Fusion protein, nucleic acid, vector, host cell, trimeric fusion protein, method for producing the fusion protein, composition, and use of the fusion protein.

Description

/ 28 Fusion protein, nucleic acid, vector, host cell, trimeric fusion protein, method for producing the fusion protein, composition, and use of the fusion protein. Technical Field

[001] The present description refers to the field of biomedical technology and, in particular, to a fusion protein used as a vaccine. Fundamentals of the Invention

[002] The global pandemic of the novel coronavirus (SARS-CoV-2) has resulted in nearly 500 million people infected and more than 6 million deaths. The SARS-CoV-2 spike (S) protein is responsible for recognizing and invading cells, in which the receptor-binding domain (RBD) contained in the S protein is the most important, being responsible for recognizing and binding to the angiotensin receptor (ACE2) to mediate the entry of the virus into cells. The mRNA vaccines against COVID-19, protein subunit vaccines, and adenovirus vector vaccines currently being developed are all designed for the SARS-CoV-2 S protein or RBD protein. The SARS-CoV-2 S protein is a trimeric protein in its natural state. Many current studies have proven that trimers of the S protein or trimers of the RBD protein are more immunogenic than monomers of the S protein or monomers of the RBD protein.In order to achieve the expression of trimeric RBD or S protein, some studies have fused Foldon tags to RBD or S protein for expression and formed a trimer form under the effect of Foldon. However, Foldon has a higher molecular weight and its immunogenicity is very strong. Immunization of the human body with a trimer with Foldon fused to RBD / S proteins will produce a strong immune response to the Foldon protein, weakening the antigenic response to SARS-CoV-2 S or RBD. Similarly, trimers formed using other tags or non-human materials still exhibit similar deficiencies. Petition 870260020750, dated 06 / 03 / 2026, page 16 / 58 / 28

[003] Therefore, other strategies still need to be developed to promote the formation of immunogen trimers, especially antigens of the new coronavirus. Summary of the Invention

[004] The inventor had already tracked and optimized a type III collagen sequence. The recombinant human collagen produced has excellent hydrophilicity and stability, its amino acid composition is 100% identical to the corresponding part of the amino acid sequence of natural collagen. It will not produce an immune response when applied to the human body. Furthermore, recombinant human collagen has a trimer structure and can be widely used in biomedicine and other industries. Because type III human collagen is an optimized repeat of human collagen peptide, the expression of its fusion with the RBD protein of the novel coronavirus or hemagglutinin of the influenza virus may destroy the trimer structure of the collagen.Furthermore, even if the RBD protein of the novel coronavirus or the hemagglutinin of the influenza virus does not destroy the trimer structure of collagen and form a trimeric RBD protein, under the influence of this collagen, the trimeric RBD may also have a structure different from the natural RBD trimer, so that an immune response in the body cannot be properly induced. Therefore, the effect of a fusion of type III collagen with the RBD protein of the novel coronavirus or with the hemagglutinin of the influenza virus as a vaccine is unpredictable. In the present description, the inventor introduced cysteine ​​into the C-terminal of recombinant human type III collagen to form a disulfide bond to stabilize the collagen. An amino acid linker is designed to fuse with the RBD protein of the novel coronavirus or with the hemagglutinin of the influenza virus, and the fusion is expressed via a eukaryotic or prokaryotic system. Finally, the trimeric fusion protein was successfully expressed.The use of this recombinant type III trimeric collagen protein. Petition 870260020750, dated 06 / 03 / 2026, p. 17 / 58 / 28 human-RBD as a vaccine to immunize mice may produce more potent binding and neutralizing antibodies than the SARS-CoV-2 RBD protein monomer or influenza virus hemagglutinin. Therefore, the description skillfully fuses the human type III collagen peptide to the novel coronavirus RBD protein or influenza virus hemagglutinin to be expressed, leading the RBD protein or influenza virus hemagglutinin to form a correct trimer conformation, enhancing the immunogenicity of the RBD or influenza virus hemagglutinin. Furthermore, since the human type III collagen sequence comes from humans, it will not induce an immune response in the human body, stimulating the specific immune response to the trimer region of the RBD or influenza virus hemagglutinin.

[005] In one aspect, the description provides a fusion protein comprising a trimerization region and an immunogenic region linked by a linker, wherein the trimerization region includes one or more repeat units, wherein the repeat unit is established in SEQ ID NO. 1 or has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the repeat unit as established in SEQ ID NO. 1, provided that the repeat unit retains trimerization function.

[006] In one embodiment, the immunogenic region is an immunogenic protein of the pathogen.

[007] In one embodiment, the pathogen is a virus, bacterium, fungus and / or parasite.

[008] In one embodiment, the immunogenic region is selected from the group consisting of an RBD region of a coronavirus, a membrane protein of HIV, or a hemagglutinin protein of an influenza virus (e.g., HA1 H1N1 protein) and immunogenic fragments thereof.

[009] In one embodiment, the trimerization region contains 10 to 20 Petition 870260020750, dated 06 / 03 / 2026, page 18 / 58 / 28 (for example, 11, 12, 13, 14, 15, 16, 17, 18 or 19) repeat units, wherein the repeat unit is established in SEQ ID NO. 1 or has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the repeat unit, as established in SEQ ID NO. 1. In one embodiment, the trimerization region comprises one or more regions in the Cterminal or sequences, wherein the region is established in SEQ ID NO. 2 or has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence, as established in SEQ ID NO. 2.

[0010] In one embodiment, the trimerization region comprises the amino acid sequence as set forth in SEQ ID NO. 3 or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the sequence as set forth in SEQ ID NO. 3.

[0011] In one embodiment, the trimerization region comprises the amino acid sequence as set forth in SEQ ID NO. 4 or SEQ ID NO. 10 or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the sequence as set forth in SEQ ID NO. 4 or SEQ ID NO. 10.

[0012] In one embodiment, the linker is (GGGS)n or (GGGGS)n or (G)n. In one embodiment, n is an integer from 1 to 10. Preferably, the linker is (GGGS)n or (GGGGS)n.

[0013] In one embodiment, the trimerization region and the immunogenicity region are at the N-terminal and C-terminal of the fusion protein, respectively.

[0014] In one embodiment, the fusion protein comprises the amino acid sequence as set forth in SEQ ID NO. 5 or 9 or a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the sequence as set forth in SEQ ID NO. 5 or 9.

[0015] In one aspect, the description provides a nucleic acid Petition 870260020750, dated 06 / 03 / 2026, page 19 / 58 / 28 comprising nucleotides encoding the fusion protein described herein. In one embodiment, the nucleic acid comprises the amino acid sequence as set forth in SEQ ID NO. 7. In one embodiment, the nucleic acid further comprises the coding sequence of a purification tag, such as a His tag, a GST tag, an MBP tag, a SUMO tag, or a NusA tag. In one embodiment, the purification tag is at the C-terminus of the fusion protein. In one embodiment, the nucleic acid further comprises nucleotides encoding a leader sequence.

[0016] In one aspect, the description provides a vector comprising the nucleic acid described herein. In one embodiment, the vector contains expression control elements operationally linked to the nucleic acid. In one embodiment, the expression control elements are promoters, terminators, and / or enhancers.

[0017] In one aspect, the description provides a host cell comprising the nucleic acid or vector described herein. In one embodiment, the host cell is a plant cell that is incapable of regenerating a plant or is not a plant cell. In one embodiment, the host cell is a eukaryotic or prokaryotic cell. In one embodiment, the eukaryotic cell is a yeast cell, an animal cell, and / or an insect cell. In one embodiment, the prokaryotic cell is an E. coli cell.

[0018] In one aspect, the description provides a trimeric fusion protein comprising the fusion protein described herein.

[0019] In one aspect, the description provides a method for producing the fusion protein or the trimeric fusion protein as described herein, comprising: cultivate the host cells described herein under appropriate culture conditions; collect the host cells and / or culture medium containing the Petition 870260020750, dated 06 / 03 / 2026, page 20 / 58 6 / 28 fusion protein or trimeric fusion protein; and purify the fusion protein or trimeric fusion protein.

[0020] In one embodiment, the fusion protein or trimeric fusion protein can be purified from the culture medium. In another embodiment, the fusion protein or trimeric fusion protein can be obtained by homogenizing the host cells and then purifying the fusion protein or trimeric fusion protein from the cell lysate.

[0021] In one aspect, the description provides compositions comprising the fusion protein, nucleic acid, vector, host cell and / or trimeric fusion protein, as described herein.

[0022] In one embodiment, the composition is a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a vaccine. In one embodiment, the composition further comprises pharmaceutically acceptable vehicles, excipients, diluents and / or adjuvants. In one embodiment, the adjuvants are aluminum adjuvants, saponin adjuvants, lipid A, monophosphoryl lipid A, trehalose dimycolate, oil emulsion adjuvants, polysaccharide adjuvants, Tolllike receptor activators and / or STING agonists. For example, the adjuvant is

[0023] In one aspect, the description provides for the use of the fusion protein, nucleic acid, vector, host cell trimeric fusion protein and / or Petition 870260020750, dated 06 / 03 / 2026, page 21 / 58 / 28 composition described herein in the manufacture of a medicament for the treatment or prevention of a pathogen-mediated condition or disease. In one embodiment, the pathogen is a virus, bacterium, fungus and / or parasite. In one embodiment, the virus is selected from the group consisting of coronaviruses, HIV and influenza viruses. In one embodiment, the coronavirus is SARS-CoV-2. In one embodiment, the influenza virus is an influenza A virus or an influenza B virus.

[0024] In one aspect, the description provides a method of vaccinating an individual, comprising administering to the individual the fusion protein, trimer or pharmaceutical composition of the present description.

[0025] Compared with the previous technique, the beneficial effects of the present description include: 1. The inventor first verified that, by introducing disulfide bonds and linking molecules, the trimerization sequence of type III collagen can still perform the trimerization function after being fused to the RBD protein of a novel coronavirus or influenza virus hemagglutinin, and the resulting trimers of the RBD or influenza virus hemagglutinin have the correct conformation, producing higher levels of neutralizing antibodies than the monomers of the RBD or influenza virus hemagglutinin.

[0026] 2. Because type III collagen is homologous to human collagen, it will not induce strong antibodies against that collagen, which may stimulate a specific immune response to the RBD protein of the novel coronavirus or hemagglutinin of the influenza virus in vivo.

[0027] 3. The present description also provides a strategy for forming trimers for other immunogens. Description of the Drawings

[0028] Figure 1 shows the map of the vector plasmid used to construct the trimeric collagen-RBD protein. Petition 870260020750, dated 06 / 03 / 2026, page 22 / 58 / 28

[0029] Figure 2 is an SDS-PAGE image of the expressed trimeric collagen-RBD protein.

[0030] Figure 3 shows the detection of the molecular weight of trimeric collagen-RBD protein using a particle size analyzer.

[0031] Figure 4 shows the mouse immunization scheme.

[0032] Figure 5 shows the detection of specific RBD-binding IgG antibody levels in sera on day 21 after immunization of mice.

[0033] Figure 6 shows the titer of SARS-CoV-2 RBD-specific IgG binding antibodies in sera on day 21 after immunization of mice.

[0034] Figure 7 shows the neutralizing antibody titers for SARS-CoV-2 pseudovirus in sera on day 21 after immunization of mice with collagen-RBD trimers with CF501 adjuvant or RBD monomers.

[0035] Figure 8 shows the neutralizing antibody titers for SARS-CoV-2 pseudovirus in sera on day 21 after immunization of mice with collagen-RBD trimers with aluminum adjuvant or RBD monomers.

[0036] Figure 9 shows the detection of levels of H1N1 HA1-specific IgG binding antibodies in sera on day 17 after immunization of mice with influenza HA1-collagen / CF501 and HA1 / CF501.

[0037] Figure 10 shows the detection of levels of H1N1 HA1-specific IgG binding antibodies in sera on day 17 after immunization of mice with influenza HA1-collagen / aluminum adjuvant and HA1 / aluminum adjuvant. Detailed Description of the Invention

[0038] In order to make the purpose, technical solutions and Petition 870260020750, dated 06 / 03 / 2026, page 23 / 58 / 28. The advantages of the present description, the technical solutions in the examples of the present description will be described clearly and completely below in conjunction with the examples of the present description. Obviously, the examples described are part of the embodiments of the present description, not all embodiments of the present description. Based on the examples of the present description, all other embodiments obtained by persons skilled in the art without creative effort fall within the scope of protection of the present description.

[0039] As used herein, the inventors used a previously discovered recombinant humanized type III collagen polypeptide as part of a fusion protein to serve as the trimerization domain. In the field of proteins, the structure and / or function of a protein depend to some extent on the amino acid sequence. Prior to the present description, the inventor assumed that adding amino acid residues to one end of the recombinant humanized type III collagen polypeptide could affect the trimerization function of the recombinant humanized type III collagen. Surprisingly, when recombinant humanized type III collagen is fused with an immunogenic peptide, it is fully capable of forming a trimerized fusion protein, and the trimerized fusion protein produces significantly enhanced immunogenicity.For example, in the Examples, the inventors demonstrated that when recombinant humanized type III collagen forms a fusion protein with SARS-CoV-2 RBD protein or influenza virus hemagglutinin, trimers can be formed. When compared to SARS-CoV-2 RBD protein or influenza virus hemagglutinin and adjuvant, this trimer, alone or in combination with adjuvants, can produce significantly improved immunogenicity and generate a higher titer of neutralizing antibodies in vivo.

[0040] As used herein, a fusion protein refers to a Petition 870260020750, dated 06 / 03 / 2026, page 24 / 58 / 28 A fusion protein is formed by the indirect linkage of two or more functional protein portions via a ligand in the structure. A fusion protein may contain a trimerization region, an immunogenic region, and an optional ligand. The position of each portion is not particularly limited. For example, the trimerization region may be at the N-terminal or C-terminal of the fusion protein, and the immunogenic region may be at the C-terminal or N-terminal of the fusion protein.

[0041] As used herein, a trimerization region refers to a functional region that can perform a trimerization function. The trimerization region may contain one or more repeating units, as set forth in SEQ ID NO. 1. Each of the repeating units may be directly linked to or separated by one or more amino acid residues. The number of repeating units may vary. For example, the number of repeating units may be from 2 to 50, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40. The trimerization region may include one or more stabilizing regions, as set forth in SEQ ID NO. 2 at the C-terminus. The trimerization region may comprise the amino acid sequence, as set forth in SEQ ID NO. 3.

[0042] As used herein, an immunogenic region refers to a protein or a fragment thereof (e.g., a fragment of an antigen) derived from a pathogen capable of eliciting an immune response in an organism, in particular a mammal, such as a human body. The immunogenic protein may be selected from the group consisting of an RBD region of a coronavirus, an HIV membrane protein (e.g., gp140), or an influenza virus hemagglutinin protein and immunogenic fragments thereof. The immunogenic region may comprise the amino acid sequence as set forth in SEQ ID NO. 4 or 10. Several prior art documents (such as CN202110676901.2 and CN202111337048.8) describe an RBD region of Petition 870260020750, dated 06 / 03 / 2026, page 25 / 58 / 28 a coronavirus and related immunogenic fragments.

[0043] The trimerization region and the immunogenicity region can be indirectly linked by means of a ligand. The type of ligand may not be particularly limited. The ligand can be (GGGS)n or (GGGGS)n or (G)n. For example, n can be an integer from 1 to 10, such as 2, 3, 4, 5, 6, 7, 8 or 9. Preferably, the ligand can be (GGGS)n or (GGGGS)n, therefore a disulfide linkage can be introduced.

[0044] In order to facilitate the production and purification of the fusion proteins described herein, additional portions may be added to the N-terminus or C-terminus of the fusion proteins described herein. For example, the fusion protein described herein may contain a secretion signal or leader region at the N-terminus to facilitate the secretion of the fusion protein into the culture medium. The fusion protein described herein may also contain a purification tag at the C-terminus, such as a His tag, a GST tag, an MBP tag, a SUMO tag, or a NusA tag to facilitate the purification of the fusion protein. For example, the fusion protein may comprise the amino acid sequence as set forth in SEQ ID NO. 6.

[0045] As used herein, nucleic acids include polynucleotides that encode proteins. The nucleic acid may be a native nucleic acid of each fusion protein fragment or it may be a codon-optimized nucleic acid to facilitate expression in the target host. The nucleic acid may comprise the sequence of SEQ ID NO. 7.

[0046] As used herein, the term “vector” is a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into the host cell by means of transformation, transduction, or transfection, so that the elements of the genetic material that it contains can be incorporated into the host cell. Petition 870260020750, dated 06 / 03 / 2026, page 26 / 58 / 28 carries that they can be expressed in the host cell. Vectors are well known to those skilled in the art, including, but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); phages, such as lambda phage or M13 phage, and animal viruses, etc. Vectors may contain a variety of expression control elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, vectors may also contain replication origin sites. Vectors may contain nucleic acids of the description to facilitate introduction into cells for expression.Vectors can contain expression control elements, such as promoters, terminators, and / or enhancers, operationally linked to the nucleic acid.

[0047] The term “host cell” refers to a cell into which nucleic acid molecules have been introduced using molecular biology techniques. These techniques include transfection with viral vectors, transformation with plasmid vectors, and the introduction of naked DNA via electroporation, lipofection, and particle gun bombardment. The host cell can be a eukaryotic cell or a prokaryotic cell. Eukaryotic cells are, for example, yeast cells, animal cells, and / or insect cells. A prokaryotic cell can be an E. coli cell.

[0048] The term “individual” refers to any human being or other animal, especially other mammals, that are subjected to prophylaxis, treatment, or diagnosis. Other mammals may include, for example, dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, rats, guinea pigs, mice, and the like.

[0049] The term “vaccine”, as used herein, refers to a suspension or solution of an antigenic part, generally containing a Petition 870260020750, dated 06 / 03 / 2026, page 27 / 58 / 28 infectious agent or parts of an infectious agent, which is injected into the body to produce immunity. The antigenic part for preparing the vaccine may be a microorganism, or a purified natural product of a microorganism, a synthetic product, or a genetically engineered protein, peptide, polysaccharide or similar product. The vaccine described herein may be an oral or parenteral vaccine, for example, a vaccine administered subcutaneously, intradermally, intravenously, intramuscularly, intra-arterially or by infusion.

[0050] The term “adjuvant” refers to any substance that enhances or modifies the immune response when mixed with an immunogen to be injected. The adjuvant may be an aluminum adjuvant, saponin adjuvant, lipid A, monophosphoryl lipid A, trehalose dimycolate, oil emulsion adjuvant, polysaccharide adjuvant, Toll-like receptor activator and / or STING agonist.

[0051] The fusion protein of the present description can be assembled into a trimeric fusion protein due to the presence of a trimerization region. The trimeric fusion protein can be a homotrimer or a heterotrimer. Preferably, the trimeric fusion protein is a homotrimer. The trimeric fusion protein of the present description can generate a higher level of neutralizing antibodies than the immunogen alone, although it has no immunogenicity against the trimerization region. The fusion protein or trimeric fusion protein of the present description can be produced by the following method, which includes: (1) culturing the host cell according to the present description under appropriate culture conditions; (2) harvesting the host cells and / or culture medium containing the fusion protein or trimeric fusion protein; and (3) purifying the fusion protein or trimeric fusion protein.There are no specific limitations regarding culture and purification conditions, and those skilled in the art can select appropriate culture and purification conditions for their needs. Petition 870260020750, dated 06 / 03 / 2026, page 28 / 58 / 28 the isolation of the fusion protein or trimeric protein of the present description.

[0052] The fusion protein, nucleic acid, vector, host cell and / or trimeric fusion protein of the description may be prepared in compositions. The composition may comprise the fusion protein, nucleic acid, vector, host cell and / or trimeric fusion protein of the description. For example, the composition may be a pharmaceutical composition, especially a vaccine. In this case, the composition may additionally comprise pharmaceutically acceptable vehicles, excipients, diluents and / or adjuvants.For example, in the case of a vaccine, the vaccine may additionally contain an aluminum adjuvant, a saponin adjuvant, a lipid A, a monophosphoryl lipid A, a trehalose dimycolate, an oil emulsion adjuvant, a polysaccharide adjuvant, a Toll-like receptor activator, and / or a STING agonist, to enhance an individual's immune response to the immunogen. The fusion protein, nucleic acid, vector, host cell, and / or trimeric fusion protein or composition of the description may be used to treat or prevent pathogen-mediated conditions or diseases. Pathogens are viruses, bacteria, fungi, and / or parasites. For example, viruses may be selected from various types of coronaviruses, HIV, and influenza viruses. For example, the fusion protein of the present description may be prepared in a vaccine and used to prepare a vaccine for the SARS-CoV-2 virus or currently circulating influenza virus. Examples

[0053] The objective, technical characteristics and beneficial effects of the present description will be described in more detail below with reference to examples, but the implementation of the present description is not limited to them. Example 1: Construction and expression of a trimeric recombinant human-RBD-derived type III collagen protein.

[0054] The amino acid sequence of recombinant type III collagen Petition 870260020750, dated 06 / 03 / 2026, page 29 / 58 / 28. The human-derived protein designed by the inventor is as set forth in SEQ ID NO. 3. The amino acid sequence of the SARS-CoV-2 RBD protein is as set forth in SEQ ID NO. 4. The amino acid sequence of the linker arm between them is as set forth in SEQ ID NO. 8. The amino acid sequence of the fusion protein, collagen-RBD protein, is as set forth in SEQ ID NO. 6. The optimized nucleotide sequence encoding the fusion protein, collagen-RBD protein, is as set forth in SEQ ID NO. 7, to which are added 8 histidine-coding nucleotides and a stop codon.

[0055] Based on the nucleotide sequence of the collagen-RBD fusion protein, GenScript Biotech Corporation (Nanjing, China) was tasked with performing a gene synthesis. The synthesized gene fragment was ligated into the pFUSE-hIgG1-Fc2 vector via the EcoR I and Nco I restriction sites (Figure 1) to obtain the pFUSE-hIgG1-Fc2 vector containing the nucleotide sequence of the collagen-RBD fusion protein. The nucleotide sequence was verified as correct after sequencing, and the plasmid was transformed into competent DH5a cells. The specific steps were as follows.

[0056] 1) A tube of competent DH5a cells was removed from 80C and placed on ice.

[0057] 2) 1 μl of the constructed plasmid was added to competent DH5a cells and placed on ice for 30 minutes.

[0058] 3) The tube containing the competent DH5a cells was subjected to thermal shock at 42°C for 90 seconds and then quickly placed on ice.

[0059] 4) After the tube had been at rest for 2 minutes, 600 μl of antibiotic-free LB medium was added, and the cell tube was shaken on a shaker at 37°C for 1 hour.

[0060] 5) 100 μl of the culture were collected and placed on a plate LB containing bleomycin. Petition 870260020750, dated 06 / 03 / 2026, page 30 / 58 / 28

[0061] 6) After colony growth, colonies from a single clone were collected and added to 5 ml of LB medium containing bleomycin. After 12 hours, 5 ml of LB medium were transferred to 500 ml of LB medium.

[0062] 7) After 8 hours, the plasmid was extracted using Plasmid. Extraction Maxi Kit (TIANGEN BIOTECH Co., LTD.).

[0063] Next, collagen fusion protein-RBD was expressed. The specific steps were as follows: 1) Well-cultured EXPi293F cells were passed to a density of 1x106 / ml.

[0064] 2) After 12 hours, the cells were transfected with the plasmid containing the nucleotide sequence encoding the collagen fusion protein-RBD according to the instructions of the EZ Trans cell transfection reagent (Life-iLab Biotech Co., Ltd.).

[0065] 3) After the cells were cultured for another 6 days, they were centrifuged at 2000 rpm and the cell supernatant was collected.

[0066] NI-NTA was used to purify collagen-RBD fusion protein and the specific steps were as follows.

[0067] 1) The collected cell supernatant was filtered through a 0.45 μm filter membrane.

[0068] 2) 1 ml of NI-NTA (Qiagen) was added to the protein purification column.

[0069] 3) 5 ml of ddH2O were added to remove the alcohol from NI-NTA.

[0070] 4) NI-NTA was equilibrated by the addition of 10 ml of a binding buffer (40 mM NaH2PO3, 500 mM NaCl, pH: 7.8).

[0071] 5) The treated NI-NTA was added to the protein supernatant and incubated at 4°C for 3 hours.

[0072] 6) The protein impurities were then washed away using the binding buffer containing 5 mM imidazole in a total volume of Petition 870260020750, dated 06 / 03 / 2026, page 31 / 58 / 28 30 ml wash.

[0073] 7) The protein of interest was eluted using binding buffer containing 40 mM imidazole.

[0074] 8) The eluted protein of interest was run on SDSPAGE.

[0075] The results are shown in Figure 2. The collagen-RBD fusion protein was successfully expressed and a high-purity collagen-RBD fusion protein was obtained. After denaturation treatment, the molecular weight was approximately 80 kDa, consistent with the expected size.

[0076] In order to verify whether the protein formed a trimer, a particle size analyzer was used to detect the molecular weight of the protein. The results showed that the predicted molecular weight of the collagen-RBD fusion protein was 255 kDa, which is approximately 3 times the molecular weight of the monomer (Figure 3). Therefore, this collagen-RBD fusion protein was a trimeric protein. Example 2: Comparison between the levels of specific RBD-binding antibodies produced by mice immunized with trimeric collagen-RBD protein and monomeric RBD protein

[0077] Materials: Balb / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The aluminum adjuvant was purchased from Thermo Scientific. The monomeric RBD-His protein was purchased from Kactus Biosystems (Shanghai) Co., Ltd. The CF501 adjuvant has the following structure and was owned by the Applicant's laboratory. Petition 870260020750, dated 06 / 03 / 2026, p. 32 / 58 18 / 28

[0078] Twenty-four 6-week-old Balb / c mice were equally divided into 4 groups, with 6 mice in each group. The first group of mice was injected intramuscularly with 5 pg of trimeric collagen-RBD protein and 20 pg of CF501 adjuvant. The second group of mice was injected intramuscularly with 5 pg of monomeric RBD protein and 20 pg of CF501 adjuvant. The third group of mice was injected intramuscularly with 5 pg of trimeric collagen-RBD protein and an equal volume of aluminum adjuvant. The fourth group of mice was injected intramuscularly with 5 pg of monomeric RBD protein and an equal volume of aluminum adjuvant. The immunization schedule for the mice is shown in Figure 4. The mice were immunized twice on days 0 and 14, respectively, and sera were collected from the mice on day 21. The mouse sera were inactivated at 56°C for 30 minutes.

[0079] The ELISA method was used to detect specific IgG antibody titers for SARS-CoV-2 RBD in mouse sera. The specific steps were as follows.

[0080] 1) 1 pg / ml of RBD-His monomeric protein (Kactus Biosystems (Shanghai) Co., Ltd) was coated onto an ELISA plate and placed at 4°C overnight for coating. Petition 870260020750, dated 06 / 03 / 2026, p. 33 / 58 / 28

[0081] 2) The ELISA plate was blocked, 200 μL of PBS containing 5% BSA were added to each well and placed at 37°C for 2 hours.

[0082] 3) PBST was used to dilute mouse sera. The mouse sera were diluted from a 100-fold dilution and then to a 3-fold dilution.

[0083] 4) Diluted mouse sera were added to the ELISA plate and incubated at 37°C for 45 minutes.

[0084] 5) The ELISA plate was washed 5 times with PBST.

[0085] 6) Rabbit anti-mouse secondary antibody labeled with HRP (Dako) was added and incubated at 37°C for 45 minutes.

[0086] 7) The ELISA plate was washed 5 times with PBST.

[0087] 8) The TMB substrate was added to develop the color for minutes.

[0088] 9) The development of the color was interrupted by the addition of H2SO4.

[0089] 10) A microplate reader was used to read OD450 and antibody titers in the sera were calculated.

[0090] The results are shown in Figure 5, Figure 6, and Table 1. After mice were immunized with CF501 / collagen-RBD trimers, the mean IgG antibody titer to SARS-CoV-2 RBD in sera reached 656,100, which was 28 times higher than the specific IgG antibody titer to RBD in sera from mice immunized with the CF501 / RBD monomer. Furthermore, IgG antibody titers in sera from mice immunized with the aluminum / collagen-RBD adjuvant trimer were also almost five times higher than those in sera from mice immunized with the aluminum / RBD adjuvant monomer. These data indicate that the trimeric collagen-RBD protein as an immunogen can potentially enhance the immunogenicity of RBD. Petition 870260020750, dated 06 / 03 / 2026, page 34 / 58 / 28

[0091] Table 1: SARS-CoV-2 RBD-specific IgG antibody titers in sera on day 21 after mouse immunization______ Mouse clustering CF501 / Collagen-RBD CF501 / RBD Aluminum adjuvant / collagen-RBD Aluminum adjuvant / RBD SARS-CoV-2 RBD-specific IgG titers (mean ± standard error) 656100±0 23100±10686 5467±1666 1133±509 Example 3: Trimeric collagen-RBD protein can induce a highly potent neutralizing antibody titer for a SARS-CoV-2 pseudovirus in mice.

[0092] In order to compare the neutralizing antibody titers for SARS-CoV-2 induced by collagen-RBD trimer and RBD monomer, the neutralizing effect on a SARS-CoV-2 pseudovirus was tested in sera after immunization of mice. The SARS-CoV-2 pseudovirus was packaged as follows.

[0093] 1) Well-developed HEK-293T cells were passed.

[0094] 2) After 24 hours of incubation, HEK-293T cells were cotransfected with a major structure plasmid PNL-4-3 and a SARS-CoV-2 envelope plasmid, PcDNA3.1-SARS-CoV-2-S (Liu, Z., Zhou, J., Xu, W. et al. A novel STING agonist-adjuvanted pan-sarbecovirus vaccine elicits potent and durable neutralizing antibody and T cell responses in mice, rabbits and NHPs. Cell Res 32, 269-287 (2022). https: / / doi.org / 10.1038 / s41422-022-00612-2) using Vigofect transfection reagent (Vigorous Biotechnology Beijing Co., Ltd.).

[0095] 3) After 12 hours of incubation, the medium was replaced with fresh DMEM medium containing 10% FBS.

[0096] 4) After an additional 48 hours of incubation, the cell supernatant was collected and the SARS-CoV-2 pseudovirus was obtained.

[0097] The neutralization experiment on the SARS-CoV-2 pseudovirus was performed and described in particular as follows.

[0098] 1) Well-cultured Huh-7 cells were placed in a Petition 870260020750, dated 06 / 03 / 2026, page 35 / 58 / 28 plate of 96 wells with 10,000 cells per well.

[0099] 2) After 8 hours of incubation, the mice's sera were diluted with DMEM.

[00100] 3) Diluted mouse sera were mixed with SARS-CoV-2 pseudovirus and incubated for 30 minutes.

[00101] 4) The pseudovirus and mouse sera were added to Huh-7 cells.

[00102] 5) After 12 hours of incubation, the medium was replaced with a new DMEM medium.

[00103] 6) The luciferase value of each well was detected using a luciferase kit (Promega Corporation) after 48 hours.

[00104] 7) Neutralizing antibody titers in the sera were calculated.

[00105] The results are shown in Figure 7, Figure 8 and Table 2. The mean titer of neutralizing antibodies to SARS-CoV-2 pseudovirus produced by trimeric collagen-RBD protein with CF501 as adjuvant reached 3620, while the mean titer of neutralizing antibodies to SARS-CoV-2 pseudovirus produced by RBD monomer with CF501 as adjuvant was only 191. Similarly, the neutralizing antibody titer in mice immunized with trimeric collagen-RBD protein with aluminum adjuvant was also higher than that of mice immunized with monomeric RBD protein with aluminum adjuvant.

[00106] Table 2: Neutralizing antibody titers for SARS-CoV-2 pseudovirus in sera on day 21 after immunization of mice__________________________________________________ Mouse clustering: CF501 / Collagen-RBD, CF501 / RBD, Aluminum Adjuvant / Collagen-RBD, Aluminum Adjuvant / RBD. Neutralizing antibody titers for SARS-CoV-2 pseudovirus (mean ± standard error): 3620±1017, 191.3±54, 198.6±22, 63.66±9. Petition 870260020750, dated 06 / 03 / 2026, p. 36 / 58 / 28 Example 4: Comparison of antibody levels that specifically bind to HA1 produced after immunization of mice with a trimeric collagen-HA1 H1N1 protein and a monomeric HA1 H1N1 protein.

[00107] Construction and expression of a recombinant type III collagen trimeric protein-HA1 H1N1

[00108] Based on the nucleotide sequence of the collagen-HA1 H1N1 fusion protein, GenScript Biotech Corporation (Nanjing, China) was tasked with performing the gene synthesis. The synthesized gene fragment was ligated into the pFUSE-hIgG1-Fc2 vector via the EcoR I and Nco I restriction sites to obtain the pFUSE-hlgG1-Fc2 vector containing the nucleotide sequence of the collagen-HA1 H1N1 fusion protein. The collagen-HA1 fusion protein was expressed according to the protein expression procedure in Example 1.

[00109] Twenty-four 6-week-old Balb / c mice were equally divided into 4 groups, with 6 mice in each group. The first group of mice was injected intramuscularly with 5 μg of trimeric collagen protein-HA1 H1N1 and 20 μg of adjuvant CF501. The second group of mice was injected intramuscularly with 5 μg of monomeric protein-HA1 H1N1 and 20 μg of adjuvant CF501. The third group of mice was injected intramuscularly with 5 μg of trimeric collagen protein-HA1 H1N1 and an equal volume of aluminum adjuvant. The fourth group of mice was injected intramuscularly with 5 μg of monomeric protein-HA1 H1N1 and an equal volume of aluminum adjuvant. The immunization schedule for the mice is shown in Figure 4. The mice were immunized twice on days 0 and 14, respectively, and sera were collected from the mice on day 21. The mouse sera were inactivated at 56°C for 30 minutes.

[00110] The ELISA method was used to detect the titers of Petition 870260020750, dated 06 / 03 / 2026, p. 37 / 58 / 28 specific IgG antibodies for H1N1 HA1 in mouse sera. The specific steps were as follows: 1) 1 μg / ml of the monomeric protein HA1 H1N1-His was coated onto an ELISA plate and placed at 4°C overnight for coating.

[00111] 2) The ELISA plate was blocked, 200 μl of PBS containing 5% BSA were added to each well and placed at 37°C for 2 hours.

[00112] 3) PBST was used to dilute mouse sera. The mouse sera were diluted from a 100-fold dilution and then to a 3-fold dilution.

[00113] 4) Diluted mouse sera were added to the ELISA plate and incubated at 37°C for 45 minutes.

[00114] 5) The ELISA plate was washed 5 times with PBST.

[00115] 6) Rabbit anti-mouse secondary antibody labeled with HRP (Dako) was added and incubated at 37°C for 45 minutes.

[00116] 7) The ELISA plate was washed 5 times with PBST.

[00117] 8) The TMB substrate was added to develop the color for minutes.

[00118] 9) The development of the color was interrupted by the addition of H2SO4.

[00119] 10) A microplate reader was used to read OD450 and antibody titers in the sera were calculated.

[00120] The results are shown in Figures 9 and 10. Whether using CF501 adjuvant or aluminum adjuvant, HA1 H1N1 protein with labeled collagen can produce higher levels of IgG antibodies to HA1 protein than HA1 H1N1 protein alone. Therefore, the collagen trimerization region or label can significantly improve the immunogenicity of antigens, regardless of whether they are novel coronavirus antigens or influenza virus antigens. Petition 870260020750, dated 06 / 03 / 2026, page 38 / 58 / 28 Sequences

[00121] Repeating unit in the trimerization region: GERGAPGFRGPAGPNGIPGEKGPAGERGAP (SEQ ID NO. 1).

[00122] The region at the C-terminal of the trimerization region: GAPGPCCGG (SEQ ID NO. 2).

[00123] Example trimerization region: GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGF RGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGE RGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAG PNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPG ERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPG EKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAP GFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPA GERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGP AGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERG APGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPN GIPGEKGPAGERGAP GAPGPCCGG (SEQ ID NO. 3).

[00124] Example immunogenic region (the amino acid sequence of the SARS-CoV-2 RBD protein): RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKR ISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGD EVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNY RYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPT NGVGYQPYRVVVLSFELLHAPATVCGPKKSTN (SEQ ID NO. 4).

[00125] Example fusion protein (without His tag): GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGF RGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGE RGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAG PNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPG Petition 870260020750, dated 06 / 03 / 2026, page 39 / 58 / 28 ERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPG EKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAP GFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPA GERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGP AGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERG APGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPN GIPGEKGPAGERGAPGAPGPCCGGGGGGSGGGGSGGGGSGGGGSRV QPTESIVRFNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYS VLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAP GQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLF RKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNG VGYQPYRVVVLSFELLHAPATVCGPKKSTN (SEQ ID NO. 5, trimerization region underlined, immunogenicity region in bold).

[00126] Example fusion protein (with His tag): GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGF RGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGE RGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAG PNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPG ERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPG EKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAP GFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPA GERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGP AGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERG APGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPN GIPGEKGPAGERGAPGAPGPCCGGGGGGSGGGGSGGGGSGGGGGSRV QPTESIVRFFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVL YNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG KIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLK PFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRV Petition 870260020750, dated 06 / 03 / 2026, page 40 / 58 / 28 VVLSFELLHAPATVCGPKKSTNHHHHHHHH (SEQ ID NO. 6, His label in bold).

[00127] Example fusion protein coding sequences: GGAGAACTGTGGAGCCCCCGGATTCAGAGGACCAGCCGGCCCCAACGGAATCCCTGGTGAGAAGGGCCCTGCCGGCGAAAGG GGCGCCCCGGGCGAGAGAGGAGCCCCTGGCTTCCGGGGCCCGGCC GGGCCTAATGGCATCCCTGGCGAGAAGGGCCCTGCTGGAGAACGG GGCGCCCCCGGCGAGCGCGGAGCTCCCGGCTTCCGGGGCCCAGCC GGGCCTAACGGCATCCCTGGCGAGAAGGGCCCTGCCGGCGAGAGG GGCGCTCCTGGGGAGAGAGGCGCCCCTGGCTTCAGAGGACCCGCC GGTCCTAATGGAATCCCCGGCGAGAAGGGCCCTGCCGGCGAAAGA GGCGCCCCTGGAGAGAGAGGCGCTCCTGGATTTAGAGGTCCAGCC GGTCCCAACGGAATCCCTGGAGAGAAGGGGCCCGCTGGCGAAAG AGGAGCCCCTGGCGAAAGAGGCGCCCCCGGATTTAGAGGACCTGC AGGCCCTAATGGAATCCCTGGCGAGAAGGGCCCTGCCGGAGAACG GGGCGCCCCTGGCGAGAGAGGTGCCCCTGGCTTTAGAGGCCCCGC TGGCCCCAACGGCATCCCTGGGGAGAAAGGCCCGGCCGGCGAACG GGGAGCCCCGGGCGAGAGGGGCGCCCCAGGATTCCGGGGCCCTGC TGGCCCCAACGGCATTCCTGGCGAAAAAGGCCCTGCCGGGGAGCG GGGCGCCCCTGGCGAAAGAGGCGCTCCTGGTTTCCGGGGCCCTGC TGGGCCTAACGGCATCCCCGGCGAAAAGGGCCCAGCCGGGGAGA GAGGAGCCCCTGGCGAGCGGGGAGCTCCTGGCTTCAGAGGCCCTG CCGGCCCTAACGGCATCCCCGGCGAAAAAGGCCCCGCCGGTGAAA GAGGGGCGCCCGGTGAAAGAGGCGCCCCTGGCTTCCGGGGCCCTG CCGGCCCAAATGGTATCCCTGGAGAAAAGGGCCCTGCAGGAGAGCGTGGCGCCCCTGGCGAACGCGGCGCCCCTGGCTTTAGAGGCCCAG CAGGCCCTAACGGCATCCCTGGCGAGAAAGGACCCGCTGGTGAAA GAGGCGCCCCAGGCGAGAGAGGGGCTCCTGGCTTCAGAGGCCCAG CCGGCCCAAACGGCATCCCTGGCGAGAAGGGCCCCGCCGGCGAAC Petition 870260020750, dated 06 / 03 / 2026, page 41 / 58 / 28 GGGGCGCCCCGGGCGAGAGAGGAGCCCCCGGTTTCCGGGGTCCCG CCGGCCCTAATGGCATCCCCGGCGAGAAAGGACCTGCCGGCGAGC GGGGCGCTCCTGGAGAGCGGGGCGCCCCTGGCTTCCGGGGCCCTG CTGGCCCTAACGGAATCCCTGGCGAAAAAGGCCCTGCCGGCGAGA GAGGCGCCCCCGGCGAGAGAGGGGCCCCAGGCTTCCGGGGACCTG CCGGCCCTAACGGCATCCCTGGCGAGAAGGGCCCTGCTGGCGAAA GAGGCGCCCCCGGAGCGCCTGGCCCCTGTTGCGGCGGAGGAGGAG GTGGATCTGGCGGCGGAGGCAGCGGAGGCGGCGGCTCTGGGGGC GGCGGCTCCAGAGTGCAGCCTACAGAGAGCATCGTGCGGTTCCCC AACATCACCAACCTGTGTCCTTTCGGTGAGGTGTTCAACGCTACCA GATTCGCCAGCGTGTACGCCTGGAATAGAAAGAGAATCAGCAATT GCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCTCTTTCAGCAC CTTCAAGTGCTATGGCGTGTCCCCTACCAAGCTGAACGACCTGTGC TTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGATGAG GTGAGACAGATCGCCCCTGGCCAGACCGGCAAGATCGCCGATTAC AACTACAAGCTGCCTGACGACTTCACCGGTTGTGTGATCGCCTGGA ACAGCAACAACCTGGACAGCAAGGTGGGCGGAAATTACAACTACA GATACAGACTGTTCAGAAAGTCCAACCTCAAGCCCTTCGAGAGAG ATATCTCTACAGAAATCTACCAGGCCGGATCTAAGCCTTGCAACG GCGTGGAAGGCTTTAACTGCTACTTCCCTCTGCAGAGCTACGGCTT TCAGCCCACCAACGGCGTTGGTTATCAACCTTACAGAGTCGTGGTCCTGAGCTTTGAGCTGCTGCACGCCCCTGCTACAGTGTGCGGCCCCA AGAAGAGCACAAACCACCACCACCACCACCACCACCATTGA (SEQ ID NO. 7).

[00128] Example ligand: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO. 8).

[00129] Collagen fusion protein-HA1 H1N1 (with His tag): GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGF RGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGE Petition 870260020750, dated 06 / 03 / 2026, page 42 / 58 / 28 RGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAG PNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPG ERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPG EKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAP GFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPA GERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGP AGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERG APGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPN GIPGEKGPAGERGAPGAPGPCCGGGGGGSGGGGSGGGGSGGGGSDTI CIGYHANNSTDTVDTIFEKNVAVTHSVNLLEDRHNGKLCKLKGIAPL QLGKCNIIGWLLGNPECDSLLPARSWSYIVETPNSENGACYPGDFIDY EELREQLSSVSSLERFEIFPKESSWPNHTFNGVTASCSHRGKSSFYRNL LWLTKKGDSYPKLTNSYVNNKGKEVLVLWGVHHPSSSDEQQSLYSN GNAYVSVASSNYNRRFTPEIAARPKVKDQHGRMNYYWTLLEPGDTII FEATGNLIAPWYAFALSRGFESGIITSNASMHECNTKCQTPQGSINSNL PFQNIHPVTIGECPKYVRSTKLRMVTGLRNIPSIQYRHHHHHHHH (SEQ ID NO. 9).

[00130] Sequência de aminoácidos da proteína HA1 do vírus influenza H1N1 (A / WSN / 1933): DTICIGYHANNSTDTVDTIFEKNVAVTHSVNLLEDRHN GKLCKLKGIAPLQLGKCNIIGWLLGNPECDSLLPARSWSYIVETPNSE NGACYPGDFIDYEELREQLSSVSSLERFEIFPKESSWPNHTFNGVTASC SHRGKSSFYRNLLWLTKKGDSYPKLTNSYVNNKGKEVLVLWGVHHP SSSDEQQSLYSNGNAYVSVASSNYNRRFTPEIAARPKVKDQHGRMNY YWTLLEPGDTIIFEATGNLIAPWYAFALSRGFESGIITSNASMHECNTK CQTPQGSINSNLPFQNIHPVTIGECPKYVRSTKLRMVTGLRNIPSIQYR (SEQ ID NO. 10). Petição 870260020750, de 06 / 03 / 2026, pág. 43 / 58

Claims

1 / 3 CLAIMS 1. Fusion protein, characterized in that it comprises a trimerization region and an immunogenicity region linked by a linker, wherein the trimerization region consists of the amino acid sequence of SEQ ID NO. 3; and the immunogenicity region is a receptor-binding domain (RBD) of a coronavirus or a hemagglutinin protein of an influenza virus.

2. Fusion protein according to claim 1, characterized in that the hemagglutinin protein of the influenza virus is the HA1 H1N1 protein.

3. Fusion protein according to claim 1, characterized in that the C-terminal of the trimerization region is linked to one or more stabilization regions, as set forth in SEQ ID NO. 2, either directly or by one or more amino acid residues.

4. Fusion protein according to claim 1 or 3, characterized in that the immunogenic region comprises the amino acid sequence as set forth in SEQ ID NO.4 or SEQ ID NO. 10, and / or the ligand is (GGGS)n or (GGGGS)n or (G)n, wherein n is an integer from 1 to 10.

5. Fusion protein according to any one of claims 1 to 4, characterized in that the trimerization region and the immunogenicity region are located at the N-terminal and C-terminal of the fusion protein, respectively.

6. Fusion protein according to claim 1, characterized in that the fusion protein comprises the amino acid sequence as set forth in SEQ ID NO. 5 or 9.

7. Nucleic acid, characterized in that it comprises a nucleotide sequence encoding the fusion protein as defined in any one of claims 1 to 6, wherein the sequence of Petition 870260072113, dated 07 / 21 / 2026, p. 12 / 17 2 / 3 nucleotides consists of the sequence SEQ ID NO: 7.8.Nucleic acid according to claim 7, characterized in that the nucleic acid further comprises a nucleotide sequence encoding a purification tag; or the nucleic acid further comprises a nucleotide sequence encoding a leader sequence.

9. Vector, characterized in that it comprises the nucleic acid as defined in claim 7 or 8.

10. Vector according to claim 9, characterized in that it comprises an expression control element operationally linked to the nucleic acid.

11. Host cell, characterized in that it comprises the nucleic acid as defined in claim 7 or 8 or the vector as defined in claim 9 or 10; wherein the host cell is a prokaryotic cell or a yeast cell.

12. Host cell according to claim 11, characterized in that the prokaryotic cell is an E. coli cell. 13.Trimeric fusion protein, characterized in that it comprises the fusion protein as defined in any one of claims 1 to 6.

14. Method for producing the fusion protein as defined in any one of claims 1 to 6 or the trimeric fusion protein as defined in claim 13, characterized in that it comprises: (1) culturing the host cell as defined in claim 11 or 12 under an appropriate culture condition; (2) harvesting host cells and / or culture medium containing the fusion protein or the trimeric fusion protein; and (3) purifying the fusion protein or the trimeric fusion protein. Petition 870260072113, dated 07 / 21 / 2026, page 13 / 17 3 / 3 15. Composition, characterized in that it comprises the fusion protein as defined in any one of claims 1 to 6, the nucleic acid as defined in claim 7 or 8, the vector as defined in claim 9 or 10, the host cell as defined in claim 11 or 12 and / or the trimeric fusion protein as defined in claim 13.

16. Composition according to claim 15, characterized in that the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier, excipient, diluent and / or adjuvant.

17. Composition according to claim 16, characterized in that the composition is a vaccine. 18.Use of the fusion protein as defined in any one of claims 1 to 6, of the nucleic acid as defined in claim 7 or 8, of the vector as defined in claim 9 or 10, of the host cell as defined in claim 11 or 12 and / or of the trimeric fusion protein as defined in claim 13, of the composition as defined in any one of claims 15 to 17, characterized in that it is used in the manufacture of a medicament for the treatment or prevention of a pathogen-mediated condition or disease, wherein the pathogen is selected from the group consisting of a coronavirus and an influenza virus.

19. Use according to claim 18, characterized in that the influenza virus is influenza A virus or influenza B virus and the coronavirus is SARS-CoV-2. Petition 870260072113, dated 07 / 21 / 2026, pp. 14 / 17.

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