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 technical 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 cases of infection and over 6 million deaths. The spike protein (S) of SARS-CoV-2 is responsible for identifying and invading cells, and the receptor binding domain (RBD) contained in the S protein is the most important, as it is responsible for identifying the angiotensin 2 (ACE2) receptor and binding to the receptor to mediate the virus' entry into cells. The currently developed novel coronavirus mRNA vaccines, protein subunit vaccines, and adenovirus vector vaccines have all been developed against the S protein or RBD protein of SARS-CoV-2. The S protein of SARS-CoV-2 is a trimeric protein in its natural state. Currently, many studies have demonstrated that the S protein trimer or RBD protein trimer has higher immunogenicity than the S protein monomer or RBD protein monomer. In order to achieve trimer protein expression of RBD or S protein, some studies have added Foldon tag to RBD or S protein for fusion expression, and formed trimer form by Foldon action. However, Foldon has a large molecular weight and is highly immunogenic. Immunization of human body with trimer in which Foldon and RBD / S protein are fused generates a strong immune response against Foldon protein, weakening the antigen response against SARS-CoV-2 S or RBD. Similarly, trimers formed using some other non-human derived tags or vehicles still have similar disadvantages.

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

[0004] The inventors have previously screened and optimized the type III collagen sequence, and the recombinant human collagen produced has very high hydrophilicity and stability, and its amino acid composition is 100% identical to the corresponding part of the amino acid sequence of natural collagen, and does not cause immune reactions when applied to the human body. In addition, since the recombinant human collagen has a trimer structure, it can be widely applied in the biopharmaceutical industry. Since the human type III collagen is a repeat of the optimized human collagen peptide fragment, it may destroy the trimer structure of the collagen after fusion expression with the RBD protein of the new coronavirus or the hemagglutinin of the influenza virus. In addition, even if the RBD protein of the new coronavirus or the hemagglutinin of the influenza virus does not destroy the collagen trimer structure and produces a trimer protein of RBD, the trimer RBD may not be able to induce the body's immune response well due to the influence of the collagen, since it is different from the natural RBD trimer structure. Therefore, the effect of fusion expression of the human type III collagen with the RBD protein of the new coronavirus or the hemagglutinin of the influenza virus as a vaccine is unexpected. In the present invention, the inventors introduce cysteine ​​into the C-terminus of the recombinant human type III collagen to generate a disulfide bond to stabilize the collagen, and then fusion-express it with the RBD protein of SARS-CoV-2 or influenza virus hemagglutinin through 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 can generate stronger binding and neutralizing antibodies than the RBD protein of SARS-CoV-2 or the hemagglutinin monomer of influenza virus.Therefore, the invention cleverly links human type III collagen peptide and the RBD protein of the new coronavirus or influenza virus hemagglutinin in series and expresses them in a fusion manner, thereby promoting the formation of a correct trimer conformation of the RBD protein or influenza virus hemagglutinin, improving the immunogenicity of the RBD or influenza virus hemagglutinin, and since the human type III collagen sequence is derived from humans, it does not induce the generation of an immune response in the human body, and promotes focusing the immune response on the trimer 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 repeat units as set forth in SEQ ID NO.1 or repeat units having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the repeat unit as set forth in SEQ ID NO.1, with the proviso that the repeat 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, a bacterium, a fungus and / or a parasite.

[0008] In one embodiment, the immunogenic region is selected from the RBD region of a coronavirus, the membrane protein of the 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 10-20 (e.g., 11, 12, 13, 14, 15, 16, 17, 18 or 19) repeat units as set forth in SEQ ID NO.1 or repeat units having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the repeat units as set forth in SEQ ID NO.1. In one embodiment, the trimerization region comprises at the C-terminus one or more sequences as set forth in SEQ ID NO.2 or sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence as set forth in SEQ ID NO.2.

[0010] In one embodiment, the trimerization region comprises an 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 an 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, respectively, of the fusion protein.

[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 according to the invention comprises nucleotides encoding a fusion protein as described herein. In one embodiment, the nucleic acid comprises the amino acid sequence shown in SEQ ID NO.7. In one embodiment, the nucleic acid further comprises a coding sequence for a purification tag, such as 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 as 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 according to the invention comprises a nucleic acid or vector as 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 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, a trimeric fusion protein according to the invention comprises a fusion protein as described herein.

[0019] In one aspect, the method of the invention for producing a fusion protein or a 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 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 pharma- ceutically 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 as shown in the formula:

[0023] [ka]

[0024] In one aspect, the invention provides a use of the fusion protein, nucleic acid, vector, host cell, trimeric fusion protein and / or composition described herein in the manufacture of a medicament, said medicament treating or preventing a pathogen-mediated condition or disease. In one embodiment, the pathogen is a virus, a bacterium, a fungus and / or a 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. Effect of the Invention

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

[0027] 1. The 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 with the novel coronavirus RBD protein or influenza virus hemagglutinin, and 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 has homology with humans, 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 description of the drawings]

[0030] [Figure 1] 1 shows the vector plasmid profile required for construction of collagen-RBD trimer protein. [Diagram 2] FIG. 1 shows an SDS-PAGE image of the expressed collagen-RBD trimer protein. [Diagram 3] The molecular weight of collagen-RBD trimer protein detected using a particle size analyzer is shown. [Figure 4] 1 shows the immunization schedule for mice. [Diagram 5] Detected levels of specific IgG-binding antibodies against SARS-CoV-2 RBD in serum of mice on day 21 after immunization. [Figure 6] Shows 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] 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 influenza H1N1 HA1 in serum of mice on day 17 after immunization with HA1-collagen / CF501 and HA1 / CF501 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 PREFERRED EMBODIMENTS

[0031] In order to make the objectives, technical means and advantages of the present invention clearer, the technical means in the embodiments of the present invention are described below clearly and completely with reference to the embodiments of the present invention, and obviously, the described embodiments are only some of the embodiments of the present invention, and not all of the embodiments. Any other embodiments that a person skilled in the art can think of based on the embodiments of the present invention without any creative effort are all within the protection scope of the present invention.

[0032] In the present specification, the inventors use the recombinant type III humanized collagen polypeptide found previously as a part of a fusion protein as a trimerization domain. In the protein region, the structure and / or function of the protein is determined to a certain extent by the amino acid sequence. Prior to the present invention, the inventors assume that adding an amino acid residue to one end of the recombinant type III humanized collagen polypeptide may affect the trimerization function of the recombinant type III humanized collagen polypeptide. Surprisingly, a trimerization fusion protein can be completely formed after fusing the recombinant type III humanized collagen polypeptide with an immunogenic peptide, and the trimerization fusion protein produces significantly improved immunogenicity. For example, in the Examples, the inventors have demonstrated that a trimer can be formed when a fusion protein is produced between the recombinant type III humanized collagen polypeptide and the SARS-CoV-2 RBD protein or the hemagglutinin of influenza virus. 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 produce 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 in a manner that aligns the reading frame via a linker. The fusion protein may include a trimerization region, an immunogenic region, and an optional linker. The location of each moiety is not particularly limited. For example, the trimerization region may be located at the N-terminus or C-terminus of the fusion protein, and the immunogenic region may be located at the C-terminus or N-terminus of the fusion protein.

[0034] As used herein, trimerization region refers to a functional region capable of achieving trimerization function. The trimerization region may comprise one or more repeat units as shown in SEQ ID NO.1. Each repeat unit 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 2-50, for example, 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 comprise one or more constant regions as shown in SEQ ID NO.2 at the C-terminus. The trimerization region may comprise an amino acid sequence as shown in 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 an organism, particularly a mammal, such as the human body. The immunogenic protein may be selected, for example, from the RBD region of coronavirus, the membrane protein of AIDS virus (e.g., gp140) or the hemagglutinin protein of influenza virus and immunogenic fragments thereof. The immunogenic region may comprise the amino acid sequence shown in SEQ ID NO.4 or SEQ ID NO.10. Several documents in the prior art (e.g., CN202110676901.2 and CN202111337048.8) disclose coronavirus RBD regions and related immunogenic fragments.

[0036] The trimerization region and the immunogenic region may be indirectly linked via a linker. The type of the linker is not particularly limited. The linker may be (GGGS)n or (GGGGS)n or (G)n. For example, n may be an integer from 1 to 10, for example, 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] Additional moieties can be added to the N-terminus or C-terminus of the fusion protein of the invention to facilitate production and purification of the fusion protein herein. For example, the fusion protein of the invention may include a secretion signal and leader region at the N-terminus to secrete the fusion protein into the medium. The fusion protein of the invention may further include a purification tag, such as a His tag, a GST tag, a MBP tag, a SUMO tag, or a NusA tag, at the C-terminus to facilitate purification of the fusion protein. For example, the fusion protein may include the amino acid sequence shown in SEQ ID NO.6.

[0038] As used herein, a nucleic acid includes a polynucleotide that encodes a protein. The nucleic acid may be the native nucleic acid of each portion of the fusion protein, or may be a codon-optimized nucleic acid, thereby facilitating expression in the target host. The nucleic acid may include the sequence shown 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 is called an expression vector if it is capable of expressing a protein encoded by an inserted polynucleotide. A vector can be introduced into a host cell by transformation, transduction or transfection, and allows 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 phages or M13 phages, and animal viruses. A vector can include 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 include an origin of replication. A vector can include the nucleic acid of the present invention for introduction into a cell and expression. A vector can include expression control elements, such as promoters, terminators, and / or enhancers, 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 gun. The host cell may be a eukaryotic or prokaryotic cell. For example, the eukaryotic cell is a yeast cell, an animal cell, and / or an insect cell. The prokaryotic cell may be an E. coli cell.

[0041] The term "subject" refers to any human or other animal, particularly other mammals, receiving 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, which is injected into the body to produce immunity. The antigenic moieties from which the vaccine is made may be microorganisms, natural products purified from microorganisms, synthetic or engineered proteins, peptides, polysaccharides or similar products. The vaccines herein may be oral or parenteral vaccines, 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 protein of the present invention can produce a trimeric fusion protein due to the presence of a trimerization region. The trimeric fusion protein may be a homotrimer or a heterotrimer. Preferably, the trimeric fusion protein is a homotrimer. The trimeric fusion protein of the present invention can produce higher neutralizing antibodies than a single immunogen and has no immunogenicity against the trimerization region. The fusion protein or trimeric fusion protein of the present invention can be produced by a method including the steps of (1) culturing the host cell of the present invention under suitable culture conditions, (2) harvesting the host cell and / or medium containing the fusion protein or trimeric fusion protein, and (3) purifying the fusion protein or trimeric fusion protein. There are no particular limitations on the culture and purification conditions, and a person skilled in the art can select suitable culture and purification conditions to separate the fusion protein or trimeric protein of the present invention.

[0045] The fusion protein, nucleic acid, vector, host cell and / or trimeric fusion protein of the present invention can be used to prepare a composition. The composition may comprise the fusion protein, nucleic acid, vector, host cell and / or trimeric fusion protein of the present invention. For example, the composition may be a pharmaceutical composition, in particular a vaccine. In this case, the composition may further comprise a pharma-ceutically acceptable vector, excipient, diluent and / or adjuvant. For example, in the case of a vaccine, the vaccine may comprise 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 to enhance the immune response of the subject to the immunogen. Such a fusion protein, nucleic acid, vector, host cell and / or trimeric fusion protein or composition of the present invention can treat or prevent a pathogen-mediated condition or disease. The pathogen may be a virus, a bacterium, a fungus and / or a parasite. 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, such as a vaccine against SARS-CoV-2 virus or influenza virus that is currently circulating. EXAMPLES

[0046] The objects, technical features and advantageous effects of the present invention will be described 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 collagen III-RBD trimer protein The amino acid sequence of the recombinant human collagen type III 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, adding therein eight histidine coding nucleotides and a stop codon.

[0048] Based on the nucleotide sequence of collagen-RBD fusion protein, we commissioned Nanjing Jinsirui Biological Company to carry out gene synthesis. The synthesized gene fragment is ligated into the pFUSE-hIgG1-Fc2 vector via EcoR I and Nco I enzyme cleavage sites (Figure 1), to obtain the pFUSE-hIgG1-Fc2 vector containing the nucleotide sequence of collagen-RBD fusion protein. After the sequence is accurate, the plasmid is transformed into DH5α competent cells. The specific steps are as follows:

[0049] 1) Take DH5α competent cells at -80℃ and place them 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 90s, 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 ul of the bacterial solution was taken and spread onto an 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 h, 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, expression of the collagen-RBD protein fusion protein is carried out. The specific steps are as follows:

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

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

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

[0060] Purify collagen-RBD protein 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 proteins were 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), and the eluted target protein was subjected to SDS-PAGE.

[0069] The results are shown in Figure 2. Collagen-RBD was successfully expressed and high-purity collagen-RBD protein was harvested. 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. As for 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 Gaika Biotechnology Co., Ltd. The 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 evenly divided into four groups, each with six mice. Group 1 was intramuscularly injected with 5 μg collagen-RBD trimer protein and 20 μg CF501 adjuvant, group 2 was intramuscularly injected with 5 μg RBD monomer protein and 20 μg CF501 adjuvant, group 3 was intramuscularly injected with 5 μg collagen-RBD trimer protein and an equal volume of aluminum adjuvant, and group 4 was intramuscularly injected with 5 μg RBD monomer protein and an equal volume of aluminum adjuvant. The immunization schedule of the mice is shown in Figure 4. The mice were immunized twice in total on days 0 and 14, respectively, and the mouse serum was collected on day 21. The mouse serum was inactivated at 56°C for 30 min.

[0074] The specific IgG antibody titer against SARS-CoV-2 RBD in mouse serum was detected by ELISA. The specific steps are as follows:

[0075] 1) 1 μg / ml of RBD-His monomer protein (Gaia octopus) 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 and incubated at 37° C. for 2 hours.

[0077] 3) Mouse serum was diluted using PBST, and the mouse serum was diluted from 100-fold to 3-fold.

[0078] 4) The diluted mouse sera were 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-conjugated 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), the color was developed with TMB substrate and incubated for 15 min.

[0083] 9) The color development was stopped by adding H2SO4.

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

[0085] The experimental results are shown in Figure 5, Figure 6 and Table 1. After mice were immunized with CF501 / collagen-RBD trimer, the average IgG antibody titer against SARS-CoV-2 RBD in the serum was 656100, 28 times higher than the RBD-specific IgG antibody titer in the serum of mice after CF501 / RBD monomer immunization. In addition, the IgG antibody titer in the serum of mice after aluminum adjuvant / collagen-RBD trimer immunization was also about 5 times higher than the IgG antibody titer in the serum of mice after aluminum adjuvant / RBD monomer immunization. These data demonstrated that collagen-RBD trimer protein as an immunogen can very 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 neutralizing antibody titers 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, the inventors detected the neutralizing effect against the SARS-CoV-2 pseudovirus in the serum of mice after immunization. The packaging steps of the SARS-CoV-2 pseudovirus are as follows:

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

[0090] 2) After 24 h, 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) were co-transfected into HEK-293T cells using Vigofect (Vigorous) transfection reagent.

[0091] 1) 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, mouse serum was diluted using DMEM.

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

[0097] 4) Mock virus 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) and the neutralizing antibody titer in serum was calculated.

[0101] The experimental results are shown in Figure 7, Figure 8 and Table 2. The average neutralizing antibody titer against SARS-CoV-2 pseudovirus produced by collagen-RBD trimer protein adjuvanted with CF501 is 3620, while the average neutralizing antibody titer against SARS-CoV-2 pseudovirus produced by RBD monomer adjuvanted with CF501 is only 191. Similarly, the mouse neutralizing antibody titer of aluminum adjuvant-collagen-RBD trimer protein group is also higher than that of 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 trimer protein and H1N1 HA1 monomer protein Construction and expression of recombinant type III collagen-H1N1 HA1 trimer protein Based on the nucleotide sequence of collagen-H1N1 HA1 fusion protein, Nanjing Jinsirui Biological Company was commissioned 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 to obtain the pFUSE-hIgG1-Fc2 vector containing the nucleotide sequence of collagen-H1N1 HA1 fusion protein. The collagen-HA1 fusion protein was expressed according to the protein expression step of Example 1.

[0104] Twenty-four 6-week-old Balb / c mice were equally divided into four groups, each with six mice. The first group was intramuscularly injected with 5 μg collagen-H1N1 HA1 trimer protein and 20 μg CF501 adjuvant, the second group was intramuscularly injected with 5 μg H1N1 HA1 monomer protein and 20 μg CF501 adjuvant, the third group was intramuscularly injected with 5 μg collagen-H1N1 HA1 trimer protein and an equal volume of aluminum adjuvant, and the fourth group was intramuscularly injected with 5 μg H1N1 HA1 monomer protein and an equal volume of aluminum adjuvant. The immunization schedule of the mice is shown in Figure 4. The mice were immunized twice in total on days 0 and 14, respectively, and the mouse serum was collected on day 21. The mouse serum was inactivated at 56°C for 30 min.

[0105] The specific IgG antibody titer against H1N1 HA1 in mouse serum was detected by ELISA. 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 and incubated at 37° C. for 2 hours.

[0108] 3) Mouse serum was diluted using PBST, and the mouse serum was diluted from 100-fold to 3-fold.

[0109] 4) The diluted mouse sera were 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-conjugated 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), the color was developed with TMB substrate and incubated for 15 min.

[0114] 9) The color development was stopped by adding H2SO4.

[0115] 10) The 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, and show that the collagen-tagged H1N1 HA1 protein can produce higher levels of IgG antibodies against the HA1 protein than the H1N1 HA1 protein alone, whether it is adjuvanted with CF501 or aluminum. Therefore, the collagen trimer tag can significantly improve the immunogenicity of the antigens, whether they are novel coronavirus antigens or influenza virus antigens.

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Claims

1. A fusion protein comprising a trimerization region and an immunogenic region linked by a linker, the trimerization region comprising one or more repeat units as shown in SEQ ID NO. 1, and the immunogenic region being an immunogenic protein of a pathogen, for example, selected from the RBD region of a coronavirus, the membrane protein of an AIDS virus, or the hemagglutinin protein of an influenza virus and immunogenic fragments thereof, preferably, the hemagglutinin protein of an influenza virus is an H1N1 HA1 protein.

2. The fusion protein according to claim 1, wherein the trimerization domain comprises 10 to 20, e.g., 11, 12, 13, 14, 15, 16, 17, 18 or 19, repeat units as shown in SEQ ID NO. 1, and optionally, at the C-terminus, one or more constant domains as shown in SEQ ID NO. 2, wherein the repeat units and the constant domain are directly linked or separated by one or more amino acid residues.

3. The fusion protein according to claim 1 or 2, wherein the trimerization domain comprises the amino acid sequence shown in SEQ ID NO. 3, and / or the immunogenic domain comprises the amino acid sequence shown in SEQ ID NO. 4 or SEQ ID NO. 10, and / or the linker is (GGGS)n or (GGGGS)n or (G)n, preferably, n is an integer from 1 to 10, e.g., 2, 3, 4, 5, 6, 7, 8 or 9.

4. The fusion protein according to any one of claims 1 to 3, wherein the trimerization domain and the immunogenic domain are located at the N-terminus and C-terminus of the fusion protein, respectively, and preferably the fusion protein comprises an amino acid sequence as shown in SEQ ID NO. 5 or SEQ ID NO.

9.

5. A nucleic acid comprising nucleotides encoding a fusion protein according to any one of claims 1 to 4, optionally further comprising nucleotides encoding a purification tag, such as a His-tag, a GST-tag, an MBP-tag, a SUMO-tag or a NusA-tag, and optionally further comprising nucleotides encoding a leader sequence, preferably comprising the sequence as shown in SEQ ID NO.

7.

6. 6. A vector comprising the nucleic acid of claim 5, and optionally comprising expression control elements, such as promoters, terminators and / or enhancers, operably linked to said nucleic acid.

7. A host cell comprising a nucleic acid according to claim 5 or a vector according to claim 6, preferably a eukaryotic or prokaryotic cell, preferably the eukaryotic cell being a yeast cell, an animal cell and / or an insect cell and / or the prokaryotic cell being an E. coli cell.

8. A trimeric fusion protein comprising the fusion protein of any one of claims 1 to 4.

9. A method for producing a fusion protein according to any one of claims 1 to 4 or a trimeric fusion protein according to claim 8, comprising the steps of: (1) culturing the host cell according to claim 7 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.

10. A composition comprising a fusion protein according to any one of claims 1 to 4, a nucleic acid according to claim 5, a vector according to claim 6, a host cell according to claim 7 and / or a trimeric fusion protein according to claim 8.

11. A pharmaceutical composition, in particular a vaccine, further comprising a pharma- ceutically acceptable vector, excipient, diluent and / or adjuvant, such as aluminum adjuvant, saponin adjuvant, lipid A, monophosphoryl lipid A, trehalose dimycolate, oil emulsion adjuvant, polysaccharide adjuvant, Toll-like receptor activator and / or STING agonist, 11. The composition of claim 10, wherein the adjuvant is of the formula: 【Chemistry 1】

12. Use of a fusion protein according to any one of claims 1 to 4, a nucleic acid according to claim 5, a vector according to claim 6, a host cell according to claim 7 and / or a trimeric fusion protein according to claim 8, a composition according to claim 10 or 11 in the manufacture of a medicament for treating or preventing a condition or disease mediated by a pathogen, preferably wherein the pathogen is a virus, a bacterium, a fungus and / or a parasite, Preferably, the virus is selected from coronavirus, AIDS virus and influenza virus; Preferably, the influenza virus is an influenza A virus or an influenza B virus; Preferably, the coronavirus is SARS-CoV-2.

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