CN117295771A - Method for improving immunogenicity of SARS-CoV-2 mutant strain ECD antigen/antigen trimer stability - Google Patents

Method for improving immunogenicity of SARS-CoV-2 mutant strain ECD antigen/antigen trimer stability Download PDF

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CN117295771A
CN117295771A CN202280034785.7A CN202280034785A CN117295771A CN 117295771 A CN117295771 A CN 117295771A CN 202280034785 A CN202280034785 A CN 202280034785A CN 117295771 A CN117295771 A CN 117295771A
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谢良志
孙春昀
张延静
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Sinocelltech Ltd
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Abstract

Relates to the field of molecular vaccine, and provides a method for improving the immunogenicity/antigen trimer stability of SARS-CoV-2 mutant strain ECD antigen and a SARS-CoV-2 mutant strain ECD immunogenic protein/peptide with improved immunogenicity/antigen trimer stability. Including but not limited to the extracellular domain (ECD) of spike protein (S protein) of SARS-CoV-2 strain, B.1 strain, B.1.1.7 strain or B.1.351 strain, having genomic sequence No. GenBank Accession No. MN908947.3, by introducing a mutation site and a homotrimer formed by trimerization assistance structure to enhance ECD antigen immunogenicity/antigen trimer stability. The vaccine further comprises a pharmaceutically acceptable adjuvant. The vaccine combination shows excellent immunogenicity in mice and cynomolgus monkeys, and can maintain humoral immunity and cellular immune response for a long time. The recombinant trimeric protein vaccine can be used for preventing SARS-CoV-2 infection related diseases.

Description

Method for improving immunogenicity of SARS-CoV-2 mutant strain ECD antigen/antigen trimer stability
Cross Reference to Related Applications
The present application claims the benefit of chinese patent application 202110606512.2 filed on 31 of 2021 and chinese patent application 202111237604.4 filed on 22 of 202110, the contents of which are incorporated herein by reference.
Technical Field
The invention relates to the field of molecular vaccinology, in particular to a method for improving the immunogenicity of an ECD antigen/antigen trimer of a SARS-CoV-2 mutant strain and an ECD immunogenic protein/peptide of the SARS-CoV-2 mutant strain with improved immunogenicity/antigen trimer stability.
Background
The novel coronavirus (SARS-CoV-2) has strong transmission capability, and the safe and effective vaccine is the most powerful technical means for controlling epidemic situation. Vaccines can be classified into the following categories, depending on the target and technology: inactivated vaccines, recombinant protein vaccines, viral vector vaccines, RNA vaccines, live attenuated vaccines, and virus-like particle vaccines.
The trimeric Spike protein (Spike, S protein) of SARS-CoV-2 is the major component of the viral envelope and plays an important role in receptor binding, fusion, viral entry and host immune defenses. SARS-CoV-2 and SARS-CoV share a common host cell receptor protein, angiotensin converting enzyme 2 (ACE 2) (Zhou, P., et al, discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin. BioRxiv, 2020.). The S protein of SARS-CoV-2 binds to the ACE2 receptor and is cleaved by host proteases into an S1 polypeptide comprising a receptor binding domain (Receptor binding domain, RBD) and an S2 polypeptide responsible for mediating fusion of the virus with the cell membrane (Hoffmann, m., et al, SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.Cell,2020.).
The RBD region of the S protein contains a major neutralizing antibody epitope that stimulates B cells to produce high titer neutralizing antibodies against RBD. In addition, the S protein also contains abundant T cell epitopes, and can induce T cells to generate specific CTL responses and clear virus infected cells. Thus, the S protein is the most critical antigen for new coronal vaccine design. Most vaccines currently designed select either the S protein or the RBD domain protein as the core immunogen.
SARS-CoV-2 is an RNA single-stranded virus, which is subject to deletion mutations and such mutations occur in repeated deletion regions of the S protein (Recurrent deletion regions, RDRs). Deletions or mutations may alter the conformation of the S protein such that antibodies induced by previous vaccine immunization reduce binding and neutralization of the mutant S protein resulting in reduced vaccine immunization effects and viral immune escape. Early D614G mutations (b.1) enhanced the affinity of the S protein for ACE2 receptor and became a rapidly prevalent strain, but did not reduce the sensitivity to neutralizing antibodies (Korber, b., et al, spike mutation pipeline reveals the emergence of a more transmissible form of SARS-CoV-2.Biorxiv, 2020:p.2020.04.29.069054; korber, b., et al, tracking Changes in SARS-CoV-2 Spike:Evidence that D614G Increases Infectivity of the COVID-19 Virus.Cell,2020.182 (4): p.812-827.e 19).
However, with the pandemic of SARS-CoV-2, 5 variants of high concern (Variants of Concern, VOC) appear worldwide: alpha (B.1.1.7), beta (B.1.351), gamma (P.1), detla (B.1.617.2) and omacron (B.1.1.529) and 2 careless variants (Variants of Interest, VOI): lambda (C.37) and Mu (B.1.621). Studies have shown that these high risk strains can increase transmissibility, exacerbate disease progression (increase hospitalization or mortality), severely reduce antibody neutralization by past infections or vaccinations, reduce therapeutic or vaccine effectiveness, or disable diagnostic assays (https:// www.cdc.gov/corenavirus/2019-ncov/cas-updates/variant-survivinil/variant-info. Alpha spreads rapidly and increases the risk of 61% related death (Davies, n.g., et al Increased mortality in community-tested cases of SARS-CoV-2 lineage B.1.1.7.Nature,2021.). The neutralization effect study showed that the neutralization ability of the convalescent plasma or vaccine immunized serum to Alpha remained essentially unchanged, whereas the neutralization ability to Beta was greatly reduced (Cele, S., et al, escape of SARS-CoV-2 501Y.V2 from neutralization by convalescent plasma.2021.; zhou, D., et al, evidence of Escape of SARS-CoV-2 variant B.1.351 from natural and vaccine-reduced sera. Cell,2021.; tada, T.,2021.https:// doi.org/10.1101/2021.02.05.003; wang, P.; et al, increased Resistance of SARS-CoV-2 Variants B.1.351 and B.1.1.7 to Antibody Neutralization.bioRxiv,2021.; wadman, M.and J.Cohen,2021.https:// doi.org/10.1136/bmj.n296; wu, K., et al, mRNA-3 vaccine induces neutralizing antibodies against spike mutants from global SARS-CoV-2 derivatives.2021. Clinical results also show that Alpha has little effect on the protective effect of the vaccine, while Beta significantly reduces the protective effect against light conditions (Madhi, S.A., et al, efficacy of the ChAdOx nCoV-19 Covid-19 Vaccine against the B.1.351 Variant.2021; shinde, V., et al, effect of NVX-CoV2373 Covid-19 Vaccine against the B.1.351 Variant.New England Journal of Medicine,2021; abu-Raddad, L.J., H.Chemaitelly, and A.A. button, effectiveness of the BNT b2 Covid-19 Vaccine against the B.1.1.7 and B.1.351 Variants.2021; karim, S.S.A., vaccines and SARS-CoV-2 variants:the urgent need for a correlate of protection.Lancet,2021.397 (10281): p.1263-1264). Compared with original virus and early variant strain, the Detla variant has stronger transmission capacity, short latency period and quick disease development process, and can reduce the protection effect of the vaccine. Omicron is the most severe variant of the mutations occurring so far, whose S protein contains about 30 amino acid mutations. These mutations result in large conformational changes in the S protein, with a large impact on infectious and immune escape. Various studies have shown that Omicron can greatly reduce the neutralizing effect induced by existing vaccines (Garcia-Beltran, w.f., et al, mRNA-based covd-19 vaccine boosters induce neutralizing immunity against SARS-CoV-2 Omicron variant.medRxiv,2021:p.2021.12.14.21267755; dejnirattisai, w., et al, reduced neutralisation of SARS-CoV-2 Omicron B.1.1.529 variant by post-immune service, lancet,2022.399 (10321): p.234-236.Araf,Y.and F.Akter,Omicron variant of SARS-CoV-2:Genomics,transmissibility,and responses to current COVID-19 vaccines.2022). Currently, omicon has spread to at least 49 countries worldwide and has replaced Delta as the dominant epidemic worldwide. The current vaccine is designed based on the sequence of an early epidemic strain (the genome sequence of the vaccine is GenBank Accession No. NC-045512), and in view of the high transmissibility of variant strains and adverse effects on the protection effect of the existing vaccine, novel vaccine with broad-spectrum and high protection effect on high-risk variant strains is urgently required.
Disclosure of Invention
Based on the above need for a vaccine with high protective effect against the novel coronavirus SARS-CoV-2 variant, the present invention provides a method for improving the immunogenicity/antigen trimer stability of an ECD antigen of a SARS-CoV-2 mutant strain by constructing an ECD antigen comprising at least any one of the amino acid sequences shown as SEQ ID No. 8, SEQ ID No. 12 or SEQ ID No. 16, or an immunogenic fragment and/or immunogenic variant thereof, whereby the ECD is in the form of a trimer in a stable prefusion conformation.
In one embodiment, the mutant strain is a high risk mutant strain comprising at least any of T19 24S, delta25/27, H49 67V, delta69/70, T95 142D, delta143/145, delta145-146, N211I, delta212/212, V213 339 346 346 371 373 376 405 408 417 452 452, 484 484 484 484 484 490 493 496 498 501 505 547 614 655 679 681 764 796 856 954 969 981F.
In one embodiment, the strain comprises at least one of a b.1 strain, a b.1.351 strain, a b.1.1.7 strain, a p.1 strain, a b.1.427 strain, a b.1.429 strain, a b.1.526 strain, a c.37 strain, a b.1.621 strain, a b.1.618 strain, a c.36.3 strain, a 20I/484Q strain, a ba.1 strain, a ba.1.1 strain, and a ba.2 strain.
In one embodiment, the ECD antigen is co-administered to the subject with one or more adjuvants selected from the group consisting of:
aluminum adjuvants, oil emulsion adjuvants, toll-like receptor (TLR) agonists, combinations of immunopotentiators, microbial adjuvants, propolis adjuvants, levamisole adjuvants, liposomal adjuvants, chinese herbal adjuvants, and small peptide adjuvants;
preferably, the oil emulsion adjuvant comprises a squalene component;
toll-like receptor (TLR) agonists comprise CpG or monophosphoryl lipid a (MPL) adsorbed on an aluminium salt; and the immune enhancer comprises QS-21 and/or MPL.
The invention also provides a method for improving the immunogenicity/antigen trimer stability of SARS-CoV-2 mutant strain ECD antigen by constructing a polynucleotide encoding a recombinant form ECD comprising at least any one of the amino acid sequences shown as SEQ ID No. 8, SEQ ID No. 12 or SEQ ID No. 16, or an immunogenic fragment and/or immunogenic variant thereof, thereby expressing a stable fusion conformation.
In one embodiment, the mutant strain is a high risk mutant strain comprising at least any one of T19 24S, delta25/27, H49 67V, delta69/70, T95 142D, delta143/145, delta145-146, N211I, delta212/212, V213 339 346 346, 371 373, 376, 405 408, 417, 452, 484 484, 493, 484, 496, 498, 501, 505, 547, 614, 655 679 681 764, 796, 856, 954, 969, 981F.
In one embodiment, the strain comprises at least one of a b.1 strain, a b.1.351 strain, a b.1.1.7 strain, a p.1 strain, a b.1.427 strain, a b.1.429 strain, a b.1.526 strain, a c.37 strain, a b.1.621 strain, a b.1.618 strain, a c.36.3 strain, a 20I/484Q strain, a ba.1 strain, a ba.1.1 strain, and a ba.2 strain.
The invention provides an ECD immunogenic protein/peptide of SARS-CoV-2 mutant strain with improved immunogenicity/antigen trimer stability, said immunogenic protein/peptide comprising at least any one of the amino acid sequences shown in SEQ ID No. 8, SEQ ID No. 12 or SEQ ID No. 16, or an immunogenic fragment and/or immunogenic variant thereof, said ECD immunogenic protein/peptide being in the form of a trimer in stable precision conformation.
In one embodiment, the mutant strain is a high risk mutant strain comprising at least any of T19 24S, delta25/27, H49 67V, delta69/70, T95 142D, delta143/145, delta145-146, N211I, delta212/212, V213 339 346 346 371 373 376 405 408 417 452 452, 484 484 484 484 484 490 493 496 498 501 505 547 614 655 679 681 764 796 856 954 969 981F.
In one embodiment, the strain comprises at least one of a b.1 strain, a b.1.351 strain, a b.1.1.7 strain, a p.1 strain, a b.1.427 strain, a b.1.429 strain, a b.1.526 strain, a c.37 strain, a b.1.621 strain, a b.1.618 strain, a c.36.3 strain, a 20I/484Q strain, a ba.1 strain, a ba.1.1 strain, and a ba.2 strain.
The invention also provides polynucleotides encoding SARS-CoV-2 mutant strain ECD immunogenic proteins/peptides having improved immunogenicity/antigen trimer stability as described above; preferably, the polynucleotide comprises the nucleotide sequence of at least any one of SEQ ID No. 7, SEQ ID No. 11 or SEQ ID No. 15.
The present invention provides an immunogenic composition comprising at least one immunogenic protein/peptide as described above, or at least one polynucleotide encoding an ECD immunogenic protein/peptide of a SARS-CoV-2 mutant strain having improved stability of the immunogenicity/antigen trimer as described above, and
any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent;
optionally, the immunogenic composition also comprises an adjuvant.
Further, the present invention provides an immunogenic composition comprising the amino acid sequences shown in SEQ ID No. 12 and SEQ ID No. 16, or an immunogenic fragment and/or immunogenic variant thereof, or the amino acid sequences shown in SEQ ID No. 8 and SEQ ID No. 16, or an immunogenic fragment and/or immunogenic variant thereof.
Still further, the present invention provides an immunogenic composition, the adjuvant of which is selected from one or more of the following: aluminum adjuvants, oil emulsion adjuvants, toll-like receptor (TLR) agonists, combinations of immunopotentiators, microbial adjuvants, propolis adjuvants, levamisole adjuvants, liposomal adjuvants, chinese herbal adjuvants, and small peptide adjuvants; preferably, the oil emulsion adjuvant comprises a squalene component;
toll-like receptor (TLR) agonists comprise CpG or monophosphoryl lipid a (MPL) adsorbed on an aluminium salt; and the immune enhancer comprises QS-21 and/or MPL.
The invention also provides the use of an immunogenic protein/peptide as described above, a polynucleotide encoding an immunogenic protein/peptide as described above and/or an immunogenic composition comprising an immunogenic protein/peptide as described above or a polynucleotide encoding said immunogenic protein/peptide for the prevention or treatment of a disease caused by a SARS-CoV-2 mutant strain; the invention also provides the use of an immunogenic protein/peptide as described above, a polynucleotide encoding an immunogenic protein/peptide as described above and/or an immunogenic composition comprising an immunogenic protein/peptide as described above or a polynucleotide encoding said immunogenic protein/peptide for the preparation of a vaccine or medicament for the prevention or treatment of a disease caused by a mutant strain of SARS-CoV-2.
Drawings
FIG. 1 is a schematic representation of the primary structure (A) and the higher structure (B, reference PDB:6 XLR) of the engineered S-ECD.
FIG. 2 shows the results of analysis of recombinant spike protein extracellular domain (S-ECD) trimer protein purity, wherein (A) is a non-reducing SDS-PAGE profile and (B) is a SEC-HPLC profile.
FIG. 3 shows a diagram of the negative electron microscopy results for recombinant spike protein extracellular domain (S-ECD) trimeric protein.
FIG. 4 shows the results of serum antibody titers after 6-8 weeks of Balb/C mice (A), 6-8 weeks of C57BL/6 mice (B) and 7-8 months old Balb/C mice (C) immunized with SCTV01C-TM8 vaccine.
FIG. 5 shows the results of serum pseudovirus neutralization titers after 6-8 weeks of Balb/C mice (A), 6-8 weeks of C57BL/6 mice (B) and 7-8 months old Balb/C mice (C) immunized with SCTV01C-TM8 vaccine.
FIG. 6 shows the statistical results of T lymphocyte numbers of splenocytes secreting IFN-. Gamma.s (A) and IL-4 (B) under different peptide pool stimulation conditions by ELISPot after immunization of 3 mouse models with SCTV01C-TM8 vaccine.
FIG. 7 shows the results of SCTV01C-TM23 vaccine cynomolgus monkey immune serum antibody titers (A) and pseudovirus neutralization titers (B) detection.
FIG. 8 shows statistical results of the T lymphocyte numbers of PBMCs secreting IFN-. Gamma.s (A) and IL-4 (B) under different peptide pool stimulation conditions following ELISPot immunization of cynomolgus monkeys with SCTV01C-TM23 vaccine.
FIG. 9 shows the results of SCTV01C-TM23 vaccine cynomolgus monkey immune serum detection against Foldon partial antibody titers.
FIG. 10 shows statistical results of T lymphocyte numbers of PBMCs secreting IFN-. Gamma.s (A) and IL-4 (B) under ELISPot assay for Foldon protein or 6P+Furin mutant engineered peptide library stimulation conditions.
FIG. 11 shows the results of SCTV01C-TM22 vaccine mouse immune serum antibody titer (A) and pseudovirus neutralization titer (B) assays.
FIG. 12 shows the results of SCTV01C-TM22 vaccine mouse immune serum pseudovirus broad spectrum neutralization potency assays.
FIG. 13 shows the results of the detection of neutralizing titers of the immune serum pseudovirus of the TM8+TM23 bivalent vaccine mice.
FIG. 14 shows statistical results of T lymphocyte numbers of splenocytes secreting IFN-. Gamma.s (A) and IL-4 (B) under different peptide pool stimulation conditions by ELISPot after immunization of mice with a TM8+TM23 bivalent vaccine.
FIG. 15 shows the results of neutralization titers of the TM22+TM23 bivalent vaccine mouse immune serum against the B.1 strain, the B.1.351 strain and the B.1.1.7 strain pseudoviruses.
FIG. 16 shows the results of the test for the neutralization titers of the bivalent vaccine mouse immune serum against the B.1.526 strain, the C.37 strain, the B.1.621 strain, the B.1.618 strain, the C.36.3 strain and the pseudovirus of the 20I/484Q strain.
FIG. 17 shows the results of neutralization titer detection of the bivalent vaccine mouse immune serum against BA.1 strain, BA.1.1 strain and BA.2 strain pseudoviruses.
Detailed Description
Definition of the definition
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For the purposes of the present invention, the following terms are further defined.
As used herein and in the appended claims, the singular forms "a," "an," "the," and "the" include plural referents unless the context clearly dictates otherwise.
The terms "comprising," "including," and "containing" are intended to include the specific components without excluding any other components. Such as "consisting essentially of … …" allows for the inclusion of other components or steps that do not detract from the novel or essential features of the invention, i.e., they exclude other non-recited components or steps that do. The term "consisting of … …" is meant to include a particular ingredient or ingredients and exclude all other ingredients.
The term "antigen" refers to a foreign substance recognized (specifically bound) by an antibody or T cell receptor, but which does not definitively induce an immune response. Foreign substances that induce specific immunity are called "immunogenic antigens" or "immunogens". "hapten" refers to an antigen that is not itself capable of eliciting an immune response (although a conjugate of several molecular haptens, or a conjugate of a hapten and a macromolecular carrier, may elicit an immune response).
A "humoral immune response" is an antibody-mediated immune response and involves the introduction and generation of antibodies that recognize and bind with some affinity to antigens in the immunogenic compositions of the invention, a "cell-mediated immune response" being an immune response mediated by T cells and/or other leukocytes. A "cell-mediated immune response" is induced by providing an epitope associated with a class I or class II molecule of the Major Histocompatibility Complex (MHC), CD1 or other atypical MHC-like molecule.
The term "immunogenic composition" refers to any pharmaceutical composition containing an antigen, such as a microorganism or component thereof, which composition can be used to elicit an immune response in an individual.
"immunogenicity" as used herein means the ability of an antigen (or epitope of an antigen), such as a coronavirus spinous process protein receptor binding region or an immunogenic composition, to elicit a humoral or cell-mediated immune response or both in a host (e.g., a mammal).
"protective" immune response refers to the ability of an immunogenic composition to elicit a humoral or cell-mediated immune response, or both, for protecting an individual from infection. The protection provided need not be absolute, i.e., it need not completely prevent or eradicate the infection, so long as there is a statistically significant improvement over a control population of individuals (e.g., infected animals that are not administered a vaccine or immunogenic composition). Protection may be limited to alleviating the severity of symptoms of infection or rapidity of onset.
"immunogenic amount" and "immunologically effective amount" are used interchangeably herein to refer to an amount of antigen or immunogenic composition sufficient to elicit an immune response (cellular (T cell) or humoral (B cell or antibody) response or both, as measured by standard assays known to those of skill in the art).
The effectiveness of an antigen as an immunogen may be measured, for example, by a proliferation assay, by a cytolytic assay, or by measuring the level of B cell activity.
The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of consecutive amino acid residues.
The terms "nucleic acid", "nucleotide" and "polynucleotide" are used interchangeably to refer to RNA, DNA, cDNA or cRNA and derivatives thereof, such as those containing a modified backbone. It is to be understood that the present invention provides polynucleotides comprising sequences complementary to the sequences described herein. "Polynucleotide" as contemplated in the present invention includes forward strands (5 'to 3') and reverse complementary strands (3 'to 5'). Polynucleotides according to the invention may be prepared in different ways (e.g., by chemical synthesis, by gene cloning, etc.), and may take various forms (e.g., linear or branched, single-or double-stranded, or hybrids thereof, primers, probes, etc.).
The term "immunogenic protein/peptide" includes polypeptides having immunological activity in the sense that upon administration to a host they are capable of eliciting an immune response against the humoral and/or cellular type of the protein. Thus, a protein fragment according to the invention comprises or essentially consists of at least one epitope or antigenic determinant. As used herein, an "immunogenic" protein or polypeptide includes the full-length sequence of a protein, an analog thereof, or an immunogenic fragment thereof. An "immunogenic fragment" refers to a fragment of a protein that contains one or more epitopes, thereby eliciting an immune response as described above.
The term "immunogenic protein/peptide" also encompasses deletions, additions and substitutions to the sequence, provided that the polypeptide functions to produce an immune response as defined herein, i.e. "immunogenic variants".
The SCTV01C recombinant protein vaccine provided by the invention is modified based on an extracellular domain (ECD, containing S1 and S2 parts) of SARS-CoV-2 spike protein. The known natural spike protein of SARS-CoV-2 is a trimeric structure, and during its production and infection function, the membrane fusion process is completed by cleavage by proteases in the Golgi apparatus and on the cell surface through the RRAR site present between S1 and S2, followed by S1 shedding, and further the S2 structure is converted from the precision conformation to the postfusion conformation, thus completing the membrane fusion (Cai, Y., J.Zhang, and T.Xaao, distinct conformational states of SARS-CoV-2 spike protein.2020.369 (6511): p.1586-1592.).
In order to obtain ECD trimers in stable precision conformation, the invention is modified based on S protein of different strain variants in three parts as follows:
1) It has now been found that antibodies with higher neutralizing activity bind to both the S1 region (specifically to the NTD and RBD regions in S1). The S1 part is kept intact, and is important for the generation of neutralizing antibodies induced by the novel crown vaccine. The Furin site is removed by modification in the SCTV01C recombinant protein vaccine, namely, the amino acid sequence from 679 to 688 is fixed to NSPGSASSVA, so that the possibility of S1 fracture and shedding is reduced.
2) Because of the allosteric propensity of S2 itself, the precision conformation of spike protein is unstable, while effective induction of neutralizing antibodies requires maintenance of the precision conformation stable, which has been demonstrated in RSV and HIV-1 vaccine studies (McLellan, J.S., et al, structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus.science,2013.342 (6158): p.592-8.; frey, G., et al Distinct conformational states of HIV-1 gp41 are recognized by neutralizing and non-nesting anti-ibodies Nat Struct Mol Biol,2010.17 (12): p.1486-91.). In current marketed vaccines, modifications of S-2P (i.e., mutation of amino acids 986 and 987 to proline) are commonly employed (Tian, J.H., et al, SARS-CoV-2 spike glycoprotein vaccine candidate NVX-CoV 2373-immunogenicity in baboons and protection in mice.2021.12 (1): p.372.; mercoado, N.B., et al, single-shot Ad26 vaccine protects against SARS-CoV-2 in rhesus macaques.2020.586 (7830): p.583-588.; corbett, K.S., et al, SARS-CoV-2 mRNA Vaccine Development Enabled by Prototype Pathogen Preparedness.bioRxiv,2020). In addition, hexaPro mutations (i.e., mutations of 817, 892, 899 and 942 amino acids into proline in addition to S-2P mutations) which effectively improve stability and do not affect its three-dimensional Structure are introduced (Hsieh, C.L., et al, structure-based Design of Prefusion-stabilized SARS-CoV-2 Spikes.bioRxiv,2020.). These mutation sites are all located at the N-terminal or Loop region of the α -helix in S2, and after mutation to the proline (P) type with this secondary structure propensity, the allosteric propensity of S2 can be effectively reduced to stabilize the presupposition conformation of S2.
3) Finally, in order to further stabilize the S-ECD trimer structure, the invention adds a trimerization module T4foldon at the C-terminal of the vaccine molecule. This module is derived from the C-terminal domain of the fibrin of the T4 bacteriophage, with 27 amino acids. T4foldon has been used in RSV vaccine candidates and proved to be safe in clinical phase I studies (Crank, M.C., A proof of concept for structure-based vaccine design targeting RSV in humans.2019.365 (6452): p.505-509.).
TABLE 1 molecular structural design modification of SCTVC 01C vaccine
TABLE 2S protein mutations of SARS-CoV-2 variant related to the invention
*https://www.who.int/en/activities/tracking-SARS-CoV-2-variants
**https://www.coronaheadsup.com/coronavirus/france-46-cases-of-b-1-1-7-with-e484q-mutation-in-bordeaux-coronavirus-outbreak-of-voc-20i-484q/
***https://en.wikipedia.org/wiki/SARS-CoV-2_Omicron_variant#Mutations
****https://www.biorxiv.org/content/10.1101/2021.05.14.444076v1
The expression vectors of the S-ECD trimer protein antigen of the B.1 strain, the B.1.351 strain and the B.1.1.7 strain which are modified are respectively constructed, and conventional purity and stability analysis is carried out on the expressed recombinant S-ECD trimer protein to prepare corresponding vaccines, namely B.1 strain SCTV01C-TM8 vaccine, B.1.351 strain SCTV01C-TM23 vaccine and B.1.1.7 strain SCTV01C-TM22 vaccine.
The ECD trimeric immunogenic proteins/peptides of the invention exhibit excellent immunogenicity in mice and cynomolgus monkeys, and can maintain long-term humoral and cellular immune responses.
Immunization of mice with the prepared B.1 strain SCTV01C-TM8 vaccine, and immunization of the B.1.351 strain SCTV01C-TM23 vaccine in cynomolgus monkeys, and immunization of the B.1.1.7 strain SCTV01C-TM22 vaccine in mice show that the three vaccines prepared by the invention can generate antibody immune responses with sufficient titer in experimental animals; the immunological evaluation of mice with SCTV 01C-TM8+SCT01C-TM 23 bivalent vaccine and SCTV 01C-TM22+SCT01C-TM 23 bivalent vaccine also shows that the bivalent vaccine has higher and similar neutralization titers on different strains, so that the bivalent vaccine has better broad-spectrum neutralization capability compared with monovalent vaccine, and the neutralization titers of the bivalent vaccine on different variant strains are far higher than the neutralization titers of serum of a convaler on SARS-CoV-2 strain with the genome sequence number of GenBank Accession No. MN 908947.3.
Examples
Example 1: new coronavirus recombinant spike protein extracellular region (S-ECD) trimer protein antigen design, construction of expression vector and protein production
1.1 B.1 Strain sequence (EPI_ISL_ 406862) based S-ECD trimeric protein (SCTV 01C-TM 8) Table
Construction of the vector
SCTV01C-TM8 contains a 3708bp gene fragment, and the target gene fragment is obtained from the template pSE-CoV2-S-ECDTM 2-T4F-primer by PCR splicing. Constructing the recombinant vector into a KpnI+NotI digested pGS3-2-SCT-1 expression vector by an In-fusion method to obtain a pGS3-2-CoV2-S-ECDTM 22-T4F-primer expression vector. The pGS3-2-CoV2-S-ECDTM 8-T4F-primer is used as a template, the SCTV01C-TM8 gene fragment is obtained through PCR amplification, and the expression vector pD2535nt-CoV2-S-ECDTM 8-T4F-primer of the SCTV01C-TM8 is obtained by constructing the expression vector into the pD2535nt-HDP stable strain expression vector of the XbaI+AscI enzyme digestion by an In-fusion method.
Amplification primers
1.2 Table of S-ECD trimeric proteins (SCTV 01C-TM 22) based on the B.1.1.7 strain sequence EPI_ISL_764238
Construction of the vector
SCTV01C-TM22 contains a 3699bp gene fragment, and the target gene fragment is obtained from the template pD2535nt-CoV2-S-ECDTM 8-T4F-primer by PCR splicing. Constructing the recombinant vector into a KpnI+NotI digested pGS5-SCT-1 expression vector by an In-fusion method to obtain a pGS5-CoV2-S-ECDTM 22-T4F-oligomer expression vector. The pGS5-CoV2-S-ECDTM 22-T4F-primer is used as a template, the SCTV01C-TM22 gene fragment is obtained through PCR amplification, and the expression vector pD2535nt-CoV2-S-ECDTM 22-T4F-primer of the SCTV01C-TM22 is obtained by constructing the expression vector into the pD2535nt-HDP stable strain expression vector of XbaI+AscI enzyme digestion by an In-fusion method.
Amplification primers
1.3S-ECD trimeric protein based on the B.1.351 strain sequence EPI_ISL_736940 (SCTV 01C-TM 23)
Construction of expression vectors
The SCTV01C-TM23 comprises 3699bp gene fragment, the target gene fragment is obtained from a template pD2535nt-CoV2-S-ECDTM 8-T4F-primer by PCR splicing, and the target gene fragment is constructed into an Xha I+Asc I enzyme-digested pD2535nt-HDP stable strain expression vector by an In-fusion method to obtain an expression vector pD2535nt-CoV2-S-ECDTM 23-T4F-primer of the SCTV01C-TM 23.
Amplification primers
1.4 Expression and purification of S-ECD trimeric proteins
The target gene constructed above is transferred into HD-BIOP3 (GS-) cells (horizons) by a chemical method, and cultured by adopting an independently developed serum-free culture medium, a cell strain with stable expression is obtained by MSX pressurized screening, and after 14 days of feeding culture, a culture supernatant is obtained by centrifugation and filtration. The culture supernatant was first captured by cation exchange chromatography (POROS XS, thermo) and eluted with high salt buffer; then adopting an anion chromatography (NanoGel-50Q, nanoMicro) combination mode and a mixed anion chromatography (Diamond MIX-A, bogurone) flow-through mode to carry out further purification, and removing impurities related to products and processes; next, viruses were inactivated and removed using low pH incubation and virus removal filtration (Planova), and finally ultrafiltration exchange with ultrafiltration membrane pack (Millipore) to citrate buffer. S-ECD trimer expression levels >500mg/L.
Example 2: analysis of purity and stability of recombinant spike protein extracellular region (S-ECD) trimer protein of novel coronavirus
2.1 analysis of recombinant S-ECD trimer protein purity
The purified recombinant S-ECD trimer protein stock solution is placed in a buffer solution which contains 1.7mM of citric acid, 8mM of sodium citrate, 300mM of sodium chloride and 0.3mg/mL of polysorbate 80 and has the pH of 6.5-7.0, the concentration of the recombinant S-ECD trimer protein stock solution is about 0.6mg/mL, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS polyacrylamide gel electrophoresis, SDS-PAGE) is used for analyzing the primary structure purity and the trimer content of the recombinant S-ECD trimer protein stock solution by size-exclusion high performance liquid chromatography (size-exclusion high performance liquid chromatograph, SEC-HPLC).
Specific procedure for SDS-PAGE: (1) preparation of SDS-PAGE gel: 3.9% of concentrated gum and 7.5% of separation gum; (2) boiling the sample at 100 ℃ for 2min, centrifuging and loading 8 mug; (3) Coomassie brilliant blue staining followed by bleaching. The SEC-HPLC operation steps are: (1) apparatus: a liquid chromatography system (Agilent company, model: agilent 1260), a water-soluble size exclusion chromatography column (Sepax company, model: SRT-C SEC-500 chromatography column); (2) mobile phase: 200mM NaH 2 PO 4 100mM Arginine,pH 6.5,0.01% isopropyl alcohol (IPA); (3) the loading amount is 80 mug; (3) The detection wavelength was 280nM, the analysis time was 35min and the flow rate was 0.15mL/min.
Recombinant SCTV01C-TM8, SCTV01C-TM22, SCTV01C-TM23 proteins are homotrimeric structures due to their non-covalent hydrophobic interactions. The non-reducing SDS-PAGE treatment resulted in monomer molecules with molecular weights of about 148kDa (FIG. 2) with purities of 98.0%, 98.8% and 97.7%, respectively. SEC-HPLC shows that the main peak purity is 96.6 percent, 95.5 percent and 96.6 percent respectively, the proportion content of aggregate and fragment is less, and the molecular weight of the main peak is 530kDa on average. FIG. 2 shows a representative test result of SCTV01C-TM 8.
2.2 morphological analysis of recombinant S-ECD trimer proteins
The morphological characteristics of the recombinant S-ECD trimeric proteins were observed using dynamic light scattering (dynamic light scattering, DLS) and negative staining electron microscopy (Negative staining electron microscopy, EM).
The specific operation steps of DLS are as follows: (1) apparatus: dynamic light scattering instrument (Wyatt Technology Co., model: dynaPro nanoStar); (2) a loading amount of 50. Mu.L; (3) After data collection, data was analyzed using dynamic 7.1.8 software
The specific operation steps of the negative-dyeing electron microscope are as follows: (1) Placing a carbon-coated support film copper net on a sealing film, dripping a drop of recombinant S-ECD trimeric protein sample (about 30 mu m) on the support film, standing for 2-5min, sucking redundant solution from the edge part by using filter paper with a tip, standing for about 10min on the filter paper by the support film (2), placing the dried support film on the sealing film, dripping a drop of uranyl acetate dye solution, dyeing for 90S, sucking redundant dye solution by using filter paper with a tip, clamping on the filter paper, drying for 3h, and observing by a microscope.
Dynamic light scattering results showed that the average diameters of the recombinant SCTV01C-TM8, SCTV01C-TM22, and SCTV01C-TM23 trimeric proteins were 18.4nm, 19.6nm, and 18.4nm, respectively.
The negative electron microscopy results confirm that the recombinant SCTV01C-TM8, SCTV01C-TM22, and SCTV01C-TM23 trimeric proteins exist predominantly in the form of relatively stable pre-fusion trimers, similar to the wild-type full-length trimeric spike protein pre-fusion form (FIG. 3), with a diameter size of about 20nm, consistent with the dynamic light scattering detection diameter size.
2.3 evaluation of recombinant S-ECD trimer protein stability
Evaluation of thermal acceleration stability
After 2 weeks of storage of the recombinant S-ECD trimeric protein at 37℃for 37T2W, the change in trimer content was analyzed by SEC-HPLC and the data are shown in tables 2-2.
The results are shown in Table 2, after the recombinant S-ECD trimer protein is accelerated for 2 weeks at 37 ℃, the SEC-HPLC trimer content change is within +/-1.5%, the aggregate and the fragment are not obviously increased, and good thermal acceleration stability is shown.
TABLE 2 recombinant S-ECD trimer protein thermally accelerated purity changes
Analysis of freeze-thaw stability
The recombinant S-ECD trimeric protein is preserved for 8 hours at the temperature of minus 80 ℃, then is transferred to the temperature of 25 ℃ for thawing for 0.5 hours (F/T-4C), and is repeatedly frozen and thawed for 4 times, and the change of the trimeric content is analyzed by using SEC-HPLC.
The results are shown in Table 3, after repeated freeze thawing of recombinant S-ECD trimer protein, SEC-HPLC trimer content change is within + -1.5%, aggregate and fragment are not increased significantly, and good freeze thawing stability is shown.
TABLE 3 repeated freeze-thaw purity variation of recombinant S-ECD trimeric proteins
Example 3: immunological evaluation of B.1 strain SCTV01C-TM8 vaccine in different mice types
3.1 vaccine preparation
The purified SCTV01C-TM8 protein was pre-diluted to 20. Mu.g/mL or 60. Mu.g/mL with PBS, and the diluted antigen was mixed with MF59 (source: china cell engineering Co., ltd., hereinafter the same) in an equal volume to obtain a final vaccine product containing MF59 with an antigen concentration of 10. Mu.g/mL or 30. Mu.g/mL.
3.2 immunization of mice
Balb/C mice at 6-8 weeks, C57BL/6 mice at 6-8 weeks, and aged Balb/C mice at 7-8 months (source: beijing Vitolith laboratory animal technologies Co., ltd.) were intramuscular injected with 0.1mL of a vaccine product containing MF59 adjuvant. The amounts of the three types of mouse immunogens were 1. Mu.g, 3. Mu.g and 3. Mu.g, respectively. A total of 3 immunizations were performed with an immunization interval of 3 weeks. Orbital sampling was performed every 1 week after the first immunization for 2 weeks, and serum was collected by centrifugation at 4500rpm for 15 minutes for subsequent serological immunoassay.
3.3 determination of mouse immune serum antibody titers and neutralization titers
SCTV01C-TM8 protein 100. Mu.L/Kong Baobei at a concentration of 5. Mu.g/mL was coated overnight at 2-8deg.C in 96-well plates. After the ELISA plate is cleaned and dried, 320 mu L/hole of blocking solution containing 2% BSA is added, and the plate is blocked for more than 1h at room temperature. SCTV01C-TM8 mouse immune serum was subjected to gradient dilution (e.g., 8000X, 16000X, 32000X, 64000X, 128000X, 256000X, 512000X, etc.) using TBST sample diluent containing 0.1% BSA, and non-immunized mouse serum diluted with the same gradient was used as negative control serum. The 96-well ELISA plate is added with 100 mu L/well serum after gradient dilution, and incubated for 1-2 h at room temperature. The plate was washed 3 times and 80ng/mL of rabbit anti-mouse IgG F (ab) was added 2 HRP detection Secondary antibody (source: jackson ImmunoResearch, hereinafter the same) 100. Mu.L/well, incubated at room temperature for 1h. Washing the plate for 5 times, addingThe substrate color development liquid is developed for 10 to 15min,2M H 2 SO 4 After termination, reading OD by ELISA 450 Immune antibody titers were calculated. Antibody titer = greater than negative serum OD 450 X 2.1 maximum dilution factor.
The immune serum with different dilution factors is added into 96-well plates at 50 mu L/well, and then 100TCID is added into 50 mu L/well 50 The pseudovirus of SARS-CoV-2 strain with genome sequence number GenBank Accession No. MN908947.3 (the pseudovirus is prepared by amplifying a replication defective vesicular stomatitis virus (namely VSV delta G-Luc-G) with a VSV-G protein gene replaced by a luciferase reporter gene in a viral genome in a cell line expressing Spike and mutant proteins thereof, prepared by China cell engineering Co., ltd., hereinafter the same), mixing and then placing at 37 ℃ C. And 5% CO 2 Incubate for 1h. The wells without serum to which the pseudoviruses were added were used as positive controls, and the wells without serum and pseudoviruses were used as negative controls. After incubation, 100. Mu.L/well was inoculated with 3X 10 4 293FT-ACE2 cells are mixed uniformly and placed at 37 ℃ and 5 percent CO 2 And (5) standing and culturing for about 20 hours in an incubator. After the completion of the culture, the culture supernatant was removed, and 1X Passive lysis buffer was added to 50. Mu.L/well, followed by mixing and cell lysis. The neutralization rate was calculated by taking 40. Mu.L/Kong Zhuairu of 96-well full-whitening chemiluminescent plate, adding luciferase substrate to 40. Mu.L/well using LB960 microplate type luminometer and measuring the luminescence value (RLU). Neutralization% = (positive control RLUs-sample RLUs)/(positive control RLUs-negative control RLUs) ×100%, IC was calculated according to the Reed-Muench formula 50 I.e. NAT 50 。
The results of antibody titers after immunization are shown in FIG. 4, and SCTV01C-TM8 induced high titers of antibody immune responses in 3 mouse models, namely, 6-8 week Balb/C mice, 6-8 week C57BL/6 mice and 7-8 month old Balb/C mice. The result of neutralization titers of the SARS-CoV-2 strain pseudovirus having genomic sequence number GenBank Accession No. Mn908947.3 is shown in FIG. 5, and SCTV01C-TM8 induces high titers of pseudovirus neutralizing antibodies in 3 mouse models. The neutralization titer of the pseudo virus after immunization is obviously higher than that of serum (HCS) of a convalescence patient with a new coronal infection on a SARS-CoV-2 strain with a genome sequence number of GenBank Accession No. MN908947.3, and the neutralization titers of the pseudo virus after 3 days and 7 days are 33.3 times, 13.9 times and 3.9 times of that of the HCS respectively.
3.4 vaccine-induced cellular immune response detection
Mouse spleen cells were isolated and 100. Mu.L/well of mouse spleen cells were inoculated into a pre-treated ELISPot well plate (source: mabtech, hereinafter the same) at a cell inoculation density of 2X 10 5 cells/wells. Then 100. Mu.L/well of RBD, S1, S2 or S protein peptide library (15 amino acids/peptide fragment, 11 amino acids overlapping each other, from Beijing Zhongke sub-optical Biotech Co., ltd., hereinafter the same) was added at a final concentration of 2. Mu.g/mL, and the mixture was placed at 37℃and 5% CO 2 Incubate in incubator for about 20h. After the incubation, the cell supernatant of the ELISpot well plate was removed, the plate was washed 5 times with PBS, and then 100 μl/well of diluted detection antibody was added. After 2h incubation, the plates were washed 5 times with PBS and 100. Mu.L/well diluted Strepitavidin-ALP (1:1000) was added. After 1h incubation at room temperature, the plates were washed 5 times with PBS, followed by 100. Mu.L/well of BCIP/NBT-plus substrate filtered with 0.45 μm filter. Color development is carried out at room temperature for 10-30 min in a dark place until clear spots appear and is stopped by deionized water. And (3) placing the ELISPot pore plate at a shade place at room temperature, naturally airing the ELISPot pore plate, and carrying out result analysis by adopting an enzyme-linked spot analyzer. Every 10 6 SFC (Spot-forming cells) of mouse spleen cells represents the number of antigen-specific IFN-gamma or IL-4 secretion-positive T cells, and data statistics were performed by GrapPad Prism software.
As a result, as shown in FIG. 6, SCTV01C-TM8 induced higher Th1 (IFN-. Gamma.) and Th2 (IL-4) cellular responses against RBD, S1, S2 and S polypeptide pools in each of the 3 mouse models after 3 days of immunization.
Example 4: immunological evaluation of SCTV01C-TM23 vaccine against 1.351 Strain in cynomolgus monkey
4.1 vaccine preparation
The purified SCTV01C-TM23 protein is pre-diluted to 120 mug/mL by PBS, and the diluted antigen is mixed with MF59 in equal volume to obtain a vaccine finished product containing MF59 with the antigen final concentration of 60 mug/mL.
4.2 cynomolgus monkey immunization
Cynomolgus monkey (source: guangxi androstane primate laboratory animal culture development Co., ltd.) is intramuscular injected with 0.5mL of a final vaccine product containing MF59 adjuvant, with an antigen content of 30. Mu.g. A total of 2 immunizations were performed with an immunization interval of 3 weeks. Venous blood sampling is carried out every 1 week after the first immunization for 2 weeks, and the non-anticoagulation centrifuge tube is placed in ice water for pre-incubation before use. After blood sample collection, transferring the blood sample into a non-anticoagulant centrifuge tube for temporary storage, and then centrifuging at the temperature of 2-8 ℃ for 10min at 3000 Xg. The isolated serum samples were subjected to subsequent serological immunoassays. Blood samples collected using anticoagulation tubes were used for routine isolation of peripheral blood lymphocytes (PBMCs) for cellular immunodetection.
4.3 determination of the immune serum antibody titers and neutralization titers of cynomolgus monkeys
ELISA was used to detect anti-SCTV 01C-TM23 or Foldon partial specific IgG antibodies in cynomolgus serum. The SCTV01C-TM23 was diluted to 2. Mu.g/mL with the coating solution and added to the ELISA plate at 100. Mu.L/well, and incubated overnight at 2-8 ℃. The plate was washed 3 times with 3 mu L/well of 2% casein-PBST blocking solution and blocked at room temperature for at least 1h. The plate was washed 3 times, 0.1% casein-PBST was used as a sample diluent to dilute the sample 1000-fold, and the immune serum was subjected to gradient dilution (e.g., 8000X, 16000X, 32000X, 64000X, 128000X, 256000X, 512000X, etc.) with 1% mixed blank cynomolgus monkey serum, 100. Mu.L/well in a typesetting, sealing plates, and incubation with shaking at room temperature for about 2 hours. Washing the plate for 3 times, diluting secondary antibody Goat pAb to Mk IgG (HRP) (source: abcam, hereinafter the same) to 15ng/mL with 0.5% casein-PBST secondary antibody diluent, adding 100 μl/well into the ELISA plate, and incubating at room temperature in the absence of light for about 1h; washing the plate for 3 times, fully and uniformly mixing the color development liquid A, B liquid according to the ratio of 1:1, and incubating for about 15 minutes at room temperature and in a dark place at 100 mu L/Kong Dianyang; 50 mu L/Kong Zhongzhi solution is added, and the antibody titer is calculated by reading with a microplate reader at a wavelength of 450 nm. Antibody titer = greater than negative serum OD 450 X 2.1 maximum dilution factor.
The immune serum was added at 50 μl/well to 96-well plates at different dilutions. Then 50 mu L/well of 100-200 TCID is added 50 The pseudovirus strain B.1.351 was mixed and incubated in a 5% CO2 incubator at 37℃for 1h. With the addition of pseudovirus serum-free cell wells as positive control, with noCell wells containing serum and pseudoviruses were negative controls. After incubation, 100. Mu.L/well was inoculated with 2X 10 4 Huh-7 cells were mixed and placed at 37℃in 5% CO 2 And (5) standing and culturing for about 20 hours in an incubator. After the completion of the culture, the culture supernatant was removed, and 1X Passive lysis buffer was added to 50. Mu.L/well, followed by mixing and cell lysis. The neutralization rate was calculated by taking 40. Mu.L/Kong Zhuairu of 96-well full-whitening chemiluminescent plate, adding luciferase substrate to 40. Mu.L/well using LB960 microplate type luminometer and measuring the luminescence value (RLU). Neutralization% = (positive control RLUs-sample RLUs)/(positive control RLUs-negative control RLUs) ×100%, IC was calculated according to the Reed-Muench formula 50 I.e. the neutralization potency NAT 50 。
As shown in FIG. 7, the SCTV01C-TM23 can induce high-titer antibody immune response in cynomolgus monkey immunization, and B.1.351 strain pseudovirus neutralization shows that the neutralization titer is gradually increased along with the time extension after immunization, and the titer peak value is reached 7-14 days after 2 days of immunization.
4.4 vaccine-induced cellular immune response detection
The literature shows that there is a conserved T cell epitope between different variants of SARS-CoV-2 (Redd, A.D., et al, CD8+ T cell responses in COVID-19 convalescent individuals target conserved epitopes from multiple prominent SARS-CoV-2 circulating variants.medRxiv,2021:p.2021.02.11.21251585; tarke, A.et al, negligible impact of SARS-CoV-2 variants on CD4+and CD8+T cell reactivity in COVID-19 exposed donors and vaccinees.bioRxiv,2021:p.2021.02.27.433180), and thus T cell immunodetection is still performed using a pool of polypeptides of SARS-CoV-2 strain sequence with genomic sequence No. GenBank Accession No. MN 908947.3. Separating monkey PBMCs by density gradient centrifugation, inoculating 100 μL/well of PBMCs cells into pretreated ELISPot orifice plate with cell inoculation density of 2.5X10 5 cells/well, then 100. Mu.L/well of RBD, S1 or S protein peptide library was added to a final concentration of 2. Mu.g/mL, and the mixture was placed at 37℃in 5% CO 2 Incubate in incubator for about 20h. After incubation, the cell supernatant of the ELISPot well plate was removed, the plate was washed 5 times with PBS, and 100. Mu.L/well was then added to the diluted final concentration of 1. Mu.g/mLAn antibody. After 2 hours incubation, the plates were washed 5 times with PBS, 100. Mu.L/well diluted Strepitavidin-ALP (1:1000) was added, and after 1 hour incubation at room temperature, the plates were washed 5 times with PBS. Subsequently 100. Mu.L/well of BCIP/NBT-plus substrate filtered with a 0.45 μm filter was added, developed to clear spots at room temperature in the dark for 10-30 min and stopped with deionized water. And (3) placing the ELISPot pore plate at a shade place at room temperature, naturally airing the ELISPot pore plate, and carrying out result analysis by adopting an enzyme-linked spot analyzer. Every 10 6 SFC (Spot-forming cells) of PBMC cells of (E) represents antigen-specific IFN-. Gamma.or TL-4 secretion-positive T cell numbers, and data statistics were performed by GrapPad Prism software.
As a result, as shown in FIG. 8, SCTV01C-TM23 induced Th1 (IFN-. Gamma.) and Th2 (IL-4) cellular responses to RBD, S1, and S polypeptide pools in cynomolgus monkeys after 7 days of 2-immunization.
4.5 Monkey immunogenicity of Foldon and mutation points in S-ECD trimer molecules
Antibody titers against Foldon after SCTV01C-TM23 immunization of cynomolgus monkeys were detected using Foldon-containing RSV F recombinant protein (RSV-F-Foldon) as coating antigen, reference example 4.3. As shown in FIG. 9, the Foldon-induced immunogenicity of the S-ECD trimer molecules was very weak, and the cynomolgus monkey' S immune titer against SCTV01C-TM23 was 54-76 times the Foldon immune titer at different time points.
The effect of Foldon introduction and mutant engineering on T cell immune responses was examined using the RSV-F-Foldon protein or a peptide library containing the "6P" mutation and Furin site mutation, as described in reference example 4. The results are shown in FIG. 10, and after 7 days of 2 days of immunization, foldon had very low T cell immune responses in cynomolgus monkeys, and "6P" and Furin site mutant engineering had no effect on the cell immune responses.
In conclusion, 3 modifications made to stabilize the S-ECD trimer structure are all relatively weak in immunogenicity and have little immune interference to the S-ECD.
Example 5: b.1.1.7 immunological evaluation of SCTV01C-TM22 vaccine against Strain in mice
5.1 vaccine preparation
The purified SCTV01C-TM22 protein is pre-diluted to 20 mug/mL by PBS, and the diluted antigen is mixed with MF59 in equal volume to obtain a vaccine finished product containing MF59 with the antigen final concentration of 10 mug/mL.
5.2 immunization of mice
6-8 weeks C57BL/6 mice (source: beijing vitamin Toril laboratory animal technologies Co., ltd.) were intramuscular injected with 0.1mL of the final vaccine product containing MF59 adjuvant, with an antigen content of 1. Mu.g. A total of 3 immunizations were performed with an immunization interval of 2 weeks. Orbital sampling was performed every 1 week after the first immunization for 2 weeks, and serum was collected by centrifugation at 4500rpm for 15 minutes for subsequent serological immunoassay.
5.3 determination of mouse immune serum antibody titers and neutralization titers
Antibody titers were determined after SCTV01C-TM22 immunization of C57BL/6 mice using SCTV01C-TM22 as coating antigen with reference to example 3.3.
50 mu L/well of 2 immune serum free of 7 days with different dilution times are added into a 96-well plate, and then 100-200 TCID is added into 50 mu L/well 50 B.1.1.7 strain pseudovirus, mixing, and placing at 37deg.C and 5% CO 2 Incubate for 1h. The wells without serum to which the pseudoviruses were added were used as positive controls, and the wells without serum and pseudoviruses were used as negative controls. After incubation, 100. Mu.L/well was inoculated with 2X 10 4 Huh-7 cells were mixed and placed at 37℃in 5% CO 2 And (5) standing and culturing for about 20 hours in an incubator. After the completion of the culture, the culture supernatant was removed, and 1X Passive lysis buffer was added to 50. Mu.L/well, followed by mixing and cell lysis. The neutralization rate was calculated by taking 40. Mu.L/Kong Zhuairu of 96-well full-whitening chemiluminescent plate, adding luciferase substrate to 40. Mu.L/well using LB960 microplate type luminometer and measuring the luminescence value (RLU). Neutralization% = (positive control RLUs-sample RLUs)/(positive control RLUs-negative control RLUs) ×100%, IC was calculated according to the Reed-Muench formula 50 I.e. the neutralization potency NAT 50 。
B.1.1.7 strain pseudovirus neutralization titers the results are shown in FIG. 11, where SCTV01C-TM22 immunization in C57BL/6 mice induced high titers of pseudovirus neutralizing antibodies. The neutralization titer after immunization is higher than that of the serum (HCS) of a convalescence patient with a new crown infection to the SARS-CoV-2 strain with the genome sequence number of GenBank Accession No. MN908947.3, and the neutralization titer of 3 days is 56.5 times of that of HCS.
5.4 Broad spectrum neutralizing Activity of SCTV01C-TM22 vaccine
The pseudovirus neutralization titers of SCTV01C-TM22 vaccine 2 immune serum against other variants (B.1 strain, B.1.351 strain, P.1 strain, and B.1.429 strain and/or B.1.427 strain) were determined by reference to example 5.3. As shown in FIG. 12, the SCTV01C-TM22 vaccine immune serum still had a higher neutralization potency for the B.1 strain, while the neutralization potency for the B.1.351 strain, the P.1 strain, and the B.1.429 strain and/or the B.1.427 strain was greatly reduced.
The neutralization activity detection result of the SCTV01C-TM22 vaccine shows that the monovalent vaccine can not generate high-titer neutralizing antibodies to variant strains, and has poor broad-spectrum neutralization activity. To cope with viral variation, the cross-protective ability of the vaccine against both the original strain and the various variant strains was enhanced, bivalent vaccines containing the S-ECD proteins of the different variant strains were prepared in the following examples, and the broad-spectrum neutralization ability of the bivalent vaccines was subjected to immune evaluation.
Example 6: immunological evaluation of SCTV 01C-tm8+sct01c-TM 23 bivalent vaccine in mice
6.1 vaccine preparation
The purified SCTV01C-TM8 and SCTV01C-TM23 proteins are pre-diluted to 20 mug/mL by PBS, and the diluted antigen is mixed with MF59 in equal volume to obtain a monovalent vaccine finished product containing MF59 with the antigen final concentration of 10 mug/mL.
The SCTV01C-TM8 and SCTV01C-TM23 proteins were pre-diluted to 40. Mu.g/mL with PBS, and the diluted antigens were mixed in equal volumes to obtain mixed antigen samples with final antigen concentrations of 20. Mu.g/mL. And mixing the mixed antigen sample with MF59 in equal volume to obtain bivalent vaccine product TM8+TM23 containing MF59 with final concentration of SCTV01C-TM8 and SCTV01C-TM23 antigen of 10 mug/mL.
6.2 immunization of mice
6-8 weeks C57BL/6 mice (source: beijing Vitolihua laboratory animal technologies Co., ltd.) were intramuscular injected with 0.1mL of a monovalent or bivalent vaccine product containing MF59 adjuvant, the monovalent vaccine antigen amount was 1. Mu.g, and the bivalent vaccine antigen amounts were 1. Mu.g, respectively. A total of 3 immunizations were performed with an immunization interval of 3 weeks. Orbital sampling was performed every 1 week after the first immunization for 2 weeks, and serum was collected by centrifugation at 4500rpm for 15 minutes for subsequent serological immunoassay.
6.3 Determination of neutralization titers of TM8+TM23 bivalent vaccine immune serum on different variants
The neutralization titers of 7-day immune sera of bivalent vaccine 2 against pseudoviruses of different variants (strain B.1, strain B.1.351, strain B.1.1.7, strain P.1, strain B.1.429 and/or strain B.1.427) were determined according to example 5.3. As shown in FIG. 13, the SCTV01C-TM8 monovalent vaccine has higher neutralization potency for B.1 strain and B.1.1.7 strain pseudoviruses, and lower neutralization potency for B.1.351 strain and P.1 strain pseudoviruses, and the reduction fold is about 5-11 times of the neutralization potency of B.1 strain. The SCTV01C-TM23 monovalent vaccine has higher neutralization titers for pseudoviruses of the B.1.351 strain and the P.1 strain, and has low neutralization titers for pseudoviruses of the B.1 strain, the B.1.1.7 strain and the B.1.429 strain and/or the B.1.427 strain, and the reduction multiple is about 4-12 times of the neutralization titers of the B.1.351 strain. The TM8+TM23 bivalent vaccine has higher and similar neutralization titers for different strains, so that the vaccine has better broad-spectrum neutralization capacity compared with a monovalent vaccine. The neutralization titer of the bivalent vaccine on different variant strains is far higher than that of the serum of a convalescent patient on SARS-CoV-2 strain with the genome sequence number of GenBank Accession No. Mn 908947.3.
6.4 vaccine-induced cellular immune response detection
T cell immune responses were examined in mice immunized 3 for 7 days using the RBD, S1, S peptide pool of SARS-CoV-2 strain sequence having genomic sequence No. GenBank Accession NC-045512 and the mixed peptide pool of SARS-CoV-2 strain S peptide pool having genomic sequence No. GenBank Accession M908947.3 and B.1.351 strain differential polypeptide (TM 23-mix) as described in reference example 3.4. As a result, as shown in FIG. 14, the SARS-CoV-2 strain peptide library having genomic sequence No. GenBank Accession No. MN908947.3 stimulated both monovalent and bivalent vaccine had higher T cell responses and similar T cell immune responses after immunization of mouse spleen cells. The S peptide library and the S+TM23-mix peptide library stimulate similar T cell responses, which indicates that the B.1.351 strain difference polypeptide has no T cell immune response, and further proves that the variant strains have conserved T cell epitopes.
Example 7: immunological evaluation of SCTV 01C-tm22+sct01c-TM 23 bivalent vaccine in mice
7.1 vaccine preparation
The purified SCTV01C-TM22 and SCTV01C-TM23 proteins are pre-diluted to 20 mug/mL by PBS, and the diluted antigen is mixed with MF59 in equal volume to obtain a monovalent vaccine finished product containing MF59 with the antigen final concentration of 10 mug/mL.
The SCTV01C-TM22 and SCTV01C-TM23 proteins were pre-diluted to 40. Mu.g/mL with PBS, and the diluted antigens were mixed in equal volumes to obtain mixed antigen samples with final antigen concentrations of 20. Mu.g/mL. And mixing the mixed antigen sample with MF59 in equal volume to obtain bivalent vaccine product TM22+TM23 containing MF59 with final concentration of SCTV01C-TM22 and SCTV01C-TM23 antigen of 10 mug/mL.
7.2 immunization of mice
6-8 weeks C57BL/6 mice (source: beijing Vitolihua laboratory animal technologies Co., ltd.) were intramuscular injected with 0.1mL of monovalent or bivalent vaccine finished product containing MF59 adjuvant, the monovalent vaccine antigen amount was 1. Mu.g, and the bivalent vaccine antigen amounts were 1. Mu.g, respectively (the bivalent vaccine antigen amounts of example 7.5 were 0.5. Mu.g, respectively). A total of 2 immunizations were performed with an immunization interval of 2 weeks. Orbital sampling was performed every 1 week after the first immunization for 2 weeks, and serum was collected by centrifugation at 4500rpm for 15 minutes for subsequent serological immunoassay.
7.3 Determination of neutralization titers of SCTV01C-TM22+TM23 bivalent vaccine immune serum on different variant strains (I)
The pseudovirus neutralization titers of bivalent vaccine 2 immune serum against 7 days for the different variants (b.1 strain, b.1.351 strain, b.1.1.7 strain) were determined with reference to example 5.3. As a result, as shown in FIG. 15, the SCTV01C-TM22 monovalent vaccine had a higher neutralization potency for the pseudoviruses of the B.1.1.7 strain and the B.1 strain, and a reduced neutralization potency for the B.1.351 strain by about 8.8 times the neutralization potency of the B.1.1.7 strain. The neutralization titer of the SCTV01C-TM23 monovalent vaccine on the B.1.351 strain is higher, and the neutralization titers of the SCTV01C-TM23 monovalent vaccine on the B.1.1.7 strain and the B.1 strain are reduced by 6.4 times and 5.1 times of the neutralization titers of the B.1.351 strain respectively. The TM22+TM23 bivalent vaccine has higher and similar neutralization titers for different strains, so that the vaccine has better broad-spectrum neutralization capacity compared with a monovalent vaccine. The neutralization titer of the bivalent vaccine on different variant strains is far higher than that of the serum of a convalescent patient on SARS-CoV-2 strain with the genome sequence number of GenBank Accession No. Mn 908947.3.
7.4 Determination of neutralizing titers of SCTV01C-TM22+TM23 bivalent vaccine immune serum against different variants (II)
The neutralization titers of 7-day immune serum of bivalent vaccine 2 against pseudoviruses of different variants of SARS-CoV-2 (B.1.526, C.37, B.1.621, B.1.618, C.36.3 and 20I/484Q strains) were determined according to reference example 5.3. As shown in FIG. 16, serum of SCTV01C-TM22+TM23 bivalent vaccine immunized group mice was used for neutralizing antibody titer NAT against pseudovirus of B.1.526 strain, C.37 strain, B.1.621 strain, B.1.618 strain, C.36.3 strain and 20I/484Q strain 50 The Geometric Mean (GMT) is raised to a certain degree compared with the monovalent vaccine SCTV01C-TM22 and SCTV01C-TM23 (the raising times are respectively 2.8 times, 2.6 times, 3.6 times, 4.2 times, 2.1 times and 0.8 times, and 1.8 times, 3.2 times, 1.5 times, 1.7 times, N/A and 2.0 times), which shows that the SCTV01C-TM22+TM23 bivalent vaccine has higher and similar neutralization titers for different strains, and therefore has better broad-spectrum neutralization capacity compared with the monovalent vaccine.
7.5 Determination of neutralizing titers of SCTV01C-TM22+TM23 bivalent vaccine immune serum against different variants (III)
The neutralization titers of the 21-day immune serum of bivalent vaccine 2 against pseudoviruses of the different variants of SARS-CoV-2Omicron (strain BA.1, strain BA.1.1, strain BA.2) were examined with reference to example 5.3. As shown in FIG. 17, serum of SCTV01C-TM22+TM23 bivalent vaccine immunized group mice was directed against Omicron BA.1 strain, BA.1.1 strain, BA.2 strain pseudovirus neutralizing antibody titer NAT 50 The Geometric Mean (GMT) was elevated to some extent compared with monovalent vaccine TM8 (18.3 fold, 25.3 fold, 10.4 fold, respectively), indicating that the SCTV01C-TM22+TM23 bivalent vaccine had higher neutralization titers against different variant strains of SARS-CoV-2Omicron, and therefore had better broad-spectrum neutralization capacity than monovalent vaccine.
In summary, bivalent vaccines have broad-spectrum neutralizing ability against different variant strains, and are hopeful to generate cross-protective ability against various variant strains, and improve the protection rate against variant infection compared with monovalent vaccines.
While the invention has been described in detail in the foregoing description and with reference to the embodiments, it is for an understanding of the nature and character of the invention to be considered as obvious and obvious to those skilled in the art, without departing from the spirit or scope of the appended claims.
All documents cited in this specification are incorporated by reference in their entirety.
Sequence list

Claims (16)

  1. A method of increasing the immunogenicity/antigen trimer stability of an ECD antigen of a mutant strain of SARS-CoV-2 by constructing an ECD antigen comprising at least any one of the amino acid sequences set forth in SEQ ID No. 8, SEQ ID No. 12 or SEQ ID No. 16, or an immunogenic fragment and/or immunogenic variant thereof, whereby the ECD is in the form of a trimer in a stable precision conformation.
  2. The method of claim 1, wherein the mutant strain is a high risk mutant strain comprising at least any of T19 24S, Δ25/27, H49 67V, Δ69/70, T95D, Δ143/145, Δ145-146, N211I, Δ212/212, V213 339 346 371 373 375 405 417 452 452 477 478 484 484 484 484 493 496 498 501 505 547 614 655 679 681 764 796 856 954 969 981F.
  3. The method of claim 1 or 2, wherein the strain comprises at least one of a b.1 strain, a b.1.351 strain, a b.1.1.7 strain, a p.1 strain, a b.1.427 strain, a b.1.429 strain, a b.1.526 strain, a c.37 strain, a b.1.621 strain, a b.1.618 strain, a c.36.3 strain, a 20I/484Q strain, a ba.1 strain, a ba.1.1 strain, and a ba.2 strain.
  4. The method of claim 1, wherein the ECD antigen is co-administered to the subject with one or more adjuvants selected from the group consisting of: aluminum adjuvants, oil emulsion adjuvants, toll-like receptor (TLR) agonists, combinations of immunopotentiators, microbial adjuvants, propolis adjuvants, levamisole adjuvants, liposomal adjuvants, chinese herbal adjuvants, and small peptide adjuvants;
    Preferably, the oil emulsion adjuvant comprises a squalene component;
    toll-like receptor (TLR) agonists comprise CpG or monophosphoryl lipid a (MPL) adsorbed on an aluminium salt; and the immune enhancer comprises QS-21 and/or MPL.
  5. A method of increasing the immunogenicity/antigen trimer stability of an ECD antigen of a mutant strain of SARS-CoV-2 by constructing a polynucleotide encoding at least any one of the amino acid sequences set forth in SEQ ID No. 8, SEQ ID No. 12 or SEQ ID No. 16, or an immunogenic fragment and/or immunogenic variant thereof, thereby expressing a stable, pre-fusion conformational form of the ECD.
  6. The method of claim 5, wherein the mutant strain is a high risk mutant strain comprising at least any of T19 24S, delta25/27, H49 67V, delta69/70, T95 142D, delta143/145, delta145-146, N211I, delta212/212, V213 339 346 371 373 375 405 417 452 452 477 478 484 484 484 484 493 496 498 501 505 547 614 655 679 681 764 796 856 954 969 981F.
  7. The method of claim 5 or 6, wherein the strain comprises at least one of a b.1 strain, a b.1.351 strain, a b.1.1.7 strain, a p.1 strain, a b.1.427 strain, a b.1.429 strain, a b.1.526 strain, a c.37 strain, a b.1.621 strain, a b.1.618 strain, a c.36.3 strain, a 20I/484Q strain, a ba.1 strain, a ba.1.1 strain, and a ba.2 strain.
  8. An immunogenic protein/peptide of SARS-CoV-2 mutant strain ECD with improved stability of an immunogenic/antigen trimer, characterized in that the immunogenic protein/peptide comprises an amino acid sequence of at least any one of SEQ ID No. 8, SEQ ID No. 12 or SEQ ID No. 16, or an immunogenic fragment and/or immunogenic variant thereof, the ECD immunogenic protein/peptide being in the form of a trimer in a stable precision conformation.
  9. The immunogenic protein/peptide of claim 8, wherein the mutant strain is a high risk mutant strain comprising at least any one of T19 24S, Δ25/27, H49 67V, Δ69/70, T95 142D, Δ143/145, Δ145-146, N211I, Δ212/212, V213 339 346 346 371 373 376 405 408 417 452 452 477 478 484 484 484 490 493 496 498 501 505 547 614 655 679 681 764 796 856 954 969 981F.
  10. The immunogenic protein/peptide of claim 8 or 9, wherein the strain comprises at least one of a b.1 strain, a b.1.351 strain, a b.1.1.7 strain, a p.1 strain, a b.1.427 strain, a b.1.429 strain, a b.1.526 strain, a c.37 strain, a b.1.621 strain, a b.1.618 strain, a c.36.3 strain, a 20I/484Q strain, a ba.1 strain, a ba.1.1 strain, and a ba.2 strain.
  11. A polynucleotide encoding the immunogenic protein/peptide of claim 8,
    preferably, the nucleotide sequence comprising at least any one of SEQ ID No. 7, SEQ ID No. 11 or SEQ ID No. 15.
  12. An immunogenic composition comprising
    At least one immunogenic protein/peptide according to claim 8, or
    At least one polynucleotide according to claim 11, and
    any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent;
    optionally, an adjuvant is included.
  13. The immunogenic composition of claim 12, comprising
    The amino acid sequences shown in SEQ ID No. 12 and SEQ ID No. 16, or immunogenic fragments and/or immunogenic variants thereof, or
    The amino acid sequences shown in SEQ ID No. 8 and SEQ ID No. 16, or an immunogenic fragment and/or immunogenic variant thereof.
  14. The immunogenic composition of claim 12 or 13, wherein the adjuvant is selected from one or more of the following:
    aluminum adjuvants, oil emulsion adjuvants, toll-like receptor (TLR) agonists, combinations of immunopotentiators, microbial adjuvants, propolis adjuvants, levamisole adjuvants, liposomal adjuvants, chinese herbal adjuvants, and small peptide adjuvants;
    Preferably, the oil emulsion adjuvant comprises a squalene component;
    toll-like receptor (TLR) agonists comprise CpG or monophosphoryl lipid a (MPL) adsorbed on an aluminium salt; and the immune enhancer comprises QS-21 and/or MPL.
  15. Use of the immunogenic protein/peptide of claim 8, the polynucleotide of claim 11 and the immunogenic composition of any one of claims 12-14 to prevent or treat a disease caused by a SARS-CoV-2 mutant strain.
  16. Use of the immunogenic protein/peptide of claim 8, the polynucleotide of claim 11 and the immunogenic composition of any one of claims 12-14 in the manufacture of a vaccine or medicament for preventing or treating a disease caused by a SARS-CoV-2 mutant strain.
CN202280034785.7A 2021-05-31 2022-05-27 Method for improving immunogenicity of SARS-CoV-2 mutant strain ECD antigen/antigen trimer stability Pending CN117295771A (en)

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