Recombinant protein capable of inhibiting human coronavirus infection in broad spectrum and application thereof

Abstract

This invention relates to the field of biomedical technology, specifically to a recombinant protein capable of broadly inhibiting human coronavirus infection and its applications. The recombinant protein contains a heptahelical repeat domain 1 of SARS-CoV-2; or, contains a heptahelical repeat domain 1 of SARS-CoV-2, with a partial addition of a portion of the CH region amino acid sequence and / or a stem helix sequence from the stem helix region of S2. The recombinant protein designed in this invention can be expressed via a prokaryotic expression system, possesses a typical α-helix structure, and can effectively bind to the HR2P peptide of SARS-CoV-2 in vitro. Furthermore, the above-mentioned recombinant proteins can effectively neutralize SARS-CoV-2 pseudovirus infection in vitro. Simultaneously, the recombinant protein can be used as an immunogen in subunit vaccine research. The recombinant protein designed in this invention has the potential to develop into a broad-spectrum entry inhibitor and broad-spectrum vaccine immunogen candidate against highly pathogenic coronaviruses and common coronaviruses, to better address the current COVID-19 pandemic and potential new highly pathogenic coronaviruses in the future.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a recombinant protein that can broadly inhibit human coronavirus infection and its applications. Background Technology

[0002] Since the outbreak of SARS-CoV-2, various COVID-19 vaccines, including inactivated virus vaccines, mRNA vaccines, and viral vector vaccines, have been approved. However, the emergence of numerous variants has posed a challenge to the already marketed COVID-19 vaccines. Recently, Qatar and Saudi Arabia have also reported cases of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infection (Chopra, H.; Islam, MA; Chandran, D.; et al. MERS-CoV recently re-emerged in Qatar, Saudi Arabia, its feasible global health risks amid FIFA World Cup 2022 and salient counteracting strategies - an update. Int J Surg, 2023). In regions where SARS-CoV-2 remains prevalent, the emergence of MERS-CoV could potentially lead to co-infection and even viral recombination (Wang, Q.; Sun, L.; Jiang, S. Potential recombination between SARS-CoV-2 and MERS-CoV: calls for the development of pan-CoV vaccines. Signal Transduct Target Ther, 2023). Furthermore, locally circulating common human coronaviruses HCoV-OC43, HCoV-229E, HCoV-NL63, and HCoV-HKU1 can also cause varying degrees of respiratory illness, posing a threat to human life. Therefore, the development of pan-coronavirus vaccines and treatments is urgently needed.

[0003] The Spike (S) protein comprises two subunits, S1 and S2. Compared to S1, S2 mediates membrane fusion and is exposed only during viral entry, making it more conserved in coronaviruses. The S2 subunit contains a fusion peptide (FP), heptapeptide repeat 1 (HR1), a central helical region (CH), a linker domain (CD), a stem helix, heptapeptide repeat 2 (HR2), and a transmembrane region (TM) (Walls, AC; Tortorici, MA; Snijder, J.; et al. Tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion. Proc Natl Acad Sci USA, 2017.). Currently, most COVID-19 vaccines are developed using the S protein or its RBD region. However, the RBD region of coronaviruses exhibits low amino acid similarity, suggesting that RBD is not a conserved, ideal immunogen for designing pan-coronavirus disease vaccines. Finding an ideal immunogen is crucial for developing a broad-spectrum vaccine for the novel coronavirus. The HR1 region is an ideal target for developing pan-coronavirus inhibitors. For example, EK1 is a peptide that targets the HR1 region and has strong inhibitory activity against SARS-CoV, MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43, and emerging SARS-CoV-2 WT, Delta, and Omicron variants (Xia, S.; Yan, L.; Xu, W.; et al. A pan-coronavirus fusion inhibitor targeting the HR1 domain of human coronavirus spike. Sci Adv, 2019. Xia S, Wang L, Jiao F, et al. SARS-CoV-2 Omicron subvariants exhibit distinct fusogenicity, but similar sensitivity, to pan-CoV fusion inhibitors. Emerg Microbes Infect, 2023.).Furthermore, a vaccine based on SARS-CoV-2 HR1 and HR2 successfully induced potent cross-neutralizing antibodies against SARS-CoV-2 and Omicron variants (Pang, W.; Lu, Y.; Zhao, Y. et al. Avariant-proof SARS-CoV-2 vaccine targeting HR1 domain in S2 subunit of spike protein. Cell Research, 2022). These results indicate that the HR1 region is a potential candidate target for designing novel entry inhibitors and pan-coronavirus vaccines. Additionally, numerous studies have shown that antibodies targeting the stem helix region can broadly neutralize β-coronaviruses (Dacon, C.; Peng, L.; Lin, TH. et al. Rare, convergent antibodies targeting the stem helix broadly neutralize diverse betacoronaviruses. Cell Host Microbe, 2022.).Crystal structure analysis revealed that in the pre-fusion conformation of the trimer S protein, the stem helix forms a surface-exposed membrane-proximal helical bundle (Wang C, van Haperen R, Gutierrez-Alvarez J, et al. A conserved immunogenic and vulnerable site on the coronavirus spike protein delineated by cross-reactive monoclonal antibodies. Nat Commun, 2021.); in the S2 post-fusion state, the middle portion of the stem helix is ​​folded into a short, surface-exposed helix, perpendicularly docking with the central coil-coil structure, playing a certain stabilizing role in the six-helix structure formed by HR1 and HR2 (Walls, AC; Tortorici, MA; Snijder, J.; et al. Tectonic conformational changes of acoronavirus spike glycoprotein promote membrane fusion. Proc Natl Acad Sci U SA, 2017.; Cai, Y.; Zhang, J.; Xiao, T.; et al. Distinct conformational states of SARS-CoV-2 spike protein. Science, 2020. More importantly, the stem helix sequence is relatively conserved in HCoVs (Sauer, MM; Tortorici, MA; Park, YJ; et al. Structural basis for broad coronavirus neutralization. Nat Struct Mol Biol, 2021.). Studies have found that vaccines targeting the MERS-CoV-2 S2 subunit can induce antibodies against the stem helix region, and these antibodies can cross-bind with the β-CoV S protein (Hsieh CL, Werner AP, Leist SR, et al. Stabilized coronavirus spike stem elicits a broadly protective antibody. Cell Rep, 2021.).These findings suggest that conserved regions in the S2 subunit, such as HR1 and stem helix regions, can be engineered as immunogen candidates for developing broad-spectrum invasion inhibitors against human coronavirus infection and broad-spectrum coronavirus vaccines to better address SARS-CoV-2 and its variants, as well as other human coronavirus infections. Summary of the Invention

[0004] The purpose of this invention is to address the problem of poor broad-spectrum efficacy of current novel coronavirus vaccines, and to provide a recombinant protein that can broadly inhibit human coronavirus infection and its applications.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A recombinant protein capable of broadly inhibiting human coronavirus infection, said recombinant protein containing a heptahelical repeat domain 1 of SARS-CoV-2; or, containing a heptahelical repeat domain 1 of SARS-CoV-2, wherein a portion of the CH region amino acid sequence and / or the stem helix sequence of the stem helix region in S2 are added to the region.

[0007] The SARS-CoV-2 heptaspiral repeat domain 1 is formed by connecting three HR1 sequences (TQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALN TLVKQLSSNFGAISSVLNDILSRLDKVE) in a forward-backward-forward sequence using connecting arms (GKGNQ and GLIDG).

[0008] The connecting arm between the forward HR1 and the reverse HR1 is GKGNQ; the connecting arm between the reverse HR1 and the forward HR1 is GLIDG;

[0009] The partial amino acid sequence of the CH region is AEVQIDRLITGRLQ;

[0010] The stem helix sequence in the stem helix region of S2 is LDSFKEELDKYFKNHTSP.

[0011] The recombinant protein is the seven-helical repeat domain 1 of SARS-CoV-2, with a His tag fused to the C-terminus for expression. The specific amino acid sequence is shown in SEQ ID NO:1, i.e., THR1.

[0012] Alternatively, the recombinant protein is a stem helix sequence in the stem helix region of S2 connected to the C-terminus of HR1 of each segment of recombinant protein THR1 via GGSSG, and the specific amino acid sequence is shown in SEQ ID NO:2, i.e., THR1S;

[0013] Alternatively, the recombinant protein is recombinant protein THR1 with a partial amino acid sequence of the CH region added to the C-terminus, and linked to the stem helix sequence of the stem helix region in S2 via GGSSG. The specific amino acid sequence is shown in SEQ ID NO:3, i.e., THR1LS.

[0014] A vector that can broadly inhibit human coronavirus infection, the vector containing the recombinant protein described above.

[0015] A host cell capable of broadly inhibiting human coronavirus infection, comprising the vector.

[0016] The use of the recombinant protein that can broadly inhibit human coronavirus infection, and the use of the recombinant protein in the preparation of medicaments for the prevention and / or treatment of coronaviruses.

[0017] The application of the recombinant protein that can broadly inhibit human coronavirus infection, and the application of the recombinant protein as an immunogen in a subunit vaccine.

[0018] The application of the recombinant protein as an immunogen in combination with adjuvants in the preparation of vaccines for the prevention and / or treatment of coronaviruses.

[0019] The application of the recombinant protein that can broadly inhibit human coronavirus infection, and the application of the recombinant protein in the preparation of coronavirus detection reagents.

[0020] Advantages of this invention:

[0021] The recombinant proteins of this invention can be expressed via a prokaryotic expression system, possess a typical α-helix structure, and can effectively bind to the HR2P peptide of SARS-CoV-2 in vitro. Furthermore, all of the above-mentioned recombinant proteins can effectively neutralize SARS-CoV-2 pseudovirus infection in vitro. THR1LS exhibits the best activity and can also effectively neutralize SARS-CoV-2 Delta, Omicorn mutant strains, and HCoVs (SARS-CoV, MERS-CoV, HCoV-229E, and HCoV-NL63) infection. Therefore, the recombinant proteins of this invention have the potential to be further developed into broad-spectrum anti-coronavirus protein drugs.

[0022] The recombinant protein of this invention can be used as an immunogen for subunit vaccines. When the above-mentioned THR1, THR1S or THR1LS proteins are mixed with aluminum adjuvants and then used to immunize Balb / c mice, both THR1S and THR1LS can elicit an immune response in vivo and produce neutralizing antibodies against SARS-CoV-2. Among them, THR1LS mixed with aluminum adjuvant has the best immunization effect.

[0023] Furthermore, this invention involves immunizing patients with a mixture of THR1LS and the novel adjuvant CF501. Compared to aluminum adjuvants, this elicits a more potent immune response, including both antibody and cellular immune responses. Post-immunization serum neutralizing antibodies effectively neutralize SARS-CoV-2 and its mutant strains, as well as various human coronavirus infections.

[0024] The recombinant protein designed in this invention has the potential to be developed into a broad-spectrum vaccine immunogen candidate against highly pathogenic coronaviruses and common coronaviruses, especially THR1LS, which can better address the COVID-19 pandemic and potential new highly pathogenic coronaviruses in the future. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the connection of recombinant proteins provided in an embodiment of the present invention;

[0026] Figure 2 This is a map of the Pet30(a) expression plasmid provided in an embodiment of the present invention.

[0027] Figure 3 SDS-PAGE electrophoresis results of the recombinant protein provided in the embodiments of the present invention;

[0028] Figure 4 The secondary structure of the recombinant protein provided in the embodiments of the present invention;

[0029] Figure 5 The binding of the recombinant protein provided in the embodiments of the present invention to SARS-CoV-2HR2P;

[0030] Figure 6 The recombinant protein provided in this embodiment of the invention exhibits inhibitory activity against SARS-CoV-2 pseudovirus;

[0031] Figure 7 The recombinant protein provided in this embodiment of the invention exhibits inhibitory activity against SARS-CoV-2 mutant pseudoviruses;

[0032] Figure 8 The recombinant protein provided in this embodiment of the invention exhibits inhibitory activity against HCoVs pseudovirus;

[0033] Figure 9 Diagram illustrating the mouse immunization strategy using a mixed aluminum adjuvant of recombinant protein THR1 / THR1S / THR1LS provided in this invention-embodiment.

[0034] Figure 10 Serum antibody levels and neutralizing antibody titers in mice immunized with a mixed aluminum adjuvant of recombinant protein THR1 / THR1S / THR1LS provided in this embodiment of the invention.

[0035] Figure 11Diagram illustrating the mouse immunization strategy using a mixture of recombinant proteins THR1 and THR1LS with CF501 adjuvant provided in this invention-embolization embodiment.

[0036] Figure 12 Serum antibody levels and neutralizing antibody titers in mice immunized with a recombinant protein THR1, THR1LS mixed with CF501 adjuvant provided in this embodiment of the invention.

[0037] Figure 13 The neutralizing effect of THR1LS mixed with CF501 adjuvant on HCoVs pseudovirus in mouse serum (Day 133) provided in this embodiment of the invention.

[0038] Figure 14 The recombinant protein THR1LS mixed with CF501 adjuvant provided in this embodiment of the invention can induce a stronger T cell response. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0040] The first aspect of this invention provides a set of recombinant proteins, wherein the recombinant proteins contain a heptahelical repeat domain 1 of SARS-CoV-2; or, contain a heptahelical repeat domain 1 of SARS-CoV-2, with a portion of the CH region amino acid sequence and / or the stem helix sequence of the S2 stem helix region added to the region. The recombinant proteins designed in this invention can be expressed via a prokaryotic expression system, possess a typical α-helix structure, and can effectively bind to the HR2P peptide of SARS-CoV-2 in vitro. Furthermore, the above-mentioned recombinant proteins can effectively neutralize SARS-CoV-2 pseudovirus infection in vitro. Among them, THR1LS exhibits the best activity and can also effectively neutralize SARS-CoV-2 Delta, Omicron mutant strains, and HCoVs (SARS-CoV, MERS-CoV, HCoV-NL63, and HCoV-229E) infection. This indicates that the recombinant proteins designed in this invention have the potential to be further developed into broad-spectrum anti-coronavirus protein drugs. Another aspect of this invention reveals that the above-mentioned recombinant proteins can be used as immunogens in subunit vaccine research. This invention involves immunizing Balb / c mice with THR1, THR1S, and THR1LS proteins respectively, after mixing them with aluminum adjuvant. Both THR1S and THR1LS elicited an immune response in vivo, producing neutralizing antibodies against SARS-CoV-2. The THR1LS-aluminum adjuvant mixture showed the best immunization effect. Furthermore, this invention discovered that immunization with a mixture of THR1LS and the novel adjuvant CF501 elicited a more potent immune response, including antibody and cellular responses, compared to aluminum adjuvant. Post-immunization, serum neutralizing antibodies effectively neutralized SARS-CoV-2 and its mutant strains, as well as various human coronavirus infections. The recombinant protein THR1LS designed in this invention has the potential to be developed into a broad-spectrum entry inhibitor and broad-spectrum vaccine immunogen candidate against highly pathogenic coronaviruses and common coronaviruses, to better address the COVID-19 pandemic and potential new highly pathogenic coronaviruses in the future.

[0041] Example 1: Design, construction, expression, and purification of recombinant proteins

[0042] Step 1: First, download the SARS-CoV-2S protein sequence from NCBI. Based on the reported HR1 region location in the literature, obtain the HR1 gene sequence. Then, construct the amino acid sequences of the recombinant proteins, naming them THR1, THR1S, and THR1LS, respectively.

[0043] THR1 comprises the HR1 and linker arms of SARS-CoV-2. It is achieved by linking three HR1 sequences in a forward-reverse-forward order using linkers (GKGNQ and GLIDG), and fusing them with a His tag at the C-terminus. Figure 1The linkage strategy shown is illustrated in SEQ ID NO: 1;

[0044] THR1S is based on THR1, with a stem helix sequence linked to the end of each HR1 sequence via GGSSG, and fused with a His tag at the C-terminus. Its amino acid sequence is shown in SEQ ID NO: 2.

[0045] THR1LS is a sequence in which a portion of the CH region is added to the end of each THR1 sequence, linked to a sequence containing the stemhelix region via GGSSG, and fused with a His tag at the C end. Its amino acid sequence is shown in SEQ ID NO: 3.

[0046] The sequence of the recombinant protein obtained above was given to BGI Genomics for codon optimization.

[0047] Step 2: The optimized gene sequence was synthesized by BGI Genomics and inserted into the Pet30(a) expression plasmid through two restriction enzyme sites, NdeI and XhoI, to obtain Pet30(a) expression plasmids for the three recombinant proteins (synthesized and provided by the company) (see [link to relevant documentation]). Figure 2 The sequence was confirmed to be correct by BGI Genomics.

[0048] Step 3: Expression and purification of the three recombinant proteins:

[0049] Step 3.1 The expression plasmids of each of the above recombinant proteins were transformed into E. coli BL21(DE3) strain, and single colonies were selected and activated overnight at 37°C in LB medium. The plasmids were then transferred to 1L of LB medium at a ratio of 1:100 and cultured. When the OD value reached 0.6, IPTG was added to a final concentration of 0.5 mmol / L, and protein expression was induced at 16°C with shaking at 220 rpm for 20 h.

[0050] Step 3.2: Collect bacterial cells by centrifuging the culture system obtained above at 6,000 rpm for 5 min. Resuspend the collected bacterial cells in PBS at pH 8.6, sonicate to disrupt, and then centrifuge at 10,000 rpm for 30 min. Bind the filtered supernatant to Ni Sepharose (GE) for 2 h. Wash impurities with buffer containing 50 mM imidazole (50 mM Tris-HCl, 500 mM NaCl, pH 8.6) and elute the target protein with buffer containing 250 mM imidazole.

[0051] Step 3.3: The protein was concentrated by ultrafiltration using an ultrafiltration tube, and the buffer was replaced with PBS. Protein integrity was then determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). (See [link to article]) Figure 3 ).

[0052] Step 3.4 Use the BCA kit to determine the protein concentration.

[0053] Test results are as follows Figure 3 As shown, all recombinant proteins were successfully expressed. The band for recombinant protein THR1 was approximately 26.7 kDa, the band for recombinant protein THR1S was approximately 31.4 kDa, and the band for recombinant protein THR1LS was approximately 39.2 kDa.

[0054]

[0055] Example 2: Detection of the secondary structure of recombinant proteins

[0056] The HR1 of SARS-CoV-2 has an α-helix structure. To detect whether the linker arm, CH region, and stemhelix region in the recombinant protein affect the secondary structure of HR1 itself, circular dichroism spectroscopy was used to detect the secondary structures of the three recombinant proteins. The specific steps are as follows:

[0057] Step 1. Dilute each recombinant protein to 15 μM with PBS at pH 7.4. Wash the quartz cuvettes used for detection, rinse twice with PBS, and then add 200 μL of PBS for baseline detection. Set the temperature to 25℃ and detect the circular dichroism signal in the PBS wavelength range of 200-260 nm.

[0058] Step 2. After washing the quartz cuvette with PBS, detect the circular dichroism signal of the recombinant protein sample under the same parameter settings.

[0059] Step 3. Convert the circular dichroism signal into a molar ellipticity curve using the number and molar concentration of amino acids in the sample (see...). Figure 4 ).

[0060] The analysis results of CD are as follows Figure 4 As shown, the recombinant proteins THR1, THR1S, and THR1LS exhibit distinct peaks at 208 nm and 220 nm, indicating that these proteins possess a typical α-helix structure.

[0061] Example 3: Detection of the binding of recombinant protein to SARS-CoV-2HR2P

[0062] Because HR1 can bind to SARS-CoV-2 HR2 to form 6-HB. Further ELISA was used to detect whether the recombinant protein obtained in the above examples could bind to the peptide HR2P derived from SARS-CoV-2 HR2. The sequence of HR2P is DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL. The specific steps are as follows:

[0063] 5 μg / ml of HR2P was coated onto ELISA plates. After blocking with PBST (containing 0.05% Tween-20 phosphate buffer) with 5% skim milk powder, the protein was serially diluted 3- or 4-fold and added to the ELISA plates, incubated at 37°C for 1 h. After washing 5 times with PBST, HRP-labeled 6×His monoclonal antibody (Proteintech) was added, and the plate was incubated at 37°C for 30 min. After washing 5 times with PBST, the substrate TMB / H2O2 was added for color development, and the reaction was terminated by adding H2SO4. OD was measured using a microplate reader. 450 , with OD 630 As a reference wavelength.

[0064] The analysis results of the HR2P peptide are as follows: Figure 5 As shown, recombinant proteins THR1, THR1S, and THR1LS can all bind to HR2P. Figure 5 ).

[0065] Example 4: Detection of the inhibitory activity of recombinant protein against SARS-CoV-2 pseudovirus

[0066] Since the above three proteins can bind to HR2P, a pseudovirus system was established to detect whether the three recombinant proteins could effectively inhibit SARS-CoV-2 infection. The specific steps are as follows:

[0067] Step 1. Pseudovirus Packaging: The constructed PcDNA3.1-SARS-CoV-2-S plasmid was co-transfected with the HIV backbone plasmid pNL4-3.Luc.RE- (owned by the applicant's laboratory, see the literature Xia S, Zhu Y, Liu M, et al. Fusion mechanism of 2019-nCoV and fusion inhibitors targeting HR1 domain in spike protein. Cell Mol Immunol, 2020.) into 293T cells (ATCC). After 48 hours, the cell supernatant was collected, which was the pseudovirus of SARS-CoV-2.

[0068] Step 2. Line 96-well plates with Caco 2 cells (ATCC), 1 × 10⁶ cells per well. 4 Cells were cultured overnight at 37°C with 5% CO2.

[0069] Step 3. Dilute each recombinant protein obtained above with MEM medium (Meilun) in 96-well plates by 3-fold or 4-fold serial dilution, then add an equal volume of SARS-CoV-2 pseudovirus, incubate at 37°C for 30 min, and then add the mixture to Caco2 cells. After 12 h, replace the cells with fresh MEM medium containing 10% FBS.

[0070] Step 4. 48 hours after medium change, add lysis buffer (Promega) to the cells and lyse for 30 minutes. Then, measure the relative abundance (Promega) of pseudoviruses in the cells using a luciferase assay system. Detect RLU values ​​using a microplate reader and calculate IC50 (see [link to relevant documentation]). Figure 6 ).

[0071] The results of the SARS-CoV-2 pseudovirus inhibition experiment showed that the recombinant protein THR1LS had the best inhibitory activity, with an IC50 of 0.17 μM. The antiviral activities of THR1 and THR1S were similar, with IC50s of 0.27 and 0.24 μM, respectively. Figure 6 ).

[0072] Example 5: Detection of the inhibitory activity of recombinant protein against SARS-CoV-2 mutant pseudoviruses

[0073] Besides the original SARS-CoV-2 strain, many different variants have emerged, especially the Omicron mutant. However, analysis revealed that the HR1 sequence of the mutant strain showed relatively little variation compared to the RBD sequence. To assess whether the recombinant protein could effectively inhibit the infection of the mutant strain, the inhibitory activities of THR1, THR1S, and THR1LS on the Delta mutant and some Omicron subvariants were further examined. The specific steps are as follows:

[0074] Step 1. Pseudovirus Packaging: The constructed PcDNA3.1-SARS-CoV-2Delta-S (or Omicron S) plasmid (see Xue S, Wang X, Wang L, et al. A novel cyclic gamma-AApeptide-based long-acting pan-coronavirus fusion inhibitor with potential oral bioavailability by targeting two sites in spike protein. Cell Discov, 2022; Liu Z, Zhou J, Wang X, et al. A pan-sarbecovirus vaccine based on RBD of SARS-CoV-2 original strain elicits potent neutralizing antibodies against XBB in non-human primates. Proc Natl Acad Sci USA, 2023) was co-transfected with the HIV backbone plasmid pNL4-3.Luc.RE- (obtained from the NIH AIDS reagent program) into 293T cells (ATCC). The cell supernatant was collected after 48 hours, which is the pseudovirus.

[0075] Step 2. Line 96-well plates with Caco 2 cells (ATCC), 1 × 10⁶ cells per well. 4 Cells were cultured overnight at 37°C with 5% CO2.

[0076] Step 3. The recombinant proteins obtained above were serially diluted 3-fold or 4-fold in 96-well plates using MEM medium (Meilun), and then the same volume of pseudovirus was added. After incubating at 37°C for 30 min, the mixture was added to Caco2 cells. After 12 h, the cells were replaced with fresh MEM medium containing 10% FBS.

[0077] Step 4. 48 hours after changing the medium, add lysis buffer (Promega) to the cells and lyse for 30 minutes. Then, measure the relative content (Promega) of pseudoviruses in the cells using a luciferase detection system. Detect the RLU value using an ELISA reader and calculate the IC50.

[0078] The results of the pseudovirus inhibition experiment were consistent with those of SARS-CoV-2. Compared with THR1 and THR1S, the recombinant protein THR1LS showed the best inhibitory activity against Delta, Omicron BA.2.75, BQ.1, XBB, and CH.1.1, with an IC50 value of [missing value]. 50 The concentrations were 0.17, 0.32, 0.20, 0.52, and 0.26 μM, respectively. Figure 7 Meanwhile, the above results indicate that THR1LS has a broad-spectrum inhibitory effect on SARS-CoV-2 variants.

[0079] Example 6: Detection of the inhibitory activity of recombinant protein against other coronavirus pseudoviruses

[0080] Furthermore, the inhibitory activity of the THR1LS protein against SARS-CoV, MERS-CoV, HCoV-229E, and HCoV-NL63 pseudoviruses was evaluated, and the specific steps are as follows:

[0081] Step 1. Pseudovirus Packaging: Plasmids encoding SARS-CoV, MERS-CoV, HCoV-NL63, and HCoV-229E S proteins (e.g., PcDNA3.1-SARS-CoV-S, Xia S, Yan L, Xu W, et al. A pan-coronavirus fusion inhibitor targeting the HR1 domain of human coronavirus spike. Sci Adv, 2019.) were co-transfected with the HIV backbone plasmid pNL4-3.Luc.RE into 293T cells (ATCC). The cell supernatant was collected after 48 hours, which was the pseudovirus.

[0082] Step 2. Line 96-well plates with Caco 2 cells (ATCC), 1 × 10⁶ cells per well. 4 Cells were cultured overnight at 37°C with 5% CO2.

[0083] Step 3. The THR1LS recombinant protein was serially diluted 3-fold or 4-fold in MEM medium (Meilun) in a 96-well plate, and then the same volume of each pseudovirus was added. After incubating at 37°C for 30 min, the mixture was added to Caco2 cells. After 12 h, the cells were replaced with fresh MEM medium containing 10% FBS.

[0084] Step 4. 48 hours after changing the medium, add lysis buffer (Promega) to the cells and lyse for 30 minutes. Then, measure the relative content (Promega) of pseudoviruses in the cells using a luciferase detection system. Detect the RLU value using an ELISA reader and calculate the IC50.

[0085] The results are as follows Figure 8 As shown, the recombinant protein THR1LS also exhibited good inhibitory activity against SARS-CoV, MERS-CoV, HCoV-229E, and HCoV-NL63, with an IC50 concentration of [missing information]. 50 The concentrations were 0.20, 0.19, 0.74, and 1.15 μM, respectively. These results indicate that THR1LS has a broad-spectrum inhibitory effect against human coronavirus infection. Example 7: Detection of antibody immune responses in mice immunized with aluminum adjuvant, using recombinant THR1, THR1S, and THR1LS proteins respectively.

[0086] Materials used in this embodiment: SPF female Balb / c mice (6 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Aluminum adjuvant was purchased from Thermo Scientific.

[0087] Step 1. Twenty Balb / c mice were randomly assigned to four groups of five each. THR1, THR1S, and THR1LS proteins were dissolved in PBS at a concentration of 0.4 mg / mL. Group 1 received an intramuscular immunization with 50 μL THR1 (20 μg) and 50 μL aluminum adjuvant; Group 2 received an intramuscular injection of 50 μL THR1S (20 μg) and 50 μL aluminum adjuvant; Group 3 received an intramuscular injection of 50 μL THR1LS (20 μg) and 50 μL aluminum adjuvant; and Group 4 received an intramuscular immunization with 100 μL of PBS. The specific immunization schedule is as follows: Figure 9 Mice were immunized three times on days 0, 28, and 108, and blood was collected from the infraorbital vein on days 42 and 122 to separate mouse serum. The mouse serum was inactivated at 56°C for half an hour and then used to detect antigen-specific binding antibodies and the neutralizing effect against coronavirus pseudoviruses.

[0088] Step 2. Detect the specific binding antibodies of THR1, THR1S, and THR1LS in the serum of mice in each group using ELISA. The specific ELISA steps are as follows: Coat 2 μg / ml of recombinant proteins THR1, THR1S, and THR1LS into ELISA plates and incubate overnight at 4°C. After blocking with PBST (containing 0.05% Tween-20 phosphate buffer) with 5% skim milk powder, serially dilute the serum 3 or 4 times with PBS in a 96-well U-plate and add it to the ELISA plate, incubating at 37°C for 1 h. After washing 5 times with PBST, add HRP-labeled rabbit anti-mouse Ig secondary antibody (Dako) and incubate at 37°C for 30 min. After washing 5 times with PBST, add the substrate TMB for color development and add H2SO4 to terminate the reaction. Detect OD using a microplate reader. 450 , with OD 630 As a reference wavelength.

[0089] Step 3. Detect the antibodies against stem-helix in the serum of mice in each group using ELISA. The specific steps are as follows: Coat 2 μg / ml of Streptavidin into an ELISA plate. After blocking with PBST (phosphate buffer containing 0.05% Tween-20) containing 5% skim milk powder, add 5 μg / ml of biotin-labeled stem-helix to the plate and incubate at 37°C for 1 h. After washing 5 times with PBST, serially dilute the serum 3-fold with PBS in a 96-well U-plate and add it to the ELISA plate, incubating at 37°C for 1 h. After washing 5 times with PBST, add HRP-labeled rabbit anti-mouse Ig secondary antibody (Dako) and incubate at 37°C for 30 min. After washing 5 times with PBST, add the substrate TMB / H2O2 for color development and add H2SO4 to terminate the reaction after 10 min. Detect OD using a microplate reader. 450 , with OD 630 As a reference wavelength.

[0090] The results of Ig antibody immune response in mouse serum at 122 days were as follows: Figure 10 As shown in Table 1 and AD, compared to the PBS control group, mice immunized with THR1, THR1S, and THR1LS three times all produced high titers of antigen-specific binding antibodies in vivo (Table 1). Furthermore, mice immunized with THR1S and THR1LS produced specific antibodies against the stem helix region (Table 2).

[0091] Table 1. Antigen-specific Ig titers in serum of mice 122 days after immunization

[0092]

[0093] Table 2. Stem Helix-specific Ig titers in serum of mice 122 days after immunization

[0094]

[0095]

[0096] Example 8: Detection of neutralizing activity of SARS-CoV-2 pseudovirus in mouse serum after immunization

[0097] The specific steps of the pseudovirus neutralization experiment on the serum of mice immunized in the above examples are as follows: Caco2 cells were seeded in 96-well plates, with 1×10 cells per well. 4Cells were cultured overnight at 37°C with 5% CO2. Inactivated mouse serum was serially diluted 3-fold with MEM in 96-well U-type plates, and an equal volume of SARS-CoV-2 pseudovirus was added. The plates were incubated at 37°C for 30 min. The serum-pseudovirus mixture was then added to Caco2 cells. Virus control wells were included (pseudovirus only). After 12 h of culture, the virus-serum mixture in the cells was replaced with fresh MEM medium containing 10% FBS. Forty-eight h after medium replacement, cells were lysed using a Promega Luciferase kit, and Luciferase activity was measured. RLU values ​​were measured using a microplate reader, and the 50% neutralizing titer (NT50) was calculated.

[0098] The calculation results are as follows Figure 10 As shown in Tables 3-4, there was no significant difference in the neutralizing effect of SARS-CoV-2 pseudovirus on day 42 in the serum of mice immunized with THR1, THR1S, and THR1LS, with neutralizing antibody titers of 55.04, 66.22, and 111.8, respectively. By day 122, the neutralizing titers of SARS-CoV-2 pseudovirus in the serum of mice immunized with THR1S and THR1LS were significantly increased, reaching 171.8 and 592.2, respectively. The highest neutralizing titer was observed after immunization with THR1LS, indicating that the HR1LS protein produced neutralizing antibodies against SARS-CoV-2 after immunization of mice.

[0099] Table 3. Neutralizing antibodies against SARS-CoV-2 pseudovirus in the serum of mice 42 days after immunization

[0100]

[0101] Table 4. Neutralizing antibodies against SARS-CoV-2 pseudovirus in the serum of mice 122 days after immunization

[0102]

[0103] Example 9 Detection of antibody immune response in mice immunized with a mixture of THR1 and THR1LS proteins and CF501 adjuvant.

[0104] Materials used in this embodiment: SPF female Balb / c mice (6 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Aluminum adjuvant was purchased from Thermo Scientific. CF501 adjuvant is a novel adjuvant discovered in our laboratory and provided by Fulgent, Inc., USA.

[0105] Step 1. Thirty SPF female Balb / c mice (6 weeks old) were randomly assigned to 5 groups, with 6 mice in each group. THR1 and THR1LS proteins were dissolved in PBS to a final concentration of 1 mg / mL. Group 1 was intramuscularly injected with 50 μL THR1 protein (50 μg) and 10 μL CF501 adjuvant (20 μg) diluted to 100 μL with PBS. Group 2 was intramuscularly injected with 50 μL THR1LS protein (50 μg) and 10 μL CF501 adjuvant (20 μg) diluted to 100 μL with PBS. Group 3 was injected with 50 μL THR1 protein (50 μg) and 50 μL aluminum adjuvant. Group 4 was injected with 50 μL THR1LS protein (50 μg) and 50 μL aluminum adjuvant. Group 5 was injected with 100 μL PBS. The specific immunization schedule is as follows: Figure 12 Mice were immunized three times on days 0, 21, and 42, and blood was collected from mice on days 35 and 56 for serum separation. Groups 2, 4, and 5 were further immunized a fourth and fifth time on days 70 and 119, respectively, and blood was collected from mice on days 84 and 133 for serum separation. The specific immunization schedule is as follows: Figure 11 Mouse serum was inactivated at 56°C for half an hour and then used to detect antigen-specific binding antibodies and their neutralizing effect on coronavirus pseudoviruses.

[0106] Step 2. Detect the specific binding antibodies of THR1 and THR1LS in the serum of mice in each group using ELISA. The specific ELISA steps are as follows: Coat 2 μg / ml of recombinant protein THR1 or THR1LS into ELISA plates and incubate overnight at 4°C. After blocking with PBST (containing 0.05% Tween-20 phosphate buffer) with 5% skim milk powder, serially dilute the serum 3 or 4 times and add it to the ELISA plate, incubating at 37°C for 1 h. After washing 5 times with PBST, add HRP-labeled rabbit anti-mouse Ig secondary antibody (Dako) and incubate at 37°C for 30 min. After washing 5 times with PBST, add the substrate TMB for color development and add H2SO4 to terminate the reaction. Detect OD using a microplate reader. 450 , with OD 630 As a reference wavelength.

[0107] Step 3. Detect the antibodies against stem-helix in the serum of mice in each group using ELISA. The specific steps are as follows: Coat 2 μg / ml of Streptavidin into an ELISA plate. After blocking with PBST (phosphate buffer containing 0.05% Tween-20) containing 5% skim milk powder, add 5 μg / ml of biotin-labeled stem-helix to the plate and incubate at 37°C for 1 h. After washing 5 times with PBST, serially dilute the serum 3-fold and add it to the ELISA plate, incubating at 37°C for 1 h. After washing 5 times with PBST, add HRP-labeled rabbit anti-mouse Ig secondary antibody (Dako) and incubate at 37°C for 30 min. After washing 5 times with PBST, add the substrate TMB / H2O2 for color development and add H2SO4 to terminate the reaction. Detect OD using a microplate reader. 450 , with OD 630 As a reference wavelength.

[0108] The results of the Ig immune response in mouse serum on day 56 are as follows: Figure 12 As shown in AB and Table 5, compared with the PBS control group, mice immunized with THR1 and THR1LS three times produced higher titers of antigen-specific binding antibodies in mice (Table 5).

[0109] Table 5. Antigen-specific Ig titers in mouse serum 56 days after immunization

[0110]

[0111] The results of Ig immune response in mouse serum on days 84 and 133 are as follows: Figure 12 As shown in CE and Tables 6-7, compared to the PBS control group, the fourth immunization with THR1LS / CF501 and THR1LS / Alum resulted in no significant increase in THR1LS-specific antibody levels compared to the third immunization (Table 6). After the fifth immunization, the THR1LS-specific antibody titer in the serum of mice in the THR1LS / CF501 group was slightly increased, while the antibody titer in the THR1LS / Alum group was significantly increased, approaching that of the THR1LS / CF501 group (Table 7). These results indicate that multiple immunizations can increase the level of specific antibodies. Antibodies against stem helix were produced in the serum of mice immunized after the fifth immunization (Table 8).

[0112] Table 6. Antigen-specific Ig titers in serum of mice 84 days after immunization

[0113]

[0114] Table 7. Antigen-specific Ig titers in mouse serum 133 days after immunization

[0115]

[0116] Table 8. Stem Helix-specific Ig titers in the serum of mice 133 days after immunization

[0117]

[0118] Example 10: Detection of neutralizing activity of SARS-CoV-2 pseudovirus against mice immunized with a mixture of THR1 and THR1LS proteins and CF501 adjuvant.

[0119] Step 1. Pseudovirus packaging: The plasmid encoding the SARS-CoV-2S protein was co-transfected with the HIV backbone plasmid pNL4-3.Luc.RE (obtained from the NIH AIDS reagent program) into 293T cells (ATCC). The cell supernatant was collected after 48 hours, which was the pseudovirus.

[0120] Step 2. Line 96-well plates with Caco 2 cells (ATCC), 1 × 10⁶ cells per well. 4 Cells were cultured overnight at 37°C with 5% CO2.

[0121] Step 3. Dilute the immunized mouse serum 3- or 4-fold with MEM medium (Meilun), add an equal volume of pseudovirus, incubate at 37°C for 30 min, and then add the mixture to Caco2 cells. After 12 h, replace the cells with fresh MEM medium containing 10% FBS.

[0122] Step 4. 48 hours after changing the medium, add lysis buffer (Promega) to the cells and lyse for 30 minutes. Then, measure the relative content (Promega) of pseudoviruses in the cells using a luciferase detection system. Detect the RLU value using an ELISA reader and calculate the IC50.

[0123] The results are as follows Figure 12As shown in FH and Tables 9-11, on day 56 after three immunizations, mice in the THR1 / CF501, THR1LS / CF501, THR1 / Alum, and THR1LS / Alum groups produced neutralizing antibodies against SARS-CoV-2 pseudovirus in their serum, with 50% neutralizing titers of 197.9, 498.5, 106.0, and 212.1, respectively. Following booster immunizations, on day 84, the neutralizing antibody titers against SARS-CoV-2 in the serum of the THR1LS / CF501 and THR1LS / Alum groups reached 535.2 and 180.4, respectively. However, by day 133, the neutralizing antibody titer against SARS-CoV-2 in the serum of the THR1LS / CF501 group reached 986.6, while the neutralizing antibody titer in the serum of the THR1LS / Alum group was only 226.4. These results indicate that immunization with a combination of THR1LS protein and the novel adjuvant CF501 can induce potent neutralizing antibodies against SARS-CoV-2 in mice.

[0124] Table 9. Neutralizing antibodies against SARS-CoV-2 pseudovirus in the serum of mice 56 days after immunization

[0125]

[0126] Table 10. Neutralizing antibodies against SARS-CoV-2 pseudovirus in the serum of mice 84 days after immunization

[0127]

[0128] Table 11. Neutralizing antibodies against SARS-CoV-2 pseudovirus in the serum of mice 133 days after immunization

[0129]

[0130] Example 11: Detection of neutralizing activity of serum from mice immunized with a mixture of THR1 and THR1LS proteins and CF501 adjuvant against other HCoVs pseudoviruses.

[0131] Step 1. Pseudovirus packaging: Plasmids encoding SARS-CoV, MERS-CoV, HCoV-NL63, and HCoV-229ES proteins were co-transfected with HIV backbone plasmid pNL4-3.Luc.RE- into 293T cells (ATCC). Cell supernatant was collected after 48 hours, which was the pseudovirus.

[0132] Step 2. Line 96-well plates with Caco 2 cells (ATCC), 1 × 10⁶ cells per well. 4 Cells were cultured overnight at 37°C with 5% CO2.

[0133] Step 3. Dilute the immunized mouse serum 3- or 4-fold with MEM medium (Meilun), add an equal volume of pseudovirus, incubate at 37°C for 30 min, then add the mixture to Caco2 cells. After 12 h, replace the cells with fresh MEM medium containing 10% FBS.

[0134] Step 4. 48 hours after changing the medium, add lysis buffer (Promega) to the cells and lyse for 30 minutes. Then, measure the relative content (Promega) of pseudoviruses in the cells using a luciferase detection system. Detect the RLU value using an ELISA reader and calculate the IC50.

[0135] The results are as follows Figure 13 As shown in Table 12, on day 133, the serum neutralizing antibodies against pseudoviruses of SARS-CoV, MERS-CoV, HCoV-NL63, and HCoV-229E in the THR1LS / CF501 group mice were 503.4, 232.1, 660.5, and 427.1, respectively. These results indicate that immunization with THR1LS mixed with CF501 adjuvant can simultaneously induce neutralizing antibodies against SARS-CoV-2, SARS-CoV, MERS-CoV, and HCoV-229E in mice.

[0136] Table 12. Serum neutralizing pseudovirus titers in mice immunized with THR1LS / CF501 group 133 days prior

[0137]

[0138] Example 12: Detection of IFN-γ in spleen cells of mice immunized with a mixture of THR1 and THR1LS proteins and CF501 adjuvant for 133 days.

[0139] Furthermore, the inventors used ELISPOT to detect IFN-γ levels in mice to assess whether a T cell response was induced. The specific steps are as follows:

[0140] Mice in the THR1LS / CF501, THR1LS / Alum, and PBS groups were euthanized after blood collection, and their spleens were harvested. The spleens were ground to prepare a spleen cell suspension. Red blood cells in the cell suspension were lysed using red blood cell lysis buffer. After multiple washes with PBS, the cells were counted. The ELISPOT plates were aseptically removed, and 10% FBS solution of 1640 was added and the plates were blocked at 37°C for 2 hours. Then, 1×10⁶ cells were added to each well of the plate. 6Cells were incubated with a 2 μg / mL SARS-CoV-2HR1 scanning peptide library (Shanghai Jier Biochemical Co., Ltd.) at 37°C for 48 h, and the cell supernatant was discarded. Cells were washed five times with PBS. Biotinylated IFN-γ antibody was added, and incubation was performed at room temperature for 2 h. After washing five times with PBS, enzyme-linked avidin was added, and incubation was performed at room temperature for 1 h. After washing five times with PBS, substrate was added for color development. Once clear spots appeared, the liquid was discarded and the cells were rinsed to terminate the process. Spot counts were performed using an ELSPOT counting instrument.

[0141] The results are as follows Figure 14 As shown, after five immunizations, the THR1LS / CF501 group exhibited the highest IFN-γ expression level in the spleen cells of mice, while the THR1LS / Alum group failed to induce a significant cellular immune response. These results indicate that immunization with the THR1LS / CF501 adjuvant not only stimulates the production of high levels of neutralizing antibodies but also elicits a strong cellular immune response in mice.

[0142] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant protein capable of broadly inhibiting human coronavirus infection, characterized in that, The amino acid sequence of the recombinant protein is shown in SEQ ID NO:

3.

2. A vector capable of broadly inhibiting human coronavirus infection, characterized in that: The vector contains the recombinant protein as described in claim 1.

3. A host cell capable of broadly inhibiting human coronavirus infection, characterized in that, The carrier as described in claim 2.

4. The use of the recombinant protein of claim 1, which can broadly inhibit human coronavirus infection, in the preparation of a medicament for the prevention and / or treatment of human coronaviruses; wherein, Human coronaviruses are SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, or HCoV-NL63.

5. The application of the recombinant protein of claim 1, which can broadly inhibit human coronavirus infection, in the preparation of an immunogen for use in a subunit vaccine; wherein, Human coronaviruses are SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, or HCoV-NL63.

6. The application according to claim 5, characterized in that: The recombinant protein was used as an immunogen, and the subunit vaccine was prepared by combining it with an adjuvant.

7. The use of the recombinant protein of claim 1, which can broadly inhibit human coronavirus infection, in the preparation of a human coronavirus detection reagent; wherein, Human coronaviruses are SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, or HCoV-NL63.

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