WO2022052984A1 - Universal sars-cov-2 vaccine and preparation method thereof - Google Patents

Universal sars-cov-2 vaccine and preparation method thereof Download PDF

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WO2022052984A1
WO2022052984A1 PCT/CN2021/117415 CN2021117415W WO2022052984A1 WO 2022052984 A1 WO2022052984 A1 WO 2022052984A1 CN 2021117415 W CN2021117415 W CN 2021117415W WO 2022052984 A1 WO2022052984 A1 WO 2022052984A1
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protein
peptide fragment
fusion protein
seq
vaccine
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PCT/CN2021/117415
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French (fr)
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Lung-Ji Chang
Cheng-Wei Chang
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Beijing Meikang Geno-Immune Biotechnology Co., Ltd.
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Definitions

  • the present disclosure belongs to the field of biological medicine, and relates to a universal SARS-CoV-2 vaccine and a preparation method thereof.
  • Spike protein S protein
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the S protein plays a same role in SARS and MERS viruses. Therefore, the S protein determines the host range and specificity of the virus, and it is an important target for antibody binding in the host, and is also a key target for the development of vaccines, therapeutic antibodies, and diagnostic reagents.
  • the virus causing COVID-19 is a newly emerged coronavirus variant.
  • the spike protein of this virus binds to ACE2 receptor with a stronger force than SARS-CoV, which explains the strong infection ability of SARS-CoV-2.
  • SARS-CoV-2 stimulates the innate immune system in an infected individual, causing the release of a large amount of cytokines in the body and causing a cytokine storm and an acute inflammatory response, thereby leading to acute respiratory distress syndrome (ARDS) and multiple organ failure.
  • ARDS acute respiratory distress syndrome
  • a great challenge in vaccine development is to find a suitable animal model.
  • non-human primate infection models are mainly used to verify the safety and efficacy of the developed vaccines, but the cost is extremely high and it takes a long time.
  • the whole virus antigen vaccine is based on the traditional inactivated virus or attenuated virus as antigen source, which has the problems of low safety and high cost;
  • the viral specific antigen vaccine is based on synthetic S protein as the antigen, while artificially synthesized S protein is mainly expressed by bacteria, yeast, cells or viral vectors, which has the problems of a length process of manufacture, single valence, easy escape of the virus, short validity period, and high cost;
  • DNA and mRNA vaccines need to enter cells and be converted into proteins to exert immune effects, and the low immunogenicity issue has been the bottleneck that the technology still cannot overcome at present time.
  • the present disclosure provides a universal SARS-CoV-2 vaccine composition and a preparation method thereof.
  • the vaccine uses immunologically modified antigen-presenting cells expressing key structural proteins and a non-structural protein of SARS-CoV-2 to induce the body to produce an immune response and form an immunological memory for the prevention and treatment of SARS-CoV-2 infection.
  • the present disclosure provides a protein or a combination of proteins for a SARS-CoV-2 vaccine.
  • the protein or combination of proteins includes any one or a combination of at least two of Spike protein (Sprotein) , Envelope protein (E protein) , Membrane protein (M protein) , Nucleocapsid protein (N protein) or ORF1a poly-protease (P protein) of SARS-CoV-2.
  • the protein or the combination of proteins for a vaccine containing key structural proteins and a non-structural protein of SARS-CoV-2 can stimulate the body to produce an immune response and form an immunological memory, with a broad-spectrum immunostimulatory effect.
  • the present disclosure provides a fusion protein for a SARS-CoV-2 vaccine.
  • the fusion protein includes an S protein signal peptide and an S protein receptor binding domain of SARS-CoV-2.
  • the S protein receptor binding domain includes the amino acid sequence as shown in SEQ ID NO: 1.
  • the S protein signal peptide includes the amino acid sequence as shown in SEQ ID NO: 2.
  • the fusion protein contains an S protein receptor binding domain (RBD) , which is the most important structure for SARS-CoV-2 to invade human cells.
  • RBD S protein receptor binding domain
  • the fusion protein helps stimulate the body to produce anti-SARS-CoV-2 antibodies having high specificity.
  • the fusion protein contains only the S protein signal peptide and the S protein receptor binding domain, and a short peptide at the carboxyl terminal is modified.
  • a designed fusion protein S includes the amino acid sequence as shown in SEQ ID NO: 3.
  • the fusion protein of the present disclosure includes the S protein signal peptide and the S protein receptor binding domain, and further includes a partial peptide fragment of the E protein and a partial peptide fragment of the M protein.
  • the partial peptide fragment of the E protein includes an amino terminal peptide fragment of the E protein.
  • the partial peptide fragment of the M protein includes a carboxyl terminal peptide fragment of the M protein.
  • the amino terminal peptide fragment of the E protein includes the amino acid sequence as shown in SEQ ID NO: 4.
  • the carboxyl terminal peptide fragment of the M protein includes the amino acid sequence as shown in SEQ ID NO: 5.
  • the fusion protein consists of an S protein signal peptide, an S protein receptor binding domain, an amino terminal peptide fragment of the E protein and a peptide fragment of the M protein.
  • a designed fusion protein SEM comprises the amino acid sequence as shown in SEQ ID NO: 6.
  • the fusion protein of the present disclosure includes the S protein signal peptide, the S protein receptor binding domain, the amino terminal peptide fragment of the E protein, and the peptide fragment of the M protein, and further includes a partial peptide fragment of the N protein and a partial peptide fragment of the ORF1a poly-protease
  • the partial peptide fragment of the N protein includes a helix-turn domain of the N protein.
  • the partial peptide fragment of the ORF1a poly-protease includes a functional domain peptide fragment of the ORF1a poly-protease.
  • the helix-turn domain of the N protein includes the amino acid sequence as shown in SEQ ID NO: 7.
  • the peptide fragment of the ORF1a poly-protease includes the amino acid sequence as shown in SEQ ID NO: 8.
  • the fusion protein consists of an S protein signal peptide, an S protein receptor binding domain, an amino terminal peptide fragment of the E protein, a peptide fragment of the M protein, a helix-turn domain of the N protein and a functional domain peptide fragment of the ORF1a poly-protease.
  • a designed fusion protein SEMNP comprises the amino acid sequence as shown in SEQ ID NO: 9.
  • a fusion protein containing several key proteins of SARS-CoV-2 is constructed based on the nucleic acid sequence of SARS-CoV-2.
  • the fusion protein produces polypeptide fragments after enzymolysis, which, after presented by antigen-presenting cells, stimulate B cells and T cells in the body to become mature B cells and T cells that are specifically directed against viral antigens, and stimulate further proliferation of activated immune cells, to achieve prevention and controlling of infections.
  • the present disclosure provides a coding gene of the fusion protein in the second aspect.
  • the coding gene comprises a nucleic acid molecule encoding an S protein signal peptide and a nucleic acid molecule encoding an S protein receptor binding domain.
  • the coding gene of the fusion protein S comprises the nucleic acid sequence as shown in SEQ ID NO: 10.
  • the coding gene further includes a nucleic acid molecule encoding the amino terminal peptide fragment of the E protein and a nucleic acid molecule encoding the peptide fragment of the M protein.
  • the encoding gene of the fusion protein SEM includes the nucleic acid sequence as shown in SEQ ID NO: 11.
  • the coding gene further includes a nucleic acid molecule encoding the helix-turn domain of the N protein and a nucleic acid molecule encoding the functional domain peptide fragment of the ORF1a poly-protease.
  • the coding gene of the fusion protein SEMNP includes the nucleic acid sequence as shown in SEQ ID NO: 12.
  • the present disclosure provides an expression vector, comprising the coding gene in the third aspect.
  • the expression vector includes any one of a lentiviral vector, a retroviral vector an adeno-associated viral vector or an adenovirus vector, preferably a lentiviral vector.
  • the present disclosure provides a recombinant lentivirus, which is obtained by packaging of the lentiviral vector in the fourth aspect and a helper plasmid in mammalian cells.
  • the present disclosure provides an antigen-presenting cell that expresses the fusion protein for a SARS-CoV-2 vaccine as described above, wherein the antigen-presenting cell further expresses a cytokine or cytokines; wherein the cytokine or cytokines include any one or a combination of at least two of Calnexin (CNX) , GM-CSF, CD80, CD86, Flt3-L, IL-2 or IL-12.
  • CNX Calnexin
  • artificial antigen presenting cells are constructed by introducing cytokines such as Calnexin (CNX) , GM-CSF, CD80, CD86, Flt3-L, IL-2 or IL-12 into myeloid-derived antigen-presenting cells using lentiviral technology.
  • cytokines such as Calnexin (CNX) , GM-CSF, CD80, CD86, Flt3-L, IL-2 or IL-12
  • the present disclosure provides a universal SARS-CoV-2 vaccine, which is an antigen-presenting cell expressing the fusion protein in the second aspect.
  • the present disclosure adopts antigen-presenting cells expressing key proteins of SARS-CoV-2 to construct cellular vaccines, which is conducive to solving the safety and high cost problems of individualized cellular infusion, and can accelerate the preparation of cellular vaccines and rapidly realize large-scale industrial production. Moreover, the present disclosure adopts mitomycin-C and gamma-ray irradiation treatment to further strengthen the safety factor, which solves the risks in preparation and safety control of vaccine cells. In addition, the present disclosure strictly regulates and controls the preparation process, and reduces costs.
  • the antigen-presenting cell expresses a cytokine or cytokines.
  • the antigen-presenting cell expresses any one or a combination of at least two of Calnexin (CNX) , GM-CSF, CD80, CD86, Flt3-L, IL-2 or IL-12.
  • CNX Calnexin
  • the prophylactic and therapeutic process of the vaccine of the present disclosure is designed to mimic the natural immune system of the body.
  • the structural and non-structural proteins of SARS-CoV-2 are exposed to stimulate the body to produce an immune response and form an immunological memory, and to produce specific antibodies against the SARS-CoV-2 proteins.
  • a stronger immunological memory may be rapidly induced when re-infected with the virus.
  • the coding gene in the third aspect is integrated into the genome of the antigen-presenting cell.
  • the antigen-presenting cell includes the expression vector in the fourth aspect and/or the recombinant lentivirus in the fifth aspect.
  • the present disclosure provides a method for preparing the vaccine in the seventh aspect, comprising:
  • a cytokine or cytokines including any one or a combination of at least two of Calnexin, GM-CSF, CD80, CD86, Flt3-L, IL-2 or IL-12
  • a minigene encoding a fusion protein composed of several structural proteins and a non-structural protein of SARS-CoV-2 is synthesized, and transduced into immunologically modified artificial antigen-presenting cells (aAPC) through a lentiviral system.
  • aAPC immunologically modified artificial antigen-presenting cells
  • the constructed cellular vaccine has a broad-spectrum immunostimulatory effect, effectively stimulates cellular immune responses and production of corresponding antibodies in the body, and can be produced on a large scale.
  • the inactivation treatment includes mitomycin-C treatment and/or gamma-ray irradiation.
  • a concentrated minigene-containing lentiviral vector LV-SEMNP, LV-SEM or LV-S is used to transduce aAPC to establish a persistent artificially synthesized antigen-presenting cell line (COVID-19-: aAPC-SEMNP, aAPC-SEM or aAPC-S) , which is inactivated by mitomycin-C and/or gamma-ray irradiation to further enhance the vaccine safety factor, to prepare a universal COVID-19/aAPC vaccine.
  • COVID-19- a persistent artificially synthesized antigen-presenting cell line
  • the present disclosure has the following beneficial effects.
  • a fusion protein containing structural proteins S protein, E protein, M protein, and N protein, and a non-structural protein ORF1a poly-protease of SARS-CoV-2, and a coding minigene thereof are constructed.
  • the minigene is transduced into immunologically modified artificial antigen-presenting cells using a lentiviral system.
  • the fusion protein is enzymatically digested in the body into several polypeptide fragments, which, after presented by antigen-presenting cells, can stimulate B cells and T cells in the body to become mature B cells and T cells that are specifically directed against viral antigens, and stimulate further proliferation of activated immune cells, to achieve prevention and controlling of infections.
  • the prophylactic and therapeutic process of the vaccine of the present disclosure is designed to mimic the natural immune system of the body.
  • the structural and non-structural proteins of SARS-CoV-2 are exposed to stimulate the body to produce an immune response and form an immunological memory, and to produce specific antibodies against the SARS-CoV-2 proteins.
  • a stronger immunological memory may be rapidly induced when re-infected with the virus, with a broad-spectrum immunostimulatory effect.
  • the present disclosure adopts a preferred antigen-presenting cell strain expressing key proteins of SARS-CoV-2 to construct cellular vaccines. It is conducive to solving the safety and high cost problems of individualized cellular infusion, accelerating the preparation time of cellular vaccines, rapidly realizing large-scale industrial production.
  • mitomycin C and/or gamma-radiation are used for inactivation treatment, which further improve (s) the safety factor of the vaccine, solve (s) the risk of vaccine cell preparation and safety control, and strictly regulate (s) and control (s) the preparation process, and the costs are reduced.
  • Figure 1 shows a schematic diagram of the preparation process of a universal SARS-CoV-2 vaccine.
  • Figure 2 shows a lentiviral vector system containing a minigene.
  • Figure 3 is a diagram showing the phenotype analysis of universal artificial vaccine cells (aAPCs) modified by lentivirus; wherein, Figure 3A shows a flow cytometric analysis comparing the expression of HLA antigens (-ABC, and -DP, DR, DQ) on cell surface between dendritic cells and aAPCs; and Figure 3B shows a flow cytometric analysis comparing the expression of CD80/CD86 and FL on cell surface and intracellular GM-CSF and IL-2 between unmodified aAPCs and lentivirus-modified aAPCs.
  • aAPCs universal artificial vaccine cells
  • Figure 4 shows the expression of viral proteins in aAPCs modified by lentiviruses containing different minigenes (aAPC-S, -SEM, -SEMNP) .
  • Figure 5A shows the amplification of T cells from Donor 1 after aAPC-SEMNP stimulation
  • Figure 5B shows the amplification of T cells from Donor 2 after aAPC-SEMNP stimulation
  • Figure 5C shows the antigenic phenotypes of CD4+ T cells after stimulation of T cells from different donors with aAPC-SEMNP
  • Figure 5D shows the antigenic phenotypes of CD8+ T cells after stimulation of T cells from different donors with aAPC-SEMNP.
  • Figure 6A shows results of an effector response assay where the universal SARS-CoV-2 vaccine aAPC-SEMNP was used to stimulate T cells from Donor 1;
  • Figure 6B shows results of an effector response test where the universal SARS-CoV-2 vaccine aAPC-SEMNP is used to stimulate T cells from Donor 2;
  • Figure 6C shows results of an effector response test where the universal SARS-CoV-2 vaccine aAPC-SEMNP is used to stimulate T cells from Donor 3; and
  • Figure 6D shows results of an effector response test where the universal SARS-CoV-2 vaccine aAPC-SEMNP is used to stimulate T cells from Donor 4.
  • SARS-CoV-2 mainly expresses four structural proteins, i.e., surface glycoprotein (Sprotein) , envelop protein (E protein) , membrane protein (M protein) , and nucleocapsid phosphoprotein (N protein) , and a functional protein, polyprotein cleavage protease (ORF1a poly-protease) .
  • Sprotein surface glycoprotein
  • E protein envelop protein
  • M protein membrane protein
  • N protein nucleocapsid phosphoprotein
  • ORF1a poly-protease polyprotein cleavage protease
  • the S protein signal peptide (SEQ ID NO: 2) and S protein receptor binding domain (SEQ ID NO: 1) were selected as the first part of the fusion protein; the amino-terminal 29 aa peptide fragment (SEQ ID NO: 4) of the E protein was selected as the second part of the fusion protein; the carboxyl-terminal 58 aa peptide fragment (SEQ ID NO: 5) of the M protein was selected as the third part of the fusion protein; the intermediate 162 aa helix-turn domain (SEQ ID NO: 7) of the N protein was selected as the fourth part of the fusion protein; and the carboxyl-terminal 123 aa peptide (SEQ ID NO: 8) of the ORF1a poly-protease was selected as the fifth part of the fusion protein.
  • the fusion protein SEMNP was truncated. Most peptides of the helix-turn domain of the N protein and all peptides of the carboxyl-terminal of the ORF1a poly-protease were deleted. A fusion protein SEM (SEQ ID NO: 6) with a length of 315 aa was obtained.
  • the fusion protein SEMNP was further truncated. Only the S protein signal peptide and the S protein receptor binding domain were retained, and a short peptide fragment at the carboxyl-terminal was modified. A fusion protein S (SEQ ID NO: 3) with a length of 228 aa was obtained.
  • the amino acid sequence of the fusion protein SEMNP was converted to a corresponding nucleic acid sequence, which was optimized to obtain the coding gene of the fusion protein SEMNP as shown in SEQ ID NO: 12.
  • Partial NP sequence at the 3' end of the coding gene of SEMNP was deleted (nt. 4990-5840, NheI-SpeI deletion) , to obtain the coding gene of fusion protein SEM as shown in SEQ ID NO: 11.
  • Partial EMNP sequence at the 3' end of the coding gene of SEMNP was deleted (nt. 4680-5850, BstBI-BstBI deletion) , to obtain the coding gene of fusion protein S as shown in SEQ ID NO: 10.
  • the coding genes of the fusion proteins S, SEM and SEMNP as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively, were artificially synthesized.
  • the above nucleic acid molecules were double digested, incubated in a 37 °C water bath for 30 min.
  • the digested products were subjected to DNA electrophoresis on a 1.5%agarose gel.
  • the digested fragments were purified and recovered using an agarose gel kit.
  • the digested fragments were inserted into the linearized lentiviral vector TYF to construct lentiviral vectors LV-S, LV-SEM and LV-SEMNP containing minigenes.
  • aAPCs were prepared using human-derived myeloid-associated cells: myelogenous leukemia AML, KG-1 or K562, for examples.
  • Cells were modified with lentivirus to express CNX, GM-CSF, CD80, CD86, Flt-L, IL-2 and IL-12 in order to enhance the antigen presentation function of aAPCs.
  • Cell strains expressing high levels of cytokines were screened as vaccine strains.
  • Peripheral blood mononuclear cells were plated in culture dishes and cultured for 2 h in AIM-V medium (Gibco-BRL, CA, USA) ;
  • Nonadherent cells were gently removed, and the culture was continued for 24 h using AIM-V medium supplemented with 50 ng/mL GM-CSF (Biosource, CA, USA) and 25 ng/mL IL-4 (Biosource, CA, USA) .
  • AIM-V medium containing 20 ng/mL IFN- ⁇ (Gentaur) , 50 ng/mL TNF- ⁇ (R&D systems, MN, USA) , 10 ng/mL IL-1 ⁇ (R&D systems, MN, USA) , 10 ng/mL IL-6 (R&D systems) and 1 ⁇ M PGE2 (Sigma-Aldrich, MO, USA) , and cultured for 24 h to generate mature dendritic cells.
  • the screened aAPC cell strains had been modified with lentivirus to express cytokines. Phenotypic analysis was performed by flow cytometry as follows.
  • aAPCs were stained with antibodies PE-anti-HLA-ABC, FITC-anti-HLA-DR, DP, DQ, FITC-anti-CD80, and FITC-anti-CD86, kept at 4 °C for 30 min, and washed twice with PBS containing 1%FBS.
  • Intracellular staining analysis was performed according to the literature. In brief, cells were treated with Monensin (Sigma) for 5 h, fixed and permeablized, followed by the addition of antibodies (BD Bioscience) against GM-CSF, IL-2, and Flt3-ligand (FL) , stained with FIX/PERM and PERM/Wash solution (BD) , and then loaded onto flow cytometry. The percentage of different antigen presenting cell subpopulations was analyzed with Flowjo software. The results are shown in Figure 3.
  • aAPCs did not express HLA-ABC and -DP, -DR, -DQ, thus the immune rejection response was reduced.
  • unmodified APCs expressed low levels of CD80 and CD86, and did not express GM-CSF and IL-2; modified aAPCs expressed high levels of CD80, CD86, GM-CSF and IL-2; unmodified APCs did not express Flt-L (FL) ; and modified aAPCs expressed a high level of FL.
  • aAPCs were transduced with concentrated minigene-containing lentiviral vectors LV-SEMNP, LV-SEM or LV-S to establish persistent artificially synthesized antigen-presenting cell strains (COVID-19-: aAPC-SEMNP, aAPC-SEM and aAPC-S) , which were then inactivated through mitomycin-C and/or gamma-ray irradiation, to produce universal SARS-CoV-2 vaccines COVID-19/aAPC-SEMNP, COVID-19/aAPC-SEM and COVID-19/aAPC-S.
  • Cell samples were lysed for 1 h with RIPA lysis solution.
  • the protein concentration was determined by a BCA method.
  • the protein concentration and the sample loading amount of the sample solution were calculated.
  • the protein solution, 6 ⁇ SDS buffer and RIPA lysis solution were proportionally prepared into a total volume of 25 ⁇ L protein loading solution and incubated at 100 °C for 5 min.
  • 10%upper and lower gels were prepared according to the Gel Fast Preparation Kit.
  • the stacking gel was run at a voltage of 70 V, and the separating gel was run at a voltage of 120 V.
  • a PVDF membrane was cut out and soaked in absolute ethanol. Filter paper used for transfer was soaked in the transfer buffer. A semi-dry transfer device was used to perform transfer.
  • the membrane was blocked in 5%BSA for 1 h at room temperature.
  • Primary antibodies of S protein, N protein, P protein and M protein were added after blocking and then incubated overnight in a refrigerator at 4°C.
  • aAPC-S cells expressed a high level of S protein (26 kD)
  • aAPC-SEMNP cell strains expressed a high level of SMENP fusion protein (66 kD)
  • aAPC-SEM cells expressed SEM fusion protein (35 kD) .
  • cytotoxic T cells were analyzed based on CD27, CD28, CD45RA, CD4 and CD8 surface staining by flow cytometry. Results are shown in Figures 5A, 5B, 5C and 5D, wherein Tcm refers to central memory T cells (CD27 + , CD28 + , CD45RA - ) , Tem refers to effector-memory T cells (CD27 +/- , CD28 +/- , CD45RA - ) , and Teff refers to terminal effector T cells (CD27 - , CD28 - , CD45RA - ) .
  • Tcm refers to central memory T cells (CD27 + , CD28 + , CD45RA - )
  • Tem refers to effector-memory T cells (CD27 +/- , CD28 +/- , CD45RA - )
  • Teff refers to terminal effector T cells (CD27 - , CD28 - , CD45RA
  • T cells from two different donors expanded after 2-3 weeks of stimulation.
  • the expanded T cells were analyzed through surface antigen staining, and it showed the expression of antigen phenotypes of memory and effector T cells.
  • Engineered immune effector cytotoxic T cells (EIE CTLs) generated after stimulation with COVID-19/aAPC vaccine were added with relevant antigenic peptides (a SEMNP mixture, S, E, M, N, P, and HIV peptides) , or artificial vaccine cells aAP-SEMNP, aAPC-SEM, or aAPC-S, and stimulated overnight. The strength of the antigen-specific response was detected using ELISPOT immunoassay.
  • EIE EIE
  • PBMCs peripheral blood mononuclear cells
  • the universal SARS-CoV-2 vaccine of the present disclosure mimics the natural immune system of the body.
  • the several polypeptide fragments formed from the fusion protein are delivered by antigen-presenting cells and stimulate the body to generate an immune response and form an immunological memory.

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Abstract

A universal SARS-CoV-2 vaccine and a preparation method thereof. The vaccine is an artificial antigen-presenting cell expressing a fusion protein of SARS-CoV-2 structural proteins S protein, E protein, M protein, N protein and non-structural protein ORF1a poly-protease P. The vaccine mimics the natural immune system of the body, and in the presence of cytokines, multiple polypeptide fragments formed by the fusion protein are presented by the antigen-presenting cells, which can stimulate the body to generate immune responses and form immunological memory, with a broad-spectrum immunostimulatory effect; the vaccine can realize rapid large-scale industrial production, with the advantages of high safety and low cost.

Description

UNIVERSAL SARS-COV-2 VACCINE AND PREPARATION METHOD THEREOF TECHNICAL FIELD
The present disclosure belongs to the field of biological medicine, and relates to a universal SARS-CoV-2 vaccine and a preparation method thereof.
BACKGROUND
Binding of Spike protein (Sprotein) to ACE2 receptor on the cell surface is key for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to infect human cells. The S protein plays a same role in SARS and MERS viruses. Therefore, the S protein determines the host range and specificity of the virus, and it is an important target for antibody binding in the host, and is also a key target for the development of vaccines, therapeutic antibodies, and diagnostic reagents.
The virus causing COVID-19 is a newly emerged coronavirus variant. The spike protein of this virus binds to ACE2 receptor with a stronger force than SARS-CoV, which explains the strong infection ability of SARS-CoV-2. SARS-CoV-2 stimulates the innate immune system in an infected individual, causing the release of a large amount of cytokines in the body and causing a cytokine storm and an acute inflammatory response, thereby leading to acute respiratory distress syndrome (ARDS) and multiple organ failure. At the present stage, the preventive effects of available vaccines or drugs to COVID-19 are limited, and vaccine development is challenging due to the limited knowledge about SARS-CoV-2. A great challenge in vaccine development is to find a suitable animal model. Currently, non-human primate infection models are mainly used to verify the safety and efficacy of the developed vaccines, but the cost is extremely high and it takes a long time.
In the existing virus vaccine technology, the whole virus antigen vaccine is based on the traditional inactivated virus or attenuated virus as antigen source, which has the problems of low safety and high cost; the viral specific antigen vaccine is based on synthetic S protein as the antigen, while artificially synthesized S protein is mainly expressed by bacteria, yeast, cells or viral vectors, which has the problems of a length process of manufacture, single valence, easy escape of the virus, short validity period, and high cost; DNA and mRNA vaccines need to enter cells and be converted into proteins to exert immune effects, and the low immunogenicity issue has been the bottleneck that the technology still cannot overcome at present time.
SUMMARY
The present disclosure provides a universal SARS-CoV-2 vaccine composition and a preparation method thereof. The vaccine uses immunologically modified antigen-presenting cells expressing key structural proteins and a non-structural protein of SARS-CoV-2 to induce the body  to produce an immune response and form an immunological memory for the prevention and treatment of SARS-CoV-2 infection.
To achieve this, the following technical solutions are used in the present disclosure.
In a first aspect, the present disclosure provides a protein or a combination of proteins for a SARS-CoV-2 vaccine. The protein or combination of proteins includes any one or a combination of at least two of Spike protein (Sprotein) , Envelope protein (E protein) , Membrane protein (M protein) , Nucleocapsid protein (N protein) or ORF1a poly-protease (P protein) of SARS-CoV-2.
In the present disclosure, the protein or the combination of proteins for a vaccine containing key structural proteins and a non-structural protein of SARS-CoV-2 can stimulate the body to produce an immune response and form an immunological memory, with a broad-spectrum immunostimulatory effect.
In a second aspect, the present disclosure provides a fusion protein for a SARS-CoV-2 vaccine. The fusion protein includes an S protein signal peptide and an S protein receptor binding domain of SARS-CoV-2.
In a specific embodiment, the S protein receptor binding domain includes the amino acid sequence as shown in SEQ ID NO: 1.
SEQ ID NO: 1:
Figure PCTCN2021117415-appb-000001
In a specific embodiment, the S protein signal peptide includes the amino acid sequence as shown in SEQ ID NO: 2.
SEQ ID NO: 2:
MFVFLVLLPLVSSQCVNLTTRTQLP.
In the present disclosure, the fusion protein contains an S protein receptor binding domain (RBD) , which is the most important structure for SARS-CoV-2 to invade human cells. The fusion protein helps stimulate the body to produce anti-SARS-CoV-2 antibodies having high specificity.
Preferably, the fusion protein contains only the S protein signal peptide and the S protein receptor binding domain, and a short peptide at the carboxyl terminal is modified. A designed fusion protein S includes the amino acid sequence as shown in SEQ ID NO: 3.
SEQ ID NO: 3:
Figure PCTCN2021117415-appb-000002
Figure PCTCN2021117415-appb-000003
The fusion protein of the present disclosure includes the S protein signal peptide and the S protein receptor binding domain, and further includes a partial peptide fragment of the E protein and a partial peptide fragment of the M protein.
Preferably, the partial peptide fragment of the E protein includes an amino terminal peptide fragment of the E protein.
Preferably, the partial peptide fragment of the M protein includes a carboxyl terminal peptide fragment of the M protein.
Preferably, the amino terminal peptide fragment of the E protein includes the amino acid sequence as shown in SEQ ID NO: 4.
SEQ ID NO: 4:
MYSFVSEETGTLIVNSVLLFLAFVVFLLV.
Preferably, the carboxyl terminal peptide fragment of the M protein includes the amino acid sequence as shown in SEQ ID NO: 5.
SEQ ID NO: 5:
PKEITVATSRTLSYYKLGASQRVAGDSGFAAYSRYRIGNYKLNTDHSSSSDNIALLVQ.
Preferably, the fusion protein consists of an S protein signal peptide, an S protein receptor binding domain, an amino terminal peptide fragment of the E protein and a peptide fragment of the M protein. A designed fusion protein SEM comprises the amino acid sequence as shown in SEQ ID NO: 6.
SEQ ID NO: 6:
Figure PCTCN2021117415-appb-000004
The fusion protein of the present disclosure includes the S protein signal peptide, the S protein receptor binding domain, the amino terminal peptide fragment of the E protein, and the peptide fragment of the M protein, and further includes a partial peptide fragment of the N protein and a partial peptide fragment of the ORF1a poly-protease
Preferably, the partial peptide fragment of the N protein includes a helix-turn domain of the N protein.
Preferably, the partial peptide fragment of the ORF1a poly-protease includes a functional domain peptide fragment of the ORF1a poly-protease.
Preferably, the helix-turn domain of the N protein includes the amino acid sequence as shown in SEQ ID NO: 7.
SEQ ID NO: 7:
Figure PCTCN2021117415-appb-000005
Preferably, the peptide fragment of the ORF1a poly-protease includes the amino acid sequence as shown in SEQ ID NO: 8.
SEQ ID NO: 8:
Figure PCTCN2021117415-appb-000006
Preferably, the fusion protein consists of an S protein signal peptide, an S protein receptor binding domain, an amino terminal peptide fragment of the E protein, a peptide fragment of the M protein, a helix-turn domain of the N protein and a functional domain peptide fragment of the ORF1a poly-protease. A designed fusion protein SEMNP comprises the amino acid sequence as shown in SEQ ID NO: 9.
SEQ ID NO: 9:
Figure PCTCN2021117415-appb-000007
In the present disclosure, a fusion protein containing several key proteins of SARS-CoV-2 is constructed based on the nucleic acid sequence of SARS-CoV-2. The fusion protein produces polypeptide fragments after enzymolysis, which, after presented by antigen-presenting cells, stimulate B cells and T cells in the body to become mature B cells and T cells that are specifically  directed against viral antigens, and stimulate further proliferation of activated immune cells, to achieve prevention and controlling of infections.
In a third aspect, the present disclosure provides a coding gene of the fusion protein in the second aspect. The coding gene comprises a nucleic acid molecule encoding an S protein signal peptide and a nucleic acid molecule encoding an S protein receptor binding domain. The coding gene of the fusion protein S comprises the nucleic acid sequence as shown in SEQ ID NO: 10.
SEQ ID NO: 10:
Figure PCTCN2021117415-appb-000008
Preferably, the coding gene further includes a nucleic acid molecule encoding the amino terminal peptide fragment of the E protein and a nucleic acid molecule encoding the peptide fragment of the M protein. The encoding gene of the fusion protein SEM includes the nucleic acid sequence as shown in SEQ ID NO: 11.
SEQ ID NO: 11:
Figure PCTCN2021117415-appb-000009
Preferably, the coding gene further includes a nucleic acid molecule encoding the helix-turn domain of the N protein and a nucleic acid molecule encoding the functional domain peptide fragment of the ORF1a poly-protease. The coding gene of the fusion protein SEMNP includes the nucleic acid sequence as shown in SEQ ID NO: 12.
SEQ ID NO: 12:
Figure PCTCN2021117415-appb-000010
In a fourth aspect, the present disclosure provides an expression vector, comprising the coding gene in the third aspect.
Preferably, the expression vector includes any one of a lentiviral vector, a retroviral vector an adeno-associated viral vector or an adenovirus vector, preferably a lentiviral vector.
In a fifth aspect, the present disclosure provides a recombinant lentivirus, which is obtained by packaging of the lentiviral vector in the fourth aspect and a helper plasmid in mammalian cells.
In a sixth aspect, the present disclosure provides an antigen-presenting cell that expresses the fusion protein for a SARS-CoV-2 vaccine as described above, wherein the antigen-presenting cell further expresses a cytokine or cytokines; wherein the cytokine or cytokines include any one or a combination of at least two of Calnexin (CNX) , GM-CSF, CD80, CD86, Flt3-L, IL-2 or IL-12.
In the present disclosure, artificial antigen presenting cells (aAPCs) are constructed by introducing cytokines such as Calnexin (CNX) , GM-CSF, CD80, CD86, Flt3-L, IL-2 or IL-12 into myeloid-derived antigen-presenting cells using lentiviral technology.
In a seventh aspect, the present disclosure provides a universal SARS-CoV-2 vaccine, which is  an antigen-presenting cell expressing the fusion protein in the second aspect.
The present disclosure adopts antigen-presenting cells expressing key proteins of SARS-CoV-2 to construct cellular vaccines, which is conducive to solving the safety and high cost problems of individualized cellular infusion, and can accelerate the preparation of cellular vaccines and rapidly realize large-scale industrial production. Moreover, the present disclosure adopts mitomycin-C and gamma-ray irradiation treatment to further strengthen the safety factor, which solves the risks in preparation and safety control of vaccine cells. In addition, the present disclosure strictly regulates and controls the preparation process, and reduces costs.
Preferably, the antigen-presenting cell expresses a cytokine or cytokines.
Preferably, the antigen-presenting cell expresses any one or a combination of at least two of Calnexin (CNX) , GM-CSF, CD80, CD86, Flt3-L, IL-2 or IL-12.
The prophylactic and therapeutic process of the vaccine of the present disclosure is designed to mimic the natural immune system of the body. In the presence of cytokines, the structural and non-structural proteins of SARS-CoV-2 are exposed to stimulate the body to produce an immune response and form an immunological memory, and to produce specific antibodies against the SARS-CoV-2 proteins. Thereby, a stronger immunological memory may be rapidly induced when re-infected with the virus.
Preferably, the coding gene in the third aspect is integrated into the genome of the antigen-presenting cell.
Preferably, the antigen-presenting cell includes the expression vector in the fourth aspect and/or the recombinant lentivirus in the fifth aspect.
In an eighth aspect, the present disclosure provides a method for preparing the vaccine in the seventh aspect, comprising:
transducing an antigen-presenting cell that expresses a cytokine or cytokines including any one or a combination of at least two of Calnexin, GM-CSF, CD80, CD86, Flt3-L, IL-2 or IL-12 with the recombinant lentivirus in the fifth aspect, and carrying out inactivation treatment, to obtain the vaccine.
In the present disclosure, a minigene encoding a fusion protein composed of several structural proteins and a non-structural protein of SARS-CoV-2 is synthesized, and transduced into immunologically modified artificial antigen-presenting cells (aAPC) through a lentiviral system. The constructed cellular vaccine has a broad-spectrum immunostimulatory effect, effectively stimulates cellular immune responses and production of corresponding antibodies in the body, and can be produced on a large scale.
Preferably, the inactivation treatment includes mitomycin-C treatment and/or gamma-ray  irradiation.
In the present disclosure, a concentrated minigene-containing lentiviral vector LV-SEMNP, LV-SEM or LV-S is used to transduce aAPC to establish a persistent artificially synthesized antigen-presenting cell line (COVID-19-: aAPC-SEMNP, aAPC-SEM or aAPC-S) , which is inactivated by mitomycin-C and/or gamma-ray irradiation to further enhance the vaccine safety factor, to prepare a universal COVID-19/aAPC vaccine.
Compared with the prior art, the present disclosure has the following beneficial effects.
(1) In the present disclosure, a fusion protein containing structural proteins S protein, E protein, M protein, and N protein, and a non-structural protein ORF1a poly-protease of SARS-CoV-2, and a coding minigene thereof are constructed. The minigene is transduced into immunologically modified artificial antigen-presenting cells using a lentiviral system. The fusion protein is enzymatically digested in the body into several polypeptide fragments, which, after presented by antigen-presenting cells, can stimulate B cells and T cells in the body to become mature B cells and T cells that are specifically directed against viral antigens, and stimulate further proliferation of activated immune cells, to achieve prevention and controlling of infections.
(2) The prophylactic and therapeutic process of the vaccine of the present disclosure is designed to mimic the natural immune system of the body. In the presence of cytokines, the structural and non-structural proteins of SARS-CoV-2 are exposed to stimulate the body to produce an immune response and form an immunological memory, and to produce specific antibodies against the SARS-CoV-2 proteins. Thereby, a stronger immunological memory may be rapidly induced when re-infected with the virus, with a broad-spectrum immunostimulatory effect.
(3) In order to realize rapid large-scale industrial production of vaccines, the present disclosure adopts a preferred antigen-presenting cell strain expressing key proteins of SARS-CoV-2 to construct cellular vaccines. It is conducive to solving the safety and high cost problems of individualized cellular infusion, accelerating the preparation time of cellular vaccines, rapidly realizing large-scale industrial production. In the vaccine preparation process, mitomycin C and/or gamma-radiation (are) is used for inactivation treatment, which further improve (s) the safety factor of the vaccine, solve (s) the risk of vaccine cell preparation and safety control, and strictly regulate (s) and control (s) the preparation process, and the costs are reduced.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1 shows a schematic diagram of the preparation process of a universal SARS-CoV-2 vaccine.
Figure 2 shows a lentiviral vector system containing a minigene.
Figure 3 is a diagram showing the phenotype analysis of universal artificial vaccine cells  (aAPCs) modified by lentivirus; wherein, Figure 3A shows a flow cytometric analysis comparing the expression of HLA antigens (-ABC, and -DP, DR, DQ) on cell surface between dendritic cells and aAPCs; and Figure 3B shows a flow cytometric analysis comparing the expression of CD80/CD86 and FL on cell surface and intracellular GM-CSF and IL-2 between unmodified aAPCs and lentivirus-modified aAPCs.
Figure 4 shows the expression of viral proteins in aAPCs modified by lentiviruses containing different minigenes (aAPC-S, -SEM, -SEMNP) .
Figure 5A shows the amplification of T cells from Donor 1 after aAPC-SEMNP stimulation; Figure 5B shows the amplification of T cells from Donor 2 after aAPC-SEMNP stimulation; Figure 5C shows the antigenic phenotypes of CD4+ T cells after stimulation of T cells from different donors with aAPC-SEMNP; and Figure 5D shows the antigenic phenotypes of CD8+ T cells after stimulation of T cells from different donors with aAPC-SEMNP.
Figure 6A shows results of an effector response assay where the universal SARS-CoV-2 vaccine aAPC-SEMNP was used to stimulate T cells from Donor 1; Figure 6B shows results of an effector response test where the universal SARS-CoV-2 vaccine aAPC-SEMNP is used to stimulate T cells from Donor 2; Figure 6C shows results of an effector response test where the universal SARS-CoV-2 vaccine aAPC-SEMNP is used to stimulate T cells from Donor 3; and Figure 6D shows results of an effector response test where the universal SARS-CoV-2 vaccine aAPC-SEMNP is used to stimulate T cells from Donor 4.
DETAILED DESCRIPTION
In order to further illustrate the technical means adopted by the present disclosure and effects thereof, the present disclosure will be further described below in conjunction with examples and drawings. It should be understood that the specific embodiments described herein are only used to explain the present disclosure, but not to limit the present disclosure.
Experiments without specific techniques or conditions noted in the examples were conducted according to techniques or conditions described in the literature in the art or a product instruction. The reagents or instruments used herein without manufacturers specified were conventional products commercially available from proper channels.
Example 1 Design of fusion protein
Comparison of the nucleic acid sequences of SARS-CoV-2 with SARS and MERS viruses reveals that SARS-CoV-2 mainly expresses four structural proteins, i.e., surface glycoprotein (Sprotein) , envelop protein (E protein) , membrane protein (M protein) , and nucleocapsid phosphoprotein (N protein) , and a functional protein, polyprotein cleavage protease (ORF1a poly-protease) .
In this example, based on the published full-length 1273 aa amino acid sequence of S protein (MN908947.3) , the S protein signal peptide (SEQ ID NO: 2) and S protein receptor binding domain (SEQ ID NO: 1) were selected as the first part of the fusion protein; the amino-terminal 29 aa peptide fragment (SEQ ID NO: 4) of the E protein was selected as the second part of the fusion protein; the carboxyl-terminal 58 aa peptide fragment (SEQ ID NO: 5) of the M protein was selected as the third part of the fusion protein; the intermediate 162 aa helix-turn domain (SEQ ID NO: 7) of the N protein was selected as the fourth part of the fusion protein; and the carboxyl-terminal 123 aa peptide (SEQ ID NO: 8) of the ORF1a poly-protease was selected as the fifth part of the fusion protein. A fusion protein SEMNP (SEQ ID NO: 9) with a length of 595 aa was obtained. Further, the length of each viral protein in the fusion protein can be increased or decreased, for example as follows.
In this example, the fusion protein SEMNP was truncated. Most peptides of the helix-turn domain of the N protein and all peptides of the carboxyl-terminal of the ORF1a poly-protease were deleted. A fusion protein SEM (SEQ ID NO: 6) with a length of 315 aa was obtained.
In this example, the fusion protein SEMNP was further truncated. Only the S protein signal peptide and the S protein receptor binding domain were retained, and a short peptide fragment at the carboxyl-terminal was modified. A fusion protein S (SEQ ID NO: 3) with a length of 228 aa was obtained.
Example 2 Design of minigene
The amino acid sequence of the fusion protein SEMNP was converted to a corresponding nucleic acid sequence, which was optimized to obtain the coding gene of the fusion protein SEMNP as shown in SEQ ID NO: 12.
Partial NP sequence at the 3' end of the coding gene of SEMNP was deleted (nt. 4990-5840, NheI-SpeI deletion) , to obtain the coding gene of fusion protein SEM as shown in SEQ ID NO: 11.
Partial EMNP sequence at the 3' end of the coding gene of SEMNP was deleted (nt. 4680-5850, BstBI-BstBI deletion) , to obtain the coding gene of fusion protein S as shown in SEQ ID NO: 10.
Example 3 Construction of lentiviral vector
The coding genes of the fusion proteins S, SEM and SEMNP as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively, were artificially synthesized. The above nucleic acid molecules were double digested, incubated in a 37 ℃ water bath for 30 min. The digested products were subjected to DNA electrophoresis on a 1.5%agarose gel. The digested fragments were purified and recovered using an agarose gel kit.
As shown in Figure 2, the digested fragments were inserted into the linearized lentiviral vector TYF to construct lentiviral vectors LV-S, LV-SEM and LV-SEMNP containing minigenes.
Example 4 Preparation of artificial antigen-presenting cells (aAPCs) and dendritic cells (DCs)
(1) Preparation of aAPCs
aAPCs were prepared using human-derived myeloid-associated cells: myelogenous leukemia AML, KG-1 or K562, for examples. Cells were modified with lentivirus to express CNX, GM-CSF, CD80, CD86, Flt-L, IL-2 and IL-12 in order to enhance the antigen presentation function of aAPCs. Cell strains expressing high levels of cytokines were screened as vaccine strains.
(2) Preparation of DCs
Peripheral blood mononuclear cells were plated in culture dishes and cultured for 2 h in AIM-V medium (Gibco-BRL, CA, USA) ;
Nonadherent cells were gently removed, and the culture was continued for 24 h using AIM-V medium supplemented with 50 ng/mL GM-CSF (Biosource, CA, USA) and 25 ng/mL IL-4 (Biosource, CA, USA) .
Cells were washed with PBS, replaced with fresh AIM-V medium containing 20 ng/mL IFN-γ (Gentaur) , 50 ng/mL TNF-α (R&D systems, MN, USA) , 10 ng/mL IL-1β (R&D systems, MN, USA) , 10 ng/mL IL-6 (R&D systems) and 1 μM PGE2 (Sigma-Aldrich, MO, USA) , and cultured for 24 h to generate mature dendritic cells.
Example 5 Phenotypic analysis of artificial antigen-presenting cells (aAPCs)
The screened aAPC cell strains had been modified with lentivirus to express cytokines. Phenotypic analysis was performed by flow cytometry as follows.
aAPCs were stained with antibodies PE-anti-HLA-ABC, FITC-anti-HLA-DR, DP, DQ, FITC-anti-CD80, and FITC-anti-CD86, kept at 4 ℃ for 30 min, and washed twice with PBS containing 1%FBS.
Intracellular staining analysis was performed according to the literature. In brief, cells were treated with Monensin (Sigma) for 5 h, fixed and permeablized, followed by the addition of antibodies (BD Bioscience) against GM-CSF, IL-2, and Flt3-ligand (FL) , stained with FIX/PERM and PERM/Wash solution (BD) , and then loaded onto flow cytometry. The percentage of different antigen presenting cell subpopulations was analyzed with Flowjo software. The results are shown in Figure 3.
As shown in Figure 3A, compared with normal immature DC cells, aAPCs did not express HLA-ABC and -DP, -DR, -DQ, thus the immune rejection response was reduced. As shown in Figure 3B, unmodified APCs expressed low levels of CD80 and CD86, and did not express GM-CSF and IL-2; modified aAPCs expressed high levels of CD80, CD86, GM-CSF and IL-2; unmodified APCs did not express Flt-L (FL) ; and modified aAPCs expressed a high level of FL.
Example 6 Preparation of a universal SARS-CoV-2 vaccine
The preparation process of the universal SARS-CoV-2 vaccine is shown in Figure 1. First, aAPCs were transduced with concentrated minigene-containing lentiviral vectors LV-SEMNP, LV-SEM or LV-S to establish persistent artificially synthesized antigen-presenting cell strains (COVID-19-: aAPC-SEMNP, aAPC-SEM and aAPC-S) , which were then inactivated through mitomycin-C and/or gamma-ray irradiation, to produce universal SARS-CoV-2 vaccines COVID-19/aAPC-SEMNP, COVID-19/aAPC-SEM and COVID-19/aAPC-S.
Example 7 Analysis of viral protein expression by universal SARS-CoV-2 vaccine (aAPC-SEMNP)
Cell samples were lysed for 1 h with RIPA lysis solution. The protein concentration was determined by a BCA method. The protein concentration and the sample loading amount of the sample solution were calculated.
According to the protein quantification results, the protein solution, 6×SDS buffer and RIPA lysis solution were proportionally prepared into a total volume of 25 μL protein loading solution and incubated at 100 ℃ for 5 min.
10%upper and lower gels were prepared according to the Gel Fast Preparation Kit. The stacking gel was run at a voltage of 70 V, and the separating gel was run at a voltage of 120 V.
According to the position of the protein molecular weight (Marker) and the size of the gel, a PVDF membrane was cut out and soaked in absolute ethanol. Filter paper used for transfer was soaked in the transfer buffer. A semi-dry transfer device was used to perform transfer.
After the transfer, the membrane was blocked in 5%BSA for 1 h at room temperature. Primary antibodies of S protein, N protein, P protein and M protein were added after blocking and then incubated overnight in a refrigerator at 4℃.
The next day, secondary antibodies were added and incubated at room temperature for 1 h. Finally, the PVDF membrane was treated with a HRP Substrate Kit and exposed on an Imager.
The results are shown in Figure 4. aAPC-S cells expressed a high level of S protein (26 kD) , aAPC-SEMNP cell strains expressed a high level of SMENP fusion protein (66 kD) , and aAPC-SEM cells expressed SEM fusion protein (35 kD) .
Example 8 Analysis of function of T cells stimulated by universal SARS-CoV-2 vaccine aAPC-SEMNP
(1) Functional test 1 of Covid-19/aAPC-stimulated T cells:
After stimulation of peripheral blood T cells by COVID-19/aAPC vaccine, cytotoxic T cells (CTLs) were analyzed based on CD27, CD28, CD45RA, CD4 and CD8 surface staining by flow cytometry. Results are shown in Figures 5A, 5B, 5C and 5D, wherein Tcm refers to central memory  T cells (CD27 +, CD28 +, CD45RA -) , Tem refers to effector-memory T cells (CD27 +/-, CD28 +/-, CD45RA -) , and Teff refers to terminal effector T cells (CD27 -, CD28 -, CD45RA -) .
As shown in Figure 5A and Figure 5B, T cells from two different donors expanded after 2-3 weeks of stimulation. As shown in Figure 5C and Figure 5D, the expanded T cells were analyzed through surface antigen staining, and it showed the expression of antigen phenotypes of memory and effector T cells.
(2) Functional test 2 of Covid-19/aAPC stimulated T cells.
Engineered immune effector cytotoxic T cells (EIE CTLs) generated after stimulation with COVID-19/aAPC vaccine were added with relevant antigenic peptides (a SEMNP mixture, S, E, M, N, P, and HIV peptides) , or artificial vaccine cells aAP-SEMNP, aAPC-SEM, or aAPC-S, and stimulated overnight. The strength of the antigen-specific response was detected using ELISPOT immunoassay.
CTLs (EIE) generated after stimulation of peripheral blood mononuclear cells (PBMCs) from 4 healthy donors with SEMNP antigen were stimulated again with Covid-19 peptides S, M, E, N, P, and SEMNP (a mixture of 5 peptides) , and different aAPC vaccine cells for overnight, and ELISpot analysis was performed.
The results are shown in Figure 6A, Figure 6B, Figure 6C and Figure 6D. Each donor expressed and produced IFN-γ after stimulation with different protein peptides, indicating that the aAPC vaccines (-S, -SEM, or -SEMNP) induced T cell-specific immune responses in large scale.
In summary, the universal SARS-CoV-2 vaccine of the present disclosure mimics the natural immune system of the body. In the presence of cytokines, the several polypeptide fragments formed from the fusion protein are delivered by antigen-presenting cells and stimulate the body to generate an immune response and form an immunological memory.
The applicant declares that the present disclosure uses the above-mentioned examples to illustrate the detailed methods of the present disclosure, but the present disclosure is not limited to the above-mentioned detailed methods, which does not mean that the present disclosure must rely on the above-mentioned detailed methods to be implemented. Those skilled in the art should understand that any improvement of the present disclosure, the equivalent replacement of each raw material of the product of the present disclosure, the addition of auxiliary components, the selection of specific methods, etc. fall within the scope of protection and disclosure of the present disclosure.

Claims (14)

  1. A protein or a combination of proteins for a SARS-CoV-2 vaccine, comprising any one or a combination of at least two of S protein, E protein, M protein, N protein or ORF1a poly-protease P of SARS-CoV-2.
  2. A fusion protein for a SARS-CoV-2 vaccine, comprising an S protein signal peptide and an S protein receptor binding domain of SARS-CoV-2.
  3. The fusion protein according to claim 2, wherein the S protein receptor binding domain includes the amino acid sequence as shown in SEQ ID NO: 1; and/or
    the S protein signal peptide includes the amino acid sequence as shown in SEQ ID NO: 2.
  4. The fusion protein according to claim 2 or 3, wherein the fusion protein consists of the S protein signal peptide and the S protein receptor binding domain;
    preferably, the fusion protein includes the amino acid sequence as shown in SEQ ID NO: 3.
  5. The fusion protein according to claim 2 or 3, wherein the fusion protein further includes a partial peptide fragment of E protein and a partial peptide fragment of M protein;
    preferably, the partial peptide fragment of the E protein includes an amino terminal peptide fragment of the E protein;
    preferably, the partial peptide fragment of the M protein includes a carboxyl terminal peptide fragment of the M protein;
    preferably, the amino terminal peptide fragment of the E protein includes the amino acid sequence as shown in SEQ ID NO: 4;
    preferably, the carboxyl terminal peptide fragment of the M protein includes the amino acid sequence as shown in SEQ ID NO: 5.
  6. The fusion protein according to claim 5, wherein the fusion protein consists of the S protein signal peptide, the S protein receptor binding domain, the amino terminal peptide fragment of the E protein, and the peptide fragment of the M protein;
    preferably, the fusion protein includes the amino acid sequence as shown in SEQ ID NO: 6.
  7. The fusion protein according to any one of claims 2-3 and 5-6, wherein the fusion protein further includes a partial peptide fragment of N protein and a partial peptide fragment of ORF1a poly-protease;
    preferably, the partial peptide fragment of the N protein includes a helix-turn domain of the N protein;
    preferably, the partial peptide fragment of the ORF1a poly-protease includes a functional domain peptide fragment (3CLpro) of the ORF1a poly-protease;
    preferably, the helix-turn domain of the N protein includes the amino acid sequence as shown in SEQ ID NO: 7;
    preferably, the peptide fragment of the ORF1a poly-protease includes the amino acid sequence as shown in SEQ ID NO: 8.
  8. The fusion protein according to claim 7, wherein the fusion protein consists of the S protein signal peptide, the S protein receptor binding domain, the amino terminal peptide fragment of the E protein, the peptide fragment of the M protein, the helix-turn domain of the N protein and the functional domain peptide fragment of the ORF1a poly-protease;
    preferably, the fusion protein includes the amino acid sequence as shown in SEQ ID NO: 9.
  9. A coding gene of the fusion protein of any one of claims 2-8, including a nucleic acid molecule encoding the S protein signal peptide and a nucleic acid molecule encoding the S protein receptor binding domain;
    preferably, the coding gene includes the nucleic acid sequence as shown in SEQ ID NO: 10;
    preferably, the coding gene further includes a nucleic acid molecule encoding the amino terminal peptide fragment of the E protein and a nucleic acid molecule encoding the peptide fragment of the M protein;
    preferably, the coding gene includes the nucleic acid sequence as shown in SEQ ID NO: 11;
    preferably, the coding gene further includes a nucleic acid molecule encoding the helix-turn domain of the N protein and a nucleic acid molecule encoding the functional domain peptide fragment of the ORF1a poly-protease;
    preferably, the coding gene includes the nucleic acid sequence as shown in SEQ ID NO: 12.
  10. An expression vector, including the coding gene of claim 9;
    preferably, the expression vector includes any one of a lentiviral vector, a retroviral vector or an adeno-associated virus vector, preferably a lentiviral vector.
  11. A recombinant lentivirus, which is obtained by packaging of the expression vector of claim 10 and a helper plasmid in mammalian cells.
  12. An antigen-presenting cell that expresses the fusion protein for a SARS-CoV-2 vaccine of any one of claims 2-8, wherein the antigen-presenting cell further expresses a cytokine or cytokines;
    wherein the cytokine or cytokines include any one or a combination of at least two of Calnexin, GM-CSF, CD80, CD86, Flt3-L, IL-2 or IL-12.
  13. A universal SARS-CoV-2 vaccine, which is an antigen-presenting cell expressing the fusion protein of any one of claims 2-8;
    preferably, the antigen-presenting cell further expresses a cytokine or cytokines;
    preferably, the cytokine or cytokines include any one or a combination of at least two of Calnexin, GM-CSF, CD80, CD86, Flt3-L, IL-2 or IL-12;
    preferably, the coding gene of claim 9 is integrated into the genome of the antigen-presenting  cell;
    preferably, the antigen-presenting cell includes the expression vector of claim 10 and/or the recombinant lentivirus of claim 11.
  14. A method for preparing the vaccine of claim 13, comprising:
    transducing an antigen-presenting cell that expresses a cytokine or cytokines including any one or a combination of at least two of Calnexin, GM-CSF, CD80, CD86, Flt3-L, IL-2 or IL-12 with the recombinant lentivirus of claim 11, and carrying out inactivation treatment, to obtain the vaccine;
    preferably, the inactivation treatment includes mitomycin-C treatment and/or gamma-ray irradiation.
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