370±0.127 nm, and 0.537±0.228 nm, respectively. Notably, the RMSF of residues 385–395 in the TLR5 complex exceeded 1 nm, suggesting high flexibility in this local region, possibly related to local conformational adaptation or dynamic binding processes. Figure 7 (D) It is important to note that a higher RMSF mainly reflects enhanced local residue fluctuations and cannot be directly equated with a critical binding region. Therefore, whether this region participates in key interactions still needs further confirmation through analysis of interface residues, hydrogen bonds, and salt bridges. Combining RMSD and Rg results, it can be seen that although the TLR2 complex has a lower local RMSF in the vaccine chain, its overall RMSD and Rg are both higher, indicating that its larger overall deviation may mainly originate from overall translation, rotation, or conformational rearrangement of the complex, rather than significant local unfolding of the vaccine chain structure. Although the TLR5 complex has high local flexibility, its overall RMSD is low, its Rg is the lowest, and its hydrogen bond count is the highest, suggesting that the complex has a stable overall conformation and a compact structure. Local flexibility may provide some space for receptor recognition or interface adaptation. The TLR4 complex exhibits the lowest RMSD and a higher number of hydrogen bonds, but its Rg is slightly higher than that of TLR5, suggesting good overall dynamic stability, but its spatial conformation is relatively more open.

[0247] In summary, the complexes formed by WLcd2 and TLR5 exhibit relatively balanced performance in terms of RMSD, Rg, and the number of hydrogen bonds, suggesting good dynamic stability, structural compactness, and interfacial interaction characteristics. While the Rg of the WLcd2-TLR4 complex is slightly higher than that of TLR5, its RMSD is the lowest and it has a higher number of hydrogen bonds, indicating smaller overall conformational deviations and more stable interfacial binding. The WLcd2-TLR2 complex, on the other hand, shows higher RMSD and Rg, but fewer hydrogen bonds, suggesting a larger overall conformational adjustment range and relatively weaker interfacial interactions. Overall, the MD simulation results support the good dynamic conformational stability and interfacial interaction characteristics of the WLcd2 complexes with TLR4 and TLR5, with the TLR5 complex showing more outstanding performance in terms of structural compactness and interfacial interactions. This provides structural and kinetic evidence for the potential stable interactions and molecular recognition characteristics between WLcd2 and immune receptors.

[0248] 1.2.5.2 MM / PBSA binding free energy

[0249] The binding free energy of WLcd2 complexes with TLR2, TLR4, and TLR5 was analyzed using the MM-PBSA method to further assess the binding tendency of the vaccine to the immune receptors from an energetics perspective. The results (Table 15) showed that the total free energy of all three complexes was negative: WLcd2-TLR2: -71.82±9.95 kcal / mol, WLcd2-TLR4: -107.38±11.48 kcal / mol, and WLcd2-TLR5: -115.82±13.72 kcal / mol, indicating a favorable binding tendency of WLcd2 with all three TLR receptors. Among them, the total free energy of the WLcd2-TLR5 complex was the most negative, followed by WLcd2-TLR4, while WLcd2-TLR2 was relatively low, indicating that WLcd2 has a stronger thermodynamic binding tendency with TLR5 and TLR4 than with TLR2.

[0250] Regarding the energy components, the van der Waals energies of all three complexes were negative, indicating that short-range nonbonded interactions significantly contribute to the stability of the complexes. The van der Waals energies of the WLcd2-TLR4 and WLcd2-TLR5 complexes were significantly higher than those of WLcd2-TLR2, suggesting a stronger contribution from short-range interactions. The electrostatic energies were also all negative, with the WLcd2-TLR5 complex exhibiting the largest absolute value (-2798.98 ± 75.42 kcal / mol), followed by WLcd2-TLR4, and the lowest for WLcd2-TLR2. This suggests that charge-charge interactions may be one of the key driving forces for the formation of favorable bonds in the WLcd2-TLR5 complex. Meanwhile, the polar solvation energies were all positive, significantly offsetting the binding free energy, especially in the WLcd2-TLR5 complex, reaching 2817.30±69.79 kcal / mol, indicating that strong electrostatic attraction is subject to significant polar solvation penalty in aqueous environment. The nonpolar solvation energies were all negative, but their values ​​were small, suggesting that their contribution to the stability of the complex was relatively limited.

[0251] The combined energy results show that although the WLcd2-TLR5 and WLcd2-TLR4 complexes exhibit a strong polar solvation cancellation effect, the combined effect of their van der Waals energy, electrostatic energy, and nonpolar solvation energy still keeps the total binding free energy at a low level. Combined with the RMSD, Rg, and hydrogen bond quantity analyses from the aforementioned MD simulations, WLcd2 may form a relatively stable receptor binding mode with TLR5 and TLR4. The TLR5 complex shows the best total binding free energy, suggesting it may have stronger receptor interaction potential; while the WLcd2-TLR2 complex has a relatively high total binding free energy, indicating relatively weaker binding stability. These results, from an energy perspective, support the possibility of a favorable interaction between WLcd2 and TLR4 / TLR5, providing a theoretical basis for further elucidating the molecular recognition characteristics and key binding residues between WLcd2 multi-epitope vaccines and immune receptors.

[0252]

[0253] 1.2.6 HLA population coverage of WLcd2

[0254] Predictive analysis based on the global HLA gene frequency database shows that the designed multi-epitope vaccine WLcd2 has a global population coverage rate of 99.97%, indicating that the vaccine has a high theoretical HLA population coverage rate and a wide potential population applicability. Figure 4-7 At the continental level, WLcd2 exhibits excellent coverage characteristics. Figure 4-7The predicted coverage rates in Europe and North America are close to or reach 100.00%, while South America also shows good performance with a coverage rate of 99.95%. These results suggest that WLcd2 has potentially broad HLA applicability in populations in Europe and North America with high CDI reported burden. At the national level, the predicted coverage rates in France, Germany, Mexico, Spain, and the United States are close to or reach 100.00%; populous countries such as China (99.84%), Japan (99.86%), and India (99.98%) also show extremely high coverage rates. Notably, Pakistan's predicted coverage rate is relatively low (87.90%), which may be related to the region's unique HLA allele distribution, suggesting that individual differences need to be considered when extending it to specific populations in South Asia. In addition, the difference between England (99.99%) and Scotland (96.88%) also reflects that even geographically proximate regions may have significant differences in HLA polymorphism distribution. Overall, the theoretical predictions of the WLcd2 vaccine based on HLA gene frequencies show that it has extremely high population coverage potential in major countries and regions around the world. However, the actual immunogenicity and protective effect may still be affected by individual immune status, antigen presentation efficiency and non-HLA genetic factors. Therefore, its broad applicability still needs to be further verified through in vitro functional experiments, more animal models and subsequent preclinical studies.

[0255] 1.2.7 Immunomimetic simulation of WLcd2 protein

[0256] This study simulated the overall immune response induced by WLcd2 vaccine using a C-IMMSIM server. Following a pre-set immunization protocol, three immunizations were performed at steps 0, 42, and 84 (corresponding to actual vaccination days 0, 14, and 28), with a total simulation duration of 350 days to analyze immunodynamic changes. Results showed that WLcd2 triggered a typical antibody response trend, with a significant enhancement effect with increasing immunization count. Peak antibody concentrations occurred after each vaccination, with the peak increasing progressively. Antibody levels reached their highest point approximately 10-14 days after the last immunization, then slowly declined and stabilized, remaining at a relative level of approximately 16,000 throughout the simulation period. Figure 8 A) suggests that the candidate vaccine may induce a relatively sustained humoral immune response in the simulated system. B) Cellular response analysis ( Figure 8 (B) showed that the number of B cells increased rapidly in the initial stage of immunization, and a memory B cell pool was quickly formed. With increasing vaccination frequency, the total B cell and memory cell populations continued to expand, suggesting that they may induce a memory-like B cell response in the simulated system. Cellular immunity level ( Figure 8The C–F simulation showed that WLcd2 can simultaneously activate TH cells, TC cells, and their subsets. TH cell activation peaked approximately 40 days post-immunization, then gradually declined and remained relatively stable, suggesting that the vaccine can induce a significant trend of helper T cell response and may promote the formation of memory B cell-related responses. TC cell activation fluctuated less overall, but showed a significant activation peak after immunization, reaching its peak approximately 14 days after the last immunization, and then returning to a resting state several tens of days later, suggesting that the vaccine may induce cytotoxic T cell-related responses. In summary, WLcd2 can simultaneously activate B cells, TH cells, and TC cells in the simulation system, and its response pattern is consistent with the expected characteristics of humoral and cellular immune responses induced by multi-epitope vaccines. This provides a computational simulation basis for the potential immunogenicity of the candidate vaccine, and the simulation results provide a theoretical basis for subsequent in vivo immunogenicity and protective effects evaluation in mice.

[0257] 1.2.8 Construction of recombinant plasmids for WLcd2 and verification of prokaryotic expression and purification

[0258] In this embodiment, the prediction results from the JCat server showed that the optimized WLcd2 cDNA sequence had a CAI value of 0.9799, a GC content of 48.97%, and an open reading frame length of 1572 bp. The target gene was directionally cloned into the pET-22b(+) expression vector, with NdeI and XhoI restriction enzyme sites selected. The theoretical length of the resulting recombinant plasmid was 6942 bp. Figure 9 A). Colony PCR verification showed that the amplified fragment size was consistent with the theoretical value. Double enzyme digestion identification results showed that the recombinant plasmid contained a target gene fragment of approximately 1572 bp and a vector backbone band of approximately 5000 bp. Figure 9 B), consistent with the expected size, indicating successful construction of the recombinant plasmid. Preliminary expression induction was performed using 0.5 mM IPTG at 37°C for 4 h. SDS-PAGE analysis showed a clear target band between 45-66.2 kDa markers, with its migration position close to the theoretical molecular weight of WLcd2 (59.31 kDa). Figure 9 C); Western blot analysis further confirmed that the band could be specifically recognized by the anti-His tag antibody, indicating that the WLcd2 protein was successfully expressed. Figure 9 D). After systematic optimization of the induction conditions, it was found that the target protein expression was maximized when the IPTG concentration was reduced to 1 mM, the induction temperature was adjusted to 15℃, and the induction time was extended to 16 h. SDS-PAGE analysis showed that WLcd2 protein was mainly enriched in the bacterial lysate, and the target band was almost undetectable in the supernatant, suggesting that the protein exists intracellularly in the form of inclusion bodies. Figure 4-10E). Although theoretical predictions suggested that WLcd2 had good solubility, actual expression results showed that it mainly existed in the form of inclusion bodies, indicating that the calculated predictions could not fully reflect the actual folding state in the prokaryotic expression system. To address the inclusion body characteristics, purification was performed using Ni-NTA affinity chromatography under denaturing conditions, followed by gradual removal of the denaturing agent via gradient dialysis to achieve protein refolding. Figure 9 F). SDS-PAGE showed that after refolding, the protein appeared as a single, distinct band between 45-66.2 kDa Markers, with migration positions largely consistent with the theoretical molecular weight of WLcd2. Gray-scale analysis showed a purity ≥95%. Figure 4-10 G). Western blotting further showed that the band could be specifically recognized by the anti-His tag antibody, verifying the successful expression, purification, and refolding of the WLcd2 protein. Figure 4-10 H). BCA quantification results showed that the final concentration of the refolded protein was approximately 0.87 mg / mL.

[0259] Example 2

[0260] 2.1 Experimental Methods

[0261] 2.1.1 Safety assessment of the fusion protein and optimization analysis of the immunization procedure

[0262] To systematically evaluate the safety and immunogenicity of the WLcd2 vaccine and determine the optimal immunization dose and schedule, this study was conducted in two phases: first, the optimal immunization dose was determined through dose gradient exploration, and then dynamic analysis of the immunization timeline and safety evaluation were carried out based on the optimal dose.

[0263] (1) Immunization dose gradient exploration experiment: Twenty wild-type BALB / c female mice aged 6–8 weeks were randomly divided into 4 groups (n=5) using a random number table: saline control group (NS), low-dose group (10 μg / mouse), medium-dose group (20 μg / mouse), and high-dose group (30 μg / mouse). Before vaccination, the WLcd2 recombinant protein was diluted to a final volume of 100 μL with sterile saline and immunized by intramuscular injection (IM). The immunization schedule was immunization once on days 0, 14, and 28, for a total of 3 times. The control group was given an equal volume of sterile saline. The weight change, activity status, and coat luster of the mice were observed and recorded daily. Mice were sacrificed 14 days after the last immunization (day 42), and serum was collected. The titer of anti-WLcd2 specific IgG antibody in the serum was detected by indirect enzyme-linked immunosorbent assay (ELISA). The optimal immunization dose was determined by comparing the antibody response intensity of different dose groups.

[0264] (2) Dynamic analysis of immune time course and safety evaluation: Based on the results of the above dose exploration experiment, the 30 μg dose with the strongest humoral immune response was selected for dynamic analysis of immune time course. Ten wild-type BALB / c female mice aged 6–8 weeks were randomly divided into two groups (n=5): NS control group and WLcd2 optimal dose group (30 μg / mouse). The immunization method and procedure were the same as in Part (1). Blood was collected from the orbital venous plexus on days 0, 14, 21, 28 and 42, and serum was separated for dynamic monitoring of anti-WLcd2 specific IgG antibodies. Among them, blood collection on days 0, 14 and 28 was completed before immunization on the same day to avoid the immediate impact of the immunization on the test results; day 21 was used to observe the changes in antibody response 7 days after the second immunization, and day 42 was used to evaluate the antibody level 14 days after the last immunization. The general condition of the mice was observed and recorded daily during the experiment.

[0265] 2.1.2 Effects of fusion proteins on mouse gut microbiota

[0266] To further evaluate the safety of the WLcd2 vaccine on the intestinal microecology of mice, fecal samples were collected before immunization (day 0), on day 7 after the last immunization (day 35), and on day 42 after treatment with mixed antibiotic water, sterile water recovery, and clindamycin pretreatment. Mice received three immunizations, 14 days apart. The vaccine group received an intramuscular injection of 30 μg WLcd2 (100 μL), while the control group received an equal volume of physiological saline (100 μL). The operation and feeding conditions were the same as those of the experimental group. To simulate the susceptible environment after antibiotics disrupt the intestinal microbiota, all mice were given a mixed antibiotic in their drinking water for 5 consecutive days starting 7 days after the last immunization. The antibiotics included kanamycin (40 mg / kg), gentamicin (3.5 mg / kg), colistin (4.2 mg / kg), metronidazole (21.5 mg / kg), and vancomycin (4.5 mg / kg). They were then fed sterile water for 2 days, and clindamycin (10 mg / kg) was administered intraperitoneally on day 41. This treatment provided a uniform microecological disturbance background for both the vaccine and control groups, facilitating a comparison of the impact of vaccination on the gut microbiota. After fecal sample collection, the 16S rRNA gene V4 region library construction and sequencing were performed by Beijing Novogene Technology Co., Ltd., using the Illumina MiSeq platform for paired-end sequencing (2×250bp). The company followed standard operating procedures for PCR amplification, barcoding, library purification, and equimolar pooling. Sequencing data underwent quality control, splicing, chimera removal, and noise reduction (DADA2) to obtain the final ASV feature table, which was then annotated in QIIME2 using the Silva 138.2 database. Subsequently, gut microbiota analysis was performed using R software, including α-diversity (Shannon index), β-diversity (PCoA analysis), and abundance of major bacterial groups (relative abundance at the phylum / genus level) to assess the potential impact of WLcd2 vaccination on the mouse gut microbiota, providing supplementary evidence for vaccine safety.

[0267] 2.1.3 The enhancing effect of adjuvants on the immunogenicity of WLcd2

[0268] To further optimize the immunization strategy of WLcd2 vaccine and evaluate the enhancing effects of different adjuvants on humoral and cellular immunity, this study designed an adjuvant compatibility experiment. Thirty healthy female BALB / c mice aged 6–8 weeks were randomly divided into 6 groups (n=5): saline control group (NS), Al(OH)3 alone group, MF59 alone group, optimal WLcd2 dose group (30 μg), WLcd2 + Al(OH)3 combined group, and WLcd2 + MF59 combined group. The adjuvant preparation schemes were as follows: the adjuvant alone group was a 1:1 (v / v) mixture of adjuvant and saline; the WLcd2 combined adjuvant group was a 1:1 (v / v) mixture of WLcd2 and adjuvant, with WLcd2 at 30 μg, then adjusted to a final volume of 100 μL with saline; the control group received 100 μL of saline. The immunization method, procedure, and general monitoring were consistent with section 2.1.1. Fourteen days after immunization, mice were sacrificed, and serum was collected. The level of anti-WLcd2 specific IgG antibody was detected by indirect ELISA to evaluate the intensity of humoral immune response. Spleen was removed under aseptic conditions, and single-cell suspension was prepared and the following tests were performed: (1) lymphocyte proliferation index was detected by CCK-8 assay; (2) IFN-γ and IL-4 levels in supernatant were detected by sandwich ELISA; (3) the ratio of CD4⁺IL-4⁺ T cells and CD8⁺IFN-γ⁺ T cells was detected by flow cytometry to systematically evaluate the characteristics of vaccine-induced cellular immune response. For specific methods, please refer to sections 2.1.9 to 2.1.12 below.

[0269] 2.1.4 Immunoprotective effects of WLcd2 with different adjuvant combinations

[0270] To evaluate the protective effect of WLcd2 in a CDI mouse model under different adjuvant combinations, an animal model of Clostridium difficile infection was established in this embodiment. Seventy-eight healthy female BALB / c mice aged 6-8 weeks were randomly divided into six groups: sham-infected group (n=16), CDI group (n=10), CDI+Al(OH)3 group (n=10), CDI+MF59 group (n=10), CDI+[WLcd2+Al(OH)3] group (n=16), and CDI+[WLcd2+MF59] group (n=16). The specific sample allocation and necropsy strategy for each group are shown in Table 16.

[0271] Immunization was administered intramuscularly, with immunizations given on days 0, 14, and 28. Seven days after the third immunization (day 35), mice were pretreated with a mixed antibiotic solution for five consecutive days to eliminate normal intestinal flora, followed by two days of sterile water. The mixed antibiotic solution was the same as described in section "2.1.2 Effects of Fusion Protein on Mouse Intestinal Flora". Twenty-four hours before Clostridium difficile infection, mice were intraperitoneally injected with clindamycin (10 mg / kg), followed by gavage inoculation with Clostridium difficile R20291 strain (200 μl / mouse, bacterial concentration 2.0 McFarland turbidity) to establish a CDI model.

[0272] During the experiment, daily monitoring of mouse weight changes, survival status, diarrhea severity, and other clinical symptoms was conducted and recorded using the standardized scoring system described in "2.1.14 Clinical Symptom Scoring" below. A humanitarian endpoint was set: if a mouse's weight decreased by more than 25% of its initial weight, or if it exhibited severe dehydration, inability to eat, or loss of mobility, euthanasia was immediately performed according to ethical guidelines. For mice that reached the humanitarian endpoint or died naturally, the highest CSS score on the day of death or euthanasia was recorded; subsequent time points after death or euthanasia were not included in the daily CSS dynamic scoring, but only in the Kaplan-Meier survival analysis. If intergroup CSS comparisons were performed, statistical analysis was based on the recorded values ​​of mice still within the observation period. On day 7 after challenge, the remaining surviving mice were sacrificed, and a systematic dissection was performed. Intestinal tissue and related samples were collected, and pathological scoring was performed according to the standards in "2.1.15 Histopathological Scoring Criteria" below to comprehensively evaluate the enhancing effect of the adjuvant on the vaccine's immunoprotective efficacy.

[0273]

[0274] 2.1.5 Mouse tissue sampling

[0275] 2.1.5.1 Mouse serum collection

[0276] Mice were first anesthetized using isoflurane inhalation or 1% pentobarbital intraperitoneal injection. The disappearance of pain reflexes was used as a marker of successful anesthesia to ensure the mice were in a stable anesthetized state for subsequent procedures. Blood was collected via orbital and ocular sampling. After collection, the blood was incubated on ice for 1 hour, then centrifuged at 4°C and 3000 rpm for 15 minutes. The supernatant serum was separated, aliquoted into 200 μL EP tubes, and stored at -80°C for later use.

[0277] 2.1.5.2 Mouse Tissue Sampling

[0278] Immediately after blood was collected from the eyeballs, the mice were euthanized using either cervical dislocation or an overdose of isoflurane. Once cardiac and respiratory arrest was confirmed, the mice were fixed supine on a clean dissecting board, and the chest, abdomen, and limb fur were thoroughly disinfected with 75% ethanol. Subsequently, abdominal organs (including the spleen, liver, kidneys, and colon) and thoracic organs (heart and lungs) were harvested.

[0279] 2.1.6 Hemolysis test

[0280] (1) Preparation of red blood cell suspension: Collect venous blood from mice, centrifuge at 3000 rpm for 15 minutes, and discard the plasma. Resuspend the red blood cells in PBS, centrifuge at 3000 rpm for 10 minutes and wash 3-5 times until the supernatant is colorless. Prepare a 4% red blood cell suspension with PBS for later use.

[0281] (2) Experimental groups: Negative control group: 4% red blood cell solution + PBS; Positive control group: 4% red blood cell suspension + ddH2O; Experimental group: 4% red blood cell suspension + WLcd2 (0.2, 0.4, 0.8 mg / mL); The volume ratio of each group was 1:1.

[0282] (3) Incubation and detection: After incubation at 37℃ for 2 hours, centrifuge at 4℃ and 3000rpm for 10 minutes. Take 100μL of the supernatant into a 96-well plate and measure the absorbance at 540 nm.

[0283] (4) Calculation of results:

[0284] Hemolysis rate (%) = (OD of experimental group - OD of negative control group) / (OD of positive control group - OD of negative control group) × 100%

[0285] Judgment criteria: Hemolysis rate <5% is considered no hemolysis, 5%-10% is considered mild hemolysis, and >10% is considered significant hemolysis.

[0286] 2.1.7 Detection of serum liver and kidney function biochemical indicators in mice

[0287] Serum ALT, AST, TBIL, ALB, BUN, Cr, and other indicators in mice were tested by Wuhan Saiweier Biotechnology Co., Ltd., and measured using the Chemray240 fully automated biochemical analyzer from Shenzhen Leidu Life Science Technology Co., Ltd.

[0288] 2.1.8 Hematoxylin-eosin (HE) staining

[0289] (1) Tissue sample processing: The tissue samples were immediately placed in 4% paraformaldehyde fixative and fixed at 4°C for 48 hours. After fixation, the samples were sent to Wuhan Pinofi Biotechnology Co., Ltd. for routine paraffin embedding and sectioning to a thickness of 4 μm.

[0290] (2) Baking, dewaxing and hydration: Place the paraffin sections in a 65℃ constant temperature oven and bake for 30-60 minutes until the sections are completely dry and the paraffin is melted. Then, immerse the sections in xylene I and xylene II in sequence for 5-10 minutes each to completely dewax them. After dewaxing, hydrate them with a gradient of ethanol: 100%, 95%, 85%, and 70% ethanol for 3 minutes each. Finally, rinse the sections with running distilled water for 1 minute, drain the water and proceed to the staining step.

[0291] (3) Hematoxylin staining: Place the sections in hematoxylin staining solution for 8 minutes, then rinse with running water for 15 minutes. Soak the sections in differentiation solution for 10 seconds, then rinse with running water for 10 minutes until the cell nuclei are clearly blue;

[0292] (4) Eosin staining: Place the section in eosin staining solution and stain for 1 minute. If staining is difficult, add 1-2 drops of glacial acetic acid to every 100 mL of staining solution to adjust the pH of the staining solution, promote staining and enhance staining stability, and prevent discoloration;

[0293] (5) Dehydration and clearing: Pass the sections through 70%, 85%, 95% ethanol and anhydrous ethanol in sequence, 1 minute for each step; then immerse them in xylene I and xylene II for clearing, 2 minutes for each step;

[0294] (6) Mounting and preservation: Gently wipe away excess xylene around the slide with filter paper, add an appropriate amount of neutral resin to cover the tissue slide, and quickly cover it with a coverslip to prevent air bubbles from forming. After mounting, place the slide in a fume hood to allow the xylene to evaporate and the resin to solidify, and then transfer it to a slide box and store it at 4°C in the dark for later use.

[0295] 2.1.9 Determination of mouse serum-specific IgG antibody levels

[0296] ① Antigen coating and blocking: WLcd2 recombinant protein was coated at a concentration of 5 μg / mL onto 96-well microplates (100 μL per well) and incubated overnight at 4°C. After coating, the liquid was discarded, and the plates were washed three times with TBST for 30 seconds each. Subsequently, 250 μL of blocking buffer was added to each well, and the plates were blocked at 37°C for 1 hour. After blocking, the plates were dried, and the plates were washed three times with TBST for 30 seconds each.

[0297] ② Antibody incubation: Add serum sample of appropriate dilution to the first well, followed by serial dilutions, and incubate at 37°C for 1 hour. After incubation, dry the plate and wash four times with TBST for 30 seconds each time. Add 100 μL of HRP-labeled goat anti-mouse IgG (1:10000 dilution) to each well and incubate at 37°C for 30 minutes. Wash the plate four more times for 30 seconds each time.

[0298] ③ Color development and detection: Add 100 μL of TMB chromogenic solution to each well, incubate at 37°C in the dark for 15 minutes, then add 100 μL of stop solution to terminate the reaction. Measure the absorbance at 450 nm and 630 nm using a microplate reader. Positive result criteria: based on OD... 450 -OD 630 As a correction absorbance value, a sample is considered positive when its corrected OD value is greater than 2.1 times that of the negative control well.

[0299] 2.1.10 Lymphocyte proliferation experiment

[0300] (1) Spleen acquisition: After euthanizing the mice by cervical dislocation, the spleen was soaked in 75% ethanol for 5 minutes and placed on a sterile operating table. The abdominal skin was cut open to expose the spleen. The spleen was separated along the connective tissue on the lower side of the spleen and removed intact. The spleen was immediately placed in pre-cooled PBS solution.

[0301] (2) Splenic lymphocyte acquisition: Place the spleen on a 100 μm cell filter sieve and add a small amount of PBS or complete culture medium to maintain a moist environment. Gently grind the spleen using a syringe plunger to release the cells into a centrifuge tube below the sieve, while simultaneously washing with culture medium to ensure complete cell release and formation of a single-cell suspension through the sieve. Centrifuge the resulting cell suspension at 4°C and 500×g for 5 min and discard the supernatant. Add 1 mL of erythrocyte lysis buffer to the cell pellet, gently pipette to mix, and lyse for about 2 min, then add ice-cold PBS to terminate the lysis. Centrifuge at 4°C and 500×g for 5 min and discard the supernatant; if erythrocyte lysis is incomplete, repeat the lysis once. Resuspend the cell pellet in PBS and wash by centrifugation at 4°C and 500×g for 5 min, repeating the washing once. Discard the supernatant and resuspend the cells in RPMI-1640 medium containing 10% fetal bovine serum (FBS), count and adjust to 2×10⁶ cells / mL. 6 / mL for later use;

[0302] (3) Lymphocyte stimulation and proliferation assay: Spleen lymphocyte suspension was seeded into 96-well cell culture plates, 200 μL per well. The experimental groups were stimulated with WLcd2 recombinant protein (2 μg / well), and a PBS negative control group and a complete culture medium blank control group were included. Each group was divided into 3 biological replicates, with 2 replicates per replicate. After incubation at 37℃ and 5% CO2 for 48 hours, 20 μL of LCK-8 reagent was added to each well, and incubation continued for 6 hours. The absorbance at 450 nm was measured using a microplate reader to assess cell proliferation levels.

[0303] 2.1.11 Detection of IFN-γ and IL-4 levels in lymphocyte supernatant

[0304] (1) Collection of spleen lymphocyte supernatant: Adjust the prepared spleen lymphocyte suspension to 2×10 6Cells were seeded at a density of 10 μg / mL in 24-well cell culture plates. The experimental groups were treated with WLcd2 recombinant protein to a final concentration of 10 μg / mL for in vitro restimulation. A PBS negative control group and a complete culture medium blank control group were also included. After incubation at 37°C, 5% CO2, and saturated humidity for 48 hours, the cell culture supernatant was collected and centrifuged at 3000 rpm for 10 minutes at 4°C to remove cell debris. The supernatant was then aliquoted into 200 μL EP tubes and stored at -80°C for later analysis.

[0305] (2) ELISA detection: The secretion levels of IFN-γ and IL-4 in the supernatant were detected by sandwich ELISA. QuantiCyto® ELISA kit was used in this experiment.

[0306] 2.1.12 Flow cytometry

[0307] (1) Cell stimulation and collection: Take 2 mL of spleen lymphocyte suspension (approximately 2 × 10⁻⁶ cells / mL) 6 Cells were seeded in 12-well cell culture plates, with 10 μg of recombinant WLcd2 protein added to each well for pre-stimulation and incubated for 1 hour. Then, 2 μL of eBioscience™ Cell Stimulation Cocktail was added per well, and the plates were incubated at 37°C and 5% CO2 for an appropriate time to induce cell activation. After culture, cells were collected into 2 mL EP tubes, centrifuged at 4°C and 400 ×g for 5 minutes, the supernatant was discarded, and the cells were retained for subsequent staining steps.

[0308] (2) Surface and intracellular staining:

[0309] ① Washing and Fc receptor blocking: Add 1 mL of cell staining buffer and mix thoroughly. Centrifuge at 400×g for 5 minutes at 4°C, discard the supernatant, and retain approximately 100 μL of cell suspension. Add 2 μL of Fc receptor blocking agent and incubate on ice for 10 minutes to reduce nonspecific binding.

[0310] ② Live / dead staining: Add 0.3 μL of live / dead dye and incubate on ice in the dark for 15 minutes. Then wash with 1 mL of cell staining buffer, centrifuge at 400×g for 5 minutes at 4°C, discard the supernatant, and retain about 100 μL of cell suspension.

[0311] ③ Surface labeling and staining: Add 0.5 μL each of fluorescently labeled antibodies CD3, CD4, and CD8, mix well in the dark to a total volume of 100 μL, and incubate on ice in the dark for 30 minutes. After incubation, wash with 1 mL of cell staining buffer, centrifuge at 400×g for 5 minutes at 4°C, discard the supernatant, and retain 100 μL of cell suspension.

[0312] ④ Fixation and cell disruption: Add 300 μL of fixation buffer and incubate on ice in the dark for 30 minutes to fix the cells. Then add 2 mL of intracellular wash buffer, centrifuge at 500 × g for 5 minutes at 4°C, discard the supernatant, and retain 50 μL of cell suspension.

[0313] ⑤ Intracellular labeling and staining: Add 0.5 μL each of fluorescently labeled antibodies IL-4 and IFN-γ, then add 50 μL of Wash Buffer and mix well. Incubate on ice in the dark for 30 minutes. Then, wash with 1 mL of Wash Buffer to dilute 10-fold, centrifuge at 500×g for 5 minutes at 4°C, discard the supernatant, and retain 100 μL of cell resuspended. After staining, store the sample in the dark and perform flow cytometry analysis within 4 hours to assess intracellular cytokine expression levels.

[0314] 2.1.13 RNA extraction and qPCR experiment

[0315] (1) RNA extraction: TransZol Up Plus RNAKit from Beijing TransGen Biotechnology Co., Ltd. was used.

[0316] (2) RNA concentration and purity determination: RNA concentration and purity were determined using an ultra-micro spectrophotometer. First, the measurement center was washed with 1 μL of enzyme-free water and zeroed. Then, 1 μL of RNA sample was added to the measurement center, and the OD was measured. 260 / 280 and OD 260 / 230 The ratio was used to calculate and record the RNA concentration.

[0317] (3) Reverse transcription experiment: The Thermo Fisher Scientific reverse transcription kit was used and the procedure was followed according to the instructions. The reaction volume was 20 μL. First, the required RNA volume was calculated to ensure that each reaction system contained 1 μg of RNA.

[0318] ① Prepare the reverse transcription system according to the kit instructions, as shown in Table 17.

[0319]

[0320] ②PCR program settings (reverse transcription program), as shown in Table 18.

[0321]

[0322] (4) qPCR experiment: Yundan Bio's SYBR Green qPCR Master Mix was used for detection.

[0323] ① Primer design: Specific primers were designed based on the target gene sequence. The primers were 18-25 bp in length, with a GC content of 40%-60% and a Tm value close to 72℃. The primers were synthesized, purified, and their concentrations determined by Sangon Biotech (Shanghai) Co., Ltd. The specific sequences are shown in Table 19.

[0324]

[0325] ② Preparation of the reaction system (total volume 20 μL, as shown in Table 20): Thaw the cDNA sample, primers, and SYBR qPCR Master Mix (frozen at -20℃) on ice and prepare them. All experimental procedures were performed on ice to ensure sample stability. The cDNA sample was diluted 5-fold before the experiment. After mixing all components, gently vortex and briefly incubate to ensure the system was homogeneous and free of air bubbles. qPCR amplification and real-time fluorescence detection were then performed.

[0326]

[0327] ③ Set up the qPCR program as shown in Table 21: Place the prepared reaction system into the qPCR instrument for qPCR, and calculate the relative expression level of the target gene using the 2⁻ΔΔCt method.

[0328]

[0329] 2.1.14 Clinical Symptom Scoring

[0330] The total clinical symptom score ranges from 0 to 12 points, covering three dimensions: stool characteristics, behavioral manifestations, and weight changes. Disease severity is graded as follows: normal (0-2 points), mild (3-5 points), moderate (6-8 points), and severe (9-12 points).

[0331]

[0332] 2.1.15 Histopathological Scoring Criteria

[0333]

[0334] 2.2 Experimental Results

[0335] 2.2.1 Security Analysis of WLcd2

[0336] To systematically evaluate the in vivo safety of the WLcd2 multiepitope vaccine, this study comprehensively assessed its in vitro hemolytic activity, serum biochemical indicators, and histopathological findings. First, the potential toxicity of the vaccine to erythrocytes was evaluated using a hemolysis assay. Results showed that no significant hemolysis was observed in erythrocytes treated with different concentrations of WLcd2, and the difference was not significant compared to the saline control group. Figure 10 A and Figure 10 B) indicates that WLcd2 does not have hemolytic toxicity. Subsequently, serum was collected from mice that received three different doses of WLcd2 (10, 20, and 30 μg / mouse) intramuscularly to detect liver and kidney function-related indicators, including ALT, AST, TBIL, ALB, BUN, and Cr. The results showed that there were no statistically significant differences in the above indicators between the WLcd2 vaccine groups and the blank control group at each dose (P>0.05), indicating that WLcd2 did not cause significant liver and kidney function damage within the experimental dose range. Figure 10 C to Figure 10 H). Furthermore, HE staining and histological observation of the heart, liver, lungs, and kidneys of mice immunized with different doses of WLcd2 showed that all organs were structurally intact and morphologically normal, with no obvious inflammatory cell infiltration, tissue necrosis, or other pathological changes. Figure 10 I). The overall results indicate that WLcd2 has good biocompatibility in experimental mice, with no hemolytic toxicity, abnormal liver or kidney function, or damage to major organs, providing a reliable safety basis for subsequent evaluation of immunogenicity and protective efficacy.

[0337] 2.2.2 WLcd2 had no significant effect on the gut microbiota of mice.

[0338] Figure 11 A shows the experimental design and fecal sample collection time points for the WLcd2 vaccine in mice, including pre-vaccination, day 7 after the last immunization (post-vacc), and post-anti (including mixed antibiotic water, sterile water recovery, and clindamycin treatment). Alpha diversity analysis (Shannon index, reflecting community richness and evenness) showed no significant differences between the WLcd2 group and the control group at any of the three time points (P > 0.05). Figure 11 B) indicates that vaccination did not significantly alter community diversity. β-diversity PCoA analysis showed high overlap in clustering between the WLcd2 group and the control group at all time points. Under the same time points and pretreatment conditions, no significant additional community structure shift was observed between the WLcd2 group and the control group. Figure 11 C). No significant changes in the composition of the major bacterial communities were observed in the relative abundance analyses at the phylum and genus levels in the vaccine group. Figure 11 D and Figure 11 E). In summary, under the same antibiotic pretreatment background, no significant additional shift in gut microbiota structure was observed between the WLcd2 group and the control group, further supporting the safety of WLcd2 at the gut microbiota level.

[0339] 2.2.3 WLcd2 vaccine significantly induces dose-dependent humoral immune response

[0340] To comprehensively evaluate the humoral immune characteristics of the WLcd2 vaccine, this study conducted a systematic experiment on the effects of immunization dose, immunization schedule, and adjuvant compatibility on antibody induction efficacy. Figure 12 A). In a dose-dependent experiment, each mouse was intramuscularly injected with 10, 20, and 30 μg of vaccine, respectively. Blood samples were collected 14 days after the completion of the three immunizations, and serum anti-WLcd2 specific IgG levels were measured by ELISA. The results showed that the antibody response increased significantly in a dose-dependent manner, with the 30 μg dose group exhibiting the highest level of specific antibodies. Figure 12 B and Figure 12 (C) This indicates that the dose is optimal in terms of humoral immunogenicity. To dynamically monitor the temporal changes in the humoral immune response, blood collection was conducted at the following times: before immunization (day 0), 14 days after the first dose (day 14), 7 days after the second dose (day 21), 14 days after the second dose (day 28), and 14 days after the third dose (day 42). Blood collection was completed in the morning before immunization, and immunization was performed in the afternoon to ensure the continuity and accuracy of the data. Figure 12 D). The results showed that a significantly higher than baseline IgG response could be detected 14 days after the first immunization; antibody titers increased further on day 21; and decreased on day 28, suggesting a possible natural decline in antibody levels during the interval between two immunizations; however, on day 42 (14 days after the third booster immunization), antibody titers increased significantly again, exceeding the level on day 28, confirming that repeated exposure could effectively activate memory B cell responses and maintain a relatively long-lasting humoral immune response. This kinetic characteristic was highly consistent with the predictions of previous C-ImmSim immunization simulations (Figure 9). In the adjuvant synergistic experiment, 30 μg WLcd2 was immunized in combination with Al(OH)3 or MF59 emulsion, with a saline control group and groups receiving the vaccine or adjuvant alone. The results showed that the serum anti-WLcd2 IgG level in the combined adjuvant group was significantly higher than that in the control group and the group receiving the vaccine alone ( Figure 12 E and Figure 12 (F) The antibody levels in the WLcd2+Al(OH)3 and WLcd2+MF59 groups were approximately 16-32 times and 8-16 times higher, respectively, compared to the group receiving 30 μg WLcd2 alone. This suggests that both Al(OH)3 and MF59 can significantly enhance the humoral immune response to WLcd2, with Al(OH)3 showing a superior enhancing effect compared to MF59. In conclusion, the WLcd2 vaccine can induce a dose-dependent, programmed cumulative specific humoral immune response in mouse models, and the adjuvant combination significantly amplifies its immunogenicity. Specifically, in terms of total IgG induction, Al(OH)3 showed a stronger enhancing effect than MF59, providing experimental evidence for subsequent vaccine immunoprotective studies and optimization of adjuvant combinations.

[0341] 2.2.4 WLcd2 vaccine significantly induces Th1 / Th2-related cellular immune responses

[0342] To systematically evaluate the antigen-specific cellular immune response to the WLcd2 vaccine, this study used lymphocyte proliferation assays, cytokine ELISA, and flow cytometry to detect the response. Figure 13 A). Negative control results showed that the control group (NS) and the adjuvant alone group (Al(OH)3 or MF59) had reduced lymphocyte proliferation ( Figure 13 B), IFN-γ / IL-4 secretion by spleen cells ( Figure 13 C and 13D) and the proportion of T cell subsets ( Figure 13 E and Figure 13 There were no significant differences in aspects F), indicating that under the conditions of this experiment, the adjuvant alone did not induce detectable significant non-specific immune activation, providing a reliable baseline for vaccine efficacy assessment. Compared with the control group, the 30 μg WLcd2 immunization group showed a significant lymphocyte proliferation response after in vitro antigen restimulation, with significantly increased levels of IFN-γ and IL-4 in spleen cells. Flow cytometry further showed a significant increase in the proportion of CD4⁺IL-4⁺ and CD8⁺IFN-γ⁺ T cells, indicating that WLcd2 can effectively induce antigen-specific Th1 / Th2 mixed cellular immune responses. Adjuvant-assisted immunization experiments showed that the Al(OH)3+WLcd2 group was significantly higher than the vaccine-only group in terms of lymphocyte proliferation, IFN-γ / IL-4 secretion, and the proportions of CD4⁺IL-4⁺ and CD8⁺IFN-γ⁺ T cells. The MF59+WLcd2 group also showed enhanced lymphocyte proliferation and cytokine secretion, but the proportion of CD4⁺IL-4⁺ T cells did not increase significantly. This may be related to the fact that short-term flow cytometry stimulation (6h) did not fully reflect the effect of MF59 on the Th2 response. There were no significant differences in lymphocyte proliferation, IFN-γ, IL-4, and the proportions of CD8⁺IFN-γ⁺ T cells between the two adjuvant-assisted groups. However, the proportion of CD4⁺IL-4⁺ in the Al(OH)3 group was significantly higher than that in the MF59 group, suggesting that the Al(OH)3-assisted group may have a certain advantage in CD4⁺IL-4⁺ T cell responses. In summary, the WLcd2 vaccine can effectively induce antigen-specific Th1 / Th2 mixed cellular immune responses, and adjuvant combination can further enhance this response. Among them, Al(OH)3 showed better performance in promoting CD4⁺ T cell activation in the early stage, providing experimental basis for the optimization of adjuvant combinations for multi-epitope vaccines.

[0343] 2.2.5 Analysis of the immunoprotective effect of WLcd2

[0344] To systematically evaluate the protective effect of WLcd2 vaccine combined with different adjuvants against CDI-infected mice, this study randomly divided 6-8 week old female BALB / c mice into six groups: a mock-infected group, a CDI group, a CDI+Al(OH)3 group, a CDI+MF59 group, a CDI+[WLcd2+Al(OH)3] group, and a CDI+[WLcd2+MF59] group. Mice were immunized with the vaccine or adjuvant according to the established immunization protocol, and then challenged with Clostridium difficile strain R20291. During the experiment, the mice's mental state, activity level, body weight, fecal condition, and survival were dynamically monitored. Figure 14 A).

[0345] 2.2.5.1 WLcd2 combined with adjuvant can significantly improve the survival rate of CDI-infected mice.

[0346] Survival analysis showed that all participants in the CDI model group and the adjuvant-only groups (CDI+Al(OH)3, CDI+MF59) died within the 7-day observation period, with the peak mortality occurring within 72 hours post-infection. In contrast, the vaccine combined with adjuvant showed significant protective effects, with the survival rate of the CDI+[WLcd2+Al(OH)3] group reaching 90% and the survival rate of the CDI+[WLcd2+MF59] group reaching 80%, both significantly higher than the model group and the adjuvant-only group; however, the difference in survival rate between the two vaccine combination groups did not reach statistical significance. Figure 14 B and Figure 14 C).

[0347] 2.2.5.2 WLcd2 combined with adjuvant can significantly improve clinical symptoms in CDI-infected mice.

[0348] Dynamic weight monitoring showed that the weight fluctuation of mice in the sham-infected control group was less than 5%, and the weight remained stable overall. However, the weight of mice in the CDI infection group and the adjuvant-only group decreased significantly on days 2-3 after challenge, dropping to 75%-85% of the initial level, and there was no statistically significant difference between the groups (P>0.05), suggesting that adjuvant alone failed to alleviate the weight loss and related disease symptoms caused by infection. Figure 15 A to Figure 15 C). In contrast, the weight loss in the vaccine combined with adjuvant group was significantly slower, and the percentage of body weight at 48h and 72h was significantly higher than that in the CDI group and the adjuvant-only group, suggesting that WLcd2 combined with adjuvant can significantly reduce CDI infection-related weight loss. The change in CSS was consistent with the trend of weight change. Figure 15 D to Figure 15F). Both the CDI group and the adjuvant-only group showed a significant increase in CSS scores within 24 hours of infection, rapidly reaching 9–11 points, indicating severe clinical symptoms. Subsequently, all mice died or reached the humanitarian endpoint within 72 hours, and dynamic CSS recording was discontinued accordingly. There was no statistically significant difference between the CDI group and the adjuvant-only group. In contrast, the overall CSS of the vaccine combined with adjuvant group was significantly lower than that of the CDI model group and the adjuvant-only group. Surviving mice gradually recovered to mild or asymptomatic levels in the later stages of infection, but a few mice progressed to severe illness and died. In summary, the WLcd2 vaccine combined with MF59 or Al(OH)3 adjuvant not only significantly improved the survival rate of CDI-infected mice but also significantly reduced infection-related weight loss and clinical symptoms, while adjuvant alone did not show a significant protective effect. This suggests that the WLcd2 combined with adjuvant has good protective potential in reducing the severity of acute CDI disease.

[0349] 2.2.5.3 WLcd2 vaccine combined with adjuvant can alleviate colonic injury in CDI mice.

[0350] To further evaluate the alleviating effect of WLcd2 vaccine on CDI-induced intestinal damage, morbid mice were necropsed within 10 minutes after euthanasia. Mice surviving to day 7 (sham infection group and vaccine combined with adjuvant group) were uniformly euthanized and necropsed at the experimental endpoint. To visually observe the differences in HE staining of the colon between dead and surviving mice in the vaccine combined with adjuvant group, this study pre-dissected a portion of the vaccine-immunized group mice at the same time point to match the necropsies of the CDI group and the adjuvant control group. Specific procedures are described in section "2.1.4 Immunoprotective Effect of WLcd2 under Different Adjuvant Combinations" (Table 16). The results showed that the colonic mucosa structure of the sham infection group mice was intact, the epithelium was neatly arranged, the crypts were clear, no obvious inflammatory infiltration was observed in the lamina propria, and there was no edema or hemorrhage in the submucosa. The pathological scores at 48h and 72h were both 0-1 points. Figure 16 A to Figure 16 D). In contrast, all mice in the CDI group died within 72 hours of infection, and their colonic tissue showed typical CDI pathological changes: extensive inflammatory cell infiltration in the mucosa and submucosa, epithelial cell necrosis and shedding leading to crypt structure destruction, submucosal edema, bloody contents visible in the intestinal lumen, and a small number of lymphocytes and granulocytes infiltrating the muscular and serosa layers. The pathological score was significantly higher than that of the sham infection group. Figure 16 A to Figure 16D). There was no significant difference in colonic pathological damage between the adjuvant-only groups (CDI+MF59, CDI+Al(OH)3) and the CDI group, suggesting that adjuvant alone had no significant alleviating effect on CDI-induced intestinal damage, providing a reliable negative control for the subsequent vaccine protection effect. The vaccine combined with adjuvant showed a significant protective effect: the colonic mucosa structure of mice in the CDI+[WLcd2+MF59] group (48h, 72h) was relatively intact, with only focal epithelial cell necrosis and mild submucosal edema, and the pathological score was significantly lower than that of the adjuvant-only group ( Figure 16 A to Figure 16 D); By the experimental endpoint, the colon tissue of surviving mice was nearly normal, with intact mucosal epithelium, clear crypt structure, and no significant difference in pathological score compared to the sham-infected group. Figure 16 E and Figure 16 F). The CDI+[WLcd2+Al(OH)3] group showed a similar protective trend, with a slightly lower natural mortality rate, and the degree of intestinal repair in surviving mice on day 7 was comparable to that in the MF59 combined group. Figure 16 A Figure 16 F). In summary, the colonic pathological analysis of each group showed that the WLcd2 vaccine combined with adjuvant significantly reduced early intestinal inflammation and tissue damage, providing favorable conditions for intestinal tissue repair in surviving mice. Overall, the WLcd2 vaccine combined with adjuvant effectively improved the survival rate of CDI-infected mice, alleviated disease severity, and created conditions for the recovery of intestinal function.

[0351] 2.2.5.4 WLcd2 combined with adjuvant can reduce the mRNA expression of pro-inflammatory factors in the colon of CDI mice.

[0352] To further evaluate the regulatory effect of WLcd2 vaccine combined with adjuvant on the local inflammatory microenvironment of the colon in CDI mice, this study used qPCR technology to detect the mRNA levels of chemokine CXCL1 and key pro-inflammatory mediators IL-1β, IL-6 and TNF-α in the colon tissue of each experimental group. Figure 17 The experimental samples included mice that died naturally at different time points after infection and surviving mice that underwent pre-planned dissection to reflect the changing trends of colonic inflammation at different time points. Results showed that, compared with the Mock control group, the expression of inflammation-related genes in all infection groups (CDI group, adjuvant alone group, and vaccine combination group) was significantly upregulated at 48h and 72h, suggesting that CDI can strongly activate pro-inflammatory signals in the colon. Further inter-group comparisons showed that the levels of pro-inflammatory factor mRNA in the vaccine combination adjuvant group (CDI+[WLcd2+MF59], CDI+[WLcd2+Al(OH)3]) at 48h were significantly lower than those in the CDI group and the adjuvant alone group. Figure 17 A to Figure 17 D), the data from 72 hours show a similar trend ( Figure 17 E to Figure 17The H) suggests that this immunization strategy can suppress infection-induced intestinal inflammation and has a certain degree of persistence. There was no significant difference between the two adjuvant combination groups. Combined with HE histological observation, although early-surviving mice in the vaccine combination group still exhibited focal epithelial cell necrosis, mild submucosal edema, and scattered inflammatory cell infiltration, the transcriptional levels of pro-inflammatory factors significantly decreased. This suggests that the WLcd2 vaccine combined with adjuvant can inhibit the pro-inflammatory signaling cascade to a certain extent in the early stage of infection, creating a favorable immune microenvironment for the subsequent repair of damaged intestinal mucosa. In conclusion, the combined application of WLcd2 vaccine with Al(OH)3 or MF59 can significantly downregulate the mRNA expression levels of pro-inflammatory factors in the colon of CDI mice, demonstrating good potential for local inflammation regulation, and providing experimental evidence for elucidating its immunoprotective mechanism and advancing subsequent preclinical studies.

[0353] The above descriptions are merely several exemplary embodiments of the present invention and are not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any equivalent or related embodiments obtained by those skilled in the art through minor modifications or variations of the disclosed technical content without departing from the scope of the present invention fall within the scope of the present invention.

Claims

1. A pharmaceutical composition comprising a fusion protein and an immune adjuvant, wherein, The fusion protein comprises an epitope fusion peptide having HTL epitope clusters, CTL epitope clusters, and B-cell epitope clusters; wherein: The HTL epitope cluster includes one or more HTL epitopes selected from those shown as SEQ ID NO. 1 to SEQ ID NO. 7; The CTL epitope cluster includes one or more CTL epitopes selected from those shown in SEQ ID NO. 8 to SEQ ID NO. 17; The B-cell epitope cluster comprises one or more B-cell epitopes selected from those shown in SEQ ID NO. 18 to SEQ ID NO. 26; preferably, the B-cell epitope cluster is located at the C-terminus of the epitope fusion peptide.

2. The pharmaceutical composition according to claim 1, wherein, The HTL epitope cluster comprises HTL epitopes as shown in SEQ ID NO. 1 to SEQ ID NO. 7; preferably, the HTL epitopes as shown in SEQ ID NO. 1 to SEQ ID NO. 7 are connected in series in sequence; more preferably, the amino acid sequence of the HTL epitope cluster comprises or is composed of the amino acid sequence shown in SEQ ID NO.

27. And / or, the CTL epitope cluster comprises CTL epitopes as shown in SEQ ID NO. 8 to SEQ ID NO. 17; preferably, the CTL epitopes as shown in SEQ ID NO. 8 to SEQ ID NO. 17 are connected in series in sequence; more preferably, the amino acid sequence of the CTL epitope cluster comprises or is composed of the amino acid sequence shown in SEQ ID NO.

28. And / or, the B-cell epitope cluster comprises B-cell epitopes as shown in SEQ ID NO. 18 to SEQ ID NO. 26; preferably, the B-cell epitopes as shown in SEQ ID NO. 18 to SEQ ID NO. 26 are sequentially connected in series; more preferably, the amino acid sequence of the B-cell epitope cluster comprises or is composed of the amino acid sequence shown in SEQ ID NO.

29.

3. The pharmaceutical composition according to claim 1 or 2, wherein, The amino acid sequence of the epitope fusion peptide comprises or consists of the amino acid sequence shown in SEQ ID NO. 30 or SEQ ID NO.

31.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein, The fusion protein further includes a panepitope region and an adjuvant region sequentially connected to the N-terminus of the epitope fusion peptide; Preferably, the generic bit region includes PADRE; Preferably, the adjuvant region comprises one or more selected from the following: cholera enterotoxin B subunit (CTXB), type II heat-sensitive enterotoxin C subunit (LT-IIC), type II heat-sensitive enterotoxin B subunit (LT-IIB), 50S ribosome L7 / L12, β-defensin, RS09, and Pam2Cys; Optionally, the fusion protein further includes a 6×His tag linked to the C-terminus of the epitope fusion peptide.

5. The pharmaceutical composition according to claim 4, wherein, The fusion protein contains or is composed of the following amino acid sequence: (1) The amino acid sequence shown in any one of SEQ ID NO. 32 to SEQ ID NO. 35; (2) An amino acid sequence obtained by deleting, substituting, or adding one or more amino acids, such as 1, 2, 3, 4, or 5, from any of the amino acid sequences shown in any of SEQ ID NO. 32 to SEQ ID NO. 35; or (3) An amino acid sequence that has more than 90% homology with any of the amino acid sequences shown in SEQ ID NO. 32 to SEQ ID NO.

35.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein, The immune adjuvant is selected from aluminum salt adjuvants, emulsion adjuvants, TLR receptor agonist adjuvants, saponin adjuvants, or combinations thereof; Preferably, the immune adjuvant is selected from aluminum hydroxide (Al(OH)3), aluminum phosphate, MF59, MPLA, CpG oligodeoxynucleotide, Poly(I:C), QS-21, or a combination thereof; More preferably, the immune adjuvant is aluminum hydroxide (Al(OH)3) or MF59.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein, The immune adjuvant is aluminum hydroxide (Al(OH)3); preferably, the mass ratio of the fusion protein to aluminum hydroxide is 1:(1-10), more preferably 1:1; Alternatively, the immune adjuvant is MF59; preferably, the volume ratio of the fusion protein to MF59 is 1:(0.5-2), more preferably 1:

1.

8. Use of the pharmaceutical composition according to any one of claims 1 to 7 in the preparation of a medicament, wherein, The drug is used to prevent and / or treat Clostridium difficile infection or diseases caused by Clostridium difficile infection.

9. A vaccine for the prevention and / or treatment of Clostridium difficile infection or diseases caused by Clostridium difficile infection, comprising an effective amount of the pharmaceutical composition according to any one of claims 1 to 7, and optionally a pharmaceutically acceptable carrier or excipient.

10. The use according to claim 8 or the vaccine according to claim 9, wherein, The diseases caused by Clostridium difficile infection are selected from one or more of the following: Clostridium difficile-associated diarrhea, Clostridium difficile-associated colitis, pseudomembranous colitis, toxic megacolon, intestinal perforation, sepsis, and septic shock.

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