CN115894713B - Heterotrimeric fusion proteins, compositions and uses thereof - Google Patents

Abstract

Heterotrimeric fusion proteins, compositions, and uses thereof are disclosed herein. The heterotrimeric fusion protein, namely PF-D trimer, has a stable novel pre-fusion conformation of coronavirus S protein; on the other hand, the application provides a composition which comprises PF-D trimer and an adjuvant, wherein the adjuvant comprises CpG1018 and aluminum hydroxide, the adjuvant has the effects of reducing the using amount of the heterotrimeric fusion protein as vaccine antigen, rapidly activating an immune system, enhancing immune response and the like, and the composition effectively combines the functions of the two, is used for preparing efficient vaccines or medicines, and can greatly improve the effects of the vaccines or medicines prepared in the prior art in defending and treating infectious diseases.

Description

Heterotrimeric fusion proteins, compositions and uses thereof

Technical Field

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

Background

The emergence of the novel coronavirus (SARS-CoV-2), which has caused a global new coronavirus pandemic, has until now still produced a significant impact on global public health. The continuous spread of new coronal pneumonia epidemic situation makes the human research on the sudden epidemic situation enter the stage of white thermalization.

SARS-CoV-2 is the seventh member of the coronaviridae family currently discovered to infect humans, and SARS-CoV-2 is a single-stranded positive strand RNA with a membrane envelope, the largest RNA virus. SARS-CoV-2 plays a major role in the invasion of human body by the spike glycoprotein (S protein for short). The S protein is a very large trimeric transmembrane glycoprotein, has a large amount of glycosylation modification, forms a special corolla structure on the surface of the virus, is the most important surface membrane protein of coronavirus, and contains two subunits S1 and S2. Wherein S1 mainly comprises a receptor binding domain (Receptor binding domain, RBD) responsible for recognizing the receptor of the cell. S2 contains the essential elements required for the membrane fusion process. The S protein bears the function of combining virus with host cell membrane receptor and membrane fusion, and is an important action site of host neutralizing antibody and a key target point of vaccine design. After the S protein is combined with a receptor on the surface of a cell, the viral envelope and the cell membrane are fused together in a homeotropic manner, so that genetic materials in the virus are injected into the cell, and the purpose of infecting the cell is achieved.

Upon binding of the S protein to the receptor of the host cell, dissociation of the S1 and S2 subunits occurs. The conformation of the S2 subunit becomes a highly stable post-fusion structure, so that the S protein has both pre-and post-fusion conformations. After fusion of the S protein with the recipient cell, the RBD with the immunogenic epitope moiety at the outermost end of the envelope of the S protein polypeptide is moved underneath to be masked by the binding to the recipient, while the non-antigenic epitope moiety is steadily exposed at the outer end. The conventionally prepared novel coronavirus S protein of the vaccine, namely the recombinant novel coronavirus S protein, only generates a strong and stable conformation after fusion, so that the immunogenicity of the vaccine antigen can be greatly reduced, and the vaccine is difficult to play a role.

Adjuvants, also called immunoadjuvants (immunologic adjuvant), refer to substances that non-specifically enhance or alter the specific immune response of the body to a matching antigen, enhance the immunogenicity of an antigen or alter the type of immune response, but are not antigenic in nature. If the vaccine component is only an antigen component, the immunogenicity of the vaccine component is often not strong enough, and the vaccine component cannot induce enough immune response when injected into a human body and is insufficient to resist invasion of a corresponding pathogen, so that the vaccine component is generally injected together with an adjuvant to play a role in sufficient protection when applied.

The most commonly used adjuvants at present are aluminum adjuvants, mainly comprising three types of aluminum phosphate, aluminum hydroxide and aluminum potassium sulfate, wherein the most commonly used adjuvants are aluminum hydroxide and aluminum phosphate. Aluminum adjuvants are widely used in, for example, HPV vaccines, inactivated poliovirus vaccines, bai-broken vaccines, hepatitis a vaccines, hepatitis b vaccines, rabies vaccines, anthrax vaccines, haemophilus influenzae type b conjugate vaccines, streptococcus pneumoniae vaccines, meningitis vaccines, veterinary vaccines (such as foot-and-mouth disease virus vaccines and botulinum vaccines), and the like. The immunogenicity enhancement effect of different adjuvants on different vaccines is different, the safety of the adjuvants on human bodies is also different, various adjuvant varieties exist on the market, and the screening of proper varieties also becomes a current research hotspot.

In addition, the novel coronaviruses have been mutated to form a variety of variants, delta and Omicron strains are the best known, but the vaccine strains currently used on the market are all along the original strains, so the vaccines provided by the prior art have limited effect on inhibiting Delta and Omicron strains.

For the above reasons, development of recombinant proteins which have stable structures and properties, such as a virus surface glycoprotein trimer structure before binding to a host cell receptor, and development of effective vaccines or drugs by selecting an adjuvant suitable for enhancing the immunogenicity of the recombinant proteins in the body, and inhibition or treatment of novel coronavirus pneumonia, particularly novel coronavirus pneumonia caused by Delta strains and Omicron strains and by respective variant strains, are needed to be a key and difficult point for solving the above technical problems.

Disclosure of Invention

Most multimeric proteins, the monomers of which possess a functional domain of a polypeptide that plays an important role in constructing and stabilizing the oligomeric conformation of the protein body, and in forming multimeric proteins, such as multimeric proteins like dimers, trimers, etc., the functional domains are divided into homologous and heterologous functional domains. The present application provides a recombinant fusion protein formed from protein monomers containing a heterologous functional domain (hereinafter "heterotrimeric domain"), i.e., a heterotrimeric fusion protein. The heterotrimeric fusion protein has stable protein conformation, and on the basis, successful synthesis of the heterotrimeric fusion protein provides a viable solution for defending or treating novel coronavirus pneumonia, such as in the preparation of effective vaccine antigens or immune drugs (e.g., antibody drugs, functional cytokines).

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

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

the PF-D trimer comprises 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 encoding 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 mutating the original novel coronavirus Delta strain S protein and deleting the membrane penetrating region fragment thereof; the amino acid sequence of the S protein of the original novel coronavirus Delta strain is shown as SEQ ID NO.5, and the nucleotide sequence of the nucleic acid for encoding the S protein of the original novel coronavirus Delta strain is shown as SEQ ID NO. 6.

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

Further, the recombinant expression vector is a recombinant plasmid, which comprises a nucleic acid molecule of the 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 for preparing a heterotrimeric fusion protein according to the first aspect, comprising the steps of:

constructing the recombinant expression vector of the second aspect;

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

culturing the engineered cell so that it secretes the produced protein; and

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

Further, the protein separation and purification process comprises the following steps:

preparing a heterotrimeric domain monoclonal antibody;

capturing the heterotrimeric fusion protein by immunoaffinity chromatography with said monoclonal antibody; and obtaining the purified heterotrimeric fusion protein by Tangential Flow Filtration (TFF) and nanofiltration (Poll).

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

Further, the adjuvant is composed 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 preparation of a novel coronavirus pneumonitis vaccine or medicament.

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

Further, the Delta strain and 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:

the present application relates to heterotrimeric fusion proteins, compositions and uses thereof. The heterotrimeric fusion protein provided by the application, namely PF-D trimer, has a stable novel pre-fusion conformation of coronavirus S protein; in addition, the application provides a composition comprising the PF-D trimer and an adjuvant, wherein the adjuvant comprises CpG1018 and aluminum hydroxide, the adjuvant has the effects of reducing the using amount of the heterotrimeric fusion protein as vaccine antigen, rapidly activating an immune system, enhancing immune response and the like, the heterotrimeric fusion protein composition effectively combines the functions of the two, and is used for preparing efficient vaccines or medicines, and the prepared vaccines or medicines have better effect of defending or treating novel coronary pneumonia caused by Delta strain or Omicron strain infection compared with the vaccines or medicines prepared by the prior art.

Drawings

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

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

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 the spike structure of the PF-D trimer and a negative staining electron microscope of the PF-D trimer provided in the example of the present application; wherein A is a schematic diagram of the spike structure of the PF-D trimer, and B is a negative staining-electron microscope image of the PF-D trimer.

FIG. 5 is a graph showing the time for 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 collection time for Sprague-Dawley rats provided in the examples of the present application.

Fig. 7 is a timing chart of syrian hamster immunization, blood collection, and spleen cell collection provided in the examples of the present application.

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

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

FIG. 10 shows the in vivo PF-D-trimer antibody titers of C57BL/6 mice at 35 days of immunization provided in the examples of the present application.

FIG. 11 is an ELISPot analysis of Th1 IFN-gamma in C57BL/6 mice stimulated with PF-D-trimer antigen as provided in the examples herein.

FIG. 12 shows PF-D-trimer antibody titres in Sprague-Dawley rats at 41 days of immunization provided in the examples of the present application.

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

FIG. 14 is a comparison of the neutralization activity of serum from Sprague-Dawley rats provided in the examples of the present application against SARS-CoV-2Delta strain, BA.2.12.1 and BA.4/5 pseudoviruses.

FIG. 15 shows PF-D-trimer antibody titres in syrian golden hamsters at day 41 of immunization provided by the practice of the present application.

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

FIG. 17 is an ELISPot analysis of the Th1 IFN-gamma of syrian golden hamster following stimulation with PF-D-trimer antigen provided in the examples of the present application.

FIG. 18 is a comparison of neutralization activities of serum from immunized syrian hamsters provided in the examples of this application against ancestor WA1 strain, delta strain, and Omicron BA1 strain.

FIG. 19 is a comparison of neutralization activity of serum from immunized syrian hamsters provided in the examples of this application against SARS-CoV-2Delta strain, BA.2.12.1 and BA.4/5 pseudoviruses.

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

FIG. 21 is a graph showing survival after intranasal infection of a SARS-CoV-2Delta strain with K18-hACE 2H 11 mice as provided in the examples of the present application.

FIG. 22 shows viral load analysis in lung tissue at necropsy (day 3 after challenge) of K18-hACE 2H 11 mice 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 will be further described in detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application. Reagents not specifically and individually described in this application are all conventional reagents and are commercially available; methods which are 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 the claims of the present invention and the above drawings are used for distinguishing similar objects, and are not necessarily used for describing a particular sequence or order, nor do they substantially limit the technical features that follow. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in sequences other than those illustrated or otherwise 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 the 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 main 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) trimeric domain. In this application, expression of the heterotrimeric fusion protein can be performed by constructing a recombinant expression vector for a fusion gene comprising the gene sequences of the S protein and the TD trimer domain, and transferring the recombinant expression vector to a host cell.

The term "adjuvant" refers to a nonspecific immunopotentiator that, when injected with an antigen or pre-injected into the body, enhances the body's immune response to the antigen or alters the type of immune response. The mechanism of enhancing immune response by adjuvant is to prolong the retention time of antigen in organism by changing the physical shape of antigen; stimulating the ability of mononuclear phagocytes to present antigen; stimulating lymphocyte differentiation and increasing the ability to amplify immune response. The adjuvant has many kinds, and common adjuvants are aluminum hydroxide adjuvant, corynebacterium pumilum, 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 host cells, and consists of the complete envelope outer structural domain of the S protein of the SARS-CoV-2 virus Delta strain, the C-terminal end of the fusion protein is fused with the TD trimer structural domain, and the TD trimer structural domain is a functional structural domain formed by a polypeptide and can be fused with the S protein of the novel coronavirus Delta strain to form a stable trimer 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 encoding the PF-D trimer fusion protein is shown as SEQ ID NO.2; the PF-D trimer comprises recombinant S protein, and the sequence of the recombinant S protein is shown as SEQ ID NO. 3; the nucleotide sequence of the nucleic acid for encoding the recombinant S protein is shown as SEQ ID NO.4; the recombinant S protein is obtained by mutating S1/S2 of original SARS-CoV-2Delta strain S protein (the amino acid sequence is shown as SEQ ID NO.5, and can be downloaded from GISAID, the accession number is EPI\UISL\U 1970349), deleting S protein membrane penetrating region fragments of SARS-CoV-2Delta strain S protein, and the obtained recombinant S protein is S protein in a C end segment truncated form, and retains two subunits S1 and S2, and retains all functional regions except the membrane penetrating region fragments, so as to maximally retain epitope on the Delta strain S protein.

PF-D-trimer is a fusion protein that mimics the natural trimeric pre-fusion structure of the S protein of the strain S of SARS-CoV-2 virus, and as found on the viral surface, consists of the complete ectodomain of the S protein of the strain S-CoV-2, with the C-terminus fused to the TD trimerization domain.

3. Preparation of heterotrimeric fusion proteins (PF-D trimers)

The embodiment of the application provides a preparation method of PF-D trimer, which comprises the following steps: 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 so that it secretes the produced protein; and separating and purifying the protein to obtain the heterotrimeric fusion protein.

Further, the protein separation and purification process comprises the following steps: preparing a TD trimer structural domain monoclonal antibody; capturing the heterotrimeric fusion protein by immunoaffinity chromatography with said monoclonal antibody; and obtaining the purified PF-D trimer by Tangential Flow Filtration (TFF) and nanofiltration (Poll).

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

In the examples of the present application, the antigen analysis and the codon optimization method are combined, and the DNA encoding the extracellular domain part of spike protein (S protein) on the membrane of the SARS-CoV-2 virus Delta strain is codon optimized to be expressed in Chinese hamster ovary cells (hereinafter referred to as "CHO cells"). The recombinant linear targeting plasmid capable of expressing PF-D trimer, namely recombinant plasmid pGENT1.0-DGV-Cov, is obtained by artificially synthesizing DNA (optimized) encoding the extracellular domain portion of S protein, adding HindIII cleavage site (recognition site is "5'-A ≡AGCTT-3'") by a conventional technical method of a person skilled in the art, purifying and linking with T4 ligase, and then inserting the DNA fragment encoding recombinant S protein (after mutation) into recombinant plasmid pGENT1.0-DGV plasmid, wherein the recombinant plasmid contains gene sequence encoding the PF-D trimer fusion protein.

In the examples of this application, recombinant linearized targeting plasmids expressing PF-D trimer were transfected into CHO K1 GenS cells to create a stable pool of CHO cells, CHO cells producing high titer products were screened out, and placed into a bioreactor, and the high secretion of protein, PF-D trimer, was sustained in the CHO cell medium by a serum-free semi-continuous (batch) cell culture process.

In the examples herein, at the end of the CHO cell growth phase, the cell supernatant was collected by depth filtration (filtration equipment available from saint Li Sisi taidi inc); purifying the filtered clarified medium, comprising: preparing a TD trimerization domain monoclonal antibody (alpha-TD trimerization domain monoclonal antibody), performing immunoaffinity chromatography on the TD trimerization domain monoclonal antibody to capture PF-D-trimer, and inactivating the PF-D-trimer by low pH treatment. The immunoaffinity chromatography eluate is concentrated and diafiltered using Tangential Flow Filtration (TFF) and final nanofiltration (Pall) to obtain purified SARS-CoV-2 recombinant S protein active material, i.e., purified PF-D-trimer.

In the examples herein, alpha-TD trimerization domain monoclonal antibodies were prepared in Sf9 insect cell lines using a recombinant baculovirus system. The preparation process of the alpha-TD trimerization domain monoclonal antibody comprises the steps of transferring Sf9 cells in a Working Cell Bank (WCB) into a shake flask for reviving and amplifying, transferring the Sf9 cells into a bioreactor (purchased from Applikon corporation) and culturing by using a serum-free medium (purchased from Beijing Ding biological Vbase). After Sf9 cells are infected with baculovirus inoculum, antibodies are secreted during Sf9 cell culture. At the end of the infection period Sf9 cells were collected by centrifugation. The clarified medium was purified by MabSelect-prism A packing (available from Cytiva, si. Tuo.) affinity chromatography according to the manufacturer's instructions. The affinity chromatography eluate was concentrated and diafiltered using tangential flow filtration (filtration equipment available from saigaku Li Sisi taidi co., ltd.). To prepare the immunoaffinity resin, an α -TD trimerization domain monoclonal antibody was coupled to NHS-activated Sepharose 4Fast Flow filler (available from sitohon, cytova) according to the manufacturer's instructions.

Results and analysis: the high-level production process of PF-D-trimer is realized by transfecting CHO cells with an expression vector containing a cDNA (containing optimized CHO codons) optimizing sequence encoding the PF-D-trimer, selecting CHO cells with optimal performance, optimizing the culture process. The process can make PF-D-trimer as secreted protein reach over 500 mg/L. As shown in fig. 1, the 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 of the reducing (+DTT) and non-reducing (-DTT) phases. The purified PF-D-trimer is a self-binding homotrimer stabilized by interchain disulfide bonds. As can be seen from fig. 2, the side-by-side analysis of PF-D-trimer for two purification batches demonstrated reproducibility of the purification process, with both batches having similar apparent purity levels after silver staining. Under reducing conditions, the PF-D-trimer was in a single form and had a molecular weight of about 170kDa. Under non-reducing conditions, 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

SARS-CoV-2S glycoprotein trimer fusion proteins, PF-D trimers, were analyzed by size exclusion chromatography (SEC-HPLC) using shimadzu LC-2030HPLC (available from shimadzu corporation) and analytical SRT SEC-3007.8 x 300mm columns (available from Sepax corporation). The HPLC detection conditions were: phosphate Buffered Saline (PBS) was used as the mobile phase, OD 280nm, detection time 20 min, flow rate 1 ml/min.

Results and analysis: PF-D-trimer was obtained by elution of the alpha-TD trimerized monoclonal antibody immunoaffinity chromatography, and the obtained PF-D-trimer was evaluated by SEC-HPLC, which resulted in a molecular weight of about 700kDa, with a purity of >97.9%, without significant aggregation or fragmentation (as shown in FIG. 3).

5. Negative staining transmission electron microscope analysis

To characterize the PF-D-trimeric protein samples immunoaffinity purified by alpha-TD trimerized monoclonal antibody, 20 μl of the samples were added dropwise to a 200 mesh grid (purchased from Fang Hua thin film copper mesh) and incubated at room temperature for 10 minutes. The mesh 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 HT7800 transmission electron microscope (available from Hitachi Corp.).

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

6. Heterotrimeric fusion protein compositions

In the examples herein, the heterotrimeric fusion protein composition is used for the preparation of a vaccine or medicament against a tumour or other immunological disease, the composition having two main components.

The first component is the aforementioned heterotrimeric fusion protein; namely PF-D Trimer (PF-D-Trimer).

The second component is an adjuvant. In the embodiment of the application, the adjuvant is taken as a foreign body relative to a host cell, and can play a role in early warning of the immune system of an organism together with an antigen (heterotrimeric fusion protein) or enhance a dangerous signal of the antigen invading the organism, so that the immune system is promoted to generate a strong reaction, and the immunogenicity of the antigen is improved; altering the immune response properties; the antigen amount and the number of times of immunizing agents required by successful immunization are reduced; improving immune response of people with low immune function. The mechanism of action of immunoadjuvants can be divided into: sustained release of antigen re-injection site (antigen depot effect); up-regulating a variety of cytokines and chemokines; recruiting immune cells to the injection site; enhancing antigen uptake and presentation; activating antigen presenting cells (antigen presenting cells, APCs), promoting transport of their mature presenting antigens to draining lymph nodes; activating inflammatory corpuscles, etc. In general, the immune adjuvant can be injected into the body to participate in the immunity in advance or simultaneously with the antigen, and the adjuvant can complete the function within 2-3 days after the immunity, and the action site is the injection site and drainage lymph node coexisting with the antigen.

Different antigen types can achieve the best effect by selecting different adjuvants, and the inventor aims at the heterotrimeric fusion protein in the embodiment of the application, adopts CpG 1018+aluminum hydroxide which contains CpG1018 and aluminum hydroxide as the adjuvants, and can achieve high efficiency in application effect. CpG1018 consists of a cytosine-phosphate guanine (CpG) base sequence, a motif that is a synthetic form of DNA that mimics bacterial and viral genetic material.

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

7. identification of the immunity and Effect of heterotrimeric fusion protein compositions

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

In the examples herein, the animals tested included 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 laboratory research center (Hubei Laboratory Research Center) without Specific Pathogen (SPF); H11-K18-hACE2 male mice (6-8 weeks) purchased from Jiangsu Jiuzhikang Biotech Co. Animals were free to drink and eat in a controlled environment with a light/dark cycle of 12 hours and temperature control of: the temperature is 16-26 ℃ and the humidity is controlled at 40-70%.

7.1 immunization and detection procedure for mice

All animal experiments were performed in SPF experiments.

(1) 28C 57BL/6 mice were randomly divided into 4 groups, and three vaccinations (shown in FIG. 5) were performed on day 0, day 14 and day 28, respectively, and the vaccinations and dosages were as shown in Table 1 below:

TABLE 1

Serum was collected from immunized mice by orbital bleeding on days 21 and 35 (as shown in fig. 5), and PF-D-Trimer (using a novel coronavirus S protein Trimer fusion protein, hereinafter abbreviated as "S-TD-oligomer") specific IgG endpoint geometric mean titer (Geometric Mean Ttiter, GMT) was detected by ELISA. Mice were sacrificed by cervical dislocation on day 51 and their spleens were taken (as shown in fig. 5) for ELISpot analysis.

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

TABLE 2

Serum was collected on day 41 (as shown in fig. 6) and assayed for S-TD-oligomer specific IgG endpoint Geometric Mean Titer (GMT) by ELISA.

(3) Syrian hamsters were randomly divided into 7 groups and three intramuscular Immunizations (IM) were performed on day 0, day 22, and day 90, respectively, as shown in fig. 7, with the vaccinations and doses shown in table 3 below:

TABLE 3 Table 3

Serum was collected on day 42 and day 110 (as shown in fig. 7) and the geometric mean titer of S-TD-primer specific IgG endpoint (GMT) was detected by ELISA. After mice were sacrificed on day 110, their spleens were taken (as shown in fig. 7) and subjected to ELISpot analysis.

7.2 ELISA detection

The specific IgG antibody endpoint GMTs was determined by ELISA as follows: a96-well ELISA plate (Costar) coated with recombinant SARS-CoV-2Delta strain S protein antigen (North Biol Co.) was used, and ELISA buffer was used at 37 ℃Sealing for 120 minutes; serum from the above mice was continuously added dropwise to a 96-well ELISA plate (Costar Co.) with 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), each well was added with HRP-conjugated ELISA buffer with horseradish peroxidase to 1:10000 dilutions of diluted goat anti-mouse IgG (proteontech), goat anti-rat IgG (proteontech) or goat anti-hamster IgG (Shanghai ring Biotechnology Co., ltd.) were incubated for 30 min at 37 ℃; after washing the ELISA plate 3 times with the buffer solution, TMB (3, 3', 5' -tetramethylbenzidine) substrate (Beijing Soy) was added to each well for color reaction, 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 multi-mode microplate reader (sammer fly). The positive judgment threshold (Cut-Off value) of the GMTs at the IgG end point is negative control OD average value+3 times negative control OD standard deviation, the sample OD value is larger than the threshold, and the sample is positive, otherwise, the sample is negative;

statistical analysis was performed using GraphPad Prism v 8.0.1.

7.3 ELISpot analysis

Spleen cells were isolated from C57BL/6 mice on day 51 post-inoculation and from Syrian hamsters on day 110 post-inoculation, and S protein-specific T cell assays were performed using ELISPOTPLUS mouse IFN-. Gamma.kit or Elispopplus hamster IFN-. Beta.kit (Mabtech) according to the manufacturer' S instructions. In short, 3 to 5X 10 ⁵ Individual splenocytes/wells were mixed with 4 μg of SARS-CoV-2 virus b.1.617.2 variant spinous process protein (south kyavizeter biotechnology limited)/well and incubated at 37 ℃; after 48 hours, wash 5 times with PBS buffer, add probe antibody at a concentration of 1. Mu.g/. Mu.L, and incubate at room temperature for 2 hours; rinse 5 times with PBS buffer, add 1:1000 dilution of alkaline phosphatase labeled streptavidin, at room temperature for 1 hour; washing 5 times with PBS buffer, adding a chromogenic solution (BCIP/NBT plus), and incubating for 10 minutes at room temperature; the color development was stopped with deionized water. The number of spots in the ELISpot wells was analyzed using an ELISpot reader system (AID Elispont reader, automatin Diagnostika GmbH company). The result data were analyzed using GraphPad Prism 8.0.2 software.

7.4 pseudo-virus based neutralization assay

Modified on the basis of the procedure of Nie et al for NT50 measurement. Briefly, immunized Sprague-Dawley rats or syrian hamsters were serially diluted 3 times with an initial dilution of 1:33.33 and at 37℃with SARS-CoV-2 pseudovirus (2X 10) ⁴ TCID ₅₀ Per mL) 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 pseudoviral 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 was defined as the dilution fold that resulted in greater than 50% inhibition of pseudoviral transduction compared to the control group.

7.5 SARS-CoV-2 neutralization assay

Vero E6 cells (2.5X10) ⁴ Cells/well) were seeded in 96-well plates and incubated overnight. Taking serum, inactivating for 30 minutes at 56 ℃, diluting with serum-free DMEM medium, and further diluting continuously with an initial dilution factor (dilution multiple) of 8; the diluted serum WAs then mixed with SARS-CoV-2 virus (WA 1-Hu-1, delta Strain YJ20210701-01, omicron Strain 249099, center for disease prevention control, hubei province, at a ratio of 1:1, 100 TCID) ₅₀ 100. Mu.L and incubated at 37℃for 1 hour; the diluted serum/virus mixture was added to Vero cells and incubated with 5% co at 37 °c ₂ Incubating for four days; cytopathic effects (CPE) of each serum dilution were monitored every 24 hours by inverted microscopy; the neutralization endpoint 50% was calculated by the Reed-Muench method (Reed, l.j. And Muench, h., 1938), i.e., the dilution of serum that protected 50% of the cells from CPE, to obtain the neutralizing antibody titer for each serum.

7.6 Immune and toxicity attack protection study of K18-hACE 2H 11 transgenic mice

The K18-hACE 2H 11 male transgenic mice were divided into 5 vaccinated groups, as shown in FIG. 8, blood was collected before immunization as a pre-immunization control; subcutaneous inoculations were made twice on day 0 and 21, respectively; serum was collected on day 28 to detect SARS-CoV-2 specific IgG endpoint GMT. On day 35, mice were transferred to BSL3 laboratory (institute of martial arts, chinese, virus). The inoculation reagents and inoculum size are shown in Table 4 below:

TABLE 4 Table 4

In the challenge protection study, 1×10 mice each ⁵ pfu (50. Mu.l) of the SARS-CoV-2Delta live virus (CRST: 1633.06.IVCAS 6.7593) was infected nasally by drops. Three animals were used as control groups and inoculated with the same volume of PBS; body weight changes and survival assays 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.

The virus titer in lung tissue was determined by cell culture infection (TCID 50) as follows:

plaque analysis was performed to determine viral load. Vero E6 cells were grown at 10 days in advance 1 day ⁵ Density of individual cells/well was seeded into 24-well plates; taking 0.1g of lung tissue and adding 1mL of PBS buffer to prepare a lung homogenate; the next day, for each sample, 100 μl of serial dilutions of 10-fold lung homogenate supernatant were added dropwise to the infected cells and incubated for 1 hour at 37 ℃; removing the virus dilution and adding 1% methylcellulose; after incubation of the well plate at 37 ℃ for 4 days, the upper layer in the well was discarded, 1mL of fixation 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 viral spots was counted. Plaque forming units per mL (PFU/mL) were determined using the following formula: (# plaque x dilution factor)/0.1 mL. Plaque-free scoringIs that<1 and is used to calculate the lower detection limit. The PFU/mL values were adjusted to 1mL and 1g of tissue volume to calculate plaque forming units per gram of tissue. The result data were analyzed using GraphPad Prism 8.0.2 software.

7.7 results of composition immunization and efficacy identification tests:

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, supra) +aluminum hydroxide+CpG 1018 adjuvant was first evaluated in C57BL/6 mice. Serum from these mice was assessed for the amount of anti-spinous process protein IgG. Mouse serum injected with PBS formulation showed only background level of titer. As shown in FIG. 9, at 21 days, the mutual GMT titers of the antibodies in the 5. Mu.g, 10. Mu.g and 20. Mu.g groups reached 6.5X10, respectively ⁵ 、4.6×10 ⁵ And 1.2X10 ⁶ . There was a significant difference in antibody titers between the 10 μg group and the 20 μg group (p=0.0329), and there was 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 in the 5. Mu.g, 10. Mu.g and 20. Mu.g groups reached 9.3X10, respectively ⁵ 、1.1×10 ⁶ And 1.2X10 ⁶ There was no significant difference in antibody titers between groups. These results indicate that the second immunization enhances the antibody response even though the 20 μg group had the same GMT on day 21 and day 35.

The mice were further tested for immune serum, whether the vaccine adjuvant system provided herein was able to induce Th1 responses in vaccinated mice. As shown in FIG. 11, the Th1 gamma interferon (IFN-. Gamma.) response was 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-gamma produced by T cells in the 5. Mu.g, 10. Mu.g, and 20. Mu.g groups compared to the PBS control group. These results indicate that the PF-D-trimer provided herein is capable of promoting development and activation of Th1 cells in C57BL/6 mice.

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

The examples of this application examined the group consisting of PF-D-trimer + aluminum hydroxide + CpG1018 adjuvantImmunogenicity of the compounds in Sprague-Dawley rats. As shown in FIG. 12, the immune serum was evaluated for the amount of anti-spinous process protein IgG, and the mutual GMT titer of 50. Mu.g and 100. Mu.g antibodies was 10 ⁷ There were no significant differences between the two groups of rats above. The 100 μg dose group may give higher antibody titers, as all animals in the group showed equivalent titers.

From the results of fig. 12, it can be seen that since two doses of 50 μg and 100 μg produced high antibody titers in Sprague-Dawley rats, and there was no significant difference, the examples of the present application used a smaller dose, i.e., 50 μg, to verify the neutralizing antibody titers. During the pandemic of new coronapneumonia, a number of SARS-CoV-2 variants have emerged, including Omicron (BA.1), a highly mutated variant, highly resistant to vaccine-induced antibody neutralizationa., 2022). Thus, immune sera were further tested for their neutralizing capacity against both the Delta strain of SARS-CoV-2 and the Omacron strain. As shown in FIG. 13, ID for Delta strains and Omiclon BA.1 with an aluminum hydroxide+CpG 1018 adjuvant+50μ gPF-D-trimer composition ₅₀ GMT was 9337 and 2113, respectively. Even though antibodies against the ba.1 strain were significantly reduced, neutralizing antibodies against both strains could be generated using the PF-D-trimer formulations (compositions) provided herein. The serum was also used to evaluate its neutralizing activity against SARS-CoV-2Delta strain, BA.2.12.1 and BA.4/5 pseudoviruses, as shown in FIG. 14, its ID ₅₀ GMT is 7290, 7058, respectively.

7.7.3 heterotrimeric fusion protein compositions induce an immune response in syrian hamsters

The inventors of the present application evaluated the immunogenicity of PF-D-trimer compositions on syrian hamsters. As shown in fig. 7, by inoculating two to three times syrian hamsters with a composition containing 5 μg or 10 μg PF-D-trimer, the adjuvant in the composition comprises aluminum hydroxide and CpG1018, or only aluminum hydroxide or CpG1018, for a period of more than two months (specifically 68 days). Hamster serum immunized with all formulations (including adjuvant-free proteins)As shown in FIG. 15, the antibody geometry average value of the group containing anti-spinous protein IgG in the 5. Mu.g PF-D-trimer group and the aluminum hydroxide adjuvant alone was 8.9X10 ⁵ In the group with aluminum hydroxide and CpG1018 adjuvant, the geometric mean of the antibody was 3.6X10 ⁶ It follows that PF-D-trimer supplemented with aluminium hydroxide and CpG1018 adjuvant was used to immunize syrian hamsters, which produced higher GMT titres of antibodies in vivo; the geometric mean of the antibody in the group containing only aluminum hydroxide adjuvant was 1.1X10 in the group containing 10. Mu.g PF-D-trimer ⁶ In the group with aluminum hydroxide and CpG1018 adjuvant, the geometric mean of the antibody was 2.8X10 ⁶ The same conclusion can be drawn as for the 5. Mu.g PF-D-trimer panel.

In the group without adjuvant, i.e.inoculated with PF-D-trimer alone, the antibody still produced had a GMT titer of 2.9X10 ⁵ But significantly less effective than the adjuvant. FIG. 16 shows a comparison of the results of the second and third inoculations, from which it can be seen that, although the antibody titer of the third time is higher than that of the second time, the overall conclusions of the second and third inoculations remain consistent in the 5. Mu.g PF-D-trimer group and the 10. Mu.g PF-D-trimer group, with or without the addition of adjuvant.

The examples herein performed a Th1 IFN-gamma response test on spleen cells of immunized hamsters. As is clear from the spot count results shown in fig. 17, PBS-vaccinated hamsters with aluminum hydroxide plus CpG1018 induced partial T cell responses, but the adjuvant-free group showed a higher spot count than the PBS group in the PF-D-primer (S-TD-primer) -immunized group, and the adjuvant-added S-TD-primer showed a higher spot count, indicating that the adjuvant-added hamsters had the best stress response. 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 indicate that the PF-D-Trimer composition with aluminum hydroxide and CpG1018 adjuvant directs hamster T cells to the Th1 phenotype.

FIG. 18 shows the ancestral WA1 strain, delta strain and Omicron of SARS-CoV-2 virus inoculated three times with 5. Mu.g of a PF-D-trimer composition (containing aluminum hydroxide and CpG1018 adjuvant)Neutralization Activity results of strains Delta Strain ID ₅₀ GMT was 851, WA1 (Wuhan) strain was 342, and Omicron strain was 181. The results of the above data indicate that neutralizing antibodies against three strains, which are the most potent neutralizing against Delta strains, were produced in hamster serum induced by PF-D-trimer composition, and also have significant neutralizing effects against WA1 and Omicron strains. The above serum was also used to evaluate 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, strain BA.2.12.1 was 1200, strain BA.4/5 was 903. The above results are similar to those of experiments performed using authentic viruses. The neutralizing activity of the serum of the immunized hamster against Delta strain was higher compared to ba.2.12.1 strain and ba.4/5 strain.

Immunogenicity and protective effects of 7.7.4 heterotrimeric fusion protein compositions on K18-hACE 2H 11 mice

The inventors of the present application evaluated the immunogenicity and protective effects of PF-D-trimer and a combination of PF-D-trimer and aluminum hydroxide+CpG 1018 adjuvant in K18-hACE 2H 11 mice. hACE2 mice develop respiratory diseases similar to severe COVID-19, and are suitable models for studying immune responses.

The inventors examined the immunogenicity of the aluminum hydroxide + CpG1018 adjuvant + PF-D-trimer composition in transgenic mice and assessed the amount of anti-spinous protein IgG in immune serum, as shown in FIG. 20, with a 30 μg panel of antibody GMT titres reaching 10 ⁷ The above, while the 10 μg group was significantly higher than the control group although the titer was lower than the 30 μg group, the titer was close to 3×10 ⁶ . Adjuvant group mice (non-vaccinated with PF-D-trimer) lost weight after intranasal infection with Delta strain, and this group of mice died on day 4 or day 5 post infection (as shown in figure 21). Mice vaccinated with the PF-D-trimer composition lost weight two days prior to infection, and subsequently began to recover, and after 7 days of infection, the mice were in good condition. Viral loads in mouse lung tissue were detected on day 3 post infection, as shown in FIG. 22, with live virus titers in 10 μg and 30 μg PF-D-trimer group mice lung tissue below the minimum detection limit (100 PFU/g), while the GMT of adjuvant group mice exceeded 1×10 ⁵ PFU/g lung tissue.

Mechanism of action of 7.7.5 adjuvant

In the examples of the present application, the adjuvant used was CpG1018 and/or aluminium hydroxide. From the results of the foregoing examples, a compound adjuvant using CpG1018 and aluminum hydroxide can achieve better immune effect when used in animal immune test in combination with PF-D-trimer as described in the present application. The aluminum adjuvant in the composite adjuvant in the present application can act to deliver PF-D-trimer to the immune system to induce immunity; cpG1018 in the composite adjuvant contains immunostimulant or enhancer, and uses receptor mediated signal path to regulate immune reaction, and can enhance immunogenicity of PF-D-trimer as antigen. From the results in the previous examples, it is clear that the combined adjuvant (without antigen protein) of CpG1018 and aluminum hydroxide and PF-D-trimer (without adjuvant) described in the present application can induce Th1 immune response in animals vaccinated alone, whereas the combination of PF-D-trimer and combined adjuvant induced Th1 immune response in vaccinated animals was stronger and better.

7.7.6 immune studies on different novel coronavirus strains

With the evolution of the virus and antibody selection pressure, new SARS-CoV-2 variants have emerged as a variety of variants, including Delta strains and Omacron strains, and most published studies use vaccines based on the original WA1 strain. In the examples herein, it can be seen from the results of the Sprague-Dawley rat and Syrian hamster model immunoassays that the neutralization ability WAs significantly enhanced when the PF-D-trimer compositions provided herein were inoculated as compared to the results without inoculation, for the WA1 strain, the Delta strain, and the Omicron strain.

The foregoing is merely a preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions easily contemplated by those skilled in the art within the technical scope of the present application should be covered by the scope of the present application.

Claims (2)

1. Use of a heterotrimeric fusion protein in the preparation of a novel coronavirus pneumonitis vaccine; the amino acid sequence of the heterotrimeric fusion protein is shown as SEQ ID NO. 1, and the nucleotide sequence of the nucleic acid for encoding 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 encoding 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 mutating the original novel coronavirus Delta strain S protein and deleting the membrane penetrating region fragment thereof; the amino acid sequence of the S protein of the original novel coronavirus Delta strain is shown as SEQ ID NO. 5, and the nucleotide sequence of the nucleic acid for encoding the S protein of the original novel coronavirus Delta strain is shown as SEQ ID NO. 6; the novel coronavirus is a Delta strain or an Omicron strain.

2. The use according to claim 1, the components for preparing the novel coronavirus pneumonitis vaccine further comprising an adjuvant consisting of CpG1018 and aluminium hydroxide in a mass ratio of CpG1018: aluminum hydroxide = 2-6: 1.