Fusion proteins and their applications

Abstract

The present application relates to a fusion protein and its application. The fusion protein of the present application comprises a trimerization region and an immunogenic region linked by a linker, the trimerization region comprises one or more repeat units shown in SEQ ID NO.1, and the immunogenic region is an immunogenic protein of a pathogen, such as the RBD region of coronavirus, the membrane protein of AIDS virus, or the hemagglutinin protein of influenza virus and immunogenic fragments thereof. The trimer obtained by the present invention can produce a higher neutralizing antibody level than the immunogenic monomer, and does not induce strong antibodies against the trimerization region in the human body, and can promote the body's immune response to focus on the immunogenic region.

Description

[Technical Field]

[0001] The present invention relates to the field of biomedicine, specifically to fusion proteins as vaccines. [Background technology]

[0002] The novel coronavirus (SARS-CoV-2) has become a global pandemic, currently causing approximately 500 million infections and over 6 million deaths. The SARS-CoV-2 spike protein (S) is responsible for cell identification and entry, and the receptor-binding domain (RBD) contained in the S protein is the most important, as it recognizes and binds to the angiotensin 2 (ACE2) receptor, mediating viral entry into cells. Currently developed novel coronavirus mRNA vaccines, protein subunit vaccines, and adenovirus vector vaccines are all based on the SARS-CoV-2 S protein or RBD protein. The SARS-CoV-2 S protein is naturally a trimeric protein. Numerous studies have demonstrated that S protein trimers or RBD protein trimers have higher immunogenicity than S protein monomers or RBD protein monomers. To achieve trimeric protein expression of the RBD or S protein, some studies have used fusion expression of the RBD or S protein with a Foldon tag, resulting in the formation of a trimer via the action of Foldon. However, Foldon has a large molecular weight and is highly immunogenic. Immunization of humans with a trimer fused to Foldon and the RBD / S protein generates a strong immune response against the Foldon protein, weakening the antigenic response to the SARS-CoV-2 S or RBD. Similarly, trimers formed using several other non-human-derived tags or vehicles still suffer from similar drawbacks.

[0003] Therefore, there remains a need to develop other strategies to promote trimer formation in immunogens, especially novel coronavirus antigens. Summary of the Invention [Problem to be solved by the invention]

[0004] The inventors previously screened and optimized the type III collagen sequence. The resulting recombinant human collagen has extremely high hydrophilicity and stability, and its amino acid composition is 100% identical to the corresponding portion of the amino acid sequence of natural collagen. It does not provoke an immune response when administered to the human body. Furthermore, because the recombinant human collagen has a trimeric structure, it can be widely used in industries such as biopharmaceuticals. Because the human type III collagen is composed of a repeat of optimized human collagen peptide fragments, it may disrupt the trimeric structure of the collagen after fusion expression with the novel coronavirus RBD protein or influenza virus hemagglutinin. Furthermore, even if the novel coronavirus RBD protein or influenza virus hemagglutinin produces an RBD trimeric protein without disrupting the collagen trimeric structure, the trimeric RBD may not effectively induce an immune response due to the influence of the collagen, since the trimeric RBD differs from the natural RBD trimeric structure. Therefore, the effectiveness of fusion expression of the human type III collagen with the novel coronavirus RBD protein or influenza virus hemagglutinin as a vaccine is unexpected. In this invention, the inventors introduced a cysteine ​​residue at the C-terminus of the recombinant human type III collagen to create a disulfide bond to stabilize the collagen, and then fused it with the SARS-CoV-2 RBD protein or influenza virus hemagglutinin via a designed linker amino acid in a eukaryotic or prokaryotic system. Finally, the trimeric fusion protein was successfully expressed. Immunizing mice with the recombinant human type III collagen-RBD trimeric protein as a vaccine produced stronger binding and neutralizing antibodies than those produced by the SARS-CoV-2 RBD protein or influenza virus hemagglutinin monomer.Therefore, this invention cleverly links human type III collagen peptide with the novel coronavirus RBD protein or influenza virus hemagglutinin in series and expresses the fusion protein, thereby promoting the formation of the correct trimeric conformation of the RBD protein or influenza virus hemagglutinin, improving the immunogenicity of the RBD or influenza virus hemagglutinin.Furthermore, because the human type III collagen sequence is derived from humans, it does not induce an immune response in the human body, promoting the concentration of the immune response on the trimeric region of the RBD or influenza virus hemagglutinin. [Means for solving the problem]

[0005] In one embodiment, the fusion protein of the present invention comprises a trimerization domain and an immunogenic domain connected by a linker, wherein the trimerization domain comprises one or more repeating units as set forth in SEQ ID NO.1 or repeating units having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the repeating units as set forth in SEQ ID NO.1, provided that the repeating units retain their trimerization function.

[0006] In one embodiment, the immunogenic region is an immunogenic protein of a pathogen.

[0007] In one embodiment, the pathogen is a virus, bacterium, fungus and / or parasite.

[0008] In one embodiment, the immunogenic region is selected from the RBD region of a coronavirus, the membrane protein of an AIDS virus, or the hemagglutinin protein of an influenza virus (eg, the H1N1 HA1 protein), and immunogenic fragments thereof.

[0009] In one embodiment, the trimerization region comprises 10 to 20 (e.g., 11, 12, 13, 14, 15, 16, 17, 18, or 19) repeat units set forth in SEQ ID NO. 1, or repeat units having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a repeat unit set forth in SEQ ID NO. 1. In one embodiment, the trimerization region comprises, at its C-terminus, one or more regions set forth in SEQ ID NO. 2, or sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a sequence set forth in SEQ ID NO. 2.

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

[0011] In one embodiment, the immunogenic region comprises the amino acid sequence 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 to the sequence 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 domain and the immunogenic domain are located at the N-terminus and C-terminus of the fusion protein, respectively.

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

[0015] In one aspect, a nucleic acid of the invention comprises nucleotides encoding a fusion protein described herein. In one embodiment, the nucleic acid comprises the amino acid sequence set forth in SEQ ID NO. 7. In one embodiment, the nucleic acid further comprises a coding sequence for a purification tag, e.g., a His tag, a GST tag, a MBP tag, a SUMO tag, or a NusA tag. In one embodiment, the purification tag is located 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, a vector of the invention comprises a nucleic acid described herein. In one embodiment, the vector comprises an expression control element operably linked to the nucleic acid. In one embodiment, the expression control element is a promoter, terminator, and / or enhancer.

[0017] In one aspect, a host cell of the present invention comprises a nucleic acid or vector described herein. In one embodiment, the host cell is a plant cell that is not capable of producing a plant, or is not a plant cell. In one embodiment, the host cell is a eukaryotic cell or a 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, a trimeric fusion protein according to the invention comprises a fusion protein described herein.

[0019] In one aspect, the method of the invention for producing a fusion protein or trimeric fusion protein described herein comprises the steps of: (1) culturing a host cell described herein under suitable 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.

[0020] In one embodiment, the fusion protein or trimeric fusion protein may be purified from the culture medium, hi another embodiment, the host cells may be homogenized and the fusion protein or trimeric fusion protein may be purified from the cell lysate.

[0021] In one aspect, a composition of the invention comprises a fusion protein, a nucleic acid, a vector, a host cell and / or a trimeric fusion protein 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 a pharmaceutically acceptable vector, excipient, diluent, and / or adjuvant. In one embodiment, the adjuvant is aluminum adjuvant, saponin adjuvant, lipid A, monophosphoryl lipid A, trehalose dimycolate, oil emulsion adjuvant, polysaccharide adjuvant, Toll-like receptor activator, and / or STING agonist. For example, the adjuvant is represented by the following formula:

[0023] [ka]

[0024] In one aspect, the invention provides for the use of a fusion protein, nucleic acid, vector, host cell, trimeric fusion protein, and / or composition described herein in the manufacture of a medicament, wherein the medicament treats or prevents 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 a coronavirus, an AIDS virus, and an influenza virus. 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.

[0025] In one aspect, a method of vaccinating a subject according to the invention comprises administering to the subject a fusion protein, trimer or pharmaceutical composition of the invention. [Effects of the Invention]

[0026] Compared with the prior art, the beneficial effects of the present invention include:

[0027] 1. The present inventors have found for the first time that by introducing disulfide bonds and linker molecules, the trimerization sequence of type III collagen can still exert its trimerization function after fusing it with the novel coronavirus RBD protein or influenza virus hemagglutinin, and that the resulting RBD or influenza virus hemagglutinin trimer has a correct conformation and can produce higher levels of neutralizing antibodies than the RBD or influenza virus hemagglutinin monomer.

[0028] 2. Because type III collagen is homologous to human collagen, it does not induce strong antibodies against the collagen, and can promote the body's immune response to focus on the RBD protein of the new coronavirus or the hemagglutinin of the influenza virus.

[0029] 3. The present invention also provides a strategy for forming trimers for other immunogens. [Brief explanation of the drawings]

[0030] [Figure 1] 1 shows the vector plasmid profile required for constructing the collagen-RBD trimer protein. [Figure 2] FIG. 1 shows an SDS-PAGE image of the expressed collagen-RBD trimer protein. [Figure 3] The molecular weight of the collagen-RBD trimer protein detected using a particle size analyzer is shown. [Figure 4] 1 shows the immunization schedule for mice. [Figure 5] Figure 1 shows the levels of specific IgG-binding antibodies against SARS-CoV-2 RBD detected in the serum of mice on day 21 after immunization. [Figure 6] Specific IgG binding antibody titers against SARS-CoV-2 RBD in serum of mice on day 21 after immunization. [Figure 7] Neutralizing antibody titers against SARS-CoV-2 pseudovirus in serum from mice on day 21 after immunization with collagen-RBD trimer or RBD monomer adjuvanted with CF501. [Figure 8] Figure 1 shows neutralizing antibody titers against SARS-CoV-2 pseudovirus in serum from mice on day 21 after immunization with collagen-RBD trimer or RBD monomer using aluminum adjuvant. [Figure 9] Detected levels of specific IgG binding antibodies against H1N1 HA1 in the serum of mice on day 17 after influenza HA1-collagen / CF501, HA1 / CF501 immunization are shown. [Figure 10] Detected levels of specific IgG-binding antibodies against H1N1 HA1 in serum from mice on day 17 after influenza HA1-collagen / aluminum adjuvant and HA1 / aluminum adjuvant immunization are shown. DETAILED DESCRIPTION OF THE INVENTION

[0031] In order to clarify the purpose, technical means and advantages of the present invention, the following will clearly and completely describe the technical means in the embodiments of the present invention with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only some of the embodiments of the present invention, and not all of the embodiments. Any other embodiments that can be conceived by those skilled in the art based on the embodiments of the present invention without any creative efforts will fall within the protection scope of the present invention.

[0032] In this specification, the inventors incorporate their previously discovered recombinant type III humanized collagen polypeptide into a fusion protein and use it as a trimerization domain. In protein regions, the structure and / or function of a protein are determined to a certain extent by its amino acid sequence. Prior to the present invention, the inventors hypothesized that adding an amino acid residue to one end of a recombinant type III humanized collagen polypeptide might affect the trimerization function of the recombinant type III humanized collagen polypeptide. Surprisingly, a trimerized fusion protein can be fully formed after fusing the recombinant type III humanized collagen polypeptide with an immunogenic peptide, and the trimerized fusion protein exhibits significantly improved immunogenicity. For example, in the Examples, the inventors demonstrated that trimers can be formed when a fusion protein is produced between a recombinant type III humanized collagen polypeptide and a SARS-CoV-2 RBD protein or influenza virus hemagglutinin. Compared to SARS-CoV-2 RBD protein or SARS-CoV-2 RBD protein or influenza virus hemagglutinin and an adjuvant, the trimer, alone or in combination with an adjuvant, produces significantly improved immunogenicity and can generate higher neutralizing antibody titers in the body.

[0033] As used herein, a fusion protein refers to a protein formed by indirectly linking two or more functional protein moieties via a linker in a manner that aligns the reading frame. The fusion protein may include a trimerization domain, an immunogenic domain, and an optional linker. The location of each domain is not particularly limited. For example, the trimerization domain may be located at the N-terminus or C-terminus of the fusion protein, and the immunogenic domain may be located at the N-terminus or C-terminus of the fusion protein.

[0034] As used herein, the term "trimerization domain" refers to a functional domain capable of achieving trimerization function. The trimerization domain may contain one or more repeat units represented by SEQ ID NO. 1. The repeat units may be directly linked or separated by one or more amino acid residues. The number of repeat units may vary, for example, the number of repeat units may be 2 to 50, e.g., 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 domain may contain one or more constant domains represented by SEQ ID NO. 2 at the C-terminus. The trimerization domain may contain the amino acid sequence represented by SEQ ID NO. 3.

[0035] As used herein, an immunogenic region refers to a protein or other fragment (e.g., a fragment in an antigen) derived from a pathogen and capable of eliciting an immune response in a living organism, particularly a mammal, e.g., a human. The immunogenic protein may be selected from, for example, the RBD region of a coronavirus, the membrane protein (e.g., gp140) of an AIDS virus, or the hemagglutinin protein of an influenza virus and immunogenic fragments thereof. The immunogenic region may comprise the amino acid sequence set forth in SEQ ID NO. 4 or SEQ ID NO. 10. Several prior art documents (e.g., CN202110676901.2 and CN202111337048.8) disclose coronavirus RBD regions and related immunogenic fragments.

[0036] The trimerization domain and the immunogenic domain may be indirectly linked via a linker. The type of linker is not particularly limited. The linker may be (GGGS)n, (GGGGS)n, or (G)n. For example, n may be an integer from 1 to 10, e.g., 2, 3, 4, 5, 6, 7, 8, or 9. Preferably, the linker may be (GGGS)n or (GGGGS)n, thereby introducing a disulfide bond.

[0037] To facilitate production and purification of the fusion proteins of the present invention, additional moieties can be added to the N- or C-terminus of the fusion proteins of the present invention. For example, the fusion proteins of the present invention may include a secretion signal and leader region at the N-terminus to secrete the fusion protein into the culture medium. The fusion proteins of the present invention may further include a purification tag, such as a His tag, GST tag, MBP tag, SUMO tag, or NusA tag, at the C-terminus to facilitate purification of the fusion protein. For example, the fusion protein may include the amino acid sequence set forth in SEQ ID NO. 6.

[0038] As used herein, a nucleic acid includes a polynucleotide encoding a protein. The nucleic acid may be the native nucleic acid of each portion of the fusion protein, or may be codon-optimized to facilitate expression in the target host. The nucleic acid may comprise the sequence set forth in SEQ ID NO. 7.

[0039] As used herein, the term "vector" refers to a nucleic acid delivery tool into which a polynucleotide can be inserted. A vector capable of expressing a protein encoded by the inserted polynucleotide is called an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection, allowing the genetic material elements carried therein to be expressed in the host cell. Vectors are known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages, such as lambda phage or M13 phage, and animal viruses. A vector can contain various expression control elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. A vector can also contain a replication origin. A vector can contain the nucleic acid of the present invention for introduction into a cell and expression. A vector can contain expression control elements, such as a promoter, terminator, and / or enhancer, operably linked to the nucleic acid.

[0040] The term "host cell" refers to a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. These techniques include transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA accelerated by electroporation, lipid transfection, and particle guns. Host cells may be eukaryotic or prokaryotic. For example, eukaryotic cells are yeast cells, animal cells, and / or insect cells. Prokaryotic cells may be E. coli cells.

[0041] The term "subject" refers to any human or other animal, particularly other mammals, that are the recipient of prophylaxis, treatment, and diagnosis. Other mammals may include, for example, dogs, cats, cows, horses, sheep, pigs, goats, rabbits, rats, guinea pigs, mice, etc.

[0042] The term "vaccine" as used herein refers to a suspension or solution of antigenic moieties, usually containing an infectious agent or some part of an infectious agent, that is injected into the body to produce immunity. The antigenic moieties from which the vaccine is made may be microorganisms or natural, synthetic, or genetically engineered proteins, peptides, polysaccharides, or similar products purified from microorganisms. Vaccines herein may be oral or parenteral, e.g., vaccines administered subcutaneously, intradermally, intravenously, intramuscularly, intraarterially, or by infusion.

[0043] The term "adjuvant" refers to any substance that can be mixed with an injected immunogen to enhance or modify the immune response. The adjuvant may be an aluminum adjuvant, a saponin adjuvant, lipid A, monophosphoryl lipid A, trehalose dimycolate, an oil emulsion adjuvant, a polysaccharide adjuvant, a Toll-like receptor activator, and / or a STING agonist.

[0044] The fusion proteins of the present invention can produce trimeric fusion proteins due to the presence of a trimerization domain. The trimeric fusion proteins may be homotrimers or heterotrimers. Preferably, the trimeric fusion proteins are homotrimers. The trimeric fusion proteins of the present invention can produce higher neutralizing antibodies than immunogens alone and do not have immunogenicity against the trimerization domain. The fusion proteins or trimeric fusion proteins of the present invention can be produced by a method comprising the steps of (1) culturing host cells of the present invention under appropriate culture conditions, (2) harvesting the host cells and / or medium containing the fusion protein or trimeric fusion protein, and (3) purifying the fusion protein or trimeric fusion protein. The culture and purification conditions are not particularly limited, and those skilled in the art can select appropriate culture and purification conditions to isolate the fusion proteins or trimeric proteins of the present invention.

[0045] The fusion proteins, nucleic acids, vectors, host cells, and / or trimeric fusion proteins of the present invention can be used to prepare compositions. The compositions may include the fusion proteins, nucleic acids, vectors, host cells, and / or trimeric fusion proteins of the present invention. For example, the compositions may be pharmaceutical compositions, particularly vaccines. In this case, the compositions may further include a pharmaceutically acceptable vector, excipient, diluent, and / or adjuvant. For example, in the case of vaccines, the vaccine may include aluminum adjuvant, saponin adjuvant, lipid A, monophosphoryl lipid A, trehalose dimycolate, oil emulsion adjuvant, polysaccharide adjuvant, Toll-like receptor activator, and / or STING agonist to enhance the subject's immune response to the immunogen. Such fusion proteins, nucleic acids, vectors, host cells, and / or trimeric fusion proteins or compositions of the present invention can treat or prevent symptoms or diseases mediated by pathogens. Pathogens can be viruses, bacteria, fungi, and / or parasites. For example, the virus may be selected from various types of coronavirus, AIDS virus, and influenza virus. For example, the fusion protein of the present invention can be used to produce a vaccine against the currently circulating SARS-CoV-2 virus or influenza virus. [Example]

[0046] The objects, technical features and beneficial effects of the present invention will be explained in more detail below with reference to examples, but the embodiments of the present invention are not limited thereto.

[0047] [Example 1] Construction and expression of recombinant human type III collagen-RBD trimer protein The amino acid sequence of the recombinant human type III collagen designed by the inventors is shown in SEQ ID NO. 3. The amino acid sequence of the SARS-CoV-2 RBD protein is shown in SEQ ID NO. 4. The amino acid sequence of the intermediate linker is shown in SEQ ID NO. 8. The amino acid sequence of the collagen-RBD protein fusion protein is shown in SEQ ID NO. 6. The optimized nucleotide sequence encoding the collagen-RBD protein fusion protein is shown in SEQ ID NO. 7, with eight histidine-encoding nucleotides and a stop codon added thereto.

[0048] Based on the nucleotide sequence of the collagen-RBD fusion protein, we commissioned Nanjing Jinsirui Biotechnology Co., Ltd. to synthesize the gene. The synthesized gene fragment was ligated into the pFUSE-hIgG1-Fc2 vector via the EcoR I and Nco I enzyme cleavage sites (Figure 1), resulting in the pFUSE-hIgG1-Fc2 vector containing the nucleotide sequence of the collagen-RBD fusion protein. After accurate sequencing, the plasmid was transformed into DH5α competent cells. The specific steps are as follows:

[0049] 1) DH5α competent cells were taken from -80°C and placed on ice.

[0050] 2), 1 μl of the constructed plasmid was added to DH5α competent cells and placed on ice for 30 minutes.

[0051] 3) After heat shock at 42°C for 90 seconds, the cells were quickly placed on ice.

[0052] 4) After leaving the mixture to stand for 2 minutes, 600 μl of LB without antibiotics was added, and the mixture was placed in a shaker and shaken at 37°C for 1 hour.

[0053] 5) 100 μl of the bacterial solution was taken and spread onto a LAB plate containing bleomycin.

[0054] 6) After colonies were generated, monoclonal colonies were selected and added to 5 ml of LB containing bleomycin, and after 12 hours, the 5 ml of LB was transferred to 500 ml of LB.

[0055] 7) After 8 hours, the plasmid was extracted using a plasmid extraction kit (Maxi type) manufactured by Amane Co., Ltd.

[0056] Next, the collagen-RBD protein fusion protein is expressed. The specific steps are as follows:

[0057] 1) Density is 1x10 6 EXPi293F cells, which showed good growth, were subcultured to a concentration of 1000 kJ / ml.

[0058] 2) After 12 hours, the cells were transfected with a plasmid containing collagen-RBD-encoding nucleotides according to the instructions of the EZ Trans (Li Ji Bio) transfection reagent.

[0059] 3) The culture was continued for 6 days, centrifuged at 2000 rpm, and the cell supernatant was collected.

[0060] The collagen-RBD protein was purified using NI-NTA. The specific steps are as follows:

[0061] 1), the collected cell supernatant was filtered through a 0.45 μm filter membrane.

[0062] 2) 1 ml of NI-NTA (Qiagen) was added to the protein purification column.

[0063] 3) 5 ml of ddH2O was added to remove the alcohol in the NI-NTA.

[0064] 4), 10 ml of binding buffer (40 mM NaH2PO3, 500 mM NaCl, pH: 7.8) was added to equilibrate the NI-NTA.

[0065] 5) The treated NI-NTA was added to the protein supernatant and incubated at 4°C for 3 hours.

[0066] 6), the hybrid protein was washed using binding buffer containing 5 mM imidazole, washing a volume of 30 ml.

[0067] 7), the target protein was eluted using binding buffer containing 40 mM imidazole.

[0068] 8), the eluted target protein was subjected to SDS-PAGE.

[0069] The results are shown in Figure 2. Collagen-RBD was successfully expressed, and highly purified collagen-RBD protein was obtained. After denaturation treatment, the molecular weight was approximately 80 kDa, consistent with the expected size.

[0070] To verify whether the protein formed a trimer, we used a particle size analyzer to detect the molecular weight of the protein, and the results showed that the expected molecular weight of the collagen-RBD protein was 255 kDa, which is three times the size of the monomer (Figure 3). Therefore, the collagen-RBD is a trimeric protein.

[0071] [Example 2] Comparison of RBD-specific binding antibody levels produced by immunizing mice with collagen-RBD trimer protein and RBD monomer protein Regarding materials, Balb / c was purchased from Beijing Weitong Lihua Biotechnology Co., Ltd., aluminum adjuvant was purchased from Thermo Scientific Co., Ltd., and RBD-His monomer protein was purchased from Shanghai Thermo Scientific Biotechnology Co., Ltd. CF501 adjuvant has the following structure and is owned by the applicant's laboratory.

[0072] [ka]

[0073] Twenty-four 6-week-old Balb / c mice were equally divided into four groups, each consisting of six mice. Group 1 received an intramuscular injection of 5 μg of collagen-RBD trimer protein and 20 μg of CF501 adjuvant; Group 2 received an intramuscular injection of 5 μg of RBD monomer protein and 20 μg of CF501 adjuvant; Group 3 received an intramuscular injection of 5 μg of collagen-RBD trimer protein and an equal volume of aluminum adjuvant; and Group 4 received an intramuscular injection of 5 μg of RBD monomer protein and an equal volume of aluminum adjuvant. The immunization schedule for mice is shown in Figure 4. Mice were immunized twice, on days 0 and 14, and serum was collected on day 21. Serum was inactivated at 56°C for 30 minutes.

[0074] Specific IgG antibody titers against SARS-CoV-2 RBD in mouse serum were detected using ELISA. The specific steps are as follows:

[0075] 1) 1 μg / ml of RBD-His monomer protein (Gaika organism) was coated onto an ELISA plate overnight at 4°C.

[0076] 2) The ELISA plate was sealed, and 200 μl of PBS containing 5% BSA was added to each well, followed by incubation at 37° C. for 2 hours.

[0077] 3) Mouse serum was diluted using PBST, starting from 100-fold dilution and then 3-fold dilution.

[0078] 4) The diluted mouse serum was added to the ELISA plate and incubated at 37°C for 45 minutes.

[0079] 5) The ELISA plate was washed five times with PBST.

[0080] 6) HRP-labeled rabbit anti-mouse IgG secondary antibody (Dako) was added and incubated at 37°C for 45 min.

[0081] 7) The ELISA plate was washed five times with PBST.

[0082] 8), color was developed with TMB substrate and reacted for 15 min.

[0083] 9), and the color development was stopped by adding H2SO4.

[0084] OD450 was read using a microplate reader, and the antibody titer in the serum was calculated.

[0085] The experimental results are shown in Figures 5 and 6 and Table 1. After immunization of mice with CF501 / collagen-RBD trimer, the mean IgG antibody titer against SARS-CoV-2 RBD in the serum was 656,100, 28-fold higher than the RBD-specific IgG antibody titer in the serum of mice immunized with CF501 / RBD monomer. Furthermore, the IgG antibody titer in the serum of mice immunized with aluminum adjuvant / collagen-RBD trimer was approximately 5-fold higher than the IgG antibody titer in the serum of mice immunized with aluminum adjuvant / RBD monomer. These data demonstrate that the collagen-RBD trimer protein can be used as an immunogen to highly effectively enhance the immunogenicity of RBD.

[0086] Table 1. Specific IgG antibody titers against SARS-CoV-2 RBD in mouse serum 21 days after immunization [Table 1]

[0087] [Example 3] Collagen-RBD trimer protein can induce the production of high-potency neutralizing antibodies against SARS-CoV-2 pseudovirus in mice.

[0088] To compare the neutralizing antibody titers against SARS-CoV-2 induced by collagen-RBD trimer or RBD monomer, we detected the neutralizing effect against SARS-CoV-2 pseudovirus in the serum of immunized mice. The SARS-CoV-2 pseudovirus packaging steps were as follows:

[0089] 1) HEK-293T cells, which show good growth, were passaged.

[0090] 2) After 24 hours, HEK-293T cells were co-transfected with the backbone plasmid PNL-4-3 and the 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 (Vigorous) transfection reagent.

[0091] 1), and after 12 hours, the medium was replaced with fresh DMEM containing 10% FBS.

[0092] 2) After 48 hours, the cell supernatant was taken and used as the SARS-CoV-2 pseudovirus.

[0093] The specific steps of the SARS-CoV-2 pseudovirus neutralization experiment are as follows:

[0094] 1) Huh-7 cells, which showed good growth, were added to a 96-well plate, with 10,000 cells added to each well.

[0095] 2) After 8 hours, the mouse serum was diluted with DMEM.

[0096] 3) Diluted mouse serum was mixed with SARS-CoV-2 pseudovirus and incubated for 30 min.

[0097] 4) Pseudovirus and mouse serum were added to Huh-7 cells.

[0098] 5) After 12 hours, the medium was replaced with fresh DMEM.

[0099] 6) After 48 hours, the luciferase level in each well was detected using a Promega luciferase assay kit.

[0100] 7) The neutralizing antibody titer in the serum was calculated.

[0101] The experimental results are shown in Figures 7 and 8 and Table 2. The mean neutralizing antibody titer against SARS-CoV-2 pseudovirus produced with collagen-RBD trimer protein adjuvanted with CF501 was 3620, while the mean neutralizing antibody titer against SARS-CoV-2 pseudovirus produced with RBD monomer adjuvanted with CF501 was only 191. Similarly, the neutralizing antibody titers in mice produced with aluminum adjuvant-collagen-RBD trimer protein were also higher than those in mice produced with aluminum adjuvant-RBD monomer protein.

[0102] Table 2. Neutralizing antibody titers against SARS-CoV-2 pseudovirus in mouse serum 21 days after immunization [Table 2]

[0103] [Example 4] Comparison of HA1-specific binding antibody levels produced by immunizing mice with collagen-H1N1 HA1 trimeric protein and H1N1 HA1 monomeric protein Construction and expression of recombinant type III collagen-H1N1 HA1 trimeric protein Based on the nucleotide sequence of the collagen-H1N1 HA1 fusion protein, gene synthesis was commissioned to Nanjing Jinsirui Biotechnology Co., Ltd. The synthesized gene fragment was ligated into the pFUSE-hIgG1-Fc2 vector via the EcoR I and Nco I enzyme cleavage sites to obtain the pFUSE-hIgG1-Fc2 vector containing the nucleotide sequence of the collagen-H1N1 HA1 fusion protein. The collagen-HA1 fusion protein was expressed according to the protein expression steps of Example 1.

[0104] Twenty-four 6-week-old Balb / c mice were equally divided into four groups, each consisting of six mice. Group 1 received an intramuscular injection of 5 μg of collagen-H1N1 HA1 trimer protein and 20 μg of CF501 adjuvant; Group 2 received an intramuscular injection of 5 μg of H1N1 HA1 monomer protein and 20 μg of CF501 adjuvant; Group 3 received an intramuscular injection of 5 μg of collagen-H1N1 HA1 trimer protein and an equal volume of aluminum adjuvant; and Group 4 received an intramuscular injection of 5 μg of H1N1 HA1 monomer protein and an equal volume of aluminum adjuvant. The immunization schedule for the mice is shown in Figure 4. Mice were immunized twice, on days 0 and 14, and serum was collected on day 21. Serum was inactivated at 56°C for 30 minutes.

[0105] ELISA was used to detect the specific IgG antibody titer against H1N1 HA1 in mouse serum. The specific steps are as follows:

[0106] 1) 1 μg / ml of H1N1 HA1-His monomer protein was coated onto an ELISA plate overnight at 4°C.

[0107] 2) The ELISA plate was sealed, and 200 μl of PBS containing 5% BSA was added to each well, followed by incubation at 37° C. for 2 hours.

[0108] 3) Mouse serum was diluted using PBST, starting from 100-fold dilution and then 3-fold dilution.

[0109] 4) The diluted mouse serum was added to the ELISA plate and incubated at 37°C for 45 minutes.

[0110] 5) The ELISA plate was washed five times with PBST.

[0111] 6) HRP-labeled rabbit anti-mouse IgG secondary antibody (Dako) was added and incubated at 37°C for 45 min.

[0112] 7) The ELISA plate was washed five times with PBST.

[0113] 8), color was developed with TMB substrate and reacted for 15 min.

[0114] 9), and the color development was stopped by adding H2SO4.

[0115] 10), OD450 was read using a microplate reader, and the antibody titer in the serum was calculated.

[0116] The results are shown in Figures 9 and 10. The collagen-tagged H1N1 HA1 protein, whether adjuvanted with CF501 or aluminum, elicited higher levels of IgG antibodies against the HA1 protein than the H1N1 HA1 protein alone. Therefore, the collagen trimer tag significantly improved the immunogenicity of both novel coronavirus and influenza virus antigens.

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Claims

1. 1. A fusion protein comprising a trimerization domain and an immunogenic domain connected by a linker, wherein the trimerization domain comprises 10 to 20 repeat units represented by SEQ ID NO. 1 or an amino acid sequence having 90% to 99% identity to SEQ ID NO. 1, the repeat units being directly linked, the immunogenic domain being an immunogenic protein of a pathogen, optionally selected from an RBD domain of a coronavirus, a membrane protein of an AIDS virus, or an influenza virus hemagglutinin protein and / or an immunogenic fragment thereof, optionally wherein the influenza virus hemagglutinin protein is an H1N1 HA1 protein, and the trimerization domain further comprises one or more constant domains represented by SEQ ID NO. 2 at its C-terminus, wherein the repeat units are directly linked to the constant domain or separated from the constant domain by one or more amino acid residues.

2. 2. The fusion protein of claim 1, wherein the trimerization region comprises the amino acid sequence set forth in SEQ ID NO. 3 or an amino acid sequence having 90% to 99% identity to SEQ ID NO. 3, and / or the immunogenic region comprises the amino acid sequence set forth in SEQ ID NO. 4 or SEQ ID NO. 10 or an amino acid sequence having 90% to 99% identity to SEQ ID NO. 4 or SEQ ID NO. 10, and / or the linker is (GGGS)n or (GGGGS)n or (G)n, optionally wherein n is an integer from 1 to 10, optionally 2, 3, 4, 5, 6, 7, 8, or 9.

3. 2. The fusion protein of claim 1, wherein the trimerization domain and the immunogenic domain are located at the N-terminus and C-terminus of the fusion protein, respectively, and optionally the fusion protein comprises an amino acid sequence set forth in SEQ ID NO. 5 or SEQ ID NO. 9, or an amino acid sequence having 90% to 99% identity to SEQ ID NO. 5 or SEQ ID NO.

9.

4. A nucleic acid comprising nucleotides encoding the fusion protein of any one of claims 1 to 3, optionally further comprising nucleotides encoding a purification tag, which is optionally a His tag, a GST tag, an MBP tag, a SUMO tag or a NusA tag, optionally further comprising nucleotides encoding a leader sequence, and optionally comprising the sequence shown in SEQ ID NO. 7 or a nucleotide sequence having 90% to 99% identity to SEQ ID NO.

7.

5. 5. A vector comprising the nucleic acid of claim 4, and optionally comprising an expression control element operably linked to said nucleic acid, said expression control element optionally being a promoter, terminator and / or enhancer.

6. 5. A host cell comprising the nucleic acid of claim 4, optionally a eukaryotic or prokaryotic cell, optionally wherein the eukaryotic cell is a yeast cell, an animal cell and / or an insect cell, and / or wherein the prokaryotic cell is an E. coli cell.

7. A trimeric fusion protein comprising the fusion protein of any one of claims 1 to 3.

8. A method for producing the fusion protein according to any one of claims 1 to 3, comprising: (1) culturing host cells under suitable 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.

9. A composition comprising the fusion protein of any one of claims 1 to 3.

10. a pharmaceutical composition, in particular a vaccine, further comprising a pharmaceutically acceptable vector, excipient, diluent and / or adjuvant, optionally an aluminum adjuvant, a saponin adjuvant, lipid A, monophosphoryl lipid A, trehalose dimycolate, an oil emulsion adjuvant, a polysaccharide adjuvant, a toll-like receptor activator and / or a STING agonist; Optionally, the adjuvant is of the formula: 【Chemistry 1】

11. A composition for treating or preventing a pathogen-mediated condition or disease, comprising the fusion protein of any one of claims 1 to 3, optionally wherein the pathogen is a virus, bacterium, fungus and / or parasite; Optionally, the virus is selected from a coronavirus, an AIDS virus, and an influenza virus; Optionally, the influenza virus is an influenza A virus or an influenza B virus; Optionally, the composition, wherein the coronavirus is SARS-CoV-2.

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