CN115894713A

CN115894713A - Heterotrimeric fusion proteins, compositions and uses thereof

Info

Publication number: CN115894713A
Application number: CN202211157307.3A
Authority: CN
Inventors: 刘滨磊; 许雁; 诺曼·吉利卡
Original assignee: Wuhan Binhui Biotech Co ltd
Current assignee: Wuhan Binhui Biotech Co ltd
Priority date: 2022-09-22
Filing date: 2022-09-22
Publication date: 2023-04-04
Anticipated expiration: 2042-09-22
Also published as: CN115894713B

Abstract

Heterotrimeric fusion proteins, compositions, and uses thereof are disclosed herein. The heterotrimeric fusion protein, namely PF-D trimer, has stable and novel pre-fusion conformation of coronavirus S protein; on the other hand, the application provides a composition, the composition comprises a PF-D trimer and an adjuvant, the adjuvant comprises CpG1018 and aluminum hydroxide, the use amount of the heterotrimeric fusion protein as a vaccine antigen is reduced, an immune system is rapidly activated, immune response is enhanced, and the like, the functions of the heterotrimeric fusion protein and the vaccine antigen are effectively combined by the composition for preparing efficient vaccines or medicines, and the effects of the vaccines or medicines prepared by the prior art in defense and treatment of infectious diseases can be greatly improved.

Description

Heterotrimeric fusion proteins, compositions and uses thereof

Technical Field

The present application relates to the field of biotechnology, in particular to heterotrimeric fusion proteins, compositions and uses thereof.

Background

The emergence of the novel coronavirus (SARS-CoV-2) has, until now, still had a major impact on global public health. The outbreak of epidemic studies by humans also enters the stage of albefaction.

SARS-CoV-2 is the seventh member of the family of coronaviridae that are currently found to infect humans, and SARS-CoV-2 is a single-stranded positive-strand RNA, enveloped, and the largest RNA virus. When SARS-CoV-2 invades human body, spike glycoprotein (S protein for short) plays a great role. The S protein is a large trimeric transmembrane glycoprotein, has a large amount of glycosylation modification, forms a special corolla structure on the surface of a virus, is the most important surface membrane protein of the coronavirus, and contains two subunits S1 and S2. Wherein S1 mainly comprises Receptor Binding Domain (RBD) which is responsible for recognizing cell receptors. S2 contains the essential elements required for the membrane fusion process. The S protein has the functions of combining virus with host cell membrane receptor and fusing membrane, and is an important action site of host neutralizing antibody and a key target of vaccine design. After the S protein is combined with a receptor on the cell surface, the virus envelope and the cell membrane are integrated into a whole, so that genetic materials in the virus are injected into the cell, and the purpose of infecting the cell is achieved.

When the S protein binds to the receptor of the host cell, it causes the S1 and S2 subunits to dissociate. The conformation of the S2 subunit becomes a highly stable post-fusion structure, so that the S protein has both pre-fusion and post-fusion conformations. After the S protein is fused with receptor cells, RBDs with immune epitope structural parts, which are positioned at the outermost ends of the tunica capsularis, of the S protein polypeptide are moved to the lower part and covered due to the combination with the receptor, and non-epitope parts are stably exposed at the outer ends. The new corona vaccine prepared conventionally, namely the recombinant novel coronavirus S protein only generates strong and stable conformation after fusion, so that the immunogenicity of the vaccine antigen is greatly reduced, and the vaccine is difficult to play.

An adjuvant, also called an immunological adjuvant (immunological adjuvant), refers to a substance that can nonspecifically enhance or modify the specific immune response of the body to a matching antigen, enhance the immunogenicity of the antigen or modify the type of immune response, but is not antigenic by itself. Such as inactivated virus vaccines, subunit vaccines and recombinant protein vaccines, if only antigen components are contained in the vaccine components, the immunogenicity of the vaccine components is often not strong enough, and the vaccine components are likely to cause insufficient immune response when injected into a human body and are not sufficient to resist the invasion of corresponding pathogens, so that the vaccine components are generally required to be injected together with adjuvants to play a sufficient protection role when applied.

The most commonly used adjuvants at present are aluminum adjuvants, mainly comprising three of aluminum phosphate, aluminum hydroxide and aluminum potassium sulfate, wherein the most commonly used adjuvants are aluminum hydroxide and aluminum phosphate. The aluminum adjuvant is widely used for HPV vaccines, inactivated poliovirus vaccines, diphtheria-pertussis-tetanus vaccines, hepatitis A vaccines, hepatitis B vaccines, rabies vaccines, anthrax vaccines, haemophilus influenzae type b vaccines, haemophilus haemophilus binding vaccines, streptococcus pneumoniae vaccines, meningitis vaccines, veterinary vaccines (such as foot-and-mouth disease virus vaccines and botulism vaccines) and the like. The immunogenicity enhancing effects of different adjuvants on different vaccines are different, the safety of the adjuvants on human bodies is also different, a plurality of adjuvant varieties exist in the market, and the screening of suitable varieties also becomes the current research hotspot.

In addition, new coronaviruses have been mutated to form a variety of varieties, the Delta strain and the Omicron strain are the most famous, but the vaccine strains used in the market at present still continue to use the original strains, so that the vaccines provided by the prior art have limited effect on inhibiting the Delta strain and the Omicron strain.

For the above reasons, it is highly desirable to develop a recombinant protein having a stable structure and properties, such as a virus surface glycoprotein trimer structure before binding to a host cell receptor, and to select an adjuvant suitable for enhancing the immunogenicity of the recombinant protein in vivo, to develop an effective vaccine or drug for inhibiting or treating novel coronavirus pneumonia, particularly novel coronavirus pneumonia caused by the Delta strain and the Omicron strain and the respective variant strains, which are key and difficult points to solve the above technical problems.

Disclosure of Invention

Most multimeric proteins, the monomers of which have a functional domain formed by a polypeptide, play an important role in the formation and stabilization of oligomerization conformations of protein bodies, and in the formation of multimeric proteins, such as dimers, trimers, etc., said functional domains are the homologous and heterologous functional domains. The present application provides a recombinant fusion protein formed from protein monomers containing heterologous functional domains (hereinafter "heterotrimeric domains"), i.e., heterotrimeric fusion proteins. The heterotrimeric fusion protein has a stable protein conformation, and on the basis, the successful synthesis of the heterotrimeric fusion protein provides a feasible solution for preparing effective vaccine antigens or immunological drugs (such as antibody drugs and functional cytokines) for defending or treating novel coronavirus pneumonia.

In combination with the above concepts, the present inventors provide heterotrimeric fusion proteins, compositions and uses thereof to solve some of the above technical problems.

In a first aspect, the present application provides a heterotrimeric fusion protein, namely a PF-D Trimer (PF-D-Trimer), having an amino acid sequence shown in SEQ ID No. 1; the nucleotide sequence of the nucleic acid for coding the heterotrimeric fusion protein is shown as SEQ ID NO.2;

the PF-D trimer comprising a recombinant novel coronavirus Delta strain S protein (recombinant S protein) and a heterotrimeric domain; the amino acid sequence of the recombinant S protein is shown as SEQ ID NO. 3; the nucleotide sequence of the nucleic acid for coding the novel recombinant coronavirus Delta strain S protein is shown as SEQ ID NO.4;

the recombinant novel coronavirus Delta strain S protein is obtained by mutating the original novel coronavirus Delta strain S protein and deleting a membrane-penetrating region fragment of the mutant novel coronavirus Delta strain S protein; the amino acid sequence of the original novel coronavirus Delta strain S protein is shown as SEQ ID NO.5, and the nucleotide sequence of the nucleic acid for coding the original novel coronavirus Delta strain S protein is shown as SEQ ID NO. 6.

In a second aspect, the present application provides a recombinant expression vector expressing the heterotrimeric fusion protein of the first aspect.

Further, the recombinant expression vector is a recombinant plasmid, and comprises a nucleic acid molecule of a nucleotide sequence shown in SEQ ID NO. 2.

In a third aspect, the present application provides an engineered cell comprising the recombinant expression vector of the second aspect.

In a fourth aspect, the present application provides a method of preparing the heterotrimeric fusion protein of the first aspect, comprising the steps of:

constructing a recombinant expression vector according to the second aspect;

transfecting the recombinant expression vector into a cell to obtain an engineered cell;

culturing the engineered cell to secrete the protein; and

separating and purifying the protein to obtain the heterotrimeric fusion protein.

Further, the process of separating and purifying the protein comprises the following steps:

preparing a heterotrimeric domain monoclonal antibody;

capturing a heterotrimeric fusion protein with the monoclonal antibody by immunoaffinity chromatography; and treating by Tangential Flow Filtration (TFF) and nanofiltration (Poll) to obtain the purified heterotrimeric fusion protein.

In a fifth aspect, the present application provides a composition comprising a heterotrimeric fusion protein of the first aspect and a pharmaceutically acceptable adjuvant.

Further, the adjuvant consists of CpG1018 and aluminum hydroxide.

Further, the mass ratio of CpG1018 to aluminum hydroxide in the adjuvant is 2-6: 1.

in a sixth aspect, the present application provides a vaccine or medicament comprising a composition according to the fifth aspect.

In a seventh aspect, the present application provides the use of a heterotrimeric fusion protein according to the first aspect or a composition according to the sixth aspect in the manufacture of a novel coronavirus pneumonia vaccine or medicament.

Further, the novel coronavirus is a novel coronavirus Delta strain and/or an Omicron strain.

Further, the Delta strain and the Omicron strain also include variant strains of the respective strains.

Compared with the prior art, the application has at least one of the following beneficial effects:

heterotrimeric fusion proteins, compositions, and uses thereof are described herein. The heterotrimeric fusion proteins provided herein, namely the PF-D trimers, have a stable novel pre-coronavirus S protein fusion conformation; in addition, the application provides a composition, the composition comprises the PF-D trimer and an adjuvant, the adjuvant comprises CpG1018 and aluminum hydroxide, the usage amount of the heterotrimeric fusion protein serving as a vaccine antigen can be reduced, the immune system can be quickly activated, the immune response can be enhanced, and the like, the functions of the heterotrimeric fusion protein composition and the heterotrimeric fusion protein can be effectively combined, the heterotrimeric fusion protein composition can be used for preparing high-efficiency vaccines or medicines, and compared with the vaccines or medicines prepared by the prior art, the prepared vaccines or medicines have better effect of defending or treating novel coronary pneumonia caused by Delta strain or Omicron strain infection.

Drawings

FIG. 1 is a SDS-PAGE analysis result of PF-D trimer provided in the examples of the present application.

FIG. 2 is a comparison of SDS-PAGE analysis of PF-D trimer under reducing and non-reducing conditions as provided in the examples herein.

FIG. 3 is a graph showing the results of SEC-HPLC analysis of PF-D trimer provided in the examples of the present application.

FIG. 4 is a schematic diagram of a spike-like structure of a PF-D trimer and a negative staining electron microscope image of the PF-D trimer provided in the embodiment of the present application; wherein A is a schematic diagram of a spike-shaped structure of the PF-D tripolymer, and B is a negative dyeing-electron microscope diagram of the PF-D tripolymer.

FIG. 5 is a time chart of immunization, blood collection and spleen cell collection of C57BL/6 mice provided in the examples of the present application.

FIG. 6 is a graph of immunization and blood sampling times for Sprague-Dawley rats provided in the examples of the present application.

FIG. 7 is a graph of Syrian hamster immunization, blood collection, spleen cell collection times provided in the examples of the present application.

FIG. 8 is a graph showing the timing of immunization and blood collection of K18-hACE 2H 11 mice provided in the examples of the present application.

FIG. 9 shows the PF-D-trimer antibody titers at day 21 of immunization in C57BL/6 mice provided in the examples of the present application.

FIG. 10 is the PF-D-trimer antibody titers in C57BL/6 mice immunized at 35 days as provided in the examples of the present application.

FIG. 11 is an ELISpot assay of Th1 IFN- γ in C57BL/6 mice after PF-D-trimer antigen stimulation provided in the examples herein.

FIG. 12 shows the PF-D-trimer antibody titers at 41 days of immunization in Sprague-Dawley rats as provided in the examples herein.

FIG. 13 is a comparison of the neutralization activity of sera from immunized Sprague-Dawley rats against the ancestral WA1 strain, delta strain and Omicron BA strain as provided in the examples of the present application.

FIG. 14 is a comparison of the neutralizing activity of the serum of Sprague-Dawley rats against SARS-CoV-2Delta strain, BA.2.12.1 and BA.4/5 pseudovirus as provided in the examples herein.

FIG. 15 shows the PF-D-trimer antibody titers in Syrian hamster bodies at day 41 of immunization provided in the practice of the present application.

FIG. 16 is a comparison of the ELISA titers of PF-D-trimer binding antibodies in syrian hamster at day 42 and day 110 of immunization provided in the examples herein.

FIG. 17 is an ELISpot analysis of Syrian golden hamster Th1 IFN- γ after stimulation with PF-D-trimer antigen as provided in the examples herein.

FIG. 18 is a comparison of the neutralizing activity of sera from immunized Syrian hamsters against the ancestral WA1 strain, delta strain and Omicron BA1 strain as provided in the examples of the present application.

FIG. 19 is a comparison of the neutralizing activity of sera from immunized Syrian hamster versus SARS-CoV-2Delta strain, BA.2.12.1, and BA.4/5 pseudovirus provided in the examples of this application.

FIG. 20 shows PF-D trimer antibody titers in K18-hACE 2H 11 mice at day 28 of immunization provided in the examples of the present application.

FIG. 21 is a graph showing the survival of K18-hACE 2H 11 mice intranasally infected with SARS-CoV-2Delta strain.

FIG. 22 is a viral load analysis in lung tissue at necropsy (day 3 post challenge) in K18-hACE 2H 11 mice as provided in the examples of the present application.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of and not restrictive on the broad application. Reagents not individually specified in detail in this application are conventional and commercially available; methods not specifically described in detail are all routine experimental methods and are known from the prior art.

It should be noted that the terms "first", "second", and the like in the description and claims of the present invention and in the drawings are used for distinguishing similar objects, and do not necessarily have to be used for describing a specific order or sequence or have a substantial limitation on technical features thereafter. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

1. Interpretation of terms

The term "fusion protein" refers to an expression product of a fusion gene or a novel protein produced by fusing two or more proteins by biological, chemical, or the like methods. In the present application, the heterotrimeric fusion protein body structure is composed of two proteins (or polypeptides), one of which is a novel coronavirus S protein and the other of which is a heterotrimeric Domain polypeptide, i.e., TD (Trimerization Domain) Trimerization Domain. In the present application, expression of the heterotrimeric fusion protein can be performed by constructing a recombinant expression vector comprising a fusion gene of the gene sequences of the S protein and the TD trimer domain, and transfecting into a host cell.

The term "adjuvant" refers to a non-specific immunopotentiator which, when injected or pre-injected into a body with an antigen, enhances the body's immune response to the antigen or alters the type of immune response. The mechanism of adjuvant enhancement of immune response is to prolong the retention time of antigen in the body by changing the physical shape of antigen; stimulating the antigen presenting ability of mononuclear phagocytes; stimulate lymphocyte differentiation and increase the ability of amplifying immune response. The adjuvant is of various types, and commonly used adjuvants include aluminum hydroxide adjuvant, corynebacterium parvum, lipopolysaccharide, and cytokine.

2. Heterotrimeric fusion proteins

In the embodiment of the application, the heterotrimeric fusion protein, namely PF-D-trimer simulates the structure of the novel coronavirus S protein natural trimer before fusion with a host cell, the heterotrimeric fusion protein consists of the complete extraenvelope structural domain of the S protein of the SARS-CoV-2 virus Delta strain, the C-terminal of the heterotrimeric fusion protein is fused with a TD tripolymer structural domain, and the TD tripolymer structural domain is a functional structural domain formed by a section of polypeptide and can be fused with the novel coronavirus Delta strain S protein into a stable tripolymer structure.

In the embodiment of the application, the amino acid sequence of the PF-D trimer fusion protein is shown as SEQ ID NO. 1; the nucleotide sequence of the nucleic acid for coding the PF-D trimer fusion protein is shown in SEQ ID NO.2; the PF-D trimer comprises recombinant S protein, and the sequence of the recombinant S protein is shown in SEQ ID NO. 3; the nucleotide sequence of the nucleic acid for coding the recombinant S protein is shown as SEQ ID NO.4; the recombinant S protein is obtained by original SARS-CoV-2Delta strain S protein (the amino acid sequence is shown as SEQ ID NO.5 and can be downloaded from GISAID, and the accession number is EPI \ UISL \ U1970349) S1/S2 mutation and deletion of a transmembrane region fragment of the SARS-CoV-2Delta strain S protein, the obtained recombinant S protein is a C-segment truncated S protein, two subunits S1 and S2 are reserved, and all functional regions except the transmembrane region fragment are reserved so as to maximally reserve the epitope on the Delta strain S protein.

PF-D-trimer is a fusion protein that mimics the pre-fusion structure of the S protein native trimer of the SARS-CoV-2 Virus Delta strain, as found on the surface of the virus, and consists of the complete exodomain of the S protein of the SARS-CoV-2Delta strain, C-terminally fused to the TD trimerization domain.

3. Preparation of a heterotrimeric fusion protein (PF-D trimer)

Provided in embodiments herein are methods of preparing a PF-D trimer, comprising: constructing a recombinant expression vector of the PF-D trimer; transfecting the recombinant expression vector into a cell to obtain an engineered cell; culturing the engineered cell to secrete the protein; and separating and purifying the protein to obtain the heterotrimeric fusion protein.

Further, the process of separating and purifying the protein comprises the following steps: preparing a TD trimer structural domain monoclonal antibody; capturing a heterotrimeric fusion protein with the monoclonal antibody by immunoaffinity chromatography; and treating by Tangential Flow Filtration (TFF) and nanofiltration (Poll) to obtain the purified PF-D trimer.

In certain embodiments, the recombinant expression vector for the PF-D trimer is a recombinant plasmid and the transfected cells are Chinese Hamster Ovary (CHO) cells.

In the examples of the present application, the DNA encoding the extracellular domain portion of the spike protein (S protein) on the membrane of the Delta strain of SARS-CoV-2 virus was codon-optimized for expression in Chinese hamster ovary cells (hereinafter referred to as "CHO cells") in combination with antigen analysis and codon optimization methods. Artificially synthesizing DNA (optimized) encoding the ectodomain part of S protein, adding HindIII enzyme cutting sites (the recognition sites are ' 5' -A ℃ > AGCTT-3' ") by adopting a conventional technical method of a person skilled in the art, inserting DNA fragments encoding recombinant S protein (mutated) into recombinant plasmid pGENT1.0-DGV plasmid after purification and T4 link enzyme connection to obtain recombinant linearized targeting plasmid capable of expressing PF-D trimer, namely the recombinant plasmid pGENT1.0-DGV-Cov, wherein the recombinant plasmid comprises a gene sequence encoding the PF-D trimer fusion protein.

In the embodiment of the application, the recombinant linearized targeting plasmid expressing the PF-D trimer is transfected into CHO K1 GenS cells to generate a stable CHO cell pool, CHO cells generating high-titer products are screened out and put into a bioreactor, and the culture medium of the CHO cells is continuously and highly secreted with protein, namely the PF-D trimer, through a serum-free semi-continuous (intermittent) cell culture process.

In the examples of the present application, at the end of the CHO cell growth phase, the cell supernatant was collected by depth filtration (filtration equipment purchased from sydows teddy limited); and (3) purifying the filtered clarified culture medium, which comprises the following steps: preparing a TD trimerization domain monoclonal antibody (alpha-TD trimerization domain monoclonal antibody), capturing PF-D-trimer through immunoaffinity chromatography of the TD trimerization domain monoclonal antibody, and enabling the PF-D-trimer to lose virus activity through low-pH treatment. The immunoaffinity chromatography eluate is concentrated and diafiltered using Tangential Flow Filtration (TFF) and final nanofiltration (Pall) to obtain the purified SARS-CoV-2 recombinant S protein active substance, i.e., the purified PF-D-trimer.

In the examples of the present application, the α -TD trimerization domain monoclonal antibody was prepared in Sf9 insect cell line using a recombinant baculovirus system. The preparation process of the alpha-TD trimerization structural domain monoclonal antibody comprises the steps of transferring Sf9 cells in a Working Cell Bank (WCB) into a shake flask for revival and amplification, then transferring the Sf9 cells into a bioreactor (purchased from Applikon company), and culturing by using a serum-free medium (purchased from Vsiosci, a Beijing Dingzhi organism). After infection of Sf9 cells with baculovirus inoculum, antibodies are secreted during the culture of Sf9 cells. At the end of the infection phase, sf9 cells were collected by centrifugation. The clarified medium was purified by affinity chromatography using MabSelect-prism packings (purchased from Situola, cytiva) according to the manufacturer's instructions. The affinity chromatography eluate was concentrated and diafiltered using tangential flow filtration (filtration equipment from sandida, inc.). To prepare the immunoaffinity resin, an α -TD trimerization domain monoclonal antibody was coupled to NHS activated Sepharose 4Fast Flow packing (purchased from stethovan, cytiva) according to the manufacturer's instructions.

Results and analysis: the high-level production process of the PF-D-trimer is realized by transfecting an expression vector containing a cDNA (containing optimized CHO codons) optimizing sequence for coding the PF-D-trimer into CHO cells, selecting the CHO cells with the best performance and optimizing a culture process. The process can make the production level of PF-D-trimer as secretory protein in the culture medium reach more than 500 mg/L. As shown in figure 1, side-by-side analysis of two different batches by SDS-PAGE showed that the expression process was robust, yielding nearly identical expression levels.

FIG. 2 shows the results of SDS-PAGE analysis for reducing (+ DTT) and non-reducing (-DTT). The purified PF-D-trimer is a self-binding homotrimer stabilized by interchain disulfide bonds. As can be seen from figure 2, side-by-side analysis of two purified batches of PF-D-trimer demonstrated reproducibility of the purification process, both batches having similar apparent purity levels after silver staining. Under reducing conditions, the PF-D-trimer is in a single form and has a molecular weight of about 170kDa. Under non-reducing conditions, the PF-D-trimer appeared as a single high molecular weight form, indicating that the protein was not cleaved by proteases produced by CHO cells.

4. SEC-HPLC analysis

The SARS-CoV-2S glycoprotein trimer fusion protein, i.e., the PF-D trimer, was analyzed by size exclusion chromatography (SEC-HPLC) using Shimadzu LC-2030HPLC (from Shimadzu, japan) and analytical SRT SEC-3007.8X 300mm column (from Sepax). The HPLC detection conditions are as follows: phosphate Buffered Saline (PBS) was used as the mobile phase, OD 280nm, detection time 20 minutes, and flow rate 1 ml/min.

Results and analysis: the PF-D-trimer was obtained by elution with an alpha-TD trimerization monoclonal antibody immunoaffinity chromatography, and the PF-D-trimer obtained was evaluated by SEC-HPLC, and the PF-D-trimer resulted in a molecular weight of about 700kDa with a purity of >97.9% and no significant aggregation or fragmentation (as shown in FIG. 3).

5. Negative staining transmission electron microscopy analysis

To characterize the PF-D-trimer protein samples immunoaffinity purified by the α -TD trimerization monoclonal antibody, 20 μ l of the sample was dropped into a 200 mesh grid (purchased from Waals film copper mesh) and incubated at room temperature for 10 minutes. The grid was then negatively stained with 2% phosphotungstic acid for 3 minutes and the remaining liquid was removed with filter paper. The prepared samples were observed with a HT7800 transmission electron microscope (available from hitachi).

Results and analysis: a schematic of the PF-D-trimer is shown in FIG. 4A, which is an assembled spike trimer. FIG. 4B shows a negative stain transmission electron microscope image of a representative pre-fusion conformation containing homotrimeric spinous process proteins.

6. Heterotrimeric fusion protein compositions

In the examples of the present application, the heterotrimeric fusion protein composition is used for preparing vaccines or medicines for tumors or other immune diseases, and the composition has two main components.

The first component is the heterotrimeric fusion protein; i.e., PF-D Trimer (PF-D-Trimer).

The second component is an adjuvant. In the embodiment of the application, the adjuvant is used as a foreign body relative to host cells, and can play a role in early warning the immune system of an organism or enhancing a danger signal that an antigen invades the organism together with the antigen (heterotrimeric fusion protein), so that the immune system is prompted to have strong reaction, and the immunogenicity of the antigen is improved; altering the nature of the immune response; reducing the antigen quantity and the immunizing agent frequency required by successful immunization; improve the immune response of people with low immune function. The action mechanism of the immunological adjuvant can be divided into: sustained release at the site of antigen re-injection (antigen depot effect); up-regulation of various cytokines and chemokines; recruiting immune cells to the injection site; enhancing antigen uptake and presentation; activating Antigen Presenting Cells (APCs) to promote the mature antigen presenting cells to be transported to a drainage lymph node; activation of inflammasome, etc. Generally, the immune adjuvant can be injected into the body with antigen in advance or simultaneously to participate in immunization, the adjuvant can complete the function within 2-3 days after immunization, and the site of action is the injection site and the drainage lymph node which coexist with the antigen.

The inventor of the present application, aiming at the heterotrimeric fusion protein in the embodiment of the present application, adopts an adjuvant CpG1018+ aluminum hydroxide, which contains two adjuvant components CpG1018 and aluminum hydroxide, and can achieve high efficiency in application effect. CpG1018 consists of a cytosine phosphate guanine (CpG) base sequence, and motifs are synthetic forms of DNA that mimic bacterial and viral genetic material.

In certain embodiments, the mass ratio of CpG1018 to aluminum hydroxide selected in the adjuvant is 2 to 6:1.

7. heterotrimeric fusion protein composition immunization and effect identification

The inventors of the present application have conducted a series of studies such as antibody titer test (specificity study), ELISpot assay (enzyme-linked immunospot assay), virus neutralization test, etc. on the heterotrimeric fusion protein composition provided in the present application. In the examples of the present application, all animal experiments were performed strictly according to the requirements of the institutional animal care and use committee of Hubei province.

In the examples of the present application, the experimental animals include pathogen-free (SPF) female C57BL/6 mice (6-8 weeks old), SPF female Syrian hamsters (5-6 weeks old) and 6-week-old Sprague-Dawley rats purchased from Hubei Experimental Research Center (Hubei Laboratory Research Center); H11-K18-hACE2 male mice (6-8 weeks) were purchased from Jiangsu Jiejiaokang Biotech Co. Animals were allowed free access to water and food in a controlled environment with a light/dark cycle of 12 hours and temperature controlled at: the temperature is 16-26 ℃, and the humidity is controlled at 40-70%.

7.1 mouse immunization and detection procedure

All animal experiments were performed in SPF experiments.

(1) 28C 57BL/6 mice were randomly divided into 4 groups and three inoculations were performed on days 0, 14 and 28 (as shown in FIG. 5), with the inoculation reagents and doses shown in Table 1 below:

TABLE 1

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Sera of the immunized mice were collected by orbital bleeds on days 21 and 35 (as shown in FIG. 5), and measured for PF-D-Trimer (using a novel coronavirus S protein Trimer fusion protein, hereinafter referred to as "S-TD-Trimer") specific IgG endpoint Geometric Mean titer (Geometric Mean Ttter, GMT) by ELISA. Mice were sacrificed by cervical dislocation on day 51 and their spleens (as shown in figure 5) were removed for ELISpot analysis.

(2) 10 Sprague-Dawley rats were randomized into three groups, as shown in FIG. 6, and two intramuscular Immunizations (IM) were performed on days 0 and 28, respectively, with the inoculation reagents and doses as shown in Table 2 below:

TABLE 2

Sera were collected on day 41 (as shown in FIG. 6) and tested for S-TD-Trimer specific IgG endpoint Geometric Mean Titer (GMT) by ELISA.

(3) Three intramuscular Immunizations (IM) were performed on day 0, day 22 and day 90, as shown in fig. 7, using syrian hamsters randomly divided into 7 groups, and the inoculation reagents and doses are shown in table 3 below:

TABLE 3

Sera were collected at day 42 and day 110 (as shown in figure 7) and tested for S-TD-Trimer specific IgG endpoint Geometric Mean Titer (GMT) by ELISA. After mice were sacrificed on day 110, their spleens (as shown in FIG. 7) were removed for ELISpot analysis.

7.2 ELISA detection

Specific IgG antibody end-points GMTs were determined by ELISA as follows: taking a 96-well enzyme label plate (Costar company), coating a recombinant SARS-CoV-2Delta strain S protein antigen (Nowa biological company) on the enzyme label plate, and sealing by using an ELISA buffer solution at 37 ℃ for 120 minutes; the serum of the aforementioned mice was taken, added dropwise to a 96-well microplate (Costar Co.) after sequentially using ELISA buffer (1% BSA and 0.05% Tween-20), and 100. Mu.L was added to each well; after three washes with buffer (PBS containing 0.05% tween-20), the ELISA buffer conjugated with horseradish peroxidase HRP was added to each well at a rate of 1:10000 dilutions of goat anti-mouse IgG (Proteintech), goat anti-rat IgG (Proteintech) or goat anti-hamster IgG (Shanghai Cyclococcal Biotech Co., ltd.) were incubated at 37 ℃ for 30 minutes; after washing the microplate 3 times with the buffer solution, TMB (3, 3', 5' -tetramethylbenzidine) substrate (Beijing Solebao) was added to each well for color development, and then 2M H was added to each well ₂ SO ₄ The reaction was stopped and OD450 values were measured using a Varioskan-LUX multimode microplate reader (Sammerfet). The positive judgment threshold value (Cut-Off value) of the IgG end point GMTs is the average value of negative control OD +3 times of the standard deviation of the negative control OD, the sample OD value is larger than the threshold value, the positive judgment is carried out, otherwise, the negative judgment is carried out;

statistical analysis was performed using GraphPad Prism v8.0.1.

7.3 ELISpot assay

Spleens from C57BL/6 mice at day 51 post inoculation and Syrian hamsters at day 110 post inoculation were removed, respectively, and splenocytes isolated for S protein specific T cell detection using the ELISpopPLUS mouse IFN-. Gamma.kit or the Elispoppplus hamster IFN-. Beta.kit (Mabtech) according to the manufacturer' S instructions. Briefly, 3 to 5X 10 ⁵ Individual splenocytes/well and 4 ug SARS-CoV-2 virus B.1.617.2 variant spinous process protein (Nanjing vitamin Biotech)Company Limited)/well mixed and incubated at 37 ℃; after 48 hours, wash 5 times with PBS buffer, add 1. Mu.g/. Mu.L concentration of probe antibody, and at room temperature for 2 hours; washed 5 times with PBS buffer, added with 1: labeling streptavidin with 1000-diluted alkaline phosphatase, and incubating at room temperature for 1 hour; washed 5 times with PBS buffer, added with a developing solution (BCIP/NBT plus), and incubated at room temperature for 10 minutes; the color development was stopped with deionized water. The number of spots in the wells of the ELISpot plate was analyzed using the ELISpot reader system (AID Elispoint reader, automation diagnostic GmbH, inc.). The resulting data were analyzed using GraphPad Prism 8.0.2 software.

7.4 neutralization assay based on pseudoviruses

Modified on the basis of the procedure of Nie et al, for NT50 measurement. Briefly, sera from immunized Sprague-Dawley rats or syrian hamsters were diluted 3 times in a gradient with an initial dilution factor of 1:33.33 and reacted with SARS-CoV-2 pseudovirus (2X 10) at 37 ℃ ⁴ TCID ₅₀ /mL) was mixed and incubated for 60 minutes, serum-free DMEM medium was used as a negative control group; HEK293T-hACE2 cells were then added to each well (2X 10) ⁴ Individual cells/well) and incubated at 37 ℃ for 48 hours. Luciferase activity reflects the extent of SARS-CoV-2 pseudovirus transduction, measured using the Bio-Lite luciferase assay System (Vazyme Corp.). NT50 was calculated by the Reed-Muench method (Reed, l.j. And Muench, h., 1938), where NT50 is defined as the dilution factor that achieves greater than 50% pseudoviral transduction inhibition compared to the control group.

7.5 SARS-CoV-2 neutralization assay

Vero E6 cells (2.5X 10) ⁴ Cells/well) were seeded in 96-well plates and incubated overnight. Taking serum, inactivating the serum for 30 minutes at 56 ℃, diluting the serum in serum-free DMEM medium, wherein the initial dilution factor (dilution multiple) is 8, and further continuously diluting; the diluted serum WAs then mixed with SARS-CoV-2 virus (central for disease prevention and control in Hubei province, WA1-Hu-1, delta strain YJ20210701-01, omicron strain 249099 at a ratio of 1 ₅₀ 100 μ L and incubation at 37 ℃ for 1 hour; adding diluted serum/virus mixtureAdding to Vero cells and reacting with 5% CO at 37 ℃ ₂ Incubating for four days; monitoring cytopathic effect (CPE) of each serum dilution every 24 hours by inverted microscope; the neutralization endpoint of 50% was calculated by the Reed-Muench method (Reed, l.j. And Muench, h., 1938), i.e., the serum dilution at which 50% of the cells could be protected from CPE, to obtain the neutralizing antibody titer of each serum.

7.6 K18-hACE 2H 11 transgenic mouse immunization and challenge protection research

The K18-hACE 2H 11 male transgenic mice were divided into 5 vaccinated groups, as shown in FIG. 8, and blood was collected before immunization as a pre-immunization control; inoculating subcutaneously twice on days 0 and 21 respectively; sera were collected on day 28 to detect the SARS-CoV-2 specific IgG endpoint GMT. On day 35, mice were transferred to the BSL3 laboratory (wuhan virus institute, chinese academy of sciences). The inoculation reagents and amounts are shown in table 4 below:

TABLE 4

In challenge protection studies, 1X 10 per mouse ⁵ pfu (50. Mu.l) SARS-CoV-2Delta strain live virus (CRST: 1633.06.IVCAS 6.7593) was infected by nasal drip. Three animals were used as a control group and inoculated with the same volume of PBS; body weight change and survival determinations were monitored daily. Half of the animals were euthanized 3 days post infection (dpi) and the remaining animals of each group were followed up to day 7; lung tissue samples were collected and virus titers were determined by plaque assay.

Viral titers were determined in lung tissue by cell culture infection (TCID 50) as follows:

plaque analysis was performed to determine viral load. Vero E6 cells were plated at 10 days 1 in advance ⁵ Density of individual cells/well seeded into 24-well plates; 0.1g of lung tissue was taken and added to 1mL of PBS buffer to prepare lung homogenate; the following day, aim atFor each sample, 100. Mu.L of a 10-fold serial dilution of lung homogenate supernatant was added dropwise to infected cells and incubated at 37 ℃ for 1 hour; remove virus diluent and add 1% methylcellulose; after incubation of the well plates at 37 ℃ for 4 days, the upper layer in the wells was discarded, 1mL of fixed staining solution (3.7% formaldehyde +1% crystal violet) was added, and the cells were treated overnight at room temperature; after washing the fixed staining solution with tap water and drying, the number of virus plaques was counted. Plaque forming units per mL (PFU/mL) were determined using the following formula: (# plaques X dilution factor)/0.1 mL. Plaque free score of<1 and is used to calculate the lower detection limit. PFU/mL values were adjusted to volumes of 1mL and 1g of tissue to calculate plaque forming units per gram of tissue. Results data were analyzed using GraphPad Prism 8.0.2 software.

7.7 composition immunization and effect identification test results:

7.7.1 heterotrimeric fusion protein compositions induce C57BL/6 mouse immune responses

The immunogenicity of a composition of PF-D-Trimer (i.e., S-TD-Trimer, hereafter) + aluminum hydroxide + CpG1018 adjuvant was first assessed in C57BL/6 mice. Sera from these mice were evaluated for the amount of anti-spinous-protein IgG. Sera from mice injected with PBS formulation showed only background levels of titer. As shown in FIG. 9, the mutual GMT titers of the antibodies of the 5. Mu.g, 10. Mu.g and 20. Mu.g groups reached 6.5X 10, respectively, at day 21 ⁵ 、4.6×10 ⁵ And 1.2X 10 ⁶ . There was a significant difference in antibody titers between the 10 μ g and 20 μ g groups (p = 0.0329), and no significant difference in antibody titers between the other groups. As shown in FIG. 10, on day 35, the mutual GMT titers of the antibodies of the 5. Mu.g, 10. Mu.g and 20. Mu.g groups reached 9.3X 10, respectively ⁵ 、1.1×10 ⁶ And 1.2X 10 ⁶ There was no significant difference in antibody titers between groups. These results indicate that the second immunization enhanced the antibody response even though the GMTs were the same in the 20 μ g group at day 21 and day 35.

The mice were further tested for immune sera whether the vaccine adjuvant system provided herein could induce a Th1 response in vaccinated mice. As shown in FIG. 11, th1 gamma interferon (IFN-. Gamma.) responses were measured in spleen cells of immunized mice after stimulation with PF-D-trimer antigen. There were significant differences in the amount of anti-PF-D-trimer specific IFN- γ produced by T cells in the 5, 10, and 20 μ g groups compared to the PBS control group. These results indicate that the PF-D-trimer provided herein can promote the development and activation of Th1 cells in C57BL/6 mice.

7.7.2 heterotrimeric fusion protein compositions induce an immune response in Sprague-Dawley rats

The composition consisting of PF-D-trimer + aluminum hydroxide + CpG1018 adjuvant was tested for immunogenicity in Sprague-Dawley rats in the examples. As shown in FIG. 12, the amount of anti-spinous-protein IgG of the immune sera was evaluated, and the mutual GMT titer of the antibody of the 50. Mu.g and 100. Mu.g groups reached 10 ⁷ Above, there were no significant differences between the two groups of rats. The 100 μ g dose group may produce higher antibody titers because all animals in this group exhibit equivalent titers.

As can be seen from the results of fig. 12, since two doses of 50 μ g and 100 μ g produced high antibody titers in Sprague-Dawley rats, and there was no significant difference, the present example used a smaller dose, i.e., 50 μ g, to verify the neutralizing antibody titer therein. During the pandemic of new coronary pneumonia, many SARS-CoV-2 variants appeared, including Omicron (BA.1), a highly mutated variant that is highly resistant to vaccine-induced antibody neutralization (

a, 2022). Thus, the immune sera were further tested for their neutralizing capacity against the Delta strain of SARS-CoV-2 and against the Omicron strain. As shown in FIG. 13, the IDs of Delta strains and Omiclon BA.1 were determined using a combination of aluminum hydroxide + CpG1018 adjuvant +50 μ g PF-D-trimer ₅₀ GMTs were 9337 and 2113, respectively. Even though the antibodies against the ba.1 strain were significantly reduced, neutralizing antibodies against both strains could be generated using the PF-D-trimer formulation (composition) provided herein. Sera were also used to assess their neutralizing activity against SARS-CoV-2Delta strain, BA.2.12.1 and BA.4/5 pseudovirus, as shown in FIG. 14, their ID ₅₀ GMTs were 7290, 7058, respectively.

7.7.3 Heterotrimerization of fusion protein compositions to induce immune responses in Syrian hamsters

The inventors evaluated the immunogenicity of PF-D-trimer compositions against Syrian hamsters. As shown in FIG. 7, two to three Syrian hamsters were inoculated with a composition containing 5. Mu.g or 10. Mu.g of PF-D-trimer, in which the adjuvant contained aluminum hydroxide and CpG1018, or only aluminum hydroxide or CpG1018, for a period of more than two months (specifically, 68 days). The serum of hamsters immunized with all the preparations (including the protein without adjuvant) contained IgG anti-spinous-process protein, and as shown in FIG. 15, the geometric mean value of the antibody was 8.9X 10 in the 5. Mu.g PF-D-trimer group to which only the alumina hydroxide adjuvant was added ⁵ In the group containing aluminum hydroxide and CpG1018 adjuvant, the geometric mean value of the antibody was 3.6X 10 ⁶ It follows that PF-D-trimer with added aluminium hydroxide and CpG1018 adjuvant is used to immunise Syrian hamsters, which produce higher GMT titres of antibody in vivo; in the group containing 10. Mu.g of PF-D-trimer and only the aluminum hydroxide adjuvant, the geometric mean value of the antibody was 1.1X 10 ⁶ In the group containing aluminum hydroxide and CpG1018 adjuvant, the geometric mean value of the antibody was 2.8X 10 ⁶ The same conclusion can be drawn as for the 5. Mu.g PF-D-trimer group.

In the non-adjuvanted group, i.e.inoculated with only PF-D-trimer, the GMT titer of the antibody still produced was 2.9X 10 ⁵ But significantly less than the effect of adjuvant vaccination. FIG. 16 shows a comparison of the results of the second and third inoculations, from which it can be seen that despite the third antibody titer being higher than the second, the overall conclusion of the second and third inoculations remained the same in the 5. Mu.g PF-D-trimer group and the 10. Mu.g PF-D-trimer group, regardless of the addition of adjuvant.

The present examples were tested for Th1 IFN-. Gamma.response in spleen cells of immunized hamsters. From the dot count results shown in fig. 17, it was found that the hamster inoculated with PBS prepared by adding CpG1018 to aluminum hydroxide induced a partial T cell response, but the PF-D-Trimer (S-TD-Trimer) -immunized group had a higher number of dots than the PBS group without the adjuvant, whereas the adjuvanted S-TD-Trimer showed a higher number of dots, indicating that the hamster immunized with the adjuvant had the best stress response effect. There was no significant difference in the number of spots in the comparison between the 5. Mu.g PF-D-trimer group and the 10. Mu.g PF-D-trimer group. These data results indicate that the PF-D-Trimer composition with the addition of aluminum hydroxide and CpG1018 adjuvant directed hamster T cells to the Th1 phenotype.

FIG. 18 shows the results of neutralization activity of hamster sera inoculated three times with 5. Mu.g of a PF-D-trimer composition (containing aluminum hydroxide and CpG1018 adjuvant) against the ancestral WA1, delta and Omicron strains of SARS-CoV-2 virus, delta Strain ID ₅₀ GMT 851, WA1 (Wuhan) 342, and Omicron 181. The results of the above data indicate that the PF-D-trimer composition induced serum from hamsters produced neutralizing antibodies against three strains, which were most active in neutralizing Delta strains, while having significant neutralizing effects against WA1 and Omicron strains. The above sera were also used to assess the neutralizing activity against SARS-CoV-2Delta, BA.2.12.1 and BA.4/5 pseudotyped viruses. As shown in FIG. 19, the ID of Delta Strain ₅₀ GMT was 3378, BA.2.12.1 strain 1200, BA.4/5 strain 903. The above results are similar to those of experiments performed using authentic viruses. The serum of this immune hamster had a higher neutralizing activity against the Delta strain compared to the BA.2.12.1 strain and the BA.4/5 strain.

7.7.4 immunogenicity and protection of the heterotrimeric fusion protein composition on K18-hACE 2H 11 mice

The inventors evaluated the immunogenicity and protective effects of PF-D-trimer and PF-D-trimer in combination with aluminum hydroxide + CpG1018 adjuvant in K18-hACE 2H 11 mice. hACE2 mice develop respiratory disease similar to severe COVID-19 and are suitable models for studying immune responses.

The inventor of the application detects the immunogenicity of the aluminum hydroxide, cpG1018 adjuvant and PF-D-tripolymer composition in transgenic mice, evaluates the quantity of the anti-spinous-process protein IgG in immune serum, and as shown in figure 20, the GMT titer of the antibody of a 30 mu g group reaches 10 ⁷ The titer of the 10 mu g group is lower than that of the 30 mu g group, but is still significantly higher than that of the control group, and the titer is close to 3 × 10 ⁶ . Adjuvant group mice (uninoculated PF-D-trimer) underwent weight loss following intranasal infection with Delta StrainAnd mice in this group died on

day

4 or 5 post infection (as shown in figure 21). Mice vaccinated with the PF-D-trimer composition lost weight on the first two days of infection and subsequently began to recover, and were well-conditioned 7 days after infection. Viral load was detected in lung tissue of mice on day 3 post-infection, and as shown in FIG. 22, live virus titers in lung tissue of mice in the 10. Mu.g and 30. Mu.g PF-D-trimer groups were below the lowest detection limit (100 PFU/g), while GMT of mice in the adjuvant group exceeded 1X 10 ⁵ PFU/g lung tissue.

7.7.5 mechanism of action of adjuvant

In the examples of the present application, the adjuvant used is CpG1018 and/or aluminum hydroxide. From the results of the foregoing examples, it can be seen that the adjuvant complex using CpG1018 and aluminum hydroxide can achieve better immune effect when used in combination with the PF-D-trimer described in the present application in animal immune tests. The aluminum adjuvant in the composite adjuvant of the present application may serve to deliver PF-D-trimer to the immune system to induce immunity; cpG1018 in the composite adjuvant contains immunostimulant or intensifier, and uses receptor-mediated signal path to regulate immune response, so as to enhance the immunogenicity of PF-D-trimer as antigen. From the results of the foregoing examples, it is understood that the adjuvant combinations of CpG1018 and aluminum hydroxide (without antigen protein) and PF-D-trimer (without adjuvant) described herein induce Th1 immune responses in the vaccinated animals alone, while the combination of PF-D-trimer and adjuvant combination induced Th1 immune responses in the vaccinated animals more effectively and better.

7.7.6 immunization Studies on different novel coronavirus strains

With the evolution of the virus and the pressure of antibody selection, the new SARS-CoV-2 variants emerged in a variety of variants, including the Delta strain and the Omicron strain, while most published studies used vaccines based on the original WA1 strain. In the examples of the present application, it can be seen from the results of the Sprague-Dawley rat and Syrian hamster model immunization test that the inoculation of the PF-D-trimer composition provided in the present application WAs significantly enhanced in neutralizing ability against the WA1 strain, delta strain and Omicron strain, as compared with the non-inoculated results.

The above description is only for the preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present application should be covered within the scope of the present application.

Claims

1. A heterotrimeric fusion protein has an amino acid sequence shown as SEQ ID NO. 1; the nucleotide sequence of the nucleic acid for coding the heterotrimeric fusion protein is shown as SEQ ID NO.2;

the heterotrimeric fusion protein comprises a recombinant novel coronavirus Delta strain S protein and a heterotrimeric domain;

the amino acid sequence of the recombinant novel coronavirus Delta strain S protein is shown as SEQ ID NO.3, and the nucleotide sequence of the nucleic acid for coding the recombinant novel coronavirus Delta strain S protein is shown as SEQ ID NO.4;

the recombinant novel coronavirus Delta strain S protein is obtained by mutation of the original novel coronavirus Delta strain S protein and deletion of a transmembrane region fragment of the mutant novel coronavirus Delta strain S protein; the amino acid sequence of the original novel coronavirus Delta strain S protein is shown as SEQ ID NO.5, and the nucleotide sequence of the nucleic acid for coding the original novel coronavirus Delta strain S protein is shown as SEQ ID NO. 6.

2. A recombinant expression vector expressing the heterotrimeric fusion protein of claim 1.

3. An engineered cell comprising the recombinant expression vector of claim 2.

4. A method of preparing the heterotrimeric fusion protein of claim 1, comprising the steps of:

constructing the recombinant expression vector of claim 2;

culturing the engineered cell to secrete the protein; and

5. A composition comprising the heterotrimeric fusion protein of claim 1 and a pharmaceutically acceptable adjuvant.

6. The composition of claim 5, wherein the adjuvant consists of CpG1018 and aluminum hydroxide.

7. The composition of claim 6, wherein the mass ratio of CpG1018 to aluminum hydroxide in the adjuvant is 2-6: 1.

8. a vaccine or medicament comprising the composition of claim 7.

9. Use of the heterotrimeric fusion protein according to claim 1 or the composition according to any one of claims 5 to 7 for the preparation of a novel coronavirus pneumonia vaccine or medicament.

10. Use according to claim 9, the novel coronavirus being a Delta strain and/or an Omicron strain.

11. Use according to claim 10, the Delta strain and the Omicron strain each comprising a respective variant strain.