WO2023220842A1

WO2023220842A1 - A fusion protein as a subunit vaccine immunogen against sars-cov-2 and the preparation thereof

Info

Publication number: WO2023220842A1
Application number: PCT/CN2022/000090
Authority: WO
Inventors: Xiaolin Liu; Hongliang LIV; Wenhong Zhang; Shengli BI
Original assignee: Shenzhen Genius Biotech Service Co.,Ltd.
Priority date: 2022-05-19
Filing date: 2022-05-19
Publication date: 2023-11-23

Abstract

Provided is a fusion protein as a subunit vaccine immunogen against SARS-CoV-2 comprising of receptor binding domain (RBD) fragment of SARS-CoV-2 spike protein and Tetanus toxoid fragment P2, they are fused by a linker sequence. Also provided is a method of obtaining the fusion protein.

Description

A fusion protein as a subunit vaccine immunogen against SARS-CoV-2 and the preparation thereof

FIELD OF THE DISCLOSURE

This disclosure relates to a fusion protein as a subunit vaccine immunogen against SARS-CoV-2, which comprising the receptor binding domain (RBD) fragment of SARS-CoV-2 spike protein and tetanus toxoid P2 fragment. The disclosure also provides a method of obtaining the fusion protein and the use as vaccine for against SARS-CoV-2.

BACKGROUND

Coronavirus infectious disease (COVID-19) was claimed a pandemic by World Health Organization (WHO) . COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) . SARS-CoV-2 belong to beta-coronavirus lineages B, is highly pathogenic.

SARS-CoV-2 has four structural proteins, known as the S (spike) , E (envelope) , M (membrane) , and N (nucleocapsid) proteins. The N protein holds the RNA genome, and the S, E, and M proteins together create the viral envelope. The spike ( "S" ) protein is responsible for allowing the virus to attach to and fuse with the membrane of a host cell.

The S protein is a class I protein which are known to exist as trimers in their pre-fuse and post-fuse states. The S protein S 1 subunit mediates cellular attachment, and the S2 subunit is involved in which allows viral genome entry into the cell. The S protein has two states, a pre-fuse state and a mature/active form, achieved after proteolytic cleavage and activation.

Receptor binding domain protein (RBD) of SARS-CoV-2 S proteins are essential for vaccine and drug development. Recombinant RBD (receptor binding domain) is challenging to produce and very unstable. Most of the recombinant RBD as immunogen on the market is expressed with mammalian cells, making the recombinant version different from wild-type and potentially less useful in research or therapy.

There are more than 250 candidate vaccines in developing worldwide, while among the vaccine technologies under evaluation are inactivated virus vaccines, recombinant protein subunit vaccines and nucleic acid vaccine. Subunit vaccine might be one of the saftest candidate vaccines for its simpler in composition and less uncontrollable risk factors.

The RBD and has the potential capacity as a vaccine immunogen containing abundant T cell and B cell epitopes including neutralization epitopes. Subunit vaccines possessed high safety profile, consistent production and could induce immune response, more importantly need appropriate adjuvants to induce high level neutralization antibody and giving effective protection.

Tetanus toxoid P2 fragment as a T-helper-epitope-rich immunogen could hance effectively humoral and cellular immune responses of

immunogenic epitopes. P2 exerts the requisite immunological enhancement and promotes production of high levels of antibodies by achieving effective T-B cell reaction. P2 has been considered because it is safe and also has been commercially applied in making human vaccines. Gln830 -Glu844 of TT (P2 peptide) as a promiscuous T-helper epitope has a strong binding capacity to DR3 allele and is frequently used as T cell stimulator to enhance the immunogenicity of exogenous epitopes and induce cellular immunity for subunit vaccine. P2 peptide has only 15 amino acids, was easily expressed in the form of recombinant protein, and minimized the interference with epitope recognition and/or binding theoretically.

Although Escherichia coli is one of the most widely used hosts for the production of recombinant proteins, insoluble expression of heterologous proteins is a major bottleneck in production of these recombinant proteins. A major pathway of product loss during the refolding step is aggregation. These insoluble protein aggregates or inclusion bodies (IB′s) can be used only after refolding in vitro into soluble form having its native conformation. The inclusion bodies of different proteins have different characteristics and require a lot of optimizations for refolding individual protein. In most cases, a significant amount of precipitation is observed while refolding the proteins. This results in a great loss of overall yield of the target proteins, with approximately 40%being refolded to soluble and biologically active form.

Thus, an appropriate method is required which minimizes the loss with improved purity during the refolding step in preparation of fusion protein.

SUMMARY

The purpose of the present invention is to provide a fusion protein as a subunit vaccine immunogen against SARS-CoV-2 and the preparation thereof.

In order to achieve the above object, the present invention adopts the following technical means:

A fusion protein as a subunit vaccine immunogen against SARS-CoV-2, comprising a receptor binding domain (RBD) fragment of SARS-CoV-2 spike protein and Tetanus toxoid peptide P2, they are fused by a linker sequence.

Preferably, the amino acids sequence of the receptor binding domain (RBD) fragment of SARS-CoV-2 spike protein is shown in SEQ ID NO: 1.

Preferably, the amino acids sequence of the Tetanus toxoid peptide P2 is shown in SEQ ID NO: 2.

Preferably, the amino acids sequence of the linker is shown in SEQ ID NO: 3-7.

Preferably, the amino acids sequence of the fusion protein is shown in SEQ ID NO: 8.

Preferably, the fusion protein is obtained via a prokaryotic expression system.

Preferably, the prokaryotic expression system is E. coli.

Furtherly, the present invention also provides a method for preparation the fusion protein, the method comprising the steps of:

a) transformation of Escherichia coli with a desired gene coding for the fusion protein using a plasmid vector;

b) culturing the transformed Escherichia coli in suitable culture medium under suitable conditions,

c) isolation and purification of inclusion bodies,

d) denaturation and solubilization of inclusion bodies at high pH value ranging from 8 to 9,

e) followed by pH adjustment within a range of 6 to 8.5, preferably at 8.0 of the solubilized protein, to produce a refolded protein,

f) intermediate purification of the refolded protein using ion exchange chromatography, hydrophobic interaction chromatography to obtain ＞99.9%pure native fusion protein and

g) the semi purified protein obtained in step (f) is further purified by one or more chromatographic separation using anion exchange chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography and size exclusion chromatography to obtain the fusion protein.

Furtherly, the present invention also provides the use of the fusion protein as a subunit vaccine immunogen in preparing the vaccine against SARS-CoV-2. and

A vaccine against SARS-CoV-2, wherein the vaccine contains effective amount of the fusion protein.

Beneficial Effect of the Invention

The invention demonstrates for the first time that P2 and fragments thereof may be used as intramolecular adjuvant for enhancing immunogenicity of a target protein. Therefore, the invention provides a novel use of P2 and fragments thereof, and provides a novel method for enhancing immunogenicity of a target protein.

a novel molecule of RBD linked P2 peptide (referred to as fusion) is made, can be used as an immunogen to generate immunity to SARS-CoV-2 and to generate protective efficiency as potential subunit vaccine.

In addition, since the fusion protein of the invention exhibits a stronger immunogenicity as compared to a target protein alone, the invention provides a new option for the manufacture of a medicament or vaccine and may achieve more effective treatment and prevention of the corresponding diseases.

For example, the fusion protein of the invention comprising P2 (or a fragment thereof) and an SARS-COV-2 S1 RBD (or an immunogenic fragment thereof) exhibits a stronger immunogenicity as compared to a SARS-COV-2 S1 RBD (or an immunogenic fragment thereof) alone, and therefore the fusion protein may be useful for the manufacture of a pharmaceutical composition and more effectively prevent and treat SARS-COV-2 infection and diseases associated with SARS-COV-2 infection such as COVID-19.

DESCRIPTION OF THE DRAWINGS

FIG. 1A-1D show SDS-PAGE analytic results of expression, purification and renaturation of the fusion constructed in Example 7, wherein the sample used in FIG. 1A is a small scale expression of fusion protein, the sample used in FIG. 1B is a large scale expression of fusion protein, the sample used in FIG. 1C is fusion protein purified with renaturation and HIC. and the sample used in FIG. 1D is fusion protein purified with renaturation, HIC, IEX. Lane left: protein molecular weight marker; Lane 1: Negative control; Lane 2: none-induced bacteria; Lane 3 and Lane 4: induced bacteria; Lane 5: Total bacteria protein; Lane 6: soluble fraction; Lane 7: Aggregates; Lane 8: fusion protein in urea solution; Lane 9: Unbound fraction eluted with IEX; Lane 10: fusion protein eluted with HIC; Lane11: renatured fusion protein.

FIG. 2 shows the results of Western blotting using the fusion proteins constructed in Example 8. Left lane: protein molecular weight marker; Lane A1: fusion protein Lane A2: Positive control; Lane B1: fusion protein Lane B2: Positive control; Lane C1: fusion protein Lane C2: Positive control; Lane D1: fusion protein Lane D2: Positive control; The results showed that fusion proteins tested had significant reactivity with the SARS-COV-2-specific antibody. A: Primary antibody; B: Rabbit monoclonal antibody against RBD; C: Rat polyclonal antibody against SARS-COV-2; D: Human convalescent serum.

FIG. 3 shows binding of recombiant P2-RBD fusion Protein to P2-hACE2. Binding of the recombinant P2-RBD, RBD to hACE2 was measured with ELISA; 0.5 μg P2-RBD or RBD was coated on plate with incubation of serially diluted hACE2. Binding was detected by using HRP conjugated anti-RBD antibody. Experiments were performed in duplicate and the error bars denote ± SD, n = 2.

Fig. 4 shows binding evaluation mice sera immunized with P2-RBD vaccine on day 27 to the SARS-CoV-2 RBD as measured by ELISA, Evaluation of binding (A) and competition with hACE2 (B) of mouse sera to the SARS-CoV-2 RBD as measured by ELISA. (A) 200 ng of RBD was coated and 5-fold serially diluted serum was added after blocking. After washing, the binding was detected by HRP conjugated anti-mouse IgG antibody. (B) 200 ng of RBD were coated and 5-fold serially diluted mouse serum was added in the presence of～20 nM biotinylated hACE2 followed by PBST washing. For detection, streptavidin-HRP secondary antibody was used. Experiments were performed in duplicate and the error bars denote ± SD, n = 2. Statistical significance was defined as *: P ＜ 0.05.

Fig. 5 shows total anti-S IgG titers in mice immunized with P2-RBD with adjuvants. Sera from BALB/c mice in Fig. 5 (N=6 per group) immunized with 5 or 10 μg of immunogen with MF59 were quantifed for the total amount of anti-S IgG with ELISA

Fig. 6 shows induction of neutralizing antibodies by MF59 adjuvanted SARSCoV-2 P2-RBD 2 weeks post-second injection. BALB/c mice (N=6 per group) were immunized with 2 dose levels of bacterial cell-expressed SARS-CoV-2 P2-RBD adjuvanted with MF59 and the antisera were harvested at 2 weeks after the second injection. Te antisera were subjected to neutralization assay with pseudovirus expressing SARS-CoV-2 spike protein to determine the ID50 titers of neutralization antibodies.

Fig. 7 shows neutralization of wild-type SARS-CoV-2 virus by antibodies induced by SARS-CoV-2 P2-RBD adjuvanted with MF59. The antisera were collected as described in Fig. 6 (N=6 per group) and subjected to a neutralization assay with wild-type SARS-CoV-2 to determine neutralization antibody titers.

Fig. A-8F shows immunogenicity of CBSVX-CoV 2020 vaccine in cynomoμgus macaques. (A) Groups of cynomoμgus macaques (n = 4 per arm) were immunized

weeks

0 and 3 with 2.5 μg with 50μg MF59 or 5 g or 25 μg P2-RBD with 50 μg Matrix-M1. Anti-spike EC50 IgG titers were measured

weeks

0, 1, 3, and 5. Lines indicate anti-spike IgG titers for individual macaques in each group. (B) Anti-spike EC50 IgG serum titers week 5 in CBSVX-CoV 2020 vaccine immunized NHP compared to anti-S EC50 IgG titers in convalescent human sera. (C) ACE2 inhibition IC50 serum titers week 5 CBSVX-CoV2020 immunized macaques compared to ACE2 inhibition titers in convalescent human sera, (D) Neutralization CPE100 titers against wild type SARS-CoV-2 virus week 5 CBSVX-CoV 2020 vaccine immunized macaques compared to neutralization CPE100 titers in convalescent human sera, (E) Subgenomic RNA (sgRNA) copies in BAL

fluid days

2 and 4 post challenge SARS-CoV-2 virus in placebo and CBSVX-CoV 2020 vaccine immunized macaques. (F) sgRNA copies in nasal

swab samples days

2 and 4 post challenge with SARS-CoV-2 virus in placebo and CBSVX-CoV 2020 vaccinated macaques. Dashed horizontal line indicates the limit of detection (LOD) . ConV: Human convalescent serum. BAL: bronchoalveolar lavage.

Fig. 9 shows representative histopathology of lungs from CBSVX-CoV2020 vaccinated cynomoμgus macaques challenged with SARS-CoV-2 (CN1 strain) . (A, B, C) Microscopic findings in placebo treated animals includes eosinophils expanding the mucosa of bronchi (asterisks) , perivascular mononuclear infiltrates, and mixed inflammation (macrophages and neutrophils) within alveoli. Three out of four animals exhibited a combination of the majority of findings. (D, E, F) Microscopic findings in the group immunized with 2.5 μg P2-RBD/MF59 includes minimal to mild mononuclear perivascular infiltrates, and no changes in the bronchi. Rare foreign material was observed in the one male with mixed inflammation. (G, H, I) Histological findings in the group immunized with 5 μg P2-RBD/MF59 includes minimal to mild perivascular mononuclear or mixed cell infiltrates (1/1 males and 2/3 females) , mild to moderate mixed cell inflammation (1/3 females) , and minimal to mild alveolar macrophages (1/1 males and 3/3 females) . The female with mixed inflammation additionally observed acellular bacteria and foreign material. (J, K, L) There were no remarkable pathologic changes observed in the bronchi, vascular, or alveoli of animals vaccinated with 25 μg P2-RBD/MF59.

DESCRIPTION OF THE INVENTION

In the invention, when the sequence of Spike receptor binding domain is involved, it is described as the sequence set forth in SEQ ID NO: 1. For example, in the expression "amino acid residues from positions 308 to 548 of RBD" , amino acid residues from positions 308 to 548 refers to amino acid residues from positions 308 to 548. Therefore, in the invention, the term "RBD" intends to comprise all such polypeptides and variants, including the polypeptide set forth in SEQ ID NO: 1 and its natural or artificial variants, wherein the variants retain the biological properties of RBD, i.e. have a strong immunogenicity and no cytotoxity. In addition, when sequence fragments of RBD are described, they include not only the sequence fragments of a polypeptide set forth in SEQ ID NO: 1, but also the corresponding sequence fragments of the natural or artificial variants of the polypeptide. For example, the expression "amino acid residues from positions 308 to 548 of RBD" intends to comprise amino acid residues from positions 308 to 548 of SEQ ID NO: 1 and the corresponding fragments of the variants (natural or artificial) of a polypeptide set forth in SEQ ID NO: 1.

According to the invention, the term "P2" refers to tetanus Toxoid Peptide (aa830-844) . In the invention, the exemplary amino acid sequence of P2 is set forth in SEQ ID NO: 2. Therefore, in the invention, when the sequence of P2 is involved, it is described as the sequence set forth in SEQ ID NO: 2. For example, in the expression "amino acid residues from positions 830 to 844 of TT, amino acid residues from positions 830 to 844 refers to amino acid residues from positions 830 to 844. However, a person skilled in the art understands that mutations or variations (including, but not limited to, substitution, deletion and/or addition) may naturally occur in or are introduced artificially into SEQ ID NO: 2 without affecting the biological properties of P2.

According to the invention, the term "linker" refers to a short peptide for linking two molecules (for example, proteins) . Generally, a protein, such as a target protein 1-linker-a target protein 2, is obtained by introduction (for example, by PCR amplification or ligase) of a polynucleotide encoding the short peptide between two DNA fragments encoding two target proteins to be linked, respectively, and protein expression thereof. As well known by a person skilled in the art, linkers include, but are not limited to flexible linking peptides, such as Gly-Gly-Gly-Gly (SEQ ID NO: 3) , Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 4) , Gly-Gly-Ser-Ser (SEQ ID NO: 5) and (Gly-Gly-Gly-Gly-Ser) 3 (SEQ ID NO: 6) , Gly-Ser-Gly-Ser-Gly- (SEQ ID NO: 7) . According to the invention, the expression "corresponding sequence fragments" or "corresponding fragments" refers to fragments that are located in equal positions of sequences when the sequences are subjected to optimal alignment, namely, the sequences are aligned to obtain a highest percentage of identity. The invention is at least partially based on the inventors′ surprising discovery: after expression of P2 or a fragment thereof with a target protein (for example, SARS-COV-2 S1 protein, or an immunogenic fragment thereof) , P2 or a fragment thereof significantly enhances immunogenicity of the target protein. Namely, P2 or a fragment thereof may be used as intramolecular adjuvant for enhancing immunogenicity of a target protein by expression with the target protein.

According to the invention, the term "E. coli expression system" refers to an expression system consisting of E. coli (strain) and a vector, wherein the E. coli (strain) is available on the market, including but not limited to: GI698, ER2566, BL21 (DE3) , B834 (DE3) , BLR (DE3) , etc.

According to the invention, the term "chromatography" includes, but is not limited to:ion exchange chromatography (e.g. cation-exchange chromatography) , hydrophobic interaction chromatography, absorbent chromatography (e.g. hydroxyapatite chromatography) , gel filtration chromatography (gel exclusion chromatography) , and affinity chromatography.

A mixed mode column refers to a column with a resin that has both cation exchange properties as well as hydrophobic interactions.

According to the invention, the term "pharmaceutically acceptable carriers and/or excipients" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with subjects and active ingredients, and are well known in the art. including, but not limited to pH adjusting agents, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to: anion surfactants, cation surfactants, or non-ionic surfactants (for example, Tween-80) ; and ionic strength enhancers include, but are not limited to sodium chloride.

According to the invention, the term "intramolecular adjuvant" refers to such an adjuvant, which forms a fusion protein with a target protein (i.e. an immunogen) , is present in the same molecule as the immunogen (i.e. a S protein comprising P2 and the immunogen) , and acts as the adjuvant of the immunogen to enhance immunogenicity of the immunogen. Namely, an intramolecular adjuvant is an adjuvant capable of enhancing immunogenicity of a target protein (immunogen) fused and expressed therewith, which generally refers to a polypeptide fragment. In the invention, an intramolecular adjuvant especially refers to a Tetanus Toxoid Peptide (P2) or a fragment thereof.

In some embodiments, an immunogen can be a recombinant SARS-COV-2 RBD protein or immunogenic fragment thereof, a protein nanoparticle or virus-like particle including the recombinant SARS-COV-2 RBD or immunogenic fragment thereof, or nucleic acid or vector encoding the recombinant SARS-COV-2 RBD protein or immunogenic fragment thereof, that is capable of inducing an immune response in a mammal, such as a mammal infected or at risk of infection with a pathogen. Administration of an immunogen to a subject can lead to protective immunity and/or proactive immunity against a pathogen of interest.

In another aspect, the invention also relates to a method for preventing and/or treating SARS-COV-2 infection and/or diseases associated with SARS-COV-2 infection such as COVID-19, comprising administering an effective amount of the fusion protein of the invention or the pharmaceutical composition comprising the fusion protein, wherein the fusion protein comprises P2 or a fragment thereof and an SARS-COV-2 S1or an immunogenic fragment thereof, which are linked together, optionally via a linker.

In another aspect, the invention provides a method for enhancing immunogenicity of a target protein, comprising obtaining a fusion protein comprising P2 or a fragment thereof as defined above and the target protein, so as to enhance immunogenicity of the target protein.

In a preferred embodiment, the fusion protein may be obtained by expression of P2 or a fragment thereof with the target protein, optionally using a linker. In a preferred embodiment, the target protein is the SARS-COV-2 spike receptor binding domain or an immunogenic fragment thereof as described above.

Therefore, in an embodiment, the invention provides a method for enhancing immunogenicity of an SARS-COV-2 S1 or an immunogenic fragment thereof, comprising obtaining a fusion protein comprising P2 or a fragment thereof and an SARS-COV-2 S1 or an immunogenic fragment thereof, so as to enhance immunogenicity of the SARS-COV-2 S1 or an immunogenic fragment thereof. In a preferred embodiment, the fusion protein may be obtained by fusion expression of P2 or a fragment thereof with a SARS-COV-2 S1or an immunogenic fragment thereof, optionally using a linker.

In another aspect, the invention relates to a use of P2 or a fragment thereof in the enhancement of immunogenicity of a target protein, characterized by obtaining a fusion protein comprising P2 or a fragment thereof and the target protein.

In a preferred embodiment, the fusion protein may be obtained by fusion expression of P2 or a fragment thereof with the target protein, optionally using a linker. In a preferred embodiment, the target protein is a SARS-COV-2 spike receptor binding domain or an immunogenic fragment thereof.

Therefore, in an embodiment, the invention relates to a use of P2 or a fragment thereof in the enhancement of immunogenicity of a SARS-COV-2 S1 or an immunogenic fragment thereof, characterized by obtaining a fusion protein comprising P2 or a fragment thereof and the SARS-COV-2 S1or an immunogenic fragment thereof. In a preferred embodiment, the fusion protein may be obtained by fusion expression of P2 or a fragment thereof with the SARS-COV-2 S1 or an immunogenic fragment thereof, optionally using a linker.

Accordingly, the main embodiment of the invention provides cost effective, robust, chromatography based process for preparation of recombinant fusion protein from a prokaryotic expression system, wherein protein is expressed as inclusion bodies which comprises: a. transformation of bacterial cells with a desired gene coding for immunogen using a plasmid vector, b. culturing the transformed bacterial cells in chemically defined media supplemented with glucose as carbon source wherein pH is maintained at 5-9 and at temperature of 30-. 40℃. lysing bacterial cells by buffer thereby producing a lysate containing inclusion bodies and cellular components; d. clarifying the cell lysate by separating solids from the solution; e. isolation and purification of inclusion bodies (IBs) by using buffers selected from carbonate, bicarbonate, Tris, borate, Arginine to remove cellular contaminants to form a pellet of purified IBs; f. denaturation and solubilization of (IBs) at a high pH value ranging from 7 to 14 using at least one buffer selected from carbonate, bicarbonate, Tris, borate, Arginine buffer; g. rapid pH adjustment within a range of 6 to 8.5, preferably at 8 of solubilized immunogen using acid containing redox systems to produce refolded protein, h. intermediate purification of the refolded protein using ion exchange chromatography , hydrophobic interaction chromatography to obtain ＞99.9%pure native fusion protein and i. the semi purified protein obtained in step (h) is further purified by one or more chromatographic separations using anion exchange chromatography, hydrophobic interaction chromatography, metal &dye affinity chromatography, affinity chromatography, multimodal chromatography, hydroxyapatite chromatography and size exclusion chromatography to obtain to obtain fusion protein. Rapid pH adjustment of solubilized fusion protein is carried out using organic or inorganic acids like HCl, orthophosphoric acid, acetic acid, citric acid containing redox system like cysteine and cysteine.

In yet another embodiment of the invention, purification by chromatography is carried out using single or multi step chromatography selected from i) direct ion exchange followed by ion exchange followed by hydrophobic interaction chromatography and ii) Ion exchange followed by hydrophobic interaction chromatography (HIC) . Further the said ion exchange chromatography is an anion exchange chromatography. The anion exchange resins are selected from the group but not limited to consisting of DEAE cellulose, MonoQ, Capto Q, Eshmino Q, Gigacap Q 650M, Nuvia-Q, Cellufine Q-h, MiniQ, Source 15Q and 30Q, Q, DEAE Sepharose Fast Flow, Q Sepharose high Performance, QAE SEPHADEX. and FAST Q SEPHAROSE (GE Healthcare) , UNOsphere Q, Macro-Prep DEAE and Macro-Prep High Q from Biorad, Ceramic HyperD Q, ceramic HyperD DEAE, Toyopearl SuperQ-650S, 650M and 650C, QAE-550C and 650S, DEAE-650M and the like.

The anion exchange column is eluted at 10 to 60%of 1M NaCl in Tris-HCl buffer and the concentration of the buffer may range from 10 mM to 300 mM. The anion exchange column runs in the pH range of 6 to 9, preferably 8 to 9.

The hydrophobic interaction chromatography support is selected from the group but not limited to butyl-, phenyl-, octyl-agarose, butyl-, phenyl-, ether-organic polymer resin and phenyl sepharose and the like.

The hydrophobic interaction chromatography column runs in the pH range of 5 to 8, preferably 6 to 7.6.

The buffer used for hydrophobic interaction chromatography may be sodium or potassium phosphate containing sodium chloride ranging from 2M to 5M concentration or only sodium sulphate salt from 2M to 3M concentration.

The invention thus involves more than one subsequent purification steps, and also exploits pI value of the fusion protein in an ion exchange chromatographic step, whereby it is separated from other contaminating proteins. Finally, the quantity of the fusion protein was quantified by BCA/Bradford/Lowry Assay and visualised in 10-12%acrylamide gel (SDS-PAGE) . The identification of polypeptide is done by Western blot and similar immunoassays. The purity and integrity of purified polypeptide is measured by SDS-PAGE and HPLC methods. The yield of the protein thus expressed was 500-3000 mg/L of the culture medium and can be subsequently varied by modulating the culture additives and conditions, as well as purification steps. The method of invention provides an industrially applicable method of tuning the induction time and subsequently modulating the pH and temperature of the chromatographic steps provides simple, inexpensive, and is not laborious. It excludes need of extensive steps involving preparation of buffers or kit or working solution thereof. A very high amount and pure form of fusion protein can be achieved by the process disclosed and illustrated herein.

Ion exchange chromatography have been used to assist . refolding of denatured proteins by enclosing a single protein within micelles or isolating them on a resin and then removing the denaturant.

A further object of the invention is a composition comprising a carrier and the said fusion protein in an effective amount to elicit immune response and/or treat a SARS-COV-2 infection.

A further object of the invention is the composition as defined above for prophylactic vaccination of humans against COVID-19. A further object of the invention is the method of obtaining a polypeptide inducing functional antibodies against SARS-COV-2 (Spike protein) isolation of a polypeptide from the naturally occurring SARS-COV-2 Spike protein, consisting of: i. an S-1 subunit, forming the viral domain with a protein binding site to the host cells receptors ii. P2 subunit fragment with the N-terminal RBD peptide, b) transformation of prokaryotic cells with an expression vector comprising a nucleic acid encoding the polypeptide of a fusion) ; c) culture of transformed prokaryotic cells producing the polypeptide of a fusion) ; d) isolation and solubilization of inclusion bodies; e) refolding and purification of the said produced fusion protein.

Preferably, the prokaryotic cells are bacterial cells, particularly E. coli.

Preferably, the isolated inclusion bodies are solubilized in the D1 buffer (20 mM TrisHCl, pH 8.0; 8 M urea; 0.01%Triton X-100) . Preferably, the polypeptide solution obtained as a result of inclusion bodies solubilization is purified on a DEAE Sepharose Fast Flow bed column (Amersham Pharmacia Biotech AB) . Preferably, the polypeptide is refolded by dilution in B1 buffer (40 mM Tris-HCl pH 8.0; 100 mM NaCl) . Preferably, after refolding the polypeptide is purified on a Phenyl Sepharose 6 Fast Flow High Solution column (Amersham Pharmacia Biotech AB) .

High expression of the fusion leads to the formation of high molecular weight aggregates, often referred to as "inclusion bodies" . The inclusion bodies fall into two categories: first, paracrystalline arrays in which the protein presumably is in a stable conformation, although not necessarily native; and second, amorphous aggregates that contain partially and completely denatured proteins, as well as aberrant proteins synthesized as a result of inaccurate translation. Such aggregates of heterologous protein constitute a significant portion of the total cell protein.

Although inclusion bodies probably afford protection to proteins against endogenous proteases, they do present problems of extraction and purification, as they are very difficultly soluble in aqueous buffers. In most instances, denaturants and detergents (e.g., guanidine hydrochloride, urea, sodium dodecylsulfate (SDS) , Triton X-100) have to be used to extract the protein. For proteins of pharmaceutical interest, particularly for parenteral administration, the use of detergents and denaturants is undesirable because it is difficult to remove them completely from isolated proteins, particularly if the proteins possess extensive hydrophobic domains. Further if urea, a relatively weak denaturant, is used as the extractant, modification of some amino acid residues may occur.

Another problem in the recovery of the desired proteins which are in the form of inclusion bodies is the need, not only to separate inclusion proteins from other host cellular materials but also subsequently to remove inclusion body protein contaminants from the desired inclusion body heterologous protein. The second problem is probably due to the strong attraction that inclusion body proteins have one for another, due perhaps to ionic attractions or hydrophobic bonding.

The inventors teach a process for converting E. coli inclusion body from the natural or induced insoluble state into soluble forms. The techniques use a combination of membrane disruption methods such as sonication or homogenization under high pressure, coupled to solubilization of proteins and weak denaturants such as urea. After such treatments the recombinant proteins are refolded by buffer exchange into a relatively weak denaturant such as urea in the presence of a reducing agent such as 2-mercaptoethanol. The fusion protein are then isolated using ion exchange chromatography and hydrophobic interaction Chromatography in the presence of buffered urea. Thus, according to this patent, denaturants must necessarily be present throughout, and subsequent to, the process for recovering inclusion body proteins.

According to this patent provide a process of separating microbial proteins in bulk from nucleoprotein complexes. The process comprises disruption of the biomass by physical means in the absence of detergents of denaturating reagents. This is followed by centrifugation to remove cell debris, Next, the protein solution is dialyzed to remove salts, was further separated on hydroxyapatite columns.

It would be useful to provide a method that solves the problems in solubilizing and separating microbial recombinant heterologous proteins by using procedures which, in their various aspects, succeed in solubilizing the proteins, removing contaminating host cellular proteins, and separating individual recombinant proteins in forms that are active and appropriate in biological and immunization, and that are soluble and biologically active in the absence of any denaturant or detergent.

The insoluble recombinant protein is liberated from the host cell by employing enzymatic and/or chemical means that disrupt the strong outer cell wall membrane under conditions of pH and ionic strength such that host cell proteins will be solubilized, or at least will remain in suspension at sedimentation forces sufficient to sediment recombinant protein-containing inclusion bodies. The chemicals employed are those that are known to attack host cell walls and various macromolecular structures such as membranes and nucleoprotein bodies, thereby promoting cell lysis. Although the sedimented pellet contains predominantly the desired recombinant protein, it is also contaminated at this stage with small amounts of soluble host cell proteins that require removal.

Contaminating host cell proteins are removed from the recombinant protein aggregates by washing with a mild solution containing both detergents and a weak denaturant.

As the recombinant protein has been precipitated in vivo under cytoplasmic conditions, it is clear that conventional aqeuous solubilization techniques will fail. Hence, a more drastic means is employed to bring the inclusion bodies into solution. It has been found that a weak denaturing solution is effective in doing this. The weak denaturing reagent will also cause unfolding of the 3-dimensional structure of the desired recombinant protein.

A partial purification of the desired protein is achieved by desalting wash or by dialysis or any other method under conditions wherein the fusion protein precipitates out of solution as the concentration of denaturant is lowered by dialysis. P2-RBD and other contaminating proteins that had the host cells′ inclusion bodies as their origin still have a tendency to aggregate in the absence of high concentrations of strong denaturants. However, proteins in the denatured condition do not exhibit their normal biological activities. Thus, an alternate means was sought to maintain the P2-RBD in solution in the absence of weak denaturants. This alternative, according to the invention, comprises reacting free amino groups of the P2-RBD with 8M urea. This reaction produces a change in the overall charge of the fusion, reducing the tendency of inclusion body, recombinant proteins to aggregate in an aqueous medium, thereby producing the highly desired water soluble monomeric proteins. Following isolation in the presence of a weak denaturant, and desalting by either dialysis or gel filtration, the invention provides for fractionation of the thus recombinant proteins by molecular sieve or ion exchange chromatography or HIC.

Another aspect of this invention is its flexibility in several of the phases of the protocol for recovery of pure recombinant proteins, cell lysis can be accomplished in the absence of detergents and denaturants by a combination of enzymatic digestion and mechanical disruption. Further, preliminary purification of recombinant protein, following of solubilizing IBs, can be accomplished by protein fractionation on ion exchange chromatography column, rather than by aggregation following reduction of the concentration of 8M urea.

"Host cell" within the present context also refers to any form of the microorganisms in which expression of heterologous proteins have been induced by upward temperature shifts or other means, and is to be taken as including the entire cell culture in its growth medium, the harvested cell paste, a frozen sample of the paste, or a frozen and thawed sample of the paste, typically cells collected by centrifugation under standard conditions, e.g., up to 6,000 X g for up to 60 minutes, most preferably 4,000 X g for 30 minutes.

Because the inclusion bodies are enclosed within host cells, it is desirable first to disrupt the cells to release the inclusion bodies and make them available for recovery by, for example, centrifugation. In one aspect of the invention, partial purification of inclusion bodies is obtained simply by ensuring that the host cell debris is sufficiently disrupted to remain in the supernatant phase during low speed centrifugation. In this aspect of the invention, cells are washed and resuspended in a buffer at pH 7.8 to 8.5, preferably about 8.0 using an ionic strength of the order of 0.01M to 2M, preferably 0.1-0.2M. Any suitable salt, preferably sodium cloride, can be used to maintain the proper ionic strength level. Ionic strength, the conventional measure of ion concentration, is desired as 1/2 of the sum of the product of the concentration of each ion present times the square of the charge thereon. Any suitable buffer may be used to maintain the pH in the correct range; Tris hydrochloride (Tris. HCl) buffer is a preferred buffer for maintaining the pH between 8 and 9 because of its buffering capacity in this range and because of its biochemical inertness.

The cells, while suspended in the foregoing buffer, are then lysed by techniques commonly employed for this purpose. Cell lysis is accomplished by a combination of enzymatic and/or chemical means. Enzymes commonly employed for this purpose include "Lysing Enzyme Mixture" from staphlolyticus, deoxyribonuclease (DNAse) from bovine pancreas and/or spleen, and Lysozyme. It is preferred to employ Lysozyme plus DNAse. It is also desirable to protect the desired proteins from aP2ack by the host cell′s own proteases. This can be accomplished by contacting the lytic mixture with a protease inhibitor, such as the lung protein apoprotinin, soybean trypsin inhibitor, or phenylmethylsulfonylfluoride (PMSF) , preferably apoprotinin and PMSF.

It is also desirable to disrupt hydrophobic structures in the cell wall/membrane with a detergent, i.e., a surface active agent. Cell lysis is aided by a nonionic detergent such as the polyoxyethylene ether types, including Triton X-15 through Triton X-405, Triton N-101, Triton WR-1339, Lubrol-PX, preferably Triton X-100 in a concentration sufficiently low so as not to disrupt the inclusion bodies at the cell lysis stage. Detergent concentrations are of the order of 0.005-1%, preferably 0.1-0.2%.

When it is judged that the cells are sufficiently disrupted so that there will be remaining no, or a minimum of, cellular fragments of size sufficiently large so as to sediment, the lysis suspension is centrifuged at low speed, around 500 to 6500g, preferably 5000-6000g, in a standard centrifuge for a suitable time depending upon volume, usually for 20-30 minutes. The resulting pellet contains inclusion bodies, usually (as determined by phase contract microscopy) still contaminated with adsorbed host cell proteins and membrane fragments.

Contaminating material can be removed by repeated suspension of the inclusion body pellets in suitable buffer, preferably twice with 0.1-0.2%Triton X-100 in Tris. HCl buffer, pH 8-9; once with 0.15-2M NaCl in Tris. HCl buffer, pH 8-9; once with Tris. HCl buffer (pH 8-9) containing a chelator such as 1-10 mM ethylenediaminetetracetate (EDTA) and an ionic detergent selected from among commercially available (Pierce Chemical Co. ) SDS, LDS, CHAPS, CHAPSO, and Zwittergents, preferably Zwittergent 3-14 (0.05-0.3%, preferably 0.1-0.2%) ; and once with a weak denaturant solution such as 2-10M, preferably 6-8M, urea in Tris. HCl buffer pH 8-9.

"Denaturing solution" refers to a solution that contains a "denaturant, " "denaturant" referring herein to those chaotropic components that, in aqueous solution, are capable of changing the conformation of proteins either through altering the state of hydration, the solvent environment, or the solvent-surface interaction. Examples of denaturants include urea, but include as well detergents, i.e., surface-active agents, as listed above. Some of the listed reagents are strong denaturants, while others are weaker. The concentration of any of these will, of course, directly affect its strength and effectiveness.

Urea is the most frequently used example of a relatively weak denaturant, as even fairly high, e.g., 8M, concentrations permit the retention of some protein secondary structures. Accordingly, washing inclusion bodies with a solution of weak denaturant, e.g. urea, will remove contaminating host cell proteins, while not dissolving the inclusion bodies themselves.

Accordingly, the inclusion bodies prepared as above are dissolved in a weak denaturing solution, preferably (8M urea in Tris. HCl bufer, pH 8.0) , in the absence of a reducing agent.

According to the embodiment set out in FIG. 2, it is possible to achieve partial purification of the P2-RBD by reducing the concentration of the denaturant in an alkaline medium. This is most preferably done by dialyzing elution against (0.3M Arginine、 5mM EDTA, 5%Glyceral, 4×CB (1.5g Na2CO3, 3g NaHCO3/L) buffer. The buffer concentration is 0.01-1M, preferably 0.05-0.10M, and the pH is 8-10, preferably 8.0-8.5. The precipitated protein is recovered as a pellet by low speed centrifugation as described herein above.

Following completion of described above, This can be accomplished, according to the invention, by dialysis of the mixture against an alkaline buffer such as (0.3M Arginine、 5mM EDTA、 5%Glyceral、 4×CB (1.5g Na2CO3, 3g NaHCO3/L) three times.

After the dialysis, the fusion protein can be recovered by fractionation on chromatography matrices of either the molecular sieve or ion exchange types. Suitable molecular sieve columns include, but are not limited to, Ultragels (LKB Products) , Fractogels (Pierce Chemical Company) , and Sepharose, Sephadex and Sephacryl (Pharmacia Fine Chemicals) . In a preferred embodiment, the recombinant protein is fractionated on an Ultragel ACA gel column equilibrated with ? buffer (0.01-1.00M, pH 8.5-9.5) , and the elution profile is monitored at a wave length of 280 nm, the region of maximum ultraviolet absorption of proteins. The molecular weight of the eluted the fusion protein is estimated by reference to the elution volumes of commonly used standard proteins.

Suitable ion exchange columns include, but are not limited to, Fractogels TSK-DEAE (Pierce Chemical Company) or DEAE-Sepharose and DEAE-Sephacel (Pharmacia Fine Chemicals) . In a preferred embodiment, fusion is fractionated on a column of Fractogel TSK-DEAE-650 equilibrated in buffer (0.01-0.10M, pH 8.5-9.5) , proteins being eluted by a salt gradient from 0.3 to 1M sodium chloride in the above-described sodium borate buffer. The Fractogel TSK-DEAE ion exchanger is preferred because of its greater rigidity and consequently faster flow rates.

The fusion protein solutions eluting off the chromatography columns can either be lyophilized to recover the protein in powdered form or stored frozen, typically at -20℃ to -180℃.

Another aspect of the invention is its flexibility with regard to several steps of the protocol for the recovery of pure inclusion body recombinant proteins. In the alternative approach, host cell lysis is carried out by a combination of enzymatic and mechanical means.

Following washing of cells with Tris buffered saline, cells are suspended in PMSF (0.5-2.0M) , DP2 (0.01-1.0M) , Triton X-100 (0.05-0.3%) , EDTA (1-20 mM, in Tris. HCl buffer (0.01-0.10M, pH 8-9) , then lysed by contact with lytic enzymes together with mechanical disruption by sonic oscillation, homogenization, grinding in mills, or exposure to a pressure cell; no denaturant is used.

In another aspect of the process, partial purification of the desired protein is achieved, not by the precipitation protocol of the process, but by fractionation on a molecular sieve in the presence of a weak denaturant, preferably Sepharose 6B-CL (Pharmacia Fine Chemicals) operated in the presence of 8M urea. This matrix is particularly suitable for recovery of proteins that are insoluble or aggregated in ordinary aqueous buffer systems.

In the method of the fusion protein production according to the invention, the P2-RBD is expressed in inclusion bodies of bacteria transformed using expression vectors, in which the protein coding sequence is optimized for a bacterial expression system. In the method of fusion protein production according to the invention, a highly efficient and effective refolding method was applied with the use of amino acids: L-arginine, frequently contained in buffers for bacterial fusion protein refolding, thus lowering the cost of vaccine. Standard chromatography methods are used in the method of immunogen production according to the invention. Accordingly, the presence of metal ions in the final product, produced by the method according to the invention, is not subject to assessment and quality evaluations may be performed using the procedures developed for biopharmaceuticals, commonly produced in a bacterial expression system.

The essence of the invention is also a method of eliciting protective immune response. The method of eliciting immune response comprises vaccinations using the fusion protein obtained by the method according to the invention, by parenteral administration of the immunogen, including subcutaneous, intradermal, intramuscular or mucosal, including intranasal, via the gastrointestinal tract, and in the case of bird immunization also conjunctivally, naso-conjunctivally, in ovo. performed employing identical or different administration routes for the vaccine immunogen in a given vaccination cycle; by administrating the immunogen with adjuvants approved in human specific to the route of administration;

There is a need for new and more effective methods of folding and/or recovering fusion from a host cell culture, e.g., for the efficient and economical production of fusion in bacterial cell culture. These new and more effective methods provide for improved recovery of a highly purified biologically active properly refolded protein and that are generally applicable to manufacturing scale production of the proteins. The invention addresses these and other needs, as will be apparent upon review of the following disclosure.

In one embodiment, a process includes: (a) Lysed said fusion protein from the prokaryotic cell culture; (b) solubilizing said protein in a first buffered solution, pH greater than 8, comprising a first chaotropic agent; (c) refolding said solubilized protein in a ion exchange chromatography, comprising a chaotropic agent, two or more reducing agents for such a time and under such conditions that refolding of the fusion protein occurs; and (d) recovering said refolded fusion protein. In one embodiment, the first buffered solution and/or the second buffered solution further comprises arginine. In one embodiment, the first buffered solution comprises 20mM Tris pH8.0 , 1mM EDTA, 500mM NaCl, 10%Glyceral, 0.1%Tween-20 or NP40. In another embodiment, the first buffered solution comprises 0.1%Triton-Tris (20mM pH8.0) . final concentration. In one embodiment, the second buffered solution comprises one or more denatrurant agents, e.g. In one embodiment, the second buffered solution comprises 8M Urea, 20mM Tris pH8.0, final concentration.

The solubilization and/or refolding can be done at a variety of temperatures. In one embodiment, the incubation temperature for the solubilization and/or refolding is room temperature. The incubation time can vary according to the fusion protein being recovered and refolded. In one embodiment, the fusion protein is incubated in the first buffered solution for at least 1 hour, or 1 to 2 hours. In one embodiment, the solubilized protein is incubated in the second buffered solution for about 3 to 24 hours. In one embodiment, the isolated fusion protein is incubated in the combo buffered solution for 3 to 24 hours.

The invention additionally provides processes and methods for refolding of fusion either alone or in connection with the recovery of the fusion protein as described herein. In a particular embodiment, purification methods include clarifying the solution containing the fusion protein and contacting said refolded fusion protein in the clarified solution with a mixed mode support, a cationic chromatographic support, a first ion exchange support support, and optionally, a second hydrophobic chromatographic support or an ion exchange support; and selectively recovering or eluting the refolded fusion protein from each support. In one embodiment, clarifying the solution comprises adding detergent to a final concentration of 1%Triton, adjusting pH to about 8.0-8.5, incubating solution for 1 to 10 hours at 25-30℃, centrifuging the solution; and filtering the liquid recovered from the centrifugation step. In one embodiment, the pH is about 8.0. In another embodiment, the pH is about 8.5. It is contemplated that the steps for recovery steps can be performed in any order, e.g., sequentially or altering the order of the chromatographic supports. In certain embodiments of the invention, methods are provided for recovering and purifying refolded fusion from manufacturing or industrial scale cell culture.

Isolating fusion Protein

Insoluble, mis-folded fusion is isolated from prokaryotic host cells expressing the protein by any of a number of art standard techniques. For example, the insoluble fusion protein is isolated in a suitable isolation buffer by exposing the cells to a buffer of suitable ionic strength to solubilize most host proteins, but in which the subject protein is substantially insoluble, or disrupting the cells so as to release the inclusion bodies or the protein form the periplasmic or intracellular space and make them available for recovery by, for example, centrifugation. This technique is well known.

The prokaryotic cells are suspended in a suitable buffer. Typically the buffer consists of a buffering agent suitable for buffering at between pH 5 to 9, or about 6 to 8 and a salt. Any suitable salt, including NaCl, is useful to maintain a sufficient ionic strength in the buffered solution. Typically an ionic strength of about 0.01 to 2 M, or 0.1 to 0.2 M is employed. The cells, while suspended in this buffer, are disrupted or lysed using techniques commonly employed such as, for example, mechanical methods, e.g., Homogenizer, a French press, a bead mill, or a sonic oscillator, or by chemical or enzymatic methods.

Examples of chemical or enzymatic methods of cell disruption include spheroplast, which entails the use of lysozyme to lyse the bacterial wall and osmotic shock, which involves treatment of viable cells with a solution of high tonicity and with a cold-water wash of low tonicity to release the polypeptides. Sonication is generally used for disruption of bacteria contained in analytical scale volumes of fermentation broth. At larger scales high pressure homogenization is typically used.

After the cells are disrupted, the suspension is typically centrifuged at low speed, generally around 500 to 15,000 X g, e.g., in one embodiment of the invention about 12,000 X g is used, in a standard centrifuge for a time sufficient to pellet substantially all of the insoluble protein. Such times can be simply determined and depend on the volume being centrifuged as well as the centrifuge design. Typically about 10 minutes to 0.5 hours is sufficient to pellet the insoluble protein. In one embodiment the suspension is centrifuged at 12,000 X g for 10 minutes.

The resulting pellet contains substantially all of the insoluble protein fraction. If the cell disruption process is not complete, the pellet may also contain intact cells or broken cell fragments. Completeness of cell disruption can be assayed by resuspending the pellet in a small amount of the same buffer solution and examining the suspension with a phase contrast microscope. The presence of broken cell fragments or whole cells indicates that further sonication or other means of disruption is necessary to remove the fragments or cells and the associated non-refractile polypeptides. After such further disruption, if required, the suspension is again centrifuged and the pellet recovered, resuspended, and reexamined. The process is repeated until visual examination reveals the absence of broken cell fragments in the pelleted material or until further treatment fails to reduce the size of the resulting pellet.

The above process can be employed whether the insoluble protein is intracellular or in the periplasmic space. In one embodiment of the invention, the conditions given herein for isolating the fusion protein are directed to inclusion bodies precipitated in the periplasmic space or intracellular space and relate particularly to P2-RBD. However, the processes and procedures are thought to be applicable to fusion in general with minor modifications as noted throughout the following text. In certain embodiments of the invention, the processes and procedures are applicable to manufacturing or industrial scale production, refolding, and purification of the fusion protein.

In one embodiment, the isolated fusion protein in the pellet is incubated in a first buffered solution sufficient to substantially solubilize the fusion protein. This incubation takes place under conditions of concentration, incubation time, and incubation temperature that will allow solubilization of desired amount or substantially all the fusion protein.

The first buffered solution comprises a buffering agent suitable for maintaining the pH range of the buffer at least about 9 or greater, with the typical range being 8-9. In one embodiment, the pH for P2-RBD is pH 8. Examples of suitable buffers that will provide a pH within this latter range include TRIS (Tris [hydroxymethyl] aminomethane) , HEPPS (N- [2-arginine, lysine, and sodium borate. In one embodiment, the buffer herein includes CHES and arginine at about pH 11. In another embodiment, the buffer herein includes Tris and arginine at about pH 11. In one embodiment, the buffer herein includes CHES at about pH 11. In another embodiment, the buffer herein includes Tris at about pH 11. In certain embodiments, the first buffered solution includes a chaotropic agent.

Chaotropic agents suitable for practicing this invention include, e.g., urea and salts of guanidine or thiocyanate, e.g., urea, guanidine hydrochloride, sodium thiocyanate, etc. The amount of chaotropic agent necessary to be present in the buffer is an amount sufficient to unfold the fusion protein in solution. In certain embodiments of the invention, a chaotrope is present at about between about 1-8 molar. In one embodiment of the invention, the chaotropic agent is urea at about 8M.

The concentration of the protein in the buffered solution must be such that the protein will be substantially solubilized as determined by optical density. The exact amount to employ will depend on, e.g., the concentrations and types of other ingredients in the buffered solution, particularly the protein concentration, chaotropic agent, and the pH of the buffer. In one embodiment of the invention, the concentration of fusion protein is in the range of 0.5-5.5 mg per ml, or 1.5-5.0 mg/ml. The solubilization is typically carried out at about 0-45℃ or about 2-40℃, or about 20-40℃, or about 23-37℃ or about 25-37℃, or about 25℃for at least about one to 24 hours. Typically, the temperature is not apparently affected by salt, reducing agent and chaotropic agent levels. In certain embodiments, the solubilization is carried out at atmospheric pressure.

Measurement of the degree of solubilization in the buffered solution can be determined and is suitably carried out, for example, by turbidity determination, by analyzing fractionation between the supernatant and pellet after centrifugation, on reduced SDS-PAGE gels, by protein assay (e.g., the Bio-Rad protein assay kit) , or by HPLC.

Optionally, the disrupted cells are not centrifuged but are diluted, e.g., 1: 4, 1: 6, 1: 8 in a second buffered solution described herein (refolding buffer) . This incubation takes place under conditions of concentration, incubation time, and incubation temperature that will allow solubilization and refolding of the fusion protein. In one embodiment, about 30%or more of fusion protein is solubilized and refolded.

Refolding fusion protein

After the polypeptide is solubilized or, alternatively, the cells are disrupted, it is placed or diluted into a second buffered solution containing at least one reducing agent, and a chaotropic agent, at concentration which allow for refolding of the fusion protein,

In certain embodiments of the invention, the dialysis buffered solution contains two or more reducing agents. The polypeptide may be diluted with the refolding buffer, e.g., at least fivefold, or at least about tenfold, or about 20 fold, or about 40 fold. The conditions of this the dialysis of the soluble, unfolded protein will generally be such that desired amount or substantial or complete refolding of the protein will take place. The exact conditions will depend on, for example, the pH of the buffer and the types and concentrations of chaotropic and reducing agents present. The dialysis temperature is generally about 0-10℃ and the dialysis will generally be carried out for at least about 6 hour to about 48 hours to effect refolding. In certain embodiments, the reaction is carried out, e.g., at 4-8℃, for at least about 6 hours, for at least about 10 hours, or between about 6 and 30 hours, or between about 6 and 24 hours.

The first dialysis solution comprises a buffering agent suitable for maintaining the pH range of the buffer at least about 8 or greater than 8, with the typical range being 8-9, a chaotropic agent, and at least one reducing agent. In certain embodiments, the s first dialysis solution comprises two or more reducing agents. In one embodiment, the pH for P2-RBD is pH 8. Examples of suitable buffers that will provide a pH within this latter range include TRIS. (Tris [hydroxymethyl] aminomethane) , HEPPS (N- [2-Hydroxyethyl] piperazine-N′- [3-propane-sulfonic acid] ) , arginine, lysine, and sodium borate. In one embodiment, the second buffered solution herein comprises TRIS and arginine at about pH 8 (at about a concentration of 10 mM and 100 mM final concentration, respectively) , with two or more reducing agents and at least one chaotropic agent.

Arginine (or another positively charged amino acid) , e.g., L-arginine/HCl, can be present in the first dialysis solution and the second dialysis solution. In certain embodiments of the invention, the concentration of arginine is e.g., about 50-500 mM, about 75-300 mM, or about 100-300 mM, or about 100 mM or 300 mM final concentration, etc. In certain embodiments of the invention, the protein is in a dialysis solution at pH greater than 8 and 8-M urea, 50-500 mM arginine and 5 mM EDTA, final concentration. In one embodiment, 5%Glyceral final concentration is used. In another embodiment, 4×CB (1.5g Na2CO3, 3g NaHCO3/L ) final concentration is used. In one embodiment, the second dialysis solution comprises 6M Urea, 300 mM arginine, 1mM EDTA, 5%Glyceral, CB, pH 8, final concentration. In another embodiment, the third dialysis solution comprises 3M Urea, 300 mM arginine, 10 mM Tris, 5 mM EDTA, 5%Glyceral, 1 X CB, pH 8, final concentration. In certain embodiments of the invention, the protein is in a third dialysis solution (refolding buffered solution) at pH 8 containing 1.5 M urea, 300 mM arginine, 10 mM Tris, 5 mM EDTA, 5%Glyceral, 1XCB, pH 8, final concentration. In one embodiment, the protein is in a refolding buffer solution with 300 mM arginine, 10 mM Tris, 5 mM EDTA, 5%Glyceral, 1 X CB, pH 8, final concentration.

The dialysis solution contains at least one chaotropic agent at a concentration such that refolding of the P2-RBD protein occurs. Generally a chaotrope is present at about between about 0.5 and 2 molar final concentration. In one embodiment of the invention, the chaotropic agent herein is urea at about 0.5-2 M, 0.5-2 M, or at about 1 M, final concentration.

The lysis buffer can optionally contain additional agents such as any of a variety of non-ionic detergents such as TRITON. X-100, NONIDET P-40, the TWEEN series. The non-ionic detergent is present at about between 0.1%and 1.0%final concentration. In one example, the concentrations for non ionic detergent are between about 0.1%and 0.5%, or about 0.1%final concentration.

The degree of refolding is suitably determined by liquid chromatography analysis using e.g., chromatography column, a cation exchange other appropriate size exclusion column. Increasing correctly folded fusion peak size in the size exclusion column assay directly correlates with increasing amounts of folded, biologically active fusion protein present in the buffer. The incubation is carried out to maximize the ratio of correctly folded fusion protein to misfolded fusion protein recovered, as determined by Superdex 75 HR 10/300 GL.

In one embodiment, the quality and quantity of properly-folded P2-RBD is assessed using SEC. SEC calibration curves were constructed based on linear regression analysis of peak elution volume (Ve) versus the logarithm of the MW of reference proteins (human gamma-globulin, 158 kDa; ovalbumin, 44 kDa; horse myoglobin, 17 kDa; B12 vitamin, 1.35 kDa) . In parallel, the Ve of purified fusion protein was interpolated in the calibration plot to calculate MW and the aggregation state.

Recovery and Purification of fusion protein

Although recovery and purification of the fusion protein can employ various methods and known procedures for the separation of such proteins such as, for example, salt and solvent fractionation, adsorption with colloidal materials, gel filtration, ion exchange chromatography, affinity chromatography, immunoaffinity chromatography, electrophoresis and high performance liquid chromatography (HPLC) , an example of a clarification step and a multi-step chromatographic procedure is described. The clarification step comprises adding detergent to a final concentration of 1% (e.g., TRITON. TM. X-100) , adjusting pH to about 8.0-8.5 (or about 7.2or about 9) , incubating solution for 1 to 10 hours at 25-30℃, centrifuging the solution; and filtering liquid recovered from the centrifugation step. The multi-step chromatographic procedure comprises contacting said refolded fusion protein with a mixed mode resin, a cationic chromatographic support, a first hydrophobic chromatography support, and optionally, a second an exchange chromatography support or an size exclution support; and selectively recovering or eluting the fusion protein from each support. It is contemplated that the steps of either procedure can be performed in any order. In one embodiment of the invention, the steps are performed sequentially.

A suitable first step in the further recovery and purification of the fusion protein characteristically provides for the concentration of the fusion protein and a reduction in sample volume. For example, the first denatruated step described above, may result in a large increase in the volume of the recovered fusion protein and concommitant dilution of the protein in the refolding buffer. Suitable first chromatographic supports provide a reduction in volume of recovered fusion protein and may advantageously provide some purification of the protein from unwanted contaminating proteins. Suitable first chromatographic steps include chromatographic supports which can be eluted and loaded directly onto a second chromatographic support.

Exemplary first chromatographic supports include, but are not limited to, mixed mode resin (e.g., GE Healthcare, Corporation) , hydroxyapatite chromatographic supports, e.g., CHT ceramic type I and type II. In one embodiment of the invention, the first chromatographic supports for the purification and recovery of P2-RBD are mixed ion exchange chromatographic supports. Elution from the first chromatographic support is accomplished according to art standard practices. Suitable elution conditions and buffers will facilitate the loading of the eluted fusion protein directly onto the cationic chromatographic support as described below.

Various anionic constituents may be attached to matrices in order to form cationic supports for chromatography. Anionic constituents include carboxymethyl, sulfethyl groups, sulfopropyl groups, phosphate and sulfonate (S) . Cellulosic ion exchange resins such as SE52 SE53, SE92, CM32, CM52, CM92, P11, DE23, DE32, DE52, EXPRESS ION S and EXPRESS ION. C are available from Whatman LTD (Maids tone Kent U.K) SEPHADEX and SEPHAROSE based and cross linked ion exchangers are also known under the product names CM SEPHADEX C-25, CM SEPHADEX. C-50 and SP SEPHADEX C-25 SP SEPHADEXC-50 and SP-SEPHAROSE. High Performance, SP-SEPHAROSE. -XL SP-SEPHAROSE. Fast Flow, CM-SEPHAROSE Fast, and DEAE-SEPHAROSE. CL-6B, all available from Pharmacia AB. Examples of ion exchangers for the practice of the invention include but are not limited to, e.g., ion exchangers under the product names SEPHAROSE such as for example DEAE-SEPHAROSE support.

Elution from cationic chromatographic supports is generally accomplished by increasing salt concentrations. Because the elution from ionic columns involves addition of salt and because, as mentioned herein, HIC is enhanced in salt concentration the introduction of HIC step following the ionic step or other salt step is optionally used. In one embodiment of the invention, a cationic exchange chromatographic step precedes at least the HIC step, e.g., a first hydrophobic interaction chromatographic support and/or a second hydrophobic interaction.

Hydrophobic columns can be used in the purification of the fusion protein, e.g., in the 2. sup. nd, 3. sup. rd, and/or 4. sup. th purification steps. Hydrophobic interaction chromatography is well known in the art and is predicated on the interaction of hydrophobic portions of the molecule interacting with hydrophobic ligands attached to "chromatographic supports. " A hydrophobic ligand coupled to a matrix is variously referred to as an HIC chromatographic support, HIC gel, or HIC column and the like. It is further appreciated that the strength of the interaction between the protein and the HIC column is not only a function of the proportion of non-polar to polar surfaces on the protein but of the distribution of the non-polar surfaces as well.

A number of matrices may be employed in the preparation of HIC columns. The most extensively used is agarose, although silica and organic polymer resins may be used. Useful hydrophobic ligands include but are not limited to alkyl groups having from about 2 to about 10 carbon atoms, such as butyl, propyl, or octyl, or aryl groups such as phenyl. Conventional HIC supports for gels and columns may be obtained commercially from suppliers such as Pharmacia, Uppsala, Sweden under the product names butyI-SEPHAROSE, buty-SEPHAROS-Fast Flow, phenyl-SEPHAROSE CL-4B, octyl SEPHAROSE. TM. and phenyl SEPHAROSE FF

Ligand density is an important parameter in that it influences not only the strength of the interaction of the protein but the capacity of the column as well. The ligand density of the commercially available phenyl or octyl phenyl gels is on the order of 5-40 μmoles/ml gel bed. Gel capacity is a function of the particular protein in question as well as pH, temperature and salt concentration but generally can be expected to fall in the range of 3-20 mg/ml gel.

The choice of particular gel can be determined by the skilled artisan. In general the strength of the interaction of the protein and the HIC ligand increases with the chain length of the alkyl ligands but ligands having from about 4 to about 8 carbon atoms are suitable for most separations. A phenyl group has about the same hydrophobicity as a pentyl group, although the selectivity can be different owing to the possibility of pi-pi interaction with aromatic groups of the protein.

Adsorption of the protein to a HIC column is favored by high salt concentration, but the actual concentration can vary over a wide range depending of the nature of the protein and the particular HIC ligand chosen. In general salt concentration between about 1 and 4 M are useful.

Elution from an HIC support, whether stepwise or in the form of a gradient, can be accomplished in a variety of ways such as a) by changing the salt concentration, b) by changing the polarity of the solvent or c) by adding detergents. By decreasing salt concentrations adsorbed proteins are eluted in order of increasing hydrophobicity. Changes in polarity may be effected by additions of solvents such as ethylene glycol or isopropanol thereby decreasing the strength of the hydrophobic interactions. Detergents function as displacers of proteins and have been used primarily in connection with the purification of membrane proteins.

Example 1 Fusion protein preparation trial in lab scall

The clone design of the fusion constructed in Example 1, wherein the linker used (Linker, also referred to L for short in the present application) is a flexible fragment consisting of 15 amino acid residues, whose sequence is (SEQ ID NO: 6) ; the P2 used comprised 15 amino acids, whose sequence is set forth in SEQ ID NO: 2; RBD refers to a polypeptide comprising amino acid residues from positions 308 to 548 (aa 308-548) of SARS-COV-2 spike protein, whose sequence is set forth in SEQ ID NO: 1.

Step (i) : Synthesis of Fusion protein Gene:

Full length Fusion protein gene was optimized according to Escherichia coli codon usage. The following parameters were used for Fusion protein gene optimization: Codon Usage Bias, GC content, mRNA Secondary Structure, Custom Desired Patterns, Custom Undesired Patterns, Repeat Sequences (direct repeat, inverted repeat, and dyad repeat) , Restriction Enzyme Recognition Sites (deletion or insertion) .

Optimized Fusion protein gene was cloned at multiple cloning site of PET-3a plasmid vector using BamH 1 and Sap 1 restriction sites, generating P2-RBD, The vectors containing Fusion protein gene was transformed in Escherichia coli BL21 host and clones was selected on LB+Kanamycin plate. The presence and correctness of Fusion protein gene in PET-3a was confirmed by restriction digestion of PET-3a -P2-RBD plasmid by Age I (located in Fusion protein gene) and Nde I (located in PET-3a plasmid) . Further the sequence of P2-RBD protein was confirmed by PCR and DNA sequencing.

Step (ii) : Inclusion Bodies Preparation

One ml vial of BL21 Escherichia coli cells was inoculated into 50 ml LB+ Kanamycin media and grown overnight at 37℃, 200 rpm. Fermentation was done at 20 L scale. Escherichia coli cells were inoculated to the fermenter and cultivated at 30℃, centigrade. The culture was induced with 0.5 mM IPTG at OD600=20. After 8-12 hours post induction fermentation culture was harvested and cell pellet was prepared by centrifugation. Cell pellet was lysed mechanically in homogenizer. Inclusion body (which contains the desired protein P2-RBD was isolated by centrifugation of cell lysates. Supernatant was discarded and pellet was retained which contains Inclusion body (IBs) . The isolated inclusion bodies are purified using three wash buffers wherein first containing 20 mM Tris+250 mM NaCl, second containing 20 mM Tris+2M Urea+1%Triton X-100 and third one containing 20 mM Tris to obtain purified fusion protein inclusion bodies.

Step (iii) Denaturation

100 g Fusion protein inclusion bodies were denatured by high pH, dissolved by stirring at 120 min, clarified by centrifugation for 40 minutes, the supernatant was collected and further clarified by 0.8-0.45μMfilter. Wherein said denaturing solution contained 50 mM Tris-, pH 12. Refolding was performed by adjusting pH to 8 using 100-250 mM HCl containing 10 mM C pH 2.0 without any dilution.

Step (iv) Purification

Anion exchange column (Capto Q, first anion exchange step) equilibrated with 50 mM Tris-HCl, pH8.0 buffer contacted with refolded protein solution, followed by elution with 20-50 mM NaCl in 50 mM Tris-HCl, pH8.0 buffer.

The protein elute subjected to 10 kDa UF/DF (lth UF/DF) step to remove salt and concentrate the protein solution.

The above concentrated and diafiltered protein elute contacted with second anion exchange column (Source 30Q) equilibrated with 50 mM Tris-HCl, pH8.0 buffer, followed by linear gradient elution with 10-15 mM NaCl in 50 mM Tris-HCl, pH8.0 buffer.

The protein elution subjected to 10 kDa UF/DF (2nd UF/DF) step to remove salt and concentrate the protein solution.

The above concentrated and diafiltered protein elute toned with 3M NaCl by adding calculated solid NaCl and contacted with hydrophobic interaction column (Phenyl Sepharose FF) equilibrated with 50 mM Tris-HCl, 3M NaCl pH8.0 buffer, followed by elution with 10 mM potassium phosphate, pH7.2 buffer.

The protein elution subjected to 10 kDa UF/DF (1st UF/DF) step to remove salt and concentrate the protein solution. The protein solution is diafiltered with 10 mM potassium phosphate, 5%sucrose pH7.2 buffer.

The concentrated and diafiltered protein elute filtered through 0.22μ filtered and frozen at -70 ℃.

Intermediate purification of the refolded protein using ion exchange chromatography is carried out to obtain ＞99.9%pure and native fusion protein and the semi purified protein obtained as a result of intermediate purification is subjected to further chromatographic separation to obtain purified fusion protein.

Refolding parameters of the Fusion protein prepared according to above described procedure is compared with the Fusion protein prepared according to the conventional refolding process described herein.

Inclusion body based proteins are solubilized using chaotropic salts. The refolding is performed by lowering the concentration of the chaotropic salt, this is achieved by dilution (over 100 folds) . The dilution results in lowering the protein concentrations below 200 microgram/ml. The conventional dilution based refolding is not feasible in large scale manufacturing due to requirement of very large scale refolding vessels.

Example 2. Solubilizing and Refolding of P2-RBD Expressed in Escherichia coli

Cell Homogenization and Inclusion Body Preparation

Whole cell broth from Escherichia coli cells producing P2-RBD protein are homogenized with a microfluidizer or Niro Soavi at pressures greater than 8000 psid. The homogenate is diluted 1: 1 with 160 mM MgSO4, 0.0375 dextran sulfate and 1%TRITONX-100 prior to harvesting the pellet by centrifugation.

Solubilization and Refolding

The pellet (e.g., 1 gram) is suspended in 4 volumes (e.g., 4 ml) of solubilization buffer: 1M Urea/300 mM arginine, 10 mM Tris 5 mM EDTA, pH 8. The suspension is thoroughly mixed for 1-2 hours at room temperature (15-30℃) . Refolding is initiated by addition of 3 volumes (1: 4 v/v) of buffer per volume of solubilization buffer, which results in the final concentration of the refolding buffer being 1 M Urea, 15 mM cysteine, 0.5-2 mM DP2, 100 mM arginine, 10 mM Tris or 5 mM EDTA, pH 9-10. The mixture is stirred with mixing speed is 200-400 rpm) for 6-24 hours at room temperature. The folding is monitored by SDS-PAGE, anion exchange HPLC chromatography.

A significant reduction in the process volume (5-fold) while maintaining the yield of recovered monomers is observed by refolding in a low pH buffer containing mild levels of denaturants and reductants.

Example 3 Single Step Solubilization and Refolding of SBD-P2 Expressed in Escherichia coli

Solubilization and Refolding

The pellet is suspended in 10-39 liter volumes of refolding buffer (in this case termed "combo buffered solution" ) for every kg of cell pellet, where the combo buffered solution contains 1 M Urea, 15 mM cysteine, 0.5 or 2 mM DP2, 100 mM arginine, 10 mM Tris or CHES, 5 mM EDTA, pH8.0-8.5, final concentration. The effect of urea and arginine addition in the refolding buffered solution is that a 1-step pellet refold (combo buffered solution) at pH 8.0 for 15 hours at room temperature. The denaturant concentrations are varied as follows: (1) 1 M urea and 100 mM arginine; (2) 1 M urea (and 0 mM arginine) ; (3) 2 M urea (and 0 mM arginine) , while all the other buffer components (e.g., Tris or CHES, DP2, etc. ) remain in the same concentration. The fusion titer extracted from these is equivalent as determined by the anion exchange HPLC assay.

Solubilization and refolding incubation is conducted at room temperature for 3-24 hours and while mixing is 200-400 rpm. For example, the P2-RBD containing pellet was added to the combo buffered solution at pH 8 at a ratio of 1: 39 (pellet kg to buffer L) . Three 2.5 L reaction tanks were prepared and the mixing rate was varied for each one to achieve a kLa of (a) 0.004, (b) 0.01, (c) 0.1 min-1. The tested mixing rates in each tank were 314 rpm. The reactions were monitored over time for yield and product quality. Optionally, the incubation can be conducted at room temperature for up to about 48 hours. Optionally, P2-RBD can be stabilized in the refold buffer by adding nitrogen in place of air at the same sparging and mixing rate after 6 hours. where at 6 hours the monomer peak is diminished (thus, indicating that the refolding reaction is substantially complete) . The folding is monitored by SDS-PAGE, anion exchange HPLC chromatography columns.

Example 4 Non-Pellet Refolding of P2-RBD Protein

Escherichia coli whole cell broth producing fusion protein is homogenized in a model 15 M laboratory homogenizer Gaulin 15M (small scale) or M3 (large scale) (Gaulin Corporation, EvereP2, Mass. ) and diluted 1: 4 (v/v) in refolding buffer per volume ofhomogenate and while mixing is 200-400 rpm. The refolding buffer contains 1 M Urea, 15 mM cysteine, 2 mM DP2, 100 mM arginine, 10 mM Tris or CHES, 5 mM EDTA, pH 9-10, final concentration. Refold incubation is conducted at room temperature for 3-24 hours. The folding is monitored by anion exchange HPLC.

Example 5. Purification of P2-RBD after Refolding

Purification 1

The refold pool is clarified by adding TRITON. TM X-100 to a final concentration of 1%, adjusting to pH 8 and then centrifugation (10,000 X g for 20 minutes at 4℃) . The supernatant is then filtered (Depth filter+0.22 or 0.45 μM membrane filter) prior to capture on a mixed mode resin (CAPTOMMC, GE Healthcare, Piscataway, N.J. ) at pH 8.0 and conductivity ＜10 mS/cm. Optionally, the refold pool is diluted at least 1: 5 in equilibration loading buffer and then filtered (Depth filter+0.22 or 0.45 μM membrane filter) prior to capture on a mixed mode resin (CAPTOMMC, GE Healthcare, Piscataway, N.J. ) at pH 8.0 and conductivity ＜10 mS/cm. The packed column is equilibrated with 25 mM TRIS pH 9 prior to loading the sample on the column. The fusion is eluted from the MMC column isocratically with 1 M arginine/25 mM Tris, at pH 6-9 (e.g., pH 8.0) .

The CAPTOMMC pool is adjusted to pH 8.0 with 0.1 N sodium hydroxide and diluted with WFI to 20 mS/cm conductivity prior to loading onto a DEAE-SEPHAROSE column (equilibrated with 20 mM TRIS pH 8.0) . The is eluted using a linear salt gradient composed of 20 mM TRIS/0-1.2 M sodium acetate pH 8.0 over 10-20 column volumes (e.g., 15 column volumes) and fractions are collected (1 column volume) . The fractions with the highest absorbance 280 nm (OD max at about 42 mS/cm) typically contain ＞90%of the fusion and are pooled for further processing.

The third chromatography step includes a hydrophobic resin (e.g., Hi Propyl, J.T. Baker, Phenyl Sepharose Fast Flow, GE Healthcare, Piscataway, N.J. ) . The DEAE-Sepharose elution pool is conditioned to 50 mS/cm conductivity using either sodium acetate or sodium sulfate prior to loading onto the equilibrated column (20 mM TRIS, 1.2 M sodium acetate, pH8.0) . The P2-RBD elutes isocratically into 50 mM TRIS, pH8.0 and the pool is analyzed for remaining host cell impurities and soluble aggregates. Fractions are collected and those which contained properly-folded P2-RBD, as determined by assays described herein are pooled. Optionally, an additional chromatography step is performed, e.g., using a second hydrophobic resin (e.g., Phenyl TSK) or ion exchange resin.

Ultrafiltration/Diafiltration

The pooled P2-RBD can be ultrafiltered on a 10 kD regenerated cellulose membrane, on a lab scale TFF system to a concentration of 6 g/L (UF1) . The sample is diafiltrated with 7-14 DV (Diavolume) with 5 mM sodium succinate via TFF system to 10 g/L and then formulated at 5 g/L for storage at -80 ℃. The formulation buffer used is 5 mM sodium succinate, 275 mM trehalose dehydrate/0.01%polysorbate 20/pH 7.2.

Purification 2

The refold pool is clarified by adding TRITON X-100 to a final concentration of 1%, adjusting to pH 8.5-9.5 (e.g., pH 8.7) and holding at 25-30 ℃. for 1 to 10 hours prior to centrifugation. After processing on the centrifuge (10,000 Xg for 20 minutes at 4 ℃. ) to remove the large density particles, the recovered liquid is passed thru a series of depth filters and sterile guard (0.22 or 0.45μM membrane) filters to remove the fine particles. fusion is then captured on a mixed mode resin (CAPTOMMC, GE Healthcare, Piscataway, N.J. ) at pH 8.5 and conductivity ＜10 mS/cm. The packed column is equilibrated with 25 mM CHES pH 8.7 prior to loading the sample on the column. The RBD is eluted from the MMC column isocratically with 0.9 M L-arginine HCl/20 mM TRIS, at pH 6-9 (e.g., pH8.0) .

The CAPTOMMC pool is adjusted to pH8.0 with 0.1 N sodium hydroxide and diluted with WFI to 20 mS/cm conductivity prior to loading onto a DEAE-SEPHAROSE High Performance column (equilibrated with 25 mM TRIS pH8.0) . The P2-RBD is eluted using a linear salt gradient composed of 50 mM TRIS/0-1.2 M sodium acetate pH8.0 over 10-20 column volumes (e.g., 15 column volumes) and fractions are collected (1 column volume) . The fractions with the highest absorbance 280 nm (OD max at about 42 mS/cm) typically contain ＞90%of the RBD and are pooled for further processing. The third chromatography step includes a hydrophobic resin (e.g., Phenyl-SEPHAROSE. Fast Flow, GE Healthcare, Piscataway, N.J. ) . The DEAE-SEPHAROSE HP elution pool is loaded directly onto the equilibrated HIC column (25 mM TRIS, 0.75 M sodium acetate, pH8.0) . The RBD elutes isocratically into 50 mM TRIS, pH8.0 and the pool is analyzed for remaining host cell impurities and soluble aggregates. Fractions are collected and those which contained properly-folded P2-RBD, as determined by assays described herein are pooled. Optionally, an additional chromatography step is performed to further remove host impurities, e.g., using a second hydrophobic resin (e.g., Phenyl TSK) or ion exchange resin.

Ultrafiltration/Diafiltration

The pooled RBD can be ultrafiltered on a 10 kD regenerated cellulose membrane in commercial TFF system (Pellicon 2 caseP2es, Millipore, Billerica, Mass. ) to a concentration of 10 g/L then diafiltered with 7-14 diavolumes (eg., 10 DV) into the formulation buffer. Final conditioning produces a solution containing 5 g/L fusion in 5 mM sodium succinate, 275 mM trehalose dehydrate/0.01%polysorbate 20/pH 7.0 that can be stored at -80 ℃.

Example 6 Assays for Determining Folded and/or Purified P2-RBD Protein

In methods and processes described herein, final purity and/or activity can be assessed by peptide mapping, disulfide mapping, SDS-PAGE (both reduced and non-reduced) , circular dichroism, limulus amobocyte lysate (LAL) , Anion exchange HPLC. HPLC can be used to determine concentration and level of misfolded species) , reverse phase (rp) HPLC chromatography (e.g., rpHPLC of reduced samples can be used to determine total concentration whereas rpHPLC of native samples can assess the quality of refolded fusion) , receptor binding (for example for RBD, ACE2) , SEC Analysis, cell assays, ELISAs with SARS-COV-2 Spike antibodies, mass spec analysis, etc.

Assay to Determine Total P2-RBD Expression

rpHPLC of Reduced Samples

The quantity of expressed P2-RBD is measured using a reverse phase HPLC assay on a C 18 column. The column is equilibrated in 0.22%trifluoroacetic acid and eluted using a linear gradient of 25%to 45%acetonitrile containing 0.2%trifluoroacetic acid in 30 min with a flow rate of 1 mL/min. The eluant is monitored at 280 nm. The sample is treated and fully reduced in guanidine and DP2 prior to injection. The reduced fusion protein elutes around 26 min and the peak area is used to calculate the amount of total fusion in the sample from a known standard curve.

Assays for Refolded P2-RBD

(1) Anion Exchange HPLC Assay

The quantity of properly refolded P2-RBD trimers is determined using an analytical anion exchange column The column is equilibrated in 50 mM sodium phosphate pH8.0. At a flow rate of 1 mL/min the column is eluted using a linear gradient from 0 to 2 M sodium chloride in equilibration buffer over 60 min. The eluant is monitored at 280 nm or 214 nm. Typically, the majority of protein is eluted in the first 30 min and P2-RBD is eluted around 40 min.

(2) rp-HPLC Assay

The quality of properly refolded fusion is determined using a Zorbax 300SB-C8 column (4.6X 150 mm, 3.5 micron, by Agilent Technologies, Santa Clara, Calif. ) . The column is equilibrated in 0.1%trifluoroacetic acid and eluted using a linear gradient of 0 to 50%acetonitrile containing 0.08%trifluoroacetic acid over 50 min with a flow rate of 1 mL/min. The eluant is monitored at 214 nm. Typically, fusion elutes around 35 min and the peak profile is evaluated for the percent content of the leading edge hydrophobic species relative to the main peak. Unfolded fusion polymers elutes 2-3 min later.

Example 7. The Expression and Purification of the fusion

Expression of fusion and Purification of Inclusion Bodies

5 μl bacterial solution (prepared by Example 1) , taken from an ultralow temperature freezer at -70℃ was seeded to 5 mL liquid LB medium containing kanamycin, and then was cultured at 37℃, 180 rpm under shaking until OD600 reached about 0.5. The resultant solution was transferred to 500 ml LB medium containing kanamycin, and then was cultured at 37℃, 180 rpm under shaking for 4-5 h. When OD600 reached about 1.5, IPTG was added to a final concentration of 0.4 mM, and the bacteria were induced under shaking at 37℃ for 4 h.

After induction, centrifugation was performed at 8000 g for 5 min to collect the bacteria, and then the bacteria were re-suspended in a lysis solution at a ratio of 1 g bacteria to 10 mL lysis solution (20 mM Tris buffer pH7.2, 300 mM NaCl) , in ice-bath. The bacteria were treated with a sonicator (Sonics VCX750 Type Sonicator) (conditions: operating time 15 min, pulse 2s, intermission 4s, output power 55%) . The bacterial lysate was centrifuged at 12000 rpm, 4℃ for 5 min (the same below) , the supernatant was discarded and the precipitate (i.e. inclusion body) was kept; 2%Triton-100 of the same volume was used for washing, the result mixture was under vibration for 30 min, centrifuged, and the supernatant was discarded. The precipitate was re-suspended in Buffer I (20 mM Tris-HCl pH8.0, 100 mM NaCl, 5 mM EDTA) , under vibration for 30 min, centrifuged, and the supernatant was discarded. The precipitate was then re-suspended in 2M urea, under vibration at 37℃ for 30 min, centrifuged, the supernatant and the precipitate were obtained. The supernatant was kept; and the precipitate was re-suspended in 4M urea in the same volume, under vibration at 37℃ for 30 min, and centrifuged at 12000 rpm, 4℃for 15 min to obtain the supernatant and precipitate. The supernatant (i.e. the 4M urea-dissolved supernatant) was kept; and the precipitate was further in re-suspended in 8M urea in the same volume, under vibration at 37℃ for 30 min, and centrifuged, and the supernatant (i.e. the 8M urea-dissolved supernatant) was kept.

The SDS-PAGE analytic results of the obtained fractions (Coomassie brilliant blue staining was used for visualization, the same below, see the methods in The Molecular Cloning Experiment Guide, 2nd edition) was showed in FIG. 1. The results showed that the fusion were expressed in inclusion bodies (see FIG. 1) , and fusion were mainly dissolved in 8M urea (see FIG. 1) . The 4M urea-dissolved supernatants or the 8M urea-dissolved supernatants containing the fusion protein, were dialyzed to PBS, respectively, to get the fusion with a purity of about 80%.

The results showed that fusion could be expressed in inclusion bodies, and were dissolved in 4M and 8M urea, while other fusion were only dissolved in 8M urea. In addition, the results also showed that after dialysis and renaturation, the fusion of a purity of about 80%were obtained.

Example 8 Purification of the fusion Protein P2-RBD by Anion Exchange Chromatography

Sample: a solution of fusion protein with a purity of about 80%as obtained above.

Equipment: AKTA Explorer 100 preparative liquid chromatography system produced by GE Healthcare (i.e. the original Amersham Pharmacia Co. )

Chromatographic media: DEAE-SEPHAROSE Fast Flow (GE Healthcare Co. ) , Column Volume: 26 mm X 20 cm.

Buffer: 20 mM phosphate buffer pH 7.7+8M urea

20 mM phosphate buffer pH 7.7+8M urea+ 1M NaCl

Flow Rate: 3 mL/min

Detector Wavelength: 280 nm

Elution protocol: eluting the protein of interest with 150 mM NaCl, eluting the undesired protein with 300 mM NaCl, and collecting the fraction eluted with 150 mM NaCl.

Purification of the fusion Protein fusion by Hydrophobic Interaction Chromatography

Chromatographic media: Phenyl SEPHAROSE 6 Fast Flow (GE Healthcare Co. )

Column Volume: 26 mm X 20 cm

Buffer: 20 mM phosphate buffer pH 7.7+8Murea+0.4M (NH ₄) ₂SO ₄.

20 mM phosphate buffer pH 7.7+4M

Flow Rate: 2 mL/min

Detector Wavelength: 280 nm

Sample: the fraction eluted with 150 mM NaCl as obtained in the previous step was dialyzed to a buffer (20 mM phosphate buffer pH 7.7+8M urea+0.4 M (NH4) 2SO4 and then was used as sample.

Elution protocol: eluting the undesired protein with 0.3M (NH4) 2SO4 eluting the protein of interest with 0.1M and 0M (NH4) 2SO4 and collecting the fraction eluted with 0.1M and 0M (NH ₄) ₂SO ₄.

The fraction eluted with 0.1M and 0M (NH4) 2SO4 was dialyzed and renatured into PBS, and then 10 μl was taken for SDS-PAGE analysis, and electrophoresis bands were visualized by Coomassie brilliant blue staining. The results showed that after the above purification steps, the fusion protein had a purity of above 90%.

Example 9. Dialysis refold and re-purification.

The flow through fraction in example 8, was diluted with 8M urea (pH 8.0) in the same volume and by the addition 0.3M Arginine , 5mM EDTA , 5%glycine, 4×CB (1.5g Na2CO3, 3gNaHCO3/L) . was dialyzed against 6M urea. 0.3M Arginine, 5mM EDTA , 5%glycine, 1×CB (1.5g Na2CO3, 3gNaHCO3/L) over a period of 6h, Residual insoluble material was removed by centrifugation at 15,000 X g for 30 minutes. The clarified was chromatographed on a 10 X 30 cm column of DEAE resin equilibrated in buffer. The column was developed in this same buffer at a linear flow rate of 0.3 cm/min. After loading the extract, the column was washed with two column volumes of this buffer to elute contaminants in the void volume, fusion protein in flow through fraction. The fractions containing pure fusion were concentrated tenfold over an ultrafiltration membrane (5,000 dalton cut-off) under nitrogen pressure, exhaustively dialyzed against 0.25 mM sodium bicarbonate, 0.21 mM sodium carbonate, pH 7.0, concentrated an additional threefold, and lyophilized. The final product was essentially pure. Overall yield, based on the original fermentor titer, was 20.2%.

The part of flow through fraction in example 8 was mixed resuspended in 8M urea (pH 8.0) in the same volume containing 0.3M Arginine, 5mMEDTA, 5%glycine, 4×CB (1.5g Na2CO3, 3gNaHCO3/L) and dialysed three times against 6M, 3M, 0M urea buffer for 6h, respectively . The dialyzed extract was chromatographed at a flow rate of 1 ml/hr over a 100cm X 35 mm column of S-300 resin equilibrated in 10mmol/L Tris pH8.0. A single peak was seen to elute in the void volume of the column and contained greater than 95%pure. The relevant fractions were pooled, concentrated threefold over an ultrafiltration membrane (5000 dalton cut-off) , dialyzed extensively against 1%CB (0.25 mM sodium bicarbonate, 0.21 mM sodium carbonate) and lyophilized. The final product mg protein) represented a recovery of 14.5%based on the total amount of protein in the original extract.

The column factions containing the recombinant fusion protein fusion were then dialysed at 4 ℃ for 6 hours against a 50-fold volume e dialysis buffer in a solution of 6M urea pH 8.0. Following extensive dialysis against 3M urea buffer pH 8.0, immunogen was further purified by gel filtration on a Sephacryl S300 HR column equilibrated in 10mmol/L Tris, pH8.0 proteins eluted in a position corresponding to a Mr of about 35,000, when compared to a 30,000 dalton molecular weight standard (Fig 1D) . Approximately 0.9 g fusion (obtained from the above steps) was run on a Sephacryl S300 HR column. fusion obtained by this purification procedure was free from detectable amounts of contaminating protein and nucleic acid. The solution containing fusion was subsequently concentrated by ultrafiltration to the appropriate concentration.

Refolding was performed by adjusting urea concentration from 6M to 0M using dialysis buffer containing 0.3M Arginine, 5mM EDTA, 5%glycine, 4×CB (1.5g Na2CO3, 3gNO3/L) without any dilution by contacting with hydrophobic interaction column (Phenyl Sepharose FF) equilibrated with 50 mM Tris-HCl, 3M NaCl pH8.0 buffer, followed by elution with 10 mM potassium phosphate, pH7.2 buffer. The flow through fraction subjected to 5 kDa UF/DF (UF/DF) step to remove salt and concentrate the protein solution. The protein solution is diafiltered with 10 mM potassium phosphate, pH7.2 buffer. The concentrated and diafiltered protein filtered through 0.22μM filtered and frozen at -70 ℃.

Intermediate purification of the refolded protein using ion exchange chromatography is carried out to obtain ＞99.9%pure and the semi purified protein obtained as a result of intermediate purification is subjected to further chromatographic separation to obtain purified fusion proteins fusion protein was mixed with adjuvant in a certain proportion for adsorption.

Example 10. The P2-RBD fusion protein as vaccine immunogen evaluation in mice

Determination of the Reactivity of the fusion protein with Antibodies by Western Blotting

The reactivity of the fusion with SARS-COV-2 antibody and anti-RBD polyclonal antiserum (which was prepared by immunizing rat with RBD through methods well known in the art, and the reactivity of the serum was confirmed by commercially available RBD ) were determined by Western blotting. The dialyzed, purified and renatured P2-RBD fusion protein samples were transferred to nitrocellulose membrane for blotting after SDS-PAGE separation; 5%skimmed milk was used to block the membrane for 2 h, monoclonal or serum antibody diluted at a certain ratio was then added (monoclonal antibody was diluted at 1: 2000, and polyclonal antiserum was diluted at 1: 1000) , and the reaction was carried out for 1 h. The membrane was washed with TNT (50 mmol/L Tris Cl (pH8.0) , 150 mmol/L NaCl, 0.05%Tween 20) for three times, 10 min for each time. Goat Anti-mouse IgG antibody (HRP conjugate, Sigma-Aldrich, SL, USA) ) was then added, the reaction was carried out for 1 h, and the membrane was then washed with TNT for three times, 10 min for each time. 3, 30, 5, 50-tetramethylbenzidine (TMB) were used for visualization.

As determined by Western blotting using the fusion protein and SARS-COV-2 neutralizing monoclonal antibody were shown in FIG. 2. The results showed that all the tested fusion had significant reactivity with SARS-COV-2 antibody.

Determination of the Reactivity of the fusion with Various SARS-COV-2 Specific Antibodies by ELISA

The reactivity of the fusion proteins and the control proteins RBD (only contained Val308 -Gly548 of spike protein) with various SARS-COV-2 specific antibodies was determined by indirect ELISA. The dialyzed, purified and renatured samples were diluted in 1XPBS (1 μg/ml) , and then were added to 96-well microplate at 100 μl/well and incubated at 37℃ for 1 h. The coating solution was discarded, the plate was washed with PBST (PBS+0.05%Tween-20) once, and then the blocking solution was added at 200μl/well and incubated at 37℃ for 1 h. The blocking solution was discarded when the detection was performed, and the SARS-COV-2 monoclonal antibodies diluted at a certain ratio (when RBD and its fusion protein fusion were detected, they were diluted at 1: 10000) when the reactivity of RBD proteins was compared, the monoclonal antibodies were subjected to 10-fold serial dilution wherein 1 mg/ml was used as the initial concentration, and the polyclonal antibody at its initial concentration was subjected to dilution in the same manner) was added at 100 μl/well. The mixture was incubated at 37℃ for 1h. The plate was then washed with PBST for five times, and HRP-labeled Goat anti Mouse (1: 5000) was then added at 100μl/well and was incubated at 37℃ for 30 min; the plate was then washed with PB ST for five times, HRP substrate was then added at 100 μl/well and was incubated at 37℃for 15 min. 2M H2SO4 solution was added at 50μl/well to stop the reaction, and Microplate reader (Thermo Fisher, Mannheim, Finland) was then used to read OD450/620 value. The results of the ELISA using fusion protein with the monoclonal antibodies were shown in FIG. 3. The results showed that the reactivity of fusion protein with the antibody was not significantly enhanced, after its fusion with P2 or a fragment thereof, wherein the reactivity of P2-RBD and RBD was not enhanced significantly; the reactivity of fusion with SARS-COV-2 -specific antibody was neither retained nor enhanced, after its fusion with P2 or a fragment thereof.

Analysis of the Reactivity of the fusion Protein renatured by dialysis and purified by Chromatography

The reactivity of the fusion protein purified by dialysis and chromatography, was analyzed by indirect ELISA (see the concrete process in the previous step) . The ELISA result was shown in FIG. 3. The result showed that the reactivity of fusion with SARS-COV-2 specific antibody was comparable to that of the control protein SARS-COV-2 inactivated immunogen.

Analysis of affinity of P2-RBD and RBD to hACE2 by ELISA, both proteins were coated on a 96-well plate (Costar) at 200 ng/well in PBS overnight at 4℃. The plate was blocked using 3%skim milk for 1 h at room temperature (RT) . We then added serially diluted hACE2-mP2 (mouse P2, Sino Biological, Beijing, China) and incubated for 2 h at RT. The plates were washed 4 times with 0.05%tween in phosphate buffered saline (PBST) . Anti-mouse IgG-horseradish peroxidase (HRP) conjugated secondary antibody (Sigma-Aldrich) was added to the plate followed by incubation for 1 h at RT. After another 4 washes with PBST, the plate was incubated with a 3, 30, 5, 50 -tetramethylbenzidine substrate solution (TMB, SigmaAldrich) for 3 min. The reaction was stopped using 1 M H2SO4 followed by reading absorbance of each well at 450 nm. For detection of anti-RBD or anti- (S1 + S2) antibodies in mouse serum, the SARSCoV-2 RBD proteins or S1 + S2 (Sino Biological, Beijing, China) were coated at 200 ng/well in PBS overnight at 4 ℃. After blocking, serially diluted mouse serum were added and incubated for 2 h at RT. For antibody isotyping, the bound RBD-specific antibodies were detected by anti-mouse IgG, IgM, IgA HRP conjugated secondary antibody (Sigma-Aldrich) , respectively. For competitive ELISA, ～20 nM (4 μg/ml) biotinylated hACE2 (Sino Biological, Beijing, China) was incubated with serially diluted mouse serum, and the mixtures were added to RBD coated wells. After washing, bound hACE2 was detected by Streptavidin-HRP secondary antibody (Sigma-Aldrich) .

Analysis P2-RBD immunogenicity with Pseudovirus neutralization assay

The pseudovirus neutralization assay was performed based on previous protocols. Briefly, HIV-1 backbone based pseudovirus was packaged in 293T cells by co-transfecting with plasmid encoding SARS-CoV-2 S protein and plasmid encoding luciferase expressing HIV-1 genome (pNL4-3. luc. RE) using polyethylenimine (PEI) . Pseudovirus-containing supernatants were collected 48 h later and concentrated using Lenti-XTM concentrator kit (Takara, CA) . Pseudovirus neutralization assay was then performed by incubation of SARS-CoV-2 pseudovirus with serially diluted mouse serum for 1 h at 37 ℃, followed by addition of the mixture into pre-seed 293T-ACE2 cells. The mixture was then centrifuged at 1000 X g for 1 h at RT. The medium was replaced 4 hrs later. After 24 h, luciferase expression was determined by Bright-Glo kits (Promega, Madison, WI) and read using BioTek synergy multi-mode reader (Winooski, VT) . The 50%pseudovirus neutralizing antibody titer (NT50) was calculated.

Analysis P2-RBD immunogenicity with microneutralization assay

The standard live virus-based microneutralization (MN) assay was used. Briefly, serially five-fold (start from 1: 5) and duplicate dilutions of mouse serum were incubated with 100 pfu of SARS-CoV-2 at room temperature for 2 h before transferring into designated wells of confluent Vero E6 cells (ATCC, CRL-1586) grown in 96-well microtiter plates. Vero E6 cells cultured with medium with or without virus were included as positive and negative controls, respectively. After incubation at 37 ℃ for 4 days, individual wells were observed under the microscopy for the status of virusinduced formation of cytopathic effect (CPE) . The titer of mouse serum (NT100) was expressed as the lowest dilution folds capable of completely preventing virus-induced CPE in 100%of the wells.

Analysis P2-RBD antibody mediated inhibition Cell-Cell fusion

To test mouse serum mediated inhibition of cell fusions, the β-gal reporter gene based quantitative cell fusion assay was used. Briefly, 293T-Scells were infected with T7 polymeraseexpressing vaccinia virus (vTF7-3) , while 293T-ACE2 cells were infected with vaccinia virus (vCB21R Lac-Z) encoding T7 promotor controlled β-gal. Two hours after infection, cells were incubated with fresh medium and transferred to 37℃ for overnight incubation. The next day, 293T-S cells were pre-mixed with serially diluted mouse serum at 37℃ for 1 h followed by incubation with 293T-ACE2 cells at a 1: 1 ratio for 3 h at 37℃. Then cells were then lysed, and the β-gal activity was measured using b-galactosidase assay kit (substrate CPRG, G-Biosciences, St. Louis, MO) following the manufacturer’s protocols. Fusion inhibition percentage (sample reading, F) was normalized by maximal fusion (reading, Fmax) of 293T-S and 293T-ACE2 cells in the absence of inhibitors using this formula: Fusion inhibition %= [ (Fmax-F) / (Fmax Fblank) ] × 100%, in which Fblank refers to the OD reading of 293T-S and 293T incubation wells. Fusion inhibition percentage was plotted against serum dilution folds from which IC50 was calculated.

Wild-type SARS-CoV-2 neutralization assay.

The neutralization assay with SARS-CoV-2 virus was conducted. Vero E6 cells (2.5×10 ⁴ cells/well) were seeded in 96-well plates and incubated overnight. Sera were heated at 56 ℃ for 30 min to inactivate complement and diluted in serum-free MEM at an initial dilution factor of 20, and then further twofold serial dilutions were performed for a total of 11 dilution steps to a final dilution of 1: 40, 960. The diluted sera were mixed with an equal volume of SARS-CoV-2 virus at 100 TCID50/50μL (SARS-CoV-2 generated from isolate 2019-nCoV/CN-WH/2019 GenBank accession MT192759) and incubated at 37 ℃ for 2 h. The sera-virus mixture was then added to 96-well plate with Vero E6 cells and incubated in MEM with 2%FBS at 37 ℃ for 5 days. Afer incubation, cells were fixed by adding 4%formalin to each of the wells for 10 min and stained with 0.1%crystal violet for visualization. Results were calculated with the Reed-Muench method for log 50%end point for ID50 and log 90%end point for ID90 titers.

Analysis P2-RBD antibody with ADE assay

FcRII expressing cell lines K562 (ATCC, CCL-243) were used to perform ADE assays. Briefly, the mouse serum was serially diluted, mixed with SARS-CoV-2 pseudovirus, and incubated at 37℃ for 1 h. Then, the mixtures were added to the pre-seeded plates with K562 cells. The following infection and culturing steps were carried out as described above in the pseudovirus neutralization assay. Pseudovirus infected K562 or 293T-ACE2 cells were set as the negative and positive controls, respectively.

To evaluate the applicability of purified and refolded P2-RBD or RBD as a vaccine immunogen, produced by overexpression in E. coli, a vaccine composition was developed using P2-RBD and RBD immunization trials were performed on mice.

Mouse immunization Four groups of 8-10 week old female BALB/c mice (n = 5) were immunized twice (day 0 and day 14) subcutaneously with RBD proteins (10 μg/mouse) with or without adjuvant MF59. Group 1 was immunized with P2-RBD fusion, group 2 was immunized with P2-RBDfusion in emulsion with MF59, group 3 was immunized with RBD in emulsion with MF59, group 4 served as a control and was immunized subcutaneously with Dulbecco’s phosphatebuffered saline DPBS (GibcoTM) . Sera were collected before (prevaccination) , after 13 days, and after 27 days vaccination.

All experiments were conducted in duplicate, and data were averaged and presented as the mean + standard deviation (SD) . Significant differences were determined by one-way analysis of variance followed by Tukey’s test, using the Graphpad Prism (version 7) package. Statistical significance was defined as P ＜ 0.05.

Recombinant SARS-CoV-2 RBD (with P2) and RBD proteins were produced in bacterial cells, and then verified by SDS-PAGE and Western blot. P2-RBD showed a homogenous band (Fig. 1) while the Besides, Western blot results showed that P2-RBD with different purity can react with hACE2 ELISA binding to hACE2 further validated the qualities of both P2-RBD proteins (Fig. 3) .

Mouse immunization and sera sampling schedule. Four groups of BALB/c mice (n = 5) received same doses of the RBD vaccine or the control DPBS on day 0 and boost again on day 14. Sera were collected on day 0 (pre-vaccination) , day 13 and 27 (post-vaccination)

Four groups of 8-10 week old female BALB/c mice (n = 5) were immunized subcutaneously at day 0 and boosted at day 14 with P2-RBD, P2-RBDin emulsion with MF59 (P2-RBD + MF59) , and RBD in emulsion with MF59 (RBD + MF59) for each group at a dose of 10 μg of protein per mouse. The fourth group received injection of DPBS, which served as the negative control. On day 0 (preimmunization) , day 13 and day 27, mouse sera were collected and analyzed for RBD binding, pseudovirus and live virus neutralization, and cell-cell fusion inhibition. Anti-RBD sera from each trial group were firstly evaluated for RBD binding as measured by ELISA (Fig. 3) . The anti-RBD antibody in post immune mouse sera were also isotyped by anti-mouse IgG, IgM and IgA antibodies, respectively. The RBD antibody titers were calculated as the dilution folds that retained 50%of maximal binding signal (EC50) . The recombinant RBD was used as the detection antigen to avoid the interference of anti-human P2 antibody titers in mice. Results showed that for the sera collected at 13 and 27 days post immunization, the anti-RBD antibodies were mostly composed of the IgG isotype with only marginally detectable IgM and no detectable IgA isotype. The low IgM titer detected at day 13 and 27 may correlate to the fact that IgM is typically rapidly mounted post infection (within one week) followed by isotype switching into IgG isotype. The lack of IgA titer may result from IgA usually deriving from mucosa immunity, leading to low titers in sera. For the IgG isotype antibodies, the preimmunization sera showed no binding to RBD, while the day 13 sera from all three RBD immunized groups exhibited varying extents of binding to the RBD. Interestingly, the RBD binding titer elicited by the RBD + M59 group on day 13 was much less than titers of the P2-RBD (titer 1: 36) and P2-RBD + M59 (titer 1: 66) groups, indicating the immune stimulation roles of P2 fusion. However, for post-boosted sera on day 27, the RBD binding titers of RBD + MF59 group was significantly increased to 1: 368. In contrast, on day 27 the titers of P2-RBD (titer 1: 603) and RBDP2 + MF59 (titer 1: 1130) groups were only improved by 17-fold compared to those of day 13, indicating distinct humoral response kinetics against RBD and P2-RBDimmunogens. Interestingly, although to a lesser extent than before receiving the booster, the P2-RBDand P2-RBD+ MF59 groups exhibited 1.6 and 3 folds higher titers, over the RBD + MF59 group (P ＜ 0.05) respectively, assuring the enhancing role of P2 in elicitation antibody response by the RBD immunogen. It is also intriguing that the P2-RBD+ MF59 group exhibited slightly higher titers than the P2-RBD group, probably due to the adjuvant role of MF59. We also correlated the RBD binding titer to the full-length S ectodomain binding titer for the day 27 sera. The ELISA showed that the RBD binding sera also bound to S1 + S2 with similar titers (Fig. 4A, 4B) , which suggest that the RBD recognition antibody in the sera can also bind to full length S. hACE2 blocking is a surrogate indicator for anti-SARS-CoV-2 antibody neutralizing activity. To preliminarily evaluate the neutralizing titers of post-immunization mouse serum, we performed the hACE2 competitive ELISA, in which serially diluted mouse sera in the presence of the biotinylated hACE2 were added into RBD coated plates. Bound hACE2 was detected by the streptavidin-HRP secondary antibody. Results showed the three RBD immunogen groups developed discemable hACE2 competitive titers on day 13 compared to the PBS control group; further significantly boosted to 1: 34, 1: 65, 1: 31 for the P2-RBD, P2-RBD+ MF59, RBD + MF59 groups respectively on day 27 (Fig. 5) . Consistent with the above RBD binding titer, the P2-RBD and P2-RBD+ MF59 groups sera showed 1.1 folds and 2.1 folds higher competitive titers respectively than the RBD + MF59 group (P ＜ 0.05) , supporting the role of P2 in mounting neutralization titers. The competitive ELISA results gave the specific hACE2 blocking titers elicited by RBD immunogens, which presumably predict their neutralization activity. Next the SARS-CoV-2 S pseudotyped HIV-1 was exploited to evaluate the neutralization activity of those anti-RBD sera. The SARS-CoV-2 pseudovirus was packaged by co-transfecting HEK 293T cells with pCDNA3.1-S plasmid encoding codon-optimized full-length SARS-CoV-2-S protein and pNL4-3. luc. RE plasmid containing the luciferase expressing HIV-1 genome. Serially diluted mouse sera were pre-incubated with pseudovirus followed by infection of 293T cells stably expressing hACE2 (293T-ACE2) . As shown in Fig. 6, on day 13 all of the RBD immunized groups sera showed substantial 50%neutralizing antibody titers (NT50, 1: 63, 1: 76, 1: 10) compared to the pre-immune sera on day 0, which were largely boosted to 1: 486, 1: 2243, 1: 165 on day 27 for the P2-RBD, P2-RBD+ MF59 and RBD + MF59 groups respectively. Intriguingly, unlike the marginal differences for the RBD binding and hACE2 competitive titers across the sera of the three RBD immunized groups on day 27, the pseudovirus neutralization titers were significantly distinct with the P2-RBD+ MF59 group showing highest titers, 4.6-fold higher than the P2-RBDgroup and 13.6-fold than the RBD + MF59 group (P ＜ 0.05) . In addition to the pseudovirus neutralization, we also evaluated the live virus neutralization potency of those mouse anti-RBD sera (day 27) by using a microneutralization (MN) assay. In this assay, the cytopathic effect (CPE) of Vero E6 was observed after 4 days incubation with live virus, which was pre-mixed with the anti-RBD sera. The neutralization titer of mouse serum (NT100) was expressed as the lowest dilution folds capable of completely preventing virus-induced CPE in 100%of the wells. Consistently, the NT100 of serum in RBDP2 + MF59 group (1: 25) was higher than those of the P2-RBD and RBD + MF59 groups (1: 5) (Fig. 7) . These results clearly demonstrated that P2 fusion could significantly augment the elicitation of neutralizing antibody titers by RBD immunogen, and the adjuvant MF59 can further stimulate the antigenicity of the P2-RBD fusion proteins. Interestingly, the pseudovirus neutralization titer positively correlated with the hACE2 competition titer (Fig. 6) , demonstrating the utility of hACE2 competition ELISA in predicting neutralization titers in convalescent plasma therapy and in detecting of the presence of neutralizing antibodies in serological tests during the COVID19 pandemic. To further evaluate whether the anti-RBD sera could prevent SARS-CoV-2 S-mediated cell-cell fusion, a quantitative cell fusion assay using β-galactosidase (β-gal) as a reporter gene. 293T cell lines stably overexpressing SARS-CoV-2 S (293T-S) was constructed and hACE2 (293T-ACE2) respectively. In this assay, the 293T-S cells were infected with T7 polymeraseexpressing vTF7-3 vaccinia virus and the 293T-ACE2 cells were infected with T7 promotor controlled β-gal expressing vCB21R vaccinia virus. Therefore, β-gal expression is only allowed after cell-cell fusion, which can be quantified by monitoring β-gal activity. Serially diluted mouse sera were pre-mixed with infected 293T-S cells followed by incubation with infected 293T-ACE2 cells. The pre-vaccination sera at day 0 and the PBS control mouse sera did not inhibit the cell-cell fusion. However, the day 27 P2-RBD+ MF59 sera showed obvious cell-cell fusion inhibition with a 50%fusion inhibition antibody titers (IC50) of 1: 97, which was 3.9-fold higher than the inhibition titers of the P2-RBD (1: 25) and 4.9-fold higher than the RBD + MF59 (1: 20) group (P ＜ 0.05) . The extent of the correlation of ACE2 competition ELISA titer with the neutralizing titer (R2 = 0.8204, P = 0.0129) and competition ELISA titer with cell-cell fusion inhibition titer (R2 = 0.8854, P = 0.0051) were lower than that of neutralizing titer to cell-cell fusion inhibition titer (R2 = 0.9872, P ＜ 0.0001) . Whether the anti-RBD mouse sera can enhance SARS-CoV-2 infection of FcRII expressing K562 cells was evaluated. The results showed that SARS-CoV-2 pseudovirus alone cannot infect the K562 cells. In addition, treatment with serially diluted (ranging from 1: 250 to 1: 107) anti-RBD sera did not enhance SARS-CoV-2 pseudovirus infection, indicating that the anti-RBD sera may not promote ADE.

This eample has identified the potency of the recombinant SARS-CoV-2 RBD fusion as an immunogen in the context of the P2 fusion and MF59 adjuvant. that the P2-RBD fusion protein with MF59 adjuvant is the most potent RBD based immunogen to elicit neutralizing antibodies that can potently inhibit SARS-CoV-2 pseudovirus and live virus infection.

Example 11. The P2-RBD fusion protein as vaccine (named as CBSVX-CoV2020) immunogen evaluation in no human primate

Cynomoμgus macaque immunization

Antigen and adjuvant dose levels were selected based upon prior experience in mice and humans immunized with killed vaccine (5 μg or 10 μg) with and without MF59 (50 μg) . In this study, cynomoμgus macaques ＞ 3 years old (n = 4/group) at study initiation received 5 or 25 μg P2-RBD with 50μ g MF59 administered in 500 μL in the thigh muscle in two doses spaced 21 days apart. A separate group was immunized with a fractional dose (10 μg) P2-RBD with 25 μg MF59 in two doses spaced 21 days apart and a placebo group received formulation buffer. Serum was collected before immunization on day 0, day 21 just prior to the second immunization, and day 33.

Anti-spike (S) IgG ELISA Anti-SARS-CoV-2 spike (S) protein IgG ELISA titers were measured as described in Example 7. Serum samples were serially diluted 3-fold down starting with a 1: 100 dilution (ie, 10-2 to 10-8) and added to the coated plates at room temperature for 2 h. Following incubation, plates were washed with PBS-T and HRP-conjugated mouse antimonkey IgG (Southern Biotech, Birmingham, AL, USA) was added for 1 h. Plates were washed with PBS-T and 3, 30, 5, 50 -tetramethyl benzidine (TMB) peroxidase substrate (Sigma, St Louis, MO, USA) was added. Reactions were stopped with TMB stop solution (ScyTek Laboratories, Inc. Logan, UT) . Plates were read at OD 450 nm with a SpectraMax Plus plate reader (Molecular Devices, Sunnyvale, CA, USA) . Anti-S IgG EC50 titers were calculated by 4-parameter fitting using SoftMax Pro 6.5.1 GxP software. Individual animal anti-S IgG EC50 titers, group geometric mean titers (GMT) were plotted.

Inhibition of hACE2 receptor binding and neutralization Antibodies that block binding of hACE2 receptor to the S-protein and neutralize in a cytopathic effect assay (CPE) in Vero E6 cells were measured as described previously example 7 as the serum titer that blocks 100%CPE. Serum antibody titer at 50%binding inhibition (IC50) of hACE2 to SARS-CoV-2 S protein was determined in the SoftMax Pro program. Individual animal hACE2 receptor inhibiting titers, mean titers, and SEM were plotted. Neutralizing antibody titers were determined as the dilution of serum that inhibited 100%of CPE (CPE100) at 3 days post infection of Vero E6 cells in a 96 well plate format.

SARS-CoV-2 challenge procedure

The virus challenge study was done at CCDC within a BSL-3 containment facility. SARS-CoV-2 generated from isolate 2019-nCoV/CN-WH/2019 and expanded in Vero E6 cells for challenge stock generation. Animals were sedated and challenged with a targeted total dose of 1.1×10 ⁴ pfu SARS-CoV-2 by intranasal (IN) and intratracheal (IT) in a volume of 0.25 mL each route. BAL and nasal swabs were collected 2-and 4-days post challenge. Necropsy was performed 7 days following challenge and lung tissues collected for histopathology.

RNA subgenomic RT-PCR

The subgenomic viral mRNA (sgRNA) was measured in macaque bronchoalveolar lavage (BAL) and nasal swabs collected 2-and 4-days post challenge using RT-PCR (N: Primer: ATGCTGCAATCGTGCTACAA; R primer: GACTGCCGCCTCTGCTC; probe: /56-FAM/TCAAGGAAC/ZEN/AACATTGCCAA/3IABkFQ) . To generate a standard curve, the SARS-CoV-2 E gene sgRNA was cloned into a pcDNA3.1 expression plasmid. The insert was transcribed using an AmpliCap-Max T7 High Yield Message Maker Kit (Cellscript, Madison, WI) to obtain RNA for standards. Prior to RT-PCR, samples collected from challenged animals or standards were reversetranscribed using Superscript III VILO (Invitrogen) according to the manufacturer’s instructions. A Taqman custom gene expression assay (ThermoFisher Scientific, Rockville, MD) was designed using the sequences targeting the E gene sgRNA. Reactions were carried out on a

Quant Studio

6 and 7 Flex Real-Time PCR System (Applied Biosystems, Foster City, CA) according to the manufacturer’s specifications. Standard curves were used to calculate sgRNA in copies per mL. The quantitative assay was sensitive to 50 copies per mL. 2.8. Histopathology Animals were euthanized 7-days following SARS-CoV-2 challenge (Day 42) and lung tissues collected. Tissue were prepared for histologic examination by Experimental Pathology Laboratories, Inc. (EPL, Sterling, VA) . The lungs were fixed with 10%formalin, paraffin embedded, and sections stained with hematoxylin and eosin (H&E) for histological examination. Slides were examined for total inflammation, periarteriolar, and peribronchiolar inflammation and epithelial cell denuding. 2.9. Human COVID-19 convalescent serum Convalescent serum samples (n = 32) were provided by Dr.Bi Sheng Li (CCDC, Changping, Beijing ) . Samples were collected from COVID-19 individuals 18-79 years of age 4-6 weeks after testing positive for SARS CoV-2. Symptoms ranged from asymptomatic, mild to moderate symptoms, to severesymptoms requiring hospitalization. Sera were analyzed for antiSARS-CoV-2 S IgG, hACE2 receptor inhibition, and virus neutralizing antibody titers.

Immunogenicity of CBSVX-CoV2020 (P2-RBD) in nonhuman primates compared to COVID-19 convalescent human sera Macaques immunized with the prime/boost regimen of 2.5, 5, and 25μg CBSVX-CoV2020 with 25μg in the low and 50 μg MF59 adjuvant in the two higher doses induced anti-S IgG (EC50) antibodies at Day 21 after a single dose (GMT = 7,810, 22,386 and 21,472, respectively) . Two weeks following a booster immunization, anti-S IgG EC50 titers increased to GMT EC50 = 163,036, 335,017 and 469,739, respectively (Fig. 8A) . In contrast, SARS-CoV-2 anti-S antibody in convalescent human sera was 6.9-to 14.2-fold less with at GMT EC50 of 23,614 (Fig. 8B) . And, hACE2 receptor inhibition titers of 649, 1,410, and 1,320 in 2.5, 5, and 25 μg CBSVX-CoV2020 dose groups respectively were 5.2 -11.2-fold higher than in convalescent sera (Fig. 8C) . Finally, SARS-CoV-2 GMT neutralization antibody titers of 17,920-23,040 CPE100 in immunized macaques, were 7.9 -10.1-fold higher than in convalescent sera (Fig. 8D) .

Viral load in nasal swabs and BAL To evaluate the potential efficacy of CBSVX-CoV2020 vaccine, macaques were challenged with SARS-CoV-2 virus in upper and lower airways. Macaques in the placebo group had 9,131 sgRNA copies/mL in the BAL at 2 days post challenge and remained elevated at day 4 except for one animal. In contrast, immunized animals had no detectable sgRNA in BAL fluid other than one animal in the low dose group at day 2 which cleared replicating virus RNA by day 4 (Fig. 8E). Half of the controls had ～ 4 log10 of virus sgRNA copies in nasal swabs and in contrast, no detectable sgRNA was in the nose of CBSVX-CoV2020 vaccinated animals (Fig. 8F) .

Lung pathology

Lung tissues were collected from all animals at 7 days post challenge and sections examined for pathologic changes within the upper and lower airways. Placebo control animals had moderate to severe inflammation that involved the mucosa of the bronchi, perivascular mononuclear infiltrate with mixed infiltrates of macrophages and neutrophils within the alveoli. In contrast, there was little, or no inflammation observed in the lungs of macaques immunized with CBSVX-CoV2020 vaccine 7 days post challenge (Fig. 9) . These findings were consistent the absence of sgRNA in BAL fluids and nasal swabs of vaccinated animals by day 4 post challenge (Fig. 8E and 8F) .

Claims

A fusion protein as a subunit vaccine immunogen against SARS-CoV-2, comprising a receptor binding domain (RBD) fragment of SARS-CoV-2 spike protein and Tetanus toxoid peptide P2, they are fused by a linker sequence.
The fusion protein of claim 1, wherein the amino acids sequence of the receptor binding domain (RBD) fragment of SARS-CoV-2 spike protein is shown in SEQ ID NO: 1.
The fusion protein of claim 1, wherein the amino acids sequence of the Tetanus toxoid peptide P2 is shown in SEQ ID NO: 2.
The fusion protein of claim 1, wherein the amino acids sequence of the linker is shown in SEQ ID NO: 3-7.
The fusion protein of claim 1, wherein the amino acids sequence of the fusion protein is shown in SEQ ID NO: 8.
The fusion protein of claim 1, wherein the fusion protein is obtained via a prokaryotic expression system.
The fusion protein of claim 6, wherein the prokaryotic expression system is E. coli.
A method for preparation the fusion protein in any one of claims 1-7, the method comprising the steps of:

a) transformation of Escherichia coli with a desired gene coding for the fusion protein using a plasmid vector;

b) culturing the transformed Escherichia coli in suitable culture medium under suitable conditions,

c) isolation and purification of inclusion bodies,

d) denaturation and solubilization of inclusion bodies at high pH value ranging from 8 to 9,

e) followed by pH adjustment within a range of 6 to 8.5, preferably at 8.0 of the solubilized protein, to produce a refolded protein,

f) intermediate purification of the refolded protein using ion exchange chromatography, hydrophobic interaction chromatography to obtain ＞99.9%pure native fusion protein and

g) the semi purified protein obtained in step (f) is further purified by one or more chromatographic separation using anion exchange chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography and size exclusion chromatography to obtain the fusion protein.
The use of the fusion protein as a subunit vaccine immunogen in preparing the vaccine against SARS-CoV-2.
A vaccine against SARS-CoV-2, wherein the vaccine contains effective amount of the fusion protein in any one of claims 1-7.