COVID-19 fusion protein nucleocapsid antigen immunotherapy and method of use

Abstract

This disclosure provides recombinant fusion proteins for use in relation to the novel coronavirus (COVID-19), comprising a SARS-CoV-2 nucleocapsid protein (N-protein) fragment or an analogue linked to a human Fc fragment. Multiple embodiments include administration of the fusion protein as a booster vaccine to patients recovering from COVID-19, administration to antibody-naive patients to produce antibodies against the SARS-CoV-2 virus so that the patient can become a convalescent plasma donor, administration to patients infected with the SARS-CoV-2 virus and suffering from COVID-19 to limit the scope of infection and improve the disease, and administration as a prophylactic COVID-19 vaccine. Exemplary Fc fusion proteins and pharmaceutical formulations of exemplary Fc fusion proteins are provided in addition to methods of use and preparation.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 488,565, filed on 6 March 2023, entitled "NUCLEOCAPSID ANTIGEN IMMUNOTHERAPY FOR COVID-19 FUSION PROTEINS AND METHODS OF USE," which is incorporated herein by reference in its entirety.

[0002] Sequence List The following application includes a sequence listing electronically submitted as a Standard ST.26 compliant XML file named "ABC-047PCT.xml", created on February 15, 2024, with a file size of 23,503 bytes, the entire contents of which are incorporated herein by reference as part of this specification.

[0003] Technical field This technology relates to fusion proteins comprising a terminally cleaved SARS-CoV-2 nucleocapsid N protein or an analogue linked to a human Fc fragment, and their use in relation to the 2019 novel coronavirus (COVID-19). [Background technology]

[0004] background The following background information is provided solely to aid in understanding this technology and does not constitute a description or representation of prior art.

[0005] Fc fusion protein An Fc fusion protein is a protein or peptide, such as a therapeutic protein, in which a species-specific immunoglobulin (immunoglobin) Fc domain is linked to another peptide. As used herein, the terms “fusion protein” and “Fc fusion protein” refer to a protein comprising two or more parts covalently linked via peptide bonds, for example, from different sources (e.g., different proteins, polypeptides, cells, etc.). Fc fusion proteins are preferably covalently linked by (i) linking the genes encoding each part to form a single nucleic acid molecule, and (ii) expressing the protein encoded by the nucleic acid molecule in a host cell (e.g., HEK cells or CHO cells). Fully recombinant synthesis is preferred over methods of chemically linking the therapeutic protein and Fc fragment after they have been synthesized separately. Chemical linking and subsequent purification steps increase manufacturing complexity, reduce product yield, and increase costs.

[0006] The terms “Fc fragment,” “Fc region,” “Fc domain,” or “Fc polypeptide” are used herein to define the C-terminal region of an immunoglobulin heavy chain. An Fc fragment, region, domain, or polypeptide may be a native Fc region or a variant / mutant Fc region. The boundaries of an Fc region of an immunoglobulin heavy chain can vary, but generally consist of part or all of the hinge region of the heavy chain, the CH2 region of the heavy chain, and part or all of the CH3 region of the heavy chain. The hinge region of an Fc fragment (e.g., a canine or human Fc fragment) consists of an amino acid sequence linking the CH1 domain and the CH2 region of the heavy chain and contains one or more cysteines that form one or more inter-heavy-chain disulfide bridges for forming a homodimer of the Fc fusion protein from two identical but distinct monomers of the Fc fusion protein. The hinge region may consist of all or part of a native or non-native amino acid sequence.

[0007] The presence of the Fc domain, in addition to slowing the renal clearance of Fc fusion proteins due to their larger molecular size, extends their plasma half-life through interaction with the neonatal Fc receptor (FcRn). As a result, molecular reuse is possible in vivo, extending the activity of the linked peptide and improving the solubility and stability of the Fc fusion protein molecule. The Fc domain also allows the Fc fusion protein to interact with Fc receptors on immune cells. In some cases, therapeutic proteins or peptides are linked to the immunoglobulin (immunoglobin) Fc domain via a linker. The therapeutic protein or peptide and the linker effectively replace the variable region of the antibody while preserving the Fc region.

[0008] An Fc receptor (FcR) refers to a receptor that binds to an Fc fragment or Fc region of an antibody. In several examples, FcR is the native sequence of a canine or human FcR, and FcR is a receptor that binds to an Fc fragment or Fc region of an IgG antibody (γ receptor), and includes, but is not limited to, receptors of the Fc(γ) receptor I, Fc(γ) receptor IIa, Fc(γ) receptor IIb, and Fc(γ) receptor III subclass (including allelic variants and alternatively spliced ​​forms of these receptors). "FcR" also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG molecules to the fetus and for extending the in vivo elimination half-life of antibodies and Fc fusion proteins. In several examples, human-derived FcRs are used in vitro (e.g., in assays) to measure the binding of Fc fusion proteins containing Fc fragments of any mammalian origin to evaluate their FcR binding properties. Those skilled in the art will understand that a mammalian FcR from one species (e.g., a human FcR) may be able to conjugate in vitro with an Fc fragment from a second species (e.g., a dog FcR). [Overview of the project]

[0009] Overview of this technology The present invention describes fusion proteins, each comprising a viral nucleocapsid protein fragment and an Fc fragment, wherein the viral nucleocapsid fragment and the Fc fragment are linked by a peptide linker. In one or more embodiments, the nucleocapsid fragment comprises a SARS-CoV-2 nucleocapsid protein fragment comprising its functional fragment, analogue, or variant / mutant. In one or more embodiments, the nucleocapsid protein fragment comprises the nucleocapsid fragment of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, or its functional fragment, analogue, or variant / mutant. In one or more embodiments, the Fc fragment comprises the sequence or functional fragment of SEQ ID NO: 1. In one or more embodiments, the linker comprises SEQ ID NO: 14. In one or more embodiments, the fusion protein comprises or consists of the sequence of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, or SEQ ID NO: 20. In one or more embodiments, the fusion protein is a homodimer. In one or more embodiments, the Fc fragment is glycosylated.

[0010] This specification also describes immunogenic compositions comprising, or essentially comprising, a fusion protein and a pharmaceutically acceptable carrier in any embodiment or combination of embodiments described herein. In one or more embodiments, the fusion protein is dispersed in the carrier. In one or more embodiments, the composition further comprises an adjuvant. In one or more embodiments, the adjuvant is Montanide® ISA-720. In one or more embodiments, the fusion protein is emulsified with the adjuvant. In one or more embodiments, the emulsification is prepared in situ before administration. In one or more embodiments, the prepared emulsion is stable at refrigeration (4°C) or room temperature for at least 8 hours, preferably 24 hours. In one or more embodiments, the composition is an injectable formulation. In one or more embodiments, the composition is adapted for subcutaneous administration. In one or more embodiments, the composition is adapted for a prophylactic vaccine. In one or more embodiments, the composition is adapted for a therapeutic vaccine.

[0011] Furthermore, this specification describes various methods for increasing antibody production against an antigenic substance in a subject. These methods generally involve administering a therapeutically effective amount of a fusion protein or immunogenic composition in any embodiment or combination of embodiments described herein to the subject. In one or more embodiments, the subject has a measurable antibody titer against the antigenic substance prior to administration of the fusion protein or immunogenic composition. In one or more embodiments, the subject is antibody-naive prior to administration of the fusion protein or immunogenic composition. In one or more embodiments, the fusion protein or immunogenic composition is administered by injection. In one or more embodiments, the fusion protein or immunogenic composition is administered subcutaneously or intramuscularly. In one or more embodiments, the fusion protein or immunogenic composition is provided in unit dosage forms. In one or more embodiments, the fusion protein or immunogenic composition is administered in combination with an adjuvant. In one or more embodiments, these methods further involve preparing the fusion protein or immunogenic composition for administration, which includes pre-mixing the fusion protein or immunogenic composition with an adjuvant prior to administration. In one or more embodiments, the premixing comprises emulsifying the adjuvant and the fusion protein to form an emulsion, and administering the emulsion to the target. In one or more embodiments, the prepared emulsion is stable at refrigeration (4°C) or room temperature for at least 8 hours, preferably 24 hours, after preparation.

[0012] This specification also describes methods for inducing an immune response in a subject to a viral infection, preferably the SARS-CoV-2 virus, more preferably COVID-19. These methods generally involve administering a therapeutically effective amount of a fusion protein or immunogenic composition in any embodiment or combination of embodiments described herein to the subject. In one or more embodiments, the subject has a measurable antibody titer against the viral infection prior to administration of the fusion protein or immunogenic composition. In one or more embodiments, the subject is antibody-naive prior to administration of the fusion protein or immunogenic composition. In one or more embodiments, the fusion protein or immunogenic composition is administered by injection. In one or more embodiments, the fusion protein or immunogenic composition is administered subcutaneously or intramuscularly. In one or more embodiments, the fusion protein or immunogenic composition is provided in unit dosage forms. In one or more embodiments, the fusion protein or immunogenic composition is administered in combination with an adjuvant. In one or more embodiments, these methods further comprise preparing a fusion protein or immunogenic composition for administration, which comprises pre-mixing the fusion protein or immunogenic composition with an adjuvant before administration. In one or more embodiments, the pre-administration comprises emulsifying the adjuvant and fusion protein to form an emulsion, and administering the emulsion to the subject. In one or more embodiments, the prepared emulsion is stable at refrigeration (4°C) or room temperature for at least 8 hours, preferably 24 hours, after preparation.

[0013] This specification also describes methods for producing fusion proteins according to any embodiment or combination of embodiments described in the present invention. These methods generally involve transiently transfecting HEK293 with a nucleic acid encoding the fusion protein, and the transfected HEK293 cells express the fusion protein. In one or more embodiments, the fusion protein is further secreted by the cells into a cell culture medium, and the fusion protein is purified or isolated from the medium. Advantageously, the yield of the purified or isolated fusion protein is higher than 100 mg / L in any of the expression systems.

[0014] Furthermore, this specification describes cells manipulated to express fusion proteins according to any embodiment or combination of embodiments described in the present invention. In one or more embodiments, the cells are HEK293 cells.

[0015] As described herein, any embodiment or combination of embodiments of the present invention may be used in therapy and / or as a drug.

[0016] As described herein, any embodiment or combination of embodiments of the present invention can be used to increase antibody production in a subject.

[0017] As described herein, any embodiment or combination of embodiments of the present invention may be used in the treatment and / or prevention of viral infections, preferably SARS-CoV-2 virus infections, and more preferably COVID-19 infections.

[0018] As described herein, any embodiment or combination of embodiments of the present invention can be used as a prophylactic vaccine, a therapeutic vaccine and / or a booster vaccine.

[0019] Certain embodiments relate to a fusion protein selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 20, or a pharmaceutical composition thereof, for use in the treatment and / or prevention of viral infection, preferably SARS-CoV-2 viral infection, more preferably COVID-19 infection.

[0020] As described herein, a fusion protein or immunogenic composition according to any embodiment or combination of embodiments described in the present invention can be used in the manufacture of a medicament for the treatment and / or prevention of viral infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] [Figure 1] Figure 1 shows a schematic diagram of an insulin-Fc fusion protein homodimer. [Figure 2] Figure 2 shows a schematic diagram of an exemplary SARS-CoV-2 N-Fc fusion protein homodimer. [Figure 3] Figure 3 shows the Fc(γ) receptor I binding of the insulin-Fc fusion proteins of SEQ ID NO: 5 and SEQ ID NO: 7. [Figure 4] Figure 4 shows the titers of anti-insulin antibodies (AIA) against RHI at an average dilution of 200-fold for six beagles chemically induced with diabetes by eight consecutive administrations of the insulin-Fc fusion protein of SEQ ID NO: 5. [Figure 5] Figure 5 shows the percentage change in anti-insulin antibody (AIA) titer against RHI on day 0 of the test for six beagles chemically induced with diabetes by eight consecutive weekly administrations of the insulin-Fc fusion protein of SEQ ID NO: 5. [Figure 6] Figure 6 shows the normalized AIA titers of eight client dogs treated with diabetes with the insulin-Fc fusion protein of SEQ ID NO: 5 according to Protocol 1 of Example 18 or Protocol 2 as described in Example 19. [Figure 7]Figure 7 shows the normalized AIA value when treatment was discontinued in one dog treated with the insulin-Fc fusion protein of Sequence ID No. 5 for diabetes. [Figure 8] Figure 8 shows a graph of the number of dogs that developed AIA after being treated with the insulin-Fc fusion protein of Sequence ID No. 5, which was prepared using either the HEK transient cell pool or the CHO stable surface cell pool. [Figure 9] Figure 9 shows the normalized AIA values ​​of eight dogs treated with the insulin-Fc fusion protein of Sequence ID No. 7 for diabetes. [Figure 10] Figure 10 shows a graph of the number of dogs that developed AIA after being treated with insulin-Fc fusion protein of SEQ ID NO: 5 or SEQ ID NO: 7. [Figure 11] Figure 11 shows APC treatment of Fc-fusion proteins via the Fc(γ) receptor. [Figure 12] Figure 12 shows that when BALB / c mice were administered the insulin-Fc fusion protein of SEQ ID NO: 15 on days 0, 21, and 46, there was no anti-SP / RBD mouse IgG titer. [Figure 13] Figure 13 shows that anti-nucleocapsid protein mouse IgG titers were present when BALB / c mice were administered the insulin-Fc fusion protein of SEQ ID NO: 15 on days 0, 21, and 46. [Figure 14] Figure 14 shows a parallel sequence comparison of the SARS-CoV-2 nucleocapsid fragment of sequence number 10 and the truncated nucleocapsid fragment of sequence number 11. [Figure 15] Figure 15 shows a parallel sequence comparison of the SARS-CoV-2 nucleocapsid fragment of sequence number 10 and a further shortened nucleocapsid fragment of sequence number 12. [Figure 16] Figure 16 shows a parallel sequence comparison of the SARS-CoV-2 nucleocapsid fragments of sequence numbers 10 and 12 and the nucleocapsid fragment of sequence number 13. [Figure 17]Figure 17 shows a parallel sequence comparison of the SARS-CoV-2 nucleocapsid fragment of sequence number 10 and the nucleocapsid fragment of sequence number 8. [Figure 18] Figure 18 shows a parallel sequence comparison of the SARS-CoV-2 nucleocapsid fragment of sequence number 9 and the nucleocapsid fragment of sequence number 8. [Figure 19] Figure 19 shows a parallel sequence comparison of the SARS-CoV-2 nucleocapsid fragments of sequence numbers 8, 9, and 12, and the nucleocapsid fragment of sequence number 13. [Figure 20] Figure 20 shows a parallel sequence comparison of the SARS-CoV-2 nucleocapsid fragment of sequence number 12 and the nucleocapsid fragment of sequence number 13. [Figure 21] Figure 21 shows a 96-well microplate that could be used for a common serological test to assess existing SARS-CoV-2 antibody titers in serum. [Modes for carrying out the invention]

[0022] Detailed explanation COVID-19 COVID-19 is a severe acute respiratory illness caused by the SARS-CoV-2 virus. SARS-CoV-2 spread worldwide, infecting over 400 million people by the end of 2021. Experts agree that society cannot return to normal until, and until, a sufficient level of immunity is achieved in the population. It is estimated that at least 70% of the population needs to be infected to achieve natural herd immunity, which would result in millions of deaths worldwide—an ethically unacceptable outcome.

[0023] ACE2 receptor Angiotensin-converting enzyme 2 (ACE2) is a host cell receptor that mediates infection by SARS-CoV-2 (i.e., the receptor-binding domain of the SARS-CoV-2 spike protein binds to it, leading to infection). ACE2 is a type 1 transmembrane metallocarboxypeptidase. Polymerase chain reaction (PCR) analysis has shown that ACE2 is expressed in lung epithelium, vascular endothelium, and certain nerve cells, and appears to be responsible for the dominant clinical symptoms of COVID-19, including pulmonary, cardiovascular, and neurological complications, respectively. Based on the sequence similarity of the receptor-binding domains between SARS-CoV-2 and SARS-CoV, researchers have shown that SARS-CoV-2 can use ACE2 expressed on the surface of human cells to invade ACE2-expressing HeLa cells.

[0024] Convalescent serum for the treatment of viral patients Human convalescent serum is being used as a treatment option for COVID-19, particularly in immunocompromised patients.

[0025] Convalescent serum is a form of passive antibody therapy in which serum from infected and recovered individuals containing antiviral antibodies is transfused to susceptible or infected individuals, thereby providing them with some degree of immunity and preventing or reducing the severity of the disease. Unlike vaccines, this therapy works by inducing an immune response in the individual, causing them to produce their own specific antibodies against the virus. Experience with the SARS-CoV outbreak in 2002 and the H1N1 influenza outbreaks of 2009-2010 has shown that serum from patients who have been infected with and recovered from the virus (human convalescent serum) contains antibodies that can neutralize the virus and is useful as an intervention or preventive vaccine for patients with severe symptoms.

[0026] The use of human convalescent serum carries risks and limitations. Firstly, when blood substances are transferred from one person to another, there is a risk of unintentional infection with another disease, along with the risk of reaction to other serum components. Another challenge in using convalescent serum is that some patients who have recovered from viral illnesses do not have high levels of neutralizing antibodies. In the case of Middle East Respiratory Syndrome (MERS-CoV), another human coronavirus, three patients in South Korea were treated with convalescent serum, but only two recipients had neutralizing antibodies in their serum. Some people who have neutralizing antibodies after recovering from a viral illness do not have sufficiently high levels of neutralizing antibodies to be effective donors. Further research on SARS-CoV showed that of 99 convalescent serum samples from SARS patients, 87 had neutralizing antibodies, with a geometric mean antibody titer of 1:61. These, along with various other studies, suggest that few patients show a high titer response, and that neutralizing antibody titers decrease over time. Many companies are trying to overcome this challenge by producing recombinant antibodies rather than relying solely on antibodies from recovered patients, but production scale is insufficient, and the medical intervention required to administer effective doses to patients every few weeks to months, mostly by intravenous injection or infusion, is extremely burdensome. A major limitation of recombinant antibody therapy is that the SARS-CoV-2 virus may mutate, rendering existing monoclonal antibody therapies ineffective. In fact, as of November 2022, all six monoclonal antibody therapies approved by the FDA in the US between 2020 and 2022, which had been used to treat COVID-19, had their FDA approval revoked because they were deemed incapable of neutralizing new omicron variants of the SARS-CoV-2 virus. As a result, the use of convalescent serum in immunocompromised individuals infected with COVID-19 has been resumed, because such serums contain a wide range of antibodies and offer a broader range of treatment options than monoclonal antibodies.

[0027] However, the use of convalescent serum to treat COVID-19 patients has limitations, as it relies on preparations containing high-titer SARS-CoV-2 neutralizing antibodies. This requires a significant number of donors who have recovered from the disease or developed antibodies from vaccination or booster and can provide convalescent serum. It is difficult to determine who has already been infected and acquired some immunity. COVID-19 presents with a wide range of severity, and many people with mild symptoms may not even realize they have been infected.

[0028] However, even if we can identify recovered patients with high titer neutralizing antibodies, it is unlikely that one individual's plasma can treat several patients. Therefore, while current approaches to convalescent serum therapy may prevent or treat COVID-19 in a small number of patients, this solution does not address the greater needs of humanity during and after this pandemic.

[0029] Current vaccine overview and challenges Clinicians and researchers worldwide have developed a variety of solutions to mitigate the pandemic caused by the SARS-CoV-2 virus. These solutions include vaccines that can prevent or reduce the severity of COVID-19, as well as antiviral treatments that reduce the severity and symptoms of the disease when it occurs. What is expected in the foreseeable future is that innate and vaccine-induced immunity is unlikely to last long, and therefore, a cost-effective and safe booster vaccine, administered as frequently as every six months if necessary, is needed to maintain robust immunity among the population. Therefore, key design features for an effective prophylactic COVID-19 vaccine are as follows: i) potent ability to induce SARS-CoV-2 virus neutralizing IgG titer and a significant T helper type 1 (Th1) cell response after a single dose in both antibody-naive and pre-existing antibody-capable subjects; ii) an acceptable safety and tolerability profile with respect to inflammation caused particularly at the reactiongenicity (systemic effects) and injection site (local effects), manufacturability and cost-of-goods (COGs) that determine the frequency and level of administration, as well as a suitable supply chain route including sufficient shelf life and robust preparation and administration procedures for the test material.

[0030] Attenuated or inactivated whole-virus vaccines are a classic strategy. The major advantages of whole-virus vaccines are their inherent immunogenicity and ability to stimulate Toll-like receptors (TLRs), including TLR3, TLR7 / 8, and TLR9. However, live virus vaccines often require comprehensive additional testing to confirm their safety. This is particularly problematic for coronavirus vaccines, given the findings that infectivity increases after immunization with live or dead whole-virus SARS coronavirus vaccines. Johnson & Johnson developed the adenovirus vector AdVac®, manufactured using Janssen's PER.C6® cell line technology, to produce the lead vaccine JNJ-78436735. This technology was an attempt to create a viral vector that replaces the whole virus with an adenovirus vector carrying a portion of the SARS-CoV-2 viral DNA. However, the use of JNJ-78436735 encountered a serious serious adverse event (SAE), leading to the temporary suspension of clinical trials.

[0031] Two further hurdles in the early development of SARS coronavirus vaccines were 1) undesirable immune enhancement in the form of Th2-mediated eosinophil infiltration, and 2) the discovery of increased viral infectivity driven by ADE, which has been noted to occur after antigen-administered infection following immunization with whole-virus vaccines and complete SP vaccines. The risk of Th2-mediated eosinophil infiltration and lung lesions is still under investigation in SARS-CoV-2 infection, but has been observed in infants and animals immunized with respiratory syncytial virus (RSV) antigen-administered or whole-RSV vaccines.

[0032] ADE is an adverse feature of original SARS-CoV, dengue virus, and other viral vaccines, including Zika virus infection vaccines, where the vaccine-induced Ab concentration or affinity is too low to neutralize the viral infection and instead tends to form immune complexes with the virus that interact with Fcγ receptors on the surface of bone marrow cells via the Fc domain of the Ab. Such Abs do not neutralize the viral infection or induce Fcγ-mediated viral clearance (Li), but rather assist viral infection by directly increasing viral uptake via the Fcγ receptor or by promoting viral replication in cells by activating downstream pathways that antagonize innate immunity (review in Sun). In both ADE and Th2 immunoenhancement, there is evidence that feline IgG2a mAbs (presumably the Th2 isotype) may mediate both adverse conditions, while IgG1 mAbs (known to have strong effector function, i.e., the Th1 isotype) avoid such effects.

[0033] In addition to the risk of causing ADE and / or Th2 immunoenhancement, another challenge for viral vector vaccines is that production, whether based on chicken eggs or cell expression systems (Ewer), has relatively low manufacturable throughput and therefore high cost of goods (COG).

[0034] Alternatively, nucleic acid expression vector vaccine platforms for COVID-19 encode the spike protein (SP), a major coronavirus target antigen (Ag) that mediates the viral infection mechanism through binding to the host receptor ACE2. A monovalent mRNA vaccine encoding full-length SP has been developed by BioNTech / Pfizer and Moderna. Recently, bivalent COVID-19 vaccines from Pfizer-BioNTech and Moderna have been approved for use as boosters in many countries worldwide. These bivalent booster vaccines target the original strain and the Omicron BA.4 and BA.5 variants and were launched with the aim of eliciting a broader immune response and improving the strength and duration of protection against circulating variants. The concept of immunizing with RNA or DNA began in 1993 with promising results showing protective immunity against influenza in mice, but for decades, these findings did not translate to similar findings in humans. Furthermore, there is growing concern that many of these RNA and DNA expression vector vaccines, while non-replicating, may continue to produce a significant amount of target viral Ag endogenously even after the intended immune response has been induced, potentially leading to immune tolerance to the virus, thus making the risks associated with such current COVID-19 mRNA vaccines a reality. Other challenges with these nucleic acid vaccines include the need for frequent administration due to the low persistence of the response, and the undesirability of COG due to the cumbersome nature of chemical synthesis. In addition, because RNA is inherently unstable, the products must be stored and transported under refrigeration, making them extremely difficult to obtain in most countries worldwide. Monovalent vaccines show over 90% efficacy against the original SARS-CoV-2 virus in protection against symptomatic SARS-CoV-2 virus infection, and bivalent vaccines provide protection against several omicron variants. However, continued mutations in the spike protein could lead to decreased vaccine efficacy.

[0035] As a further option, recombinant subunit vaccines rely on inducing an immune response to SP to prevent docking with the host's target protein, ACE2. Such vaccines consist of all or part of SP, rather than the DNA or RNA encoding the protein, which is then mixed with an adjuvant to enhance the immune response. Because proteins are inherently more stable than RNA and DNA, storage and transport requirements are less stringent for subunit vaccines. Companies developing recombinant subunit vaccines include Novavax, which develops and manufactures immunogenic virus-like nanoparticles NVX-Cov2373 based on recombinant expression of SP formulated with the saponin-based adjuvant system Matrix-M®, and Clover Biopharmaceuticals, which is developing a subunit vaccine consisting of trimerized SARS-CoV-2 SP using its patented Trimer-Tag® technology. However, full-length SP-targeted Ag is known to have low expression yields in cell expression systems, and when used in SARS vaccines, it is known to induce anti-SP IgG titers against non-neutralizing SP epitopes, which may also mediate increased viral infectivity (i.e., ADE) and inflammation caused by pulmonary eosinophilia (i.e., Th2-mediated immune enhancement, described later).

[0036] A consortium led by the Texas Children's Hospital Center for Vaccine Development at Baylor College of Medicine is developing and testing a subunit vaccine consisting solely of the receptor-binding domain (RBD) of SARS SP. When formulated with alum, this RBD-based vaccine can induce high levels of protective immunity upon antigen stimulation by the same virus, in addition to avoiding ADE and immune enhancement (Hotez. P. J et al. A novel SARS immunogenic composition (2014) US20160376321). The initial finding that the RBDs of SARS and SARS-CoV-2 show more than 80% amino acid similarity and bind to the same ACE2 target provides an opportunity to develop either protein Ag as a subunit vaccine. In fact, proof-of-concept for such subunit vaccines has been successfully demonstrated with coronavirus SP / RBD Ag from MERS and SARS infections.

[0037] Each vaccine strategy has its own unique advantages and challenges that must be managed simultaneously in the most optimal way regarding manufacturing, safety, and efficacy.

[0038] The SARS-CoV-2 virus has numerous spike protein variants and subvariants. Furthermore, a significant number of spontaneously occurring mutations in the SARS-CoV-2 spike protein have been identified in SP / RBD, and these variants may enhance the ability of the virus to evade immunity conferred through natural infection or vaccination. Existing vaccines that target all or part of the SARS-CoV-2 spike protein may be less effective with the emergence of SARS-CoV-2 variants and mutations, thus reducing the effectiveness of such vaccines and highlighting the need for a new generation of SARS-CoV-2 vaccines that can prevent infection from SARS-CoV-2 variants.

[0039] This disclosure relates to methods for producing and using a novel nucleocapsid-based Fc fusion protein (SARS-CoV-2 N-Fc fusion protein) that enables the cost-effective mass production of recombinant subunit vaccines that are robust against variants of the SARS-CoV-2 virus spike protein region and can be transported and stored at mild temperatures. In particular, this disclosure relates to methods for producing and using a nucleocapsid-based Fc fusion protein for use in a prophylactic vaccine, therapeutic vaccine, or booster vaccine that is effective in inducing patients to produce antiviral antibodies against the SARS-CoV-2 virus, for example, by reducing the intracellular SARS-CoV-2 viral load, thereby reducing viral replication ability and improving symptom severity. Using a novel SARS-CoV-2 N-Fc fusion protein prophylactic vaccine to induce patients to produce endogenous antibodies targeting the SARS-CoV-2 virus is expected to be considerably more cost-effective than creating recombinant anti-SARS-CoV-2 therapeutic monoclonal antibodies and injecting them into patients (which become ineffective as the SARS-CoV-2 virus's spike protein and RBD region mutate).

[0040] The main structural proteins of the SARS-CoV-2 virus include membrane proteins (M proteins), spike proteins (S proteins), envelope proteins (E proteins), and nucleocapsid proteins (N proteins). The N protein is more conserved and less mutated than the spike protein, and it is highly immunogenic and abundantly expressed during infection (Marra MA, Jones SJ, Astell CR, et al. The Genome sequence of the SARS-associated coronavirus. Science. 2003;300(5624):1399-1404; Drosten C, Guenther S, Preiser W, et al. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Holmes KV, Enjuanes L. Virology. The SARS coronavirus: a postgenomic era. Science. 2003;300(5624):1377-1378; Rota PA, Oberste MS, Monroe SS, et al. Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science. 2003;300(5624):1394-1399; Zhu Y, Liu M, Zhao W, et al. Isolation of virus from a SARS patient and genome-wide analysis of genetic mutations related to pathogenesis and epidemiology from 47 SARS-CoV isolates. Virus Genes. 2005;30(1):93-102. Engl J Med. 2003;348(20):1967-1976.; Cong Y, Ulasli M, Schepers H, et al.Nucleocapsid protein recruitment to replication-transcription complexes plays a crucial role in coronavirus life cycle. J Virol. 2020;94(4):e01925-e19). Previous studies have shown the detection of high levels of anti-N protein antibodies in COVID-19 patents, suggesting that the N protein may stimulate the human immune response to the SARS-CoV-2 virus (Algaissi A, Alfaleh MA, Hala S, et al. SARS-CoV-2 S1 and N-based serological assays reveal rapid seroconversion and induction of specific antibody response in COVID-19 patents. Sci Rep. 2020;10(1):16561; Jiang HW, Li Y, Zhang HN, et al. SARS-CoV-2 proteome microarray for global profiling of COVID-19 specific IgG and IgM responses. Nat Commun. 2020;11(1):3581).

[0041] In one example, a novel SARS-CoV-2 N-Fc fusion protein vaccine is administered to patients infected with the SARS-CoV-2 virus and suffering from COVID-19 in order to limit the scope of infection and improve the disease. In this example, the novel SARS-CoV-2 N-Fc fusion protein is expected to stimulate the target to produce both humoral and cell-mediated immunity, inhibit viral RNA replication, and generate high levels of IgG and IgM antibodies that bind to the native viral N protein, thereby blocking viral transmission in the body. In several cases, novel SARS-CoV-2 N-Fc fusion proteins are expected to produce high levels of interferon-gamma (IFN-γ), which plays a crucial role in inducing immune responses by promoting macrophage activation, mediating antiviral and antibacterial immunity, enhancing antigen presentation, directing the activation of the innate immune system, modulating lymphocyte-endothelial interactions, adjusting the Th1 / Th2 balance, and regulating cell proliferation and apoptosis.

[0042] In one example, a pharmaceutical composition of a novel SARS-CoV-2 N-Fc fusion protein is administered as a prophylactic COVID-19 vaccine to an individual not infected with the SARS-CoV-2 virus, resulting in that individual developing their own anti-SARS-CoV-2 antibody pool and immunity.

[0043] Equivalents As used herein, the articles “a” and “an” refer to one or more grammatical objects of the article, for example, at least one. In this specification, the use of “a” or “an” in combination with the term “contains” may mean “one,” but also coincides with the meanings of “one or more,” “at least one,” and “one or more.” As used herein, the phrase “and / or” when used in a list of two or more items means that any one of the items in the list may be taken by itself, or any combination of two or more items in the list may be taken. For example, if a composition is described as containing or not containing components A, B, and / or C, the composition may contain or not contain A alone; B alone; C alone; a combination of A and B; a combination of A and C; a combination of B and C; or a combination of A, B, and C.

[0044] As used herein, “about” and “approximately” generally mean the degree of error in a measured quantity that is permissible given the nature or precision of the measurement.

[0045] As used herein, "therapeutically effective amount" or "effective amount" means an amount of a molecule, compound, complex, or substance that is effective in treating a disorder (e.g., a disorder as described herein), when administered to a subject, single or multiple times, in the treatment of the subject, or in the healing, relief, reduction, or improvement of a subject having a disorder (e.g., a disorder as described herein), in a quantity that is more effective than what would be expected in the absence of such treatment.

[0046] As used herein, the term “analog” means a compound or complex (e.g., a compound or complex described herein, e.g., a nucleocapsid) that is similar to another compound or complex but has a different chemical structure in at least one aspect.

[0047] As used herein, the term “antigen” refers to any substance that causes a patient’s immune system to produce antibodies against it. Antigens can be chemicals, bacteria, viruses, or environmental substances such as pollen, or they may be formed within the body. An example of an antigen is the SARS-CoV-2 virus.

[0048] As used herein, the terms “antibody” or “antibody molecule” refer to an immunoglobulin molecule (Ig) or an immunoactive portion of an immunoglobulin (Ig) molecule, i.e., a molecule containing an antigen-binding site that specifically binds to an antigen, for example, an antigen-binding site that engages in an immune response with the antigen. As used herein, the term “antibody domain” refers to the variable or constant region of an immunoglobulin. In the art, it has been described that human antibodies comprise several classes, for example, IgA, IgM, or IgG in the case of mammals (e.g., humans and dogs). Classes of mammalian IgG immunoglobulins can be further classified into different isotypes, such as IgGA, IgGB, IgGC, and IgGD in dogs, and IgG1, IgG2, IgG3, and IgG4 in humans. Those skilled in the art will recognize that the immunoglobulin isotypes of a given class of immunoglobulins comprise different amino acid sequences, structures, and functional properties (e.g., different binding affinities to Fc(γ) receptors or ACE2 receptors). "Specifically binding" or "immunely responding" means that an antibody reacts with one or more antigenic determinants of a desired antigen, and has lower affinity for other polypeptides, for example, does not react with other polypeptides.

[0049] As used herein, the term “dimer” refers to a protein or fusion protein comprising two covalently linked polypeptides. In several embodiments, two identical polypeptides are covalently linked (e.g., via a disulfide bond) to form a “homodimer” (illustrated schematically in Figure 1, which illustrates an insulin-Fc fusion protein for reference, and in Figure 2, which illustrates a novel SARS-CoV-2 N-Fc fusion protein). Referring to Figure 1, more specifically, the insulin polypeptide (comprising an insulin B-chain analog linked to an insulin A-chain analog via a C-chain peptide) may have one or more amino acid mutations from native insulin. The insulin peptide is linked to the Fc fragment via a linker. The disulfide bond (the actual total number of disulfide bonds may be more or less than the number shown in Figure 1) forms a homodimer from two identical Fc fusion proteins. Referring to Figure 2, more specifically, the novel SARS-CoV-2 N-Fc fusion protein may comprise a nucleocapsid protein fragment that may contain a portion of the full-length SARS-CoV-2 nucleocapsid protein. The nucleocapsid protein fragment may have one or more amino acid mutations from the native SARS-CoV-2 nucleocapsid protein. The nucleocapsid protein fragment is linked to the Fc fragment using a peptide linker. Disulfide bonds form homodimers from two identical novel SARS-CoV-2 N-Fc fusion proteins (the actual total number of disulfide bonds may be more or less than the number shown in Figure 2). Novel SARS-CoV-2 N-Fc fusion protein homodimers may be encoded by a single nucleic acid molecule, in which case the homodimer is produced intracellularly by recombination, first forming a novel SARS-CoV-2 N-Fc fusion protein monomer, and then assembling two identical novel SARS-CoV-2 N-Fc fusion protein monomers into a homodimer during further processing within the cell.

[0050] As used herein, the terms “multimer,” “multimer,” or “multimerized state” refer to non-covalent related forms of an Fc fusion protein dimer that may be in equilibrium with the Fc fusion protein dimer or may function as a permanent aggregate of the Fc fusion protein dimer (e.g., a dimer of an Fc fusion protein homodimer, a trimer of an Fc fusion protein homodimer, a tetramer of an Fc fusion protein homodimer, or a higher-order aggregate containing five or more Fc fusion protein homodimers). It is expected that the multimerized form of an Fc fusion protein may have different physical, stable, or pharmacological activities than the fusion protein homodimer.

[0051] As used herein, SARS-CoV-2 nucleocapsid-Fc fusion protein and novel SARS-CoV-2 N-Fc fusion protein (these terms can be used interchangeably) refer to a human immunoglobulin (immunoglobin) Fc domain linked to a SARS-CoV-2 nucleocapsid fragment or analogue, which is useful for generating antibodies that specifically bind to the SARS-CoV-2 N protein. For ease of reference, the term nucleocapsid, unless otherwise indicated by context, encompasses protein residues consisting of a fragment of the SARS-CoV-2 nucleocapsid protein that retains the activity of the nucleocapsid protein. As used herein, the general terms “fusion protein” and “Fc fusion protein” refer to a protein comprising multiple portions from different sources, e.g., different proteins, polypeptides, cells, etc., covalently linked via peptide bonds. Fc fusion proteins are covalently linked by (i) ligating the genes encoding each portion into a single nucleic acid molecule, and (ii) expressing the protein encoded by the nucleic acid molecule in a host cell (e.g., HEK cells or CHO cells). Complete recombination synthesis is preferred over methods in which the therapeutic protein and Fc fragments are synthesized separately and then chemically linked. Chemical linking and subsequent purification steps increase the complexity of the manufacturing process, reduce the yield of the product, and increase costs.

[0052] As used herein, the terms “vitality,” “activity,” “biological activity,” “potency,” “vitality potency,” or “biological potency” refer to the extent to which an Fc fusion protein binds to or activates a cell receptor and / or causes the production or reduction of native or foreign substances. As used herein, “in vitro activity” or “receptor activity” refers to the affinity of an Fc fusion protein to a cell receptor, generally measured by the concentration of the Fc fusion protein that causes it to reach half of its maximum binding capacity (i.e., the EC50 value). For example, the “vitality” of a novel SARS-CoV-2 N-Fc fusion protein refers to the extent to which the novel SARS-CoV-2 N-Fc fusion protein induces the production of anti-SARS-CoV-2 antibodies in a cell assay or target.

[0053] As used herein, the terms “biosynthesis,” “recombinant synthesis,” or “recombinantly produced” refer to the process by which an Fc fusion protein is expressed in a host cell by transfecting the cell with a nucleic acid molecule (e.g., a vector) encoding the Fc fusion protein (e.g., the entire Fc fusion protein is encoded by a single nucleic acid molecule). Exemplary host cells include mammalian cells, such as HEK293 cells or CHO cells. These cells can be cultured using methods standard in the art, and the expressed Fc fusion protein can be recovered from the cell culture and purified using methods standard in the art.

[0054] As used herein, the term “cell surface receptor” generally refers to molecules found on the outer surface of a cell membrane, such as proteins that interact with soluble molecules, such as molecules circulating in the blood supply. In some embodiments, cell surface receptors may include host cell receptors (e.g., ACE2 receptor) or Fc receptors (e.g., Fc(γ) receptors, e.g., Fc(γ) receptor I, or Fc neonatal receptors, e.g., FcRn) that bind to an Fc fragment or Fc region of an antibody. As used herein, “in vitro activity,” “Fc(γ) receptor activity,” “Fc(γ) receptor binding,” “FcRn receptor activity,” or “FcRn binding” refers to the affinity with which an Fc fusion protein binds to an Fc receptor (e.g., an Fc(γ) receptor or FcRn receptor), and generally refers to the concentration of the Fc fusion protein that causes the Fc fusion protein to reach half of its maximum binding (i.e., the EC50 value), as measured by an assay (e.g., an enzyme-linked immunosorbent assay (ELISA)) using an OD450nm value measured by a microplate reader.

[0055] As used herein, the terms “immunogenous” or “immunogenicity” refer to the ability of a given molecule (e.g., the Fc fusion protein of the present invention) to induce the immune system of a target subject to produce antibodies that, after administration of the molecule, can bind to all or specific portions of that molecule (i.e., anti-drug antibodies or ADAs). As used herein, the terms “neutralizing,” “neutralizing antibody,” or “neutralizing anti-drug antibody” refer to the ability of an antibody to interfere with all or part of the biological activity of the Fc fusion protein in a target subject. For example, in the case of a novel SARS-CoV-2 N-Fc fusion protein molecule (or its pharmaceutical composition) administered to a human, the hIgG-Fc portion of the molecule is endemic to humans and therefore unlikely to induce anti-hIgG-Fc antibodies, so immunogenicity refers to antibodies that bind to the SARS-CoV-2 nucleocapsid portion of the molecule. Similarly, antibodies generated by the administration of novel SARS-CoV-2 N-Fc fusion protein molecules (or their pharmaceutical compositions) are neutralized when these anti-SARS-CoV-2 antibodies inhibit the binding of SARS-CoV-2 N proteins between host cells, which is directly related to the viability of SARS-CoV-2 RBD in the subject.

[0056] As used herein, the term “monomer” refers to a protein or fusion protein comprising a single polypeptide. In some embodiments, “monomer” is a protein or fusion protein, for example, a single polypeptide comprising a nucleocapsid fragment polypeptide and an Fc fragment polypeptide, where the nucleocapsid fragment and the Fc fragment polypeptide are linked by a peptide bond to form a single polypeptide. In some embodiments, the monomer is encoded by a single nucleic acid molecule.

[0057] As used herein, “N-terminus” refers to the starting portion of a protein or polypeptide that begins with an amino acid containing the α-amino group of the amino acid (for example, a free amino covalently bonded to a carbon atom adjacent to a second carbon atom, where the second carbon atom is part of the carbonyl group of that amino acid). As used herein, as shown in Figures 1 and 2, “C-terminus” refers to the ending portion of a protein or polypeptide that ends with an amino acid containing a carboxylic acid group, where the carbon atom of the carboxylic acid is adjacent to the α-amino group of the amino acid.

[0058] As used herein, the term “carrier” refers to diluents, excipients, vehicles, etc., on which the Fc fusion protein is dispersed, emulsified, or encapsulated for administration. A suitable carrier is pharmaceutically acceptable. As used herein, “pharmaceutically acceptable” means that it can be administered to a subject without excessive toxicity, irritation, or allergic reaction, does not cause unacceptable biological effects, and does not exhibit adverse interactions with other components of the composition in which it is contained, and is not biologically or otherwise unacceptable. Pharmaceutically acceptable carriers are, as is well known to those skilled in the art, selected to minimize the degradation of the compound or other agents and to minimize adverse side effects in the subject. Pharmaceutically acceptable components include those acceptable for veterinary and human pharmaceutical uses, and depend on the route of administration. Any carrier compatible with the excipient and Fc fusion protein is usable.

[0059] As used herein, “pharmacokinetics” or “PD” generally refers to the biological effects of an Fc fusion protein in a subject. For example, as used herein, the PD of a novel SARS-CoV-2 N-Fc fusion protein refers to the time-course measurement of anti-SARS-CoV-2 antibody titers in a subject after administration of the novel SARS-CoV-2 N-Fc fusion protein.

[0060] As used herein, “pharmacokinetics” or “PK” generally refers to the characteristic interactions between an Fc fusion protein and the target body in terms of absorption, distribution, metabolism, and excretion. For example, as used herein, PK refers to the concentration of a novel SARS-CoV-2 N-Fc fusion protein in the target's blood or serum at a given time after administration of the novel SARS-CoV-2 N-Fc fusion protein. As used herein, “half-life” refers to the time it takes for the concentration of an Fc fusion protein in the target's blood or serum to reach half of its original value, as calculated from a first-order exponential decay model for drug elimination. Fc fusion proteins with larger “half-life” values ​​have a longer duration of action in the target body.

[0061] The terms “sequence identity,” “sequence homology,” “homology,” or “identical” in relation to amino acid or nucleotide sequences, as used herein, mean that when a specified contiguous segment of a mutant nucleotide or amino acid sequence is aligned and compared with the nucleotide or amino acid sequence of a reference sequence, the same nucleotide or amino acid residue is found in both the mutant and the reference sequence. Methods for determining sequence alignment and sequence identity are known in the art and include the use of Clustal Omega to organize, align, and compare sequences in terms of similarity. This software highlights each sequence position, compares the entire sequence at that position, and assigns one of the following scores: * An asterisk (") indicates a sequence location with a single, fully conserved residue; a colon (:) indicates strong intergroup conservation of similarity with a score greater than 0.5 in the Gonnet PAM 250 matrix; a period (.) indicates weak intergroup conservation of similarity with a score less than or equal to 0.5 in the Gonnet PAM 250 matrix; a dash (-) indicates a sequence gap, meaning there is no local homology within a particular set of comparisons within a specific range of sequences; and a blank space (" ") indicates little to no sequence homology with respect to a particular location of the compared sequences.

[0062] With regard to the optimal alignment of two nucleotide sequences, the contiguous segment of the mutant nucleotide sequence may have nucleotide additions or deletions relative to the reference nucleotide sequence. Similarly, for the purpose of the optimal alignment of two amino acid sequences, the contiguous segment of the mutant amino acid sequence may have amino acid residue additions or deletions relative to the reference amino acid sequence. In some embodiments, the contiguous segment used for comparison with the reference nucleotide sequence or reference amino acid sequence comprises at least 6, 10, 15, or 20 contiguous nucleotides or amino acid residues, and may contain 30, 40, 50, 100, or more nucleotides or amino acid residues. The increased sequence identity correction due to the inclusion of gaps in the mutant nucleotide sequence or amino acid sequence can be achieved by assigning a gap penalty. Methods for sequence alignment are known in the art.

[0063] In several embodiments, the determination of the percentage of identity or "homology" between two sequences is achieved using mathematical algorithms. For example, the percentage of identity of amino acid sequences is determined using a Smith-Waterman homology search algorithm that employs a gap-open penalty 12 and a gap-extension penalty 2, and an affine 6-gap search of the BLOSUM matrix 62. In several embodiments, the percentage of identity of nucleotide sequences is determined using a Smith-Waterman homology search algorithm that employs a gap-open penalty 25 and a gap-extension penalty 5. Such determination of sequence identity is achieved, for example, by TimeLogic's DeCy

[0064] As used herein, the term “homologousity” is used to compare two or more proteins by determining the location of common structural features and common spatial distributions, such as β-chains, helices, and folding. Thus, homologous protein structures are defined by spatial analysis. Measuring structural homology involves calculating the geometric-topological features of space. One approach used to generate and analyze three-dimensional (3D) protein structures is homology modeling (also called comparative modeling or knowledge-based modeling), which works by finding similar sequences based on the fact that 3D similarity reflects 2D similarity. Homologous structures do not necessarily require sequence similarity.

[0065] As used herein, the terms “subject” and “patient” are intended to include mice, non-human primates (NHPs), rabbits, dogs, and humans. Exemplary dog ​​subjects include dogs having a disease or disorder, e.g., diabetes or another disease or disorder described herein, or normal subjects. Exemplary human subjects include individuals having a disease, e.g., COVID-19 or COVID-19 including variants of SARS-CoV-2 infection, or another virus, individuals who have had a disease or disorder described herein, or normal subjects.

[0066] As used herein, the terms “titer” or “yield” refer to the amount of fusion protein product (e.g., Fc fusion protein as described herein) obtained from biosynthesis (e.g., in mammalian cells, e.g., HEK293 cells or CHO cells) per unit volume of cell culture. The amount of product may be determined at any step of the manufacturing method (e.g., before or after purification), but is expressed per unit volume of the original cell culture. As used herein, “product yield” or “total protein yield” refers to the total amount of Fc fusion protein expressed by cells and purified by at least one affinity chromatography step (e.g., protein A or protein G), and includes monomers of Fc fusion protein, homodimers of Fc fusion protein, and higher-order molecular aggregates of homodimers of Fc fusion protein. As used herein, “homodimer percent” or “% homodimer” refers to the percentage of the fusion protein product (e.g., Fc fusion protein as described herein) that is the desired homodimer. As used herein, the term “homodimer potency” refers to the product of % homodimer and the total protein yield after the protein A purification step, reported per volume of cell culture.

[0067] As used herein, the term “treatment” in a subject with a disease or disorder refers to an intervention intended to prevent the development of an infection or to alter its pathogenesis. Thus, “treatment” includes both therapeutic measures and preventive or protective measures. A therapeutic agent may directly alleviate the pathogenesis of an infection or make the infection more susceptible to treatment by other therapeutic agents or the host’s immune system. Post-treatment improvement may manifest as a reduction or disappearance of such symptoms. Therefore, this composition is useful in treating infections by preventing the development of observable clinical symptoms of the infection, and / or reducing the incidence or severity of the clinical symptoms and / or effects of the infection, and / or shortening the duration of the infection / symptoms / effects. Treating a subject with a disease or disorder may refer to administering a regimen, such as a fusion protein, such as the Fc fusion protein described herein, or a pharmaceutical composition of a fusion protein, such as the Fc fusion protein described herein, to a subject such that at least one symptom of the disease or disorder is cured, recovered, alleviated, reduced, altered, corrected, improved, or improved. Treatment may include administering an effective dose to alleviate, reduce, modify, correct, improve, enhance, or influence the disease or disorder, or its symptoms. Treatment may include administering an effective dose to produce antibodies against the disease or disorder in a normal subject or a subject who has previously suffered from the disease or disorder. Treatment may also suppress the worsening or exacerbation of the symptoms of the disease or disorder.

[0068] As used herein, “preventive vaccine” refers to a treatment that introduces an antigen to a patient with the aim of increasing or improving the subject’s immune response to the associated disease or virus by causing the patient’s immune system to produce antibodies against the antigen. In other words, a vaccinated subject will have greater resistance to the disease or illness caused by the associated virus compared to an unvaccinated subject. This resistance may be manifested by a reduction in the severity or duration of disease symptoms, a decrease or elimination of viral shedding, and, in some cases, prevention of observable symptoms of infection in the vaccinated subject. In several embodiments, a patient treated with a preventive vaccine does not have antibodies against the antigen prior to treatment with the preventive vaccine (in other words, the patient is “antibody naive”).

[0069] As used herein, “therapeutic vaccine” refers to a treatment aimed at introducing an antigen to a patient who already has a related disease or virus, so that the patient’s immune system can produce antibodies against that antigen, and the patient’s body can more effectively fight the disease or virus that the patient already has.

[0070] As used herein, “booster vaccine” refers to an additional dose of the vaccine administered after a patient has previously received an initial dose of the vaccine, or after a patient has acquired antibodies by contracting and recovering from the associated disease or virus. In some cases, additional doses of the vaccine are regularly required to “boost” the patient’s immunity to the antigen that causes the disease or virus by increasing the patient’s antigen antibody titer.

[0071] As used herein, when referring to a portion of the SARS-CoV-2 nucleocapsid, for example, an amino acid of the SARS-CoV-2 nucleocapsid fragment, the position of the cited amino acid refers to the position of the amino acid in the SARS-CoV-2 nucleocapsid of SEQ ID NO: 10. For example, when referring to a mutation at the 41st amino acid of the SARS-CoV-2 nucleocapsid fragment, it refers to the 41st amino acid of SEQ ID NO: 10, even if the SARS-CoV-2 nucleocapsid fragment consists of only a portion of SEQ ID NO: 10, for example, a portion of SEQ ID NO: 10 that begins at the 41st amino acid.

[0072] As used herein, “nucleocapsid fragment” refers to a portion of a novel SARS-CoV-2 N-Fc fusion protein comprising a portion of the SARS-CoV-2 virus nucleocapsid protein shown in SEQ ID NO: 10. In several examples, the nucleocapsid fragment is ligated to a human Fc fragment or an analogue, as shown in Figure 2.

[0073] Evidence for Fc fusion protein vaccines As mentioned above, the recombinant protein-based subunit vaccine approach has advantages over vaccine formats based on inactivated or attenuated viruses and nucleic acid vectors, including the ability to selectively use the most dominant epitope to produce a potent neutralizing AB titer, as well as safety and the possibility of multiple booster doses. Furthermore, such protein-based vaccines can be manufactured in large quantities more cost-effectively, are stable at mild temperatures, and are easy to transport and store. However, given the difficulty of recombinant SARS-CoV-2 spike protein or nucleocapsid subunit vaccines in inducing a potent protective immune response in immunologically naive human populations, antigens must be formulated with modifications and / or immunoenhancing functions to overcome the activation thresholds of naive T and B cells.

[0074] Experimental experience with insulin-Fc fusion proteins in dogs An example of a fusion protein formed by linking a therapeutic protein to an immunoglobulin (immunoglobin) Fc domain is the insulin-Fc fusion protein. This construct has been used to provide ultra-long-acting basal insulin therapy to diabetic patients. The combination of an insulin analog as a therapeutic protein and an Fc domain via a peptide linker has been shown to achieve significantly longer activity of several days in vivo. An example of an ultra-long-acting insulin-Fc fusion protein used for the treatment of diabetes in cats and dogs is described in WO2020006529A1. In the example in WO2020006529A1, the exemplary insulin analog is: FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYC (Sequence ID 4) Therefore, an exemplary linker used to ligate a therapeutic protein (i.e., an insulin analog) to the domain is, GGGGGQGGGGQGGGGQGGGGG (Sequence 3) That is the case.

[0075] An insulin-Fc fusion protein molecule suitable for ultra-long-acting therapy for diabetes in a given species (e.g., dog, cat, or human) should be able to be produced in mammalian cells, e.g., human embryonic kidney (HEK, e.g., HEK293) cells, at an acceptable titer of the desired homodimeric product (e.g., greater than 50 mg / L from transiently transfected HEK cells, greater than 75 mg / L from transiently transfected HEK cells, greater than 100 mg / L from transiently transfected HEK cells, etc.). Empirically, in Chinese hamster ovary (CHO) cells, homodimeric titers of less than 50 mg / L are unlikely to yield a commercially viable homodimeric titer that meets the stringent low-cost manufacturing requirements of veterinary products.

[0076] The canine insulin-Fc fusion proteins described herein in WO2020006529A1 (incorporated herein by reference) and herein were prepared in HEK cells according to Example 10 or in CHO cells according to Example 11. The insulin-Fc fusion proteins were purified according to Example 12. Using conventional purification methods, only compounds containing canine IgGA and canine IgGB immunoglobulin Fc fragments showed an evaluable protein yield. The structure of the insulin-Fc fusion proteins was confirmed by non-reducible and reduced CE-SDS according to Example 13, and the sequence was confirmed by LC-MS after removal of glycans according to Example 14. Purity (evaluated by the homodimer yield percentage of the fusion protein) was measured according to Example 15. The canine IgGA-type insulin-Fc fusion protein exhibited high aggregation and low biological activity, while the canine IgGB-type insulin-Fc fusion protein showed low aggregation (i.e., high homodimer %), a desired high-titer homodimer (i.e., homodimer titer greater than 50 mg / L), and a measurable level of long-lasting glucose-lowering biological activity in dogs. Therefore, the canine IgGB (SEQ ID NO: 2) immunoglobin Fc fragment is a preferred Fc fragment for insulin-Fc fusion proteins used in dogs. DCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (Sequence ID 2)

[0077] An exemplary canine ultra-long-acting insulin-Fc fusion protein comprising the insulin analog of SEQ ID NO: 4 via the peptide linker of SEQ ID NO: 3 and the canine native IgGB fragment of SEQ ID NO: 2 is as follows: FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (Sequence ID 5)

[0078] The binding of the insulin-Fc fusion protein of SEQ ID NO: 5 to Fc(γ) receptor I (RI) was evaluated according to Example 16. Since canine receptor I is not commercially available, human Fc(γ) receptor I (i.e., rhFc(γ) receptor I) was used as an alternative mammalian receptor. The OD values, which are proportional to the binding of rhFc(γ) receptor I to SEQ ID NO: 5, were plotted against the logarithmic concentration of rhFc(γ) receptor I added to each receptor, and binding curves were created using GraphPad Prism software. The results shown in Figure 3 show that the OD450 value increases as the dose of the insulin-Fc fusion protein of SEQ ID NO: 5 increases for all Fc(γ) receptors.

[0079] The in vivo pharmacodynamics (PD) after regular administration of the insulin-Fc fusion protein of SEQ ID NO: 5, produced in HEK cells according to Example 10, were evaluated according to Example 18 or Example 19. The test population consisted of six beagle dogs weighing approximately 10 kg each, with chemically induced diabetes mellitus using alloxan-streptozotocin. Anti-insulin antibody (AIA) titers were measured weekly for eight weeks in the six beagle dogs with chemically induced diabetes mellitus that were the subjects of the laboratory study of SEQ ID NO: 5 according to Example 18. Figure 4 shows the AIA titers of the six beagle dogs with chemically induced diabetes mellitus over eight weeks of continuous administration of the insulin-Fc fusion protein of SEQ ID NO: 5. Figure 5 shows the percentage change in AIA titers from day 0 in the study of the six beagle dogs with chemically induced diabetes mellitus over eight consecutive weekly administrations of the insulin-Fc fusion protein of SEQ ID NO: 5. The data shows that the AIA titer in beagle dogs did not substantially increase over eight doses of the insulin-Fc fusion protein of SEQ ID NO: 5.

[0080] Based on the results of these positive laboratory tests using chemically induced diabetic beagles according to Example 18 or Example 19, field trials were initiated using spontaneously diabetic dogs owned by actual clients, varying in age, pedigree, and severity of diabetic disease. All client dogs in the field trials had received insulin therapy with known veterinary or human insulin preparations up to the start of the trials and were administered SEQ ID NO: 5 according to Protocol 1 described in Example 18 or Protocol 2 described in Example 19.

[0081] Here, as in Example 19, AIA titers were measured weekly throughout the course of treatment. Surprisingly, in contrast to the results obtained in chemically induced diabetic beagles, several client dogs (8 / 20) in this “wild” patient population showed a significant increase in anti-insulin antibodies. A normalized AIA titer of 0.15 was considered the minimum value at which a client dog was considered immunogenic to SEQ ID NO: 5. For client dogs whose AIA titer was not zero at the start of treatment, if the AIA more than doubled after treatment with the insulin-Fc fusion protein of SEQ ID NO: 5, the insulin-Fc fusion protein of SEQ ID NO: 5 was considered immunogenic in that particular client dog. Figure 6 is a plot of normalized AIA titers for each client dog that was considered immunogenic to the insulin-Fc fusion protein of SEQ ID NO: 5 when measured weekly during the period of their treatment (according to Protocol 1 described in Example 18 or Protocol 2 described in Example 19), with treatment shown for each dog. In each of the dogs shown in Figure 6, AIA neutralized the therapeutic effect of the insulin-Fc fusion protein of SEQ ID NO: 5, rendering the client's diabetic dogs unable to control their blood glucose levels. The observed immunogenicity was even more unexpected, as the insulin analog portion of the insulin-Fc fusion protein is a nearly native peptide for dogs. Furthermore, the IgGB Fc fragment portion of the insulin-Fc fusion protein is a native Fc fragment for dogs. These results indicate that the specific activity of the canine IgGB Fc fragment was able to induce a significant and sustained increase in antibody titers specific to the therapeutic protein region (i.e., insulin) of the fusion protein. Aside from showing a significant increase in neutralizing AIA antibody titers, the dogs remained healthy throughout repeated administration, and there were no signs of anaphylaxis or cytokine storm associated with the treatment.

[0082] In some cases, when client dogs began to show high levels of AIA, administration of the insulin-Fc fusion protein SEQ ID NO: 5 was discontinued, at which point the AIA titer began to decrease (see example of dog 2 in Figure 4). Figure 7 plots the normalized AIA titer for example dog 2 with weekly administration for 12 weeks. It can be seen that the AIA titer began to increase measurably after the 4th dose (day 21), and then began to surge after the 6th dose (day 35). Administration was discontinued after the 8th dose (day 49), and no drug was administered on day 56 or 63. Figure 7 shows that the increase in AIA titer immediately slowed down, and then the AIA titer began to decrease after day 56. The administration regimen was restarted on day 70, with administration on day 70 and again on day 77. The increase in AIA titers after resuming administration on day 70 was consistent with, or even exceeded, the maximum increase in AIA antibody titers during the 7 weeks leading up to discontinuation of administration, indicating that anti-drug antibody titers recovered unexpectedly in a considerably shorter time than those accumulated during the initial series of administrations. This robust recovery response may serve as an indicator of a responsive memory immune cell population.

[0083] Another finding from field trials of diabetic dogs owned by the client was that, as shown in Figure 8, there was a clear difference between dogs treated with the insulin-Fc fusion protein of SEQ ID NO: 5 produced from CHO cells according to Example 11 and dogs treated with the insulin-Fc fusion protein of SEQ ID NO: 5 produced from HEK cells according to Example 10. The insulin-Fc fusion protein of SEQ ID NO: 5 produced from CHO cells showed a significantly higher prevalence of anti-drug antibodies.

[0084] Each IgG fragment contains a conserved asparagine (N)-glycosylation site in the CH2 domain of each heavy chain in the Fc region. Here, the notation used to refer to the conserved N-glycosylation site is "cNg" (shown in Figures 1 and 2). In therapeutic monoclonal antibodies, glycosylation is located at the conserved amino acid N297 in the CH2 region (shown in Figures 1 and 2). In the case of insulin-Fc fusion proteins, the absolute position of the cNg site from the N-terminus of the B chain of the insulin-Fc fusion protein varies depending on the length of the insulin polypeptide, the length of the linker, and the omitted amino acids in the Fc fragment preceding the cNg site. Here, the notation used to refer to the absolute position of the cNg site in a given insulin-Fc fusion protein sequence (measured counting from the N-terminus of the B chain of the insulin-Fc fusion protein) is "NB (number)". For example, if the cNg site is located at the 151st amino acid position counting from the N-terminus of the B chain, the absolute position of this site is called cNg-NB151. As a further example, if the cNg site is located at the 151st amino acid position from the N-terminus of the B chain, and the asparagine at this site is mutated to serine, this mutation is denoted as "cNg-NB151-S".

[0085] One possible difference between the fusion protein recombinantly produced in HEK cells according to Example 10 and the fusion protein recombinantly produced in CHO cells according to Example 11 is the composition of the oligosaccharide attached to the cNg site. Given that the canine IgGB isotype interacts with the Fc(γ) receptor, there may be a risk of undesirable immunogenicity after repeated injections. One way to reduce Fc(γ) interaction involves deglycosylating or avoiding glycosylation of the Fc fragment during synthesis in the host cell. For the treatment of chronic diseases such as diabetes, antibody production is clearly undesirable because antibodies neutralize the therapeutic value of the drug. This has led to attempts to create non-glycosylated canine insulin-Fc fusion proteins. One way to remove the bound glycans from the synthesized insulin-Fc fusion protein is to mutate the cNg site to completely prevent glycan binding during production in the host cell. Here, the notation used to represent the cNg mutation is cNg-(substituted amino acid). For example, if the asparagine in the cNg site is mutated to serine, this mutation is denoted as "cNg-S".

[0086] The IgGB Fc fragment of SEQ ID NO: 2, the cNg-S at position 73 (the residue in bold below), the linker of SEQ ID NO: 3, and the following insulin analogs: FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYC (Sequence ID 6) We designed a canine insulin-Fc fusion protein that contains both of these.

[0087] The resulting insulin-Fc fusion protein is shown below. FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (Sequence ID 7)

[0088] The insulin-Fc fusion protein of SEQ ID NO: 7 was prepared in HEK cells according to Example 10, or in CHO cells according to Example 11. The insulin-Fc fusion protein was purified according to Example 12. The structure of the insulin-Fc fusion protein was confirmed by non-reducible and reduced CE-SDS according to Example 13, and the sequence was confirmed by LC-MS after removal of glycans according to Example 14. Purity (assessed by the homodimer yield percentage of the fusion protein) was measured according to Example 15.

[0089] This fusion protein exhibited similar desirable in vitro and in vivo properties to SEQ ID NO: 5. As shown in Figure 3, the only difference was that the binding affinity of Fc(γ)RI to the insulin-Fc fusion protein of SEQ ID NO: 7 was significantly lower than that of the insulin-Fc fusion protein of SEQ ID NO: 5. Field trials were initiated in five diabetic client dogs of varying ages, breeds, and the severity of diabetic disease. All five client dogs in the field trials had received insulin therapy with known veterinary or human insulin preparations up to the start of the trial and were administered the insulin-Fc fusion protein of SEQ ID NO: 7 once a week.

[0090] Here, as in Example 19, AIA titers were measured weekly or as frequently as possible throughout the course of treatment. Compared to dogs that received the insulin-Fc fusion protein of SEQ ID NO: 5, none of the client dogs in this “wild” patient population showed insulin anti-drug antibodies when administered the non-glycosylated insulin-Fc fusion protein of SEQ ID NO: 7. A normalized AIA titer of 0.15 was considered the minimum value required for a client dog to be considered immunogenic to SEQ ID NO: 7. For client dogs whose AIA titer was not zero at the start of treatment, if the AIA more than doubled after treatment with the insulin-Fc fusion protein of SEQ ID NO: 7, the client dog was considered immunogenic. Figure 9 is a plot of the normalized AIA titers for each client dog measured over the treatment period, showing that none of the five client dogs in the “wild” patient population showed insulin anti-drug antibodies when administered the non-glucosylated insulin-Fc fusion protein of SEQ ID NO: 7. As shown in Figure 10, this is in contrast to dogs that received the insulin-Fc fusion protein of SEQ ID NO: 5. None of the dogs that received the non-glycosylated insulin-Fc fusion protein of SEQ ID NO: 7 showed insulin anti-drug antibodies, whereas a total of 12 dogs showed insulin anti-drug antibodies.

[0091] In summary, these results suggest that, unexpectedly, outside of experimental animal populations, certain Fc fusion proteins may induce high-titer antibodies against therapeutic peptides or protein components, and that this response can be rapidly and robustly re-induced upon subsequent presentation of the therapeutic peptide or protein component. Furthermore, these preliminary data indicate that the induction of anti-therapeutic protein or peptide antibodies is more likely to occur in individuals that have already developed an immune response to that particular therapeutic protein or peptide. These results suggest that Fc fusion proteins may induce immune tolerance to the fused therapeutic peptide or protein, in contrast to many published findings that haptens such as DNPs, nucleosides, or penicilloyl groups, when chemically bound to an IgG carrier, were highly tolerogenic hapten-carrier conjugates.

[0092] Therapeutic proteins like insulin, used to treat chronic diseases (i.e., diabetes), can be rendered ineffective by anti-insulin antibodies. Nevertheless, the aforementioned results have led to unique insights into how to effectively design Fc fusion proteins, for example, to induce and neutralize antibodies against viral antigens. Based on field trials, the desired Fc fusion protein must, at a minimum, be native to the target (e.g., human Fc or hFc for human subjects, or canine Fc or dFc for canine subjects), properly glycosylated at the Fc-cNg site, and capable of binding to the Fc(γ)I receptor. These findings offer an opportunity to develop novel therapeutic Fc fusion proteins, for example, against the novel coronavirus SARS-CoV-2.

[0093] Novel SARS-CoV-2 Fc fusion protein The COVID-19 pandemic poses a serious threat to public health. Safe and effective solutions are urgently needed to mitigate or prevent infection by its causative agent, the SARS-CoV-2 virus. A surface glycoprotein containing the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (SP) has been identified, and it has been found that SARS-CoV-2 SP / RBD strongly binds to the human and bat angiotensin-converting enzyme 2 (ACE2) receptor. SARS-CoV-2 SP / RBD is an exogenous antigen, and fusion proteins comprising this antigen and a glycosylated human immunoglobin Fc fragment (referred herein to as SARS-CoV-2-RBD-hIgG-Fc fusion proteins or SP / RBD-Fc fusion proteins) represent a promising approach for creating fusion proteins that can amplify existing antibody titers in patients or induce new antibody titers in patients with no or low immune response to SARS-CoV-2. However, because spike protein variants have evolved over time, existing vaccines targeting de-emerged variants have the challenge of being less effective or ineffective in preventing COVID-19. Faced with new mutations in the SARS-CoV-2 spike protein, methods for producing and using Fc fusion proteins for use in preventive or booster vaccines that are effective in inducing patients to produce antiviral antibodies against the SARS-CoV-2 virus would meet this urgent need and have significant public health value.

[0094] Therefore, the objective is to create a manufacturable conjugate that presents the antigen (SARS-CoV-2 nucleocapsid) to patients in a novel way that enables rapid production of high-titer anti-SARS-CoV-2 antibodies. This conjugate consists of a portion of the SARS-CoV-2 nucleocapsid protein (or an analogue thereof) and a human Fc fragment (e.g., human IgG1 or hIgG1) containing a glycosylation-prone site or residue.

[0095] As shown in Figure 2, a novel SARS-CoV-2 N-Fc fusion protein comprising a bivalent analog of the SARS-CoV-2 nucleocapsid protein recombinantly fused to the human IgG1 Fc portion would facilitate the concentrated delivery of the nucleocapsid antigen to local APCs that internalize the novel SARS-CoV-2 N-Fc fusion protein via the Fc(γ) receptor.

[0096] The complete nucleocapsid protein of SARS-CoV-2 is shown below (GenBank:QHD43416.1). MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFP (Sequence ID 10)

[0097] Previous studies using insulin-Fc fusion proteins, such as those described in WO2018107117A1 and WO2020006529A1, have demonstrated that the selection of protein sequence, linker sequence, and Fc domain composition can all potentially affect protein yield, purity, and bioactivity.

[0098] When selecting a viral protein for a novel SARS-CoV-2 N-Fc fusion protein, it is conceivable to select a subset of the virus that includes a portion of the nucleocapsid. The viral protein for a novel SARS-CoV-2 N-Fc fusion protein may consist of all or part of the SARS-CoV-2 nucleocapsid protein. The viral protein for a novel SARS-CoV-2 N-Fc fusion protein may consist of all or part of the non-nucleocapsid portion of the SARS-CoV-2 virus, for example, the spike protein portion, the M protein portion, or the E protein portion of SARS-CoV-2. In an example, the viral protein for a novel SARS-CoV-2 N-Fc fusion protein consists of all or part of the SARS-CoV-2 nucleocapsid and all or part of the non-nucleocapsid portion of the SARS-CoV-2 virus. In several cases, one or more amino acids in the nucleocapsid fragment of a novel SARS-CoV-2 N-Fc fusion protein may be mutated from their native state.

[0099] Based on experience in insulin-Fc fusion protein production, different viral protein designs result in different protein yields for novel SARS-CoV-2 N-Fc fusion proteins. For example, a good, large or short portion of the nucleocapsid protein sequence of SEQ ID NO: 10 can be selected, and specific amino acids can be mutated by choice to produce the desired viral portion of the Fc fusion protein. The yield of the protein obtained when the selected viral protein is bound to the Fc fragment can be determined experimentally. Furthermore, the length and composition of the linker that ligates the selected viral protein to the Fc fragment also affect the protein yield, as do the selection of the Fc fragment and the portion of the hinge region of the Fc fragment linked to the viral protein.

[0100] Figure 2 illustrates an exemplary novel SARS-CoV-2 N-Fc fusion protein according to this disclosure. The novel SARS-CoV-2 N-Fc fusion protein may include a peptide linker. In several examples, several amino acids in the nucleocapsid fragment of the novel SARS-CoV-2 N-Fc fusion protein are mutated from their native state. In several examples, the therapeutic protein comprising the SARS-CoV-2 nucleocapsid protein fragment is located on the N-terminal side of the Fc fragment. The novel SARS-CoV-2 N-Fc fusion protein comprises a domain oriented from the N-terminus to the C-terminus as follows: (N-terminus)-therapeutic protein-peptide linker-Fc fragment-(C-terminus) (e.g., (N-terminus)-nucleocapsid protein-peptide linker-Fc fragment-(C-terminus)). The novel SARS-CoV-2 N-Fc fusion protein nucleocapsid fragment shown in Figure 2 may or may not be well expressed as part of the fusion protein synthesized by recombination in host cells (i.e., synthesized in HEK cells according to Example 1).

[0101] In all the following descriptions, the amino acid positions cited refer to the amino acid positions of SEQ ID NO: 10 SARS-CoV-2 nucleocapsid. The entire SARS-CoV-2 nucleocapsid protein consists of 419 amino acids. The sequence and domain structure of the SARS-CoV-2 nucleocapsid protein were analyzed using IUPred2A (Baker, NA, Sept, D., Joseph, S., Holst, MJ & McCammon, JA Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl Acad. Sci. USA 98, 10037-10041 (2001)). The SARS-CoV-2 nucleocapsid protein consists of an N-terminal domain (NTD), an RNA-binding domain (RNABD), a linker domain, a dimerization domain, and a C-terminal domain (CTD) (Cubuk, J., Alston, JJ, Incicco, JJ et al. The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA. Nat Commun 12, 1936 (2021)).

[0102] As a first attempt to create a novel SARS-CoV-2 N-Fc fusion protein, a viral nucleocapsid fragment comprising an N-terminal domain (NTD), RNA-binding domain (RNABD), linker domain, and dimerization domain was selected (SEQ ID NO: 10), resulting in a nucleocapsid fragment of 364 amino acids.

[0103] The nucleocapsid fragment (SEQ ID NO: 10) was converted to the following sequence using the peptide linker SGGGSGGGS (SEQ ID NO: 14): DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 1) It was linked to a human IgG1 Fc fragment containing [the specified component].

[0104] The human IgG1 fragment of SEQ ID NO: 1 shows that the N-terminal lysine on the native human IgG1 fragment was removed in an attempt to improve production yield and purity. Furthermore, the asparagine at the cNg site on the human IgG1 fragment was preserved to maintain glycosylation during fusion protein production in host cells.

[0105] The resulting SARS-CoV-2 N-Fc fusion protein is shown below. MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTR NPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYK HWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPSGGGSGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 17)

[0106] The SARS-CoV-2 N-Fc fusion protein of Sequence ID No. 17 was produced in HEK293 cells according to Example 1. Surprisingly, this resulted in an extremely low protein yield of only 14 mg / L.

[0107] Studies have shown that the N-terminal domain of nucleocapsid proteins is disordered and flexible, and transiently interacts with the RNABD domain of nucleocapsid proteins (Cubuk, J., Alston, JJ, Incicco, JJ et al.). It was hypothesized that the presence of all NTDs contributed to the poor protein yield of SEQ ID NO: 17. In a second attempt to create a SARS-CoV-2 N-Fc fusion protein, most of the N-terminal domain of the SEQ ID NO: 10 nucleocapsid protein fragment was removed. Specifically, the SARS-CoV-2 nucleocapsid protein of SEQ ID NO: 10 was shortened, and the first 40 amino acids (amino acids 1-40 of SEQ ID NO: 10) were removed. The resulting SARS-CoV-2 nucleocapsid protein fragment consisted of amino acids 41-364 of SEQ ID NO: 10, as shown below. RPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFP (Sequence ID 11)

[0108] Using Clustal Omega, a parallel comparison was performed between the SARS-CoV-2 nucleocapsid fragment of sequence number 10 and the truncated nucleocapsid fragment of sequence number 11, as shown in Figure 14. *An asterisk (") indicates complete homology at a given sequence position across all sequences. A colon (:) indicates strong intergroup conservation of similarity with a score greater than 0.5 in the Gonnet PAM 250 matrix. A dash (-) indicates a sequence gap, meaning that no local homology exists within a particular set of comparisons within a specific range of sequences.

[0109] The nucleocapsid fragment of SEQ ID NO: 11 is linked to the following sequence via the peptide linker SGGGSGGGS (SEQ ID NO: 14): DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 1) It was linked to a human IgG1 Fc fragment containing [the specified component].

[0110] In an attempt to improve production yield and purity, the N-terminal lysine on the human IgG1 fragment was removed as shown in SEQ ID NO: 1 above, similar to the case of the SARS-CoV-2 N-Fc fusion protein of SEQ ID NO: 17. Furthermore, the asparagine at the cNg site on the human IgG1 fragment was preserved to maintain glycosylation during fusion protein production in host cells. The resulting SARS-CoV-2 N-Fc fusion protein is shown below. RPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGG SQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSR IGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPSGGGSGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 18)

[0111] The SARS-CoV-2 N-Fc fusion protein of Sequence ID No. 18 was synthesized in HEK293 cells according to Example 1. Surprisingly, even after removing the N-terminal domain in an attempt to prevent transient interaction with the RNABD domain of the nucleocapsid protein fragment, this also resulted in a low protein yield titer of 18 mg / L.

[0112] It was hypothesized that the full length of the SARS-CoV-2Fc fusion protein molecule contributes to the low protein yield. Furthermore, studies have shown that the central linker of the SARS-CoV-2 nucleocapsid is highly dynamic, and the interaction between the nucleocapsid and the dimerization domain is minimal. However, it is hypothesized that if the dimerization domain and CTD domain are completely absent, the central linker domain may self-interact or interact with the RNABD domain (Cubuk, J., Alston, JJ, Incicco, JJ et al.). Instead of removing the dimerization domain and CTD domain while retaining the entire central linker domain, a portion of the linker domain was retained. Specifically, the complete linker domain consists of amino acids 174-243, with amino acid 1 being the first amino acid in SEQ ID NO: 10. In a further attempt to produce a novel SARS-CoV-2 N-Fc fusion protein with an acceptable production yield, the linker domain portion from amino acids 174 to 208 was retained, and the 38 amino acids at the C-terminus of the linker domain were removed to obtain the nucleocapsid fragment of Sequence ID No. 12 shown below. RPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPA (Sequence ID 12)

[0113] Using Clustal Omega, a parallel comparison was performed between the SARS-CoV-2 nucleocapsid fragment of sequence number 10 and its further shortened form, sequence number 12, as shown in Figure 15. *" indicates complete homology at a given sequence position across all sequences. ":" (colon) indicates intergroup conservation of strong similarity with a score greater than 0.5 in the Gonnet PAM 250 matrix. "-" (dash) indicates a sequence gap, meaning that no local homology exists within a particular set of comparisons within a specific range of sequences.

[0114] The nucleocapsid fragment of SEQ ID NO: 12 is linked to the following sequence via the peptide linker SGGGSGGGS (SEQ ID NO: 14): DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 1) It was linked to a human IgG1 Fc fragment containing [the specified component].

[0115] In an attempt to improve production yield and purity, the N-terminal lysine on the human IgG1 fragment was removed as shown in SEQ ID NO: 1 above, similar to the case of the SARS-CoV-2 N-Fc fusion proteins of SEQ ID NO: 17 and SEQ ID NO: 18. Furthermore, the asparagine at the cNg site on the human IgG1 fragment was preserved to maintain glycosylation during fusion protein production in host cells. The resulting novel SARS-CoV-2 N-Fc fusion protein is shown below. RPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPASGGGSGGGSDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 19)

[0116] The novel SARS-CoV-2 N-Fc fusion protein of Sequence ID No. 19 was synthesized in HEK293 cells according to Example 1. Further shortened novel SARS-CoV-2 N-Fc fusion proteins resulted in a significant improvement in production yield, with a protein titer of 163 mg / L.

[0117] Considering that the presence of mutations reduces the effectiveness of vaccines utilizing the SARS-CoV-2 spike protein region, we noted that the nucleocapsid fragment of Sequence ID No. 12 (shown below) contains two amino acids that are mutated in the N-protein present in the alpha, gamma, and omicron SARS-CoV-2 mutants, specifically R203 and G204 (their native forms are highlighted in bold below), which are mutated in these variants. RPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPA (Sequence ID 12)

[0118] In an attempt to address the potential reduction in efficacy in alpha, gamma, and omicron SARS-CoV-2 mutants, the nucleocapsid fragment of SEQ ID NO: 12 was modified at these two amino acids, with arginine (R) at position 203 being replaced with lysine (K) and glycine (G) at position 203 being replaced with arginine (R), as highlighted in bold below. As a result, the nucleocapsid fragment of SEQ ID NO: 13 was obtained, shown below. RPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSKRTSPA (Sequence ID 13)

[0119] In this nucleocapsid fragment, similar to the case using SEQ ID NO: 12, the linker domain portion from amino acids 174 to 208 was retained, and the 38 amino acids at the C-terminus of the linker domain were removed to obtain the nucleocapsid fragment.

[0120] Using Clustal Omega, we performed a parallel comparison of SARS-CoV-2 nucleocapsid fragments with mutations in SEQ ID NOs. 10, 12, and 13, which are shown in Figure 16. *" indicates complete homology at a given sequence position across all sequences. ":" (colon) indicates intergroup conservation of strong similarity with a score greater than 0.5 in the Gonnet PAM 250 matrix. "-" (dash) indicates a sequence gap, meaning that no local homology exists within a particular set of comparisons within a specific range of sequences.

[0121] The nucleocapsid fragment of SEQ ID NO: 13 is linked to the following sequence via the peptide linker SGGGSGGGS (SEQ ID NO: 14): DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 1) It was linked to a human IgG1 Fc fragment containing [the specified component].

[0122] In an attempt to improve production yield and purity, the N-terminal lysine on the human IgG1 fragment was removed as shown in SEQ ID NO: 1 above, similar to the case of the SARS-CoV-2 N-Fc fusion proteins of SEQ ID NO: 17 and SEQ ID NO: 18. Furthermore, the asparagine at the cNg site on the human IgG1 fragment was preserved to maintain glycosylation during fusion protein production in host cells. The resulting novel SARS-CoV-2 N-Fc fusion protein is shown below. RPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSKRTSPASGGGSGGGSDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 20)

[0123] The novel SARS-CoV-2 N-Fc fusion protein of Sequence ID No. 20 was synthesized in HEK293 cells according to Example 1. The novel SARS-CoV-2 N-Fc fusion protein, which had a mutant amino acid mutation in the central linker region of the nucleocapsid fragment, produced a protein titer of 117 mg / L, which is acceptable according to the design target (protein yield of over 100 mg / L). However, this yield is not as high as that of the novel SARS-CoV-2 N-Fc fusion protein without a mutation in the central linker region of the nucleocapsid fragment.

[0124] In an attempt to maintain the high yield obtained in the novel SARS-CoV-2 N-Fc fusion protein construct of Sequence ID No. 19 by removing part of the dimerizing region and central linker region of the SARS-CoV-2 nucleocapsid protein, a further nucleocapsid fragment was used in which the entire central linker region was removed, leaving the complete RNABD of the nucleocapsid protein along with a small portion of the N-terminal domain (NTD). The resulting nucleocapsid fragment is shown below as Sequence ID No. 8. RSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAE (Sequence ID 8)

[0125] Using Clustal Omega, a parallel comparison of SARS-CoV-2 nucleocapsid fragments SEQ ID NO: 10 and SEQ ID NO: 8 was performed, as shown in Figure 17. * " indicates complete homology at a given sequence position across all sequences. ":" (colon) indicates intergroup conservation of strong similarity with a score greater than 0.5 in the Gonnet PAM 250 matrix. "-" (dash) indicates a sequence gap, meaning that no local homology exists within a particular set of comparisons within a specific range of sequences.

[0126] The nucleocapsid fragment of SEQ ID NO: 8 is linked to the following sequence via the peptide linker SGGGSGGGS (SEQ ID NO: 14): DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 1) It was linked to a human IgG1 Fc fragment containing [the specified component].

[0127] In an attempt to improve production yield and purity, the N-terminal lysine on the human IgG1 fragment was removed as shown in Sequence ID No. 1 above. Furthermore, the asparagine at the cNg site on the human IgG1 fragment was preserved to maintain glycosylation during fusion protein production in host cells. The resulting novel SARS-CoV-2 N-Fc fusion protein is shown below. RSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAESGGGSGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 15)

[0128] The novel SARS-CoV-2 N-Fc fusion protein of SEQ ID NO: 15 was synthesized in HEK293 cells according to Example 1. The novel SARS-CoV-2 N-Fc fusion protein, in which the central linker portion of the nucleocapsid fragment was removed, yielded a protein titer of 140 mg / L, which is an improvement over the novel SARS-CoV-2 N-Fc fusion protein (SEQ ID NO: 20) containing a mutation in the central linker portion of the nucleocapsid fragment. The novel SARS-CoV-2 N-Fc fusion protein of SEQ ID NO: 15 was synthesized in a considerably larger batch (0.5 L instead of 0.03 L) in HEK293 cells according to Example 1. Thus, in larger batches, the novel SARS-CoV-2 Fc fusion protein, in which the central linker portion of the nucleocapsid fragment was removed, produced a protein titer of 198 mg / L, demonstrating further improvement over the novel SARS-CoV-2 N-Fc fusion protein (SEQ ID NO: 20) containing a mutation in the central linker portion of the nucleocapsid fragment.

[0129] Because retaining the NTD portion may be beneficial, we made further attempts to create a novel SARS-CoV-2 N-Fc fusion protein containing a nucleocapsid fragment with only the entire N-terminal domain (NTD) and the RNABD domain. This novel SARS-CoV-2 N-Fc fusion protein used the nucleocapsid fragment of Sequence ID No. 9 shown below. MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAE (Sequence ID 9)

[0130] Using Clustal Omega, a parallel comparison of SARS-CoV-2 nucleocapsid fragments of sequence numbers 9 and 8 was performed, as shown in Figure 18. *" indicates complete homology at a given sequence position across all sequences. ":" (colon) indicates intergroup conservation of strong similarity with a score greater than 0.5 in the Gonnet PAM 250 matrix. "-" (dash) indicates a sequence gap, meaning that no local homology exists within a particular set of comparisons within a specific range of sequences.

[0131] The nucleocapsid fragment of SEQ ID NO: 9 is linked to the following sequence via the peptide linker SGGGSGGGS (SEQ ID NO: 14): DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 1) It was linked to a human IgG1 Fc fragment containing [the specified component].

[0132] In an attempt to improve production yield and purity, the N-terminal lysine on the human IgG1 fragment was removed as shown in Sequence ID No. 1 above. Furthermore, the asparagine at the cNg site on the human IgG1 fragment was preserved to maintain glycosylation during fusion protein production in host cells. The resulting novel SARS-CoV-2 N-Fc fusion protein is shown below. MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAESGGGSGGGSDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 16)

[0133] The novel SARS-CoV-2 N-Fc fusion protein, SEQ ID NO: 16, was synthesized in HEK293 cells according to Example 1. The novel SARS-CoV-2 N-Fc fusion protein, SEQ ID NO: 16, produced an acceptable protein titer of 173 mg / L.

[0134] Therefore, the four candidate novel SARS-CoV-2 N-Fc fusion proteins, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 20, met the design target of a protein yield exceeding 100 mg / L. The novel SARS-CoV-2 N-Fc fusion proteins SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 20, which were produced by recombination in HEK cells according to Example 1, were purified according to Example 2. The Fc fusion protein structure of the novel SARS-CoV-2 N-Fc fusion proteins SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 20 was confirmed according to Example 3, and sequence identification was performed according to Example 4. To obtain the homodimer titer of each of the produced novel SARS-CoV-2 N-Fc fusion proteins, the % homodimer was measured according to Example 5, and the homodimer titer was calculated by multiplying the % homodimer by the protein titer of the fusion protein produced by recombination. Table 1 below shows the homodimeric titers of the novel SARS-CoV-2 N-Fc fusion proteins, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 20, along with their protein yields.

[0135] [Table 1]

[0136] The Fc(γ) receptor I binding of SARS-CoV-2 N-Fc fusion proteins of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 20, produced in HEK293 cells according to Example 1, was measured according to Example 6. As described in Example 6, the OD450 measurements for human Fc(γ) receptor I, Fc(γ) receptor IIA, Fc(γ) receptor IIB, Fc(γ) receptor III, FcRn, and ACE2 receptor binding are expected to increase as a function of the concentration of SARS-CoV-2 N-Fc fusion protein.

[0137] Using Clustal Omega, a parallel comparison of the nucleocapsid frame domains of SEQ ID NOs. 8, 9, 12, and 13 was performed, as shown in Figure 19. *" indicates perfect homology at a given sequence location across all sequences. ":" (colon) indicates strong intergroup conservation of similarity, with a score greater than 0.5 in the Gonnet PAM 250 matrix. "-" (dash) indicates a sequence gap, meaning there is no local homology within a particular set of comparisons within a specific range of sequences, while ":", "." or a blank space indicate that the amino acid variation between sequences at a given sequence location is conserved, moderate, or very different, respectively.

[0138] To highlight amino acid mutations in the nucleocapsid region, a parallel comparison of the nucleocapsid frame domains of SEQ ID NO: 12 and SEQ ID NO: 13 was performed using Clustal Omega, as shown in Figure 20. * " indicates perfect homology at a given sequence location across all sequences. ":" (colon) indicates strong intergroup conservation of similarity, with a score greater than 0.5 in the Gonnet PAM 250 matrix. "-" (dash) indicates a sequence gap, meaning there is no local homology within a particular set of comparisons within a specific range of sequences, while ":", "." or a blank space indicate that the amino acid variation between sequences at a given sequence location is conserved, moderate, or very different, respectively.

[0139] SARS-CoV-2 N-Fc fusion protein for use as a primary vaccine SARS-CoV-2 N-Fc fusion proteins can be used as primary vaccines. In one or more embodiments, SARS-CoV-2 N-Fc fusion proteins are provided in pharmaceutical compositions. While injection of any protein can induce an immune response, the magnitude and type of the response depend heavily on the "state" of the respective immune system. For example, injecting exogenous Ag compared to autoantigens (Ag) induces a greater immune response in immune systems that maintain central and peripheral tolerance mechanisms. Furthermore, administering exogenous Ag to an immune system that has been primed for past exposure to each Ag (e.g., viral infection) elicits a faster and stronger immune response than in an Ag-naive system. There are two immunological justifications for this priming: 1) the Ag-naive immune system has naive B and T lymphocytes with a much higher activation threshold than the Ag-priming “memory” cells of the Ag-priming immune system, and the amount of Ag required for Ag-presenting antigen-presenting cells (APCs) to activate primed memory T cells is much smaller; and 2) memory T cells expand and proliferate during Ag-priming exposure, so re-exposure to injected Ag results in an essentially larger number of such cells. The dominant APCs are dendritic cells (DCs) and macrophages that present Ag, complexed with major histocompatibility complex (MHC) molecules on their surface, to T cell Ag receptors. Figure 11 is a schematic diagram illustrating the modes in which antigens can interact with antigen-presenting cells, such as dendritic cells.

[0140] APCs can influence both the "magnitude" and "type" of the response to Ag. B cells are directly involved in the immune response through humoral immunity (antibody production) and also in the T cell immune response as specific APCs that selectively capture antigens and present them to T cells. Both of these B cell functions are achieved by the activation of surface B cell receptors (BCRs), which are membrane-bound antibodies that specifically bind to specific antigens. Polyvalent soluble antigens, such as Fc fusion homodimers containing specific antigens, can be recognized by BCRs and activated. Therefore, SARS-CoV-2 N-Fc fusion protein homodimers can activate B cells and increase antibody production through antigen-specific BCR activation, and can also lead to increased recognition and responsiveness of T cells specifically directed to RBD epitopes through B cell-mediated APC activity. Thus, these fusion proteins activate both humoral and cellular immunity after administration. More specifically, induced Th1 (cellular) immunity activates the body's cell-killing mechanisms, such as cytotoxic T cells, NK cells, and macrophages, which target cells already infected with the virus. Simultaneously, in Th2 immunity, T helper cells stimulate B cells to proliferate and differentiate into plasma cells that secrete antigen-specific antibodies. These antibodies help control infection by one of the following: (i) neutralization of the virus by binding of antibodies to the RBD site on the viral surface necessary for cell entry, in which case the antibodies also help eliminate the virus bound to the antibodies via phagocytosis directed to Fc; (ii) antibody-directed cytotoxicity (ADC), which occurs when antibodies bind to antigens presented on the surface of infected cells and instruct NK cells to destroy them; or (iii) possibly neutralization of viral internal antigens exposed within the cell, in which case the antibodies and the exposed internal viral environment come into contact, for example, within the cell.

[0141] Adjuvant In some cases, a Th1 cell response is required to clear most viral and bacterial infections, where a virus-like or bacterial-like substance (essentially non-Ag) causes APCs to express key cytokines and surface costimulatory molecules that cause T cells to become Th1-type during Ag presentation. In fact, this activation of APCs forms the conceptual basis for many immunoenhancing substances called adjuvants. The dominant APCs are dendritic cells (DCs) and macrophages that present Ag, complexed with major histocompatibility complex (MHC) molecules on their surface, to T cell Ag receptors. These APCs can influence both the "magnitude" and "type" of the response to Ag. Some adjuvants are designed to trick the immune system into reacting as if the injected vaccine Ag were part of an ongoing infection (i.e., infectious agents provide such naturally occurring viral or bacterial adjuvants). Therefore, in addition to facilitating the development of a Th1 response to effectively eliminate each infection, adjuvants enhance the Ag-presenting ability necessary to activate APCs and overcome the high activation threshold of naive T cells. Such T cells are crucial aids to B cells that specifically bind to each Ag in order to produce Ag-specific antibody (Ab) titers.

[0142] Fc fusion proteins used as primary vaccines can be co-administered with adjuvants to enhance or otherwise alter the target immune response. Adjuvants activate APCs to enhance Ag presentation ability, which is necessary to overcome the high activation threshold of naive T cells, in addition to shaping the development of a Th1 response to effectively eliminate each infection. In several cases, known adjuvants can be used in pharmaceutical compositions of SARS-CoV-2 N-Fc fusion proteins to enhance the induction of anti-SARS-CoV-2 antibodies. Known adjuvants include those used in human clinical trials over the past decade for respiratory viral infections, including trivalent or monovalent influenza vaccines, pandemic H1N1, H5N1, and SARS-CoV vaccines.

[0143] Examples of adjuvants usable in the pharmaceutical compositions disclosed herein include, but are not limited to, oil-in-water, amorphous aluminum hydroxyphosphate (AAHS), aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate (Alum), Freund's adjuvant (complete and / or imperfect), squalene, AS02, AS03, AS04, MF59, AS01B, QS-21, and CpG. Examples include 1018, ISCOMS, Montanide® ISA-51, Montanide® ISA-720, polylactide coglycolide (PLG), monophosphoryl lipid A (MPL), Detox, AGP[RC-529], DC_Chol, OM-174 (lipid A derivative), CpG motif (synthetic oligonucleotide containing immunostimulatory CpG motif), modified LT and CT, hGM-CSF, hIL-12, imudaptin, gold particles, and other inactive vehicles, as well as various experimental adjuvants from Advax (Australia), such as AddaVax (Invivogen) or other Advax-based vaccine adjuvants.

[0144] In some examples, the selected adjuvants may be MF59 (Novartis) and AS-03 (GlaxoSmithKline). Customized formulations of equivalents such as MF59 (Novartis), AddaVax (Invivogen), or other Advax-based vaccine adjuvants from Vaxine Pvt Ltd. (Australia) can be used in pharmaceutical compositions of the SARS-CoV-2 N-Fc fusion protein. In preferred embodiments, the SARS-CoV-2 N-Fc fusion protein is co-administered with Montanide® ISA-720 adjuvant to enhance or otherwise alter the immune response in the target. Many different adjuvants used in respiratory viral infections have been comprehensively tested in seasonal trivalent influenza vaccines, Australian pandemic H1N1 and H5N1 vaccines (Protein Sciences), and more recently in NIH-supported human influenza vaccine trials conducted by Sanofi Pasteur in the United States, and can also be used in pharmaceutical compositions of the SARS-CoV-2 N-Fc fusion protein.

[0145] In one or more embodiments, the SARS-CoV-2 N-Fc fusion protein formulation is prepared in situ for administration. In one embodiment, the SARS-CoV-2 N-Fc fusion protein is mixed with an adjuvant in situ under sterile mixing conditions. In one embodiment, the SARS-CoV-2 N-Fc fusion protein and the adjuvant are thoroughly mixed and / or emulsified to prepare a homogeneous emulsion for administration to a subject. The SARS-CoV-2 N-Fc fusion protein adjuvant formulation or its pharmaceutical composition is administered to the patient by subcutaneous (sc) or intramuscular (im) injection because the subcutaneous or intramuscular space contains a high concentration of dendritic cells (DCs), making the sc or im injection site more likely to induce a strong antibody response.

[0146] As described above, in some cases, it may be advantageous to use an adjuvant in the pharmaceutical composition to increase the amount of anti-SARS-CoV-2 antibody titer measured according to Example 7 and / or the viral neutralizing ability of the anti-SARS-CoV-2 antibody titer measured according to Example 9. The use of an adjuvant may be particularly advantageous for antibody-naive patients who do not have a basal immune response to the virus or RNABD. Furthermore, adjuvants may be advantageous for elderly subjects who experience age-related changes in immune function, so-called immunosenescence, which are the result of changes in multiple levels of the immune system over time. Once a patient has acquired measurable antibodies, upon re-antigen stimulation with the SARS-CoV-2 virus, the patient shows extremely rapid acquisition of anti-SARS-CoV-2 antibodies to induce self-defense against COVID-19.

[0147] Primary SARS-CoV-2 N-Fc fusion vaccine evaluated in mice The efficacy of the exemplary SARS-CoV-2 N-Fc fusion protein or its pharmaceutical composition can first be evaluated in a mouse immunoassay, following the procedure of Example 9, for its ability to induce a high titer anti-nucleocapsid protein IgG titer. BALB / c mice are a widely used relevant animal model for preclinical immunogenicity evaluation of vaccines. This strain produces a robust Ab response when immunized with adjuvant and non-adjuvant vaccine candidates. Furthermore, mouse-specific reagents are widely available for evaluating the dynamics and characterization of various immune responses to vaccination, including relevant Ab isotypes and T cell responses (e.g., Th1 and Th2 responses). Accordingly, the BALB / c mouse model is selected to evaluate the immunogenicity of SARS-CoV-2 N-Fc fusion protein vaccines in terms of Ag dose, adjuvant enhancement, administration route, and the frequency of administration required to achieve an optimal Ab response.

[0148] In short, target mice (e.g., BALB / c mice) are injected three times at predetermined intervals (e.g., on days 0, 21, and 46) with an exemplary SARS-CoV-2 N-Fc fusion protein (with or without Montanide® ISA 720 adjuvant) or its pharmaceutical composition, and serum is collected at regular intervals (every 7 days from day 14).

[0149] Following the administration of the SARS-CoV-2 N-Fc fusion protein of SEQ ID NO: 15 to N=7 BALB / c mice in one or more doses according to Example 8, anti-SP / RBD SARS-CoV-2 IgG antibody titers were measured according to Example 7. As expected, as shown in Figure 12, since the exemplary SARS-CoV-2 N-Fc fusion protein of SEQ ID NO: 15 does not contain any portion of the SP / RBD of SARS-CoV-2, no measurable anti-SARS-CoV-2 antibody titer was observed after treatment.

[0150] The serum anti-SARS-CoV-2 nucleocapsid protein antibody titer is measured according to the procedure in Example 9.

[0151] Without adjuvant, at a dose level of 10 μg, the SARS-CoV-2 N-Fc fusion protein of SEQ ID NO: 15 induced a measurable anti-SARS-CoV-2 nucleocapsid protein antibody titer starting approximately 40 days after the initial injection on day 0. Without adjuvant, at a dose level of 10 μg, the SARS-CoV-2 N-Fc fusion proteins of SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 20 are expected to induce a measurable anti-SARS-CoV-2 nucleocapsid protein antibody titer starting approximately 40 days after the initial injection on day 0.

[0152] As previously described, adjuvants activate APCs to enhance Ag presentation ability, which is necessary to overcome the high activation threshold of naive T cells, in addition to facilitating the development of the Th1 response to effectively eliminate each infection. When the SARS-CoV-2 N-Fc fusion protein of SEQ ID NO: 15 was used in combination with Montanide® ISA 720 adjuvant (30% / 70% v / v), the anti-SARS-CoV-2 nucleocapsid protein antibody titer, which began around 40 days after the initial injection on day 0, was higher compared to the SARS-CoV-2 N-Fc fusion protein without adjuvant. When SARS-CoV-2 N-Fc fusion proteins of SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 20 are combined with Montanide® ISA 720 adjuvant (30% / 70% v / v), anti-SARS-CoV-2 nucleocapsid protein antibody titers beginning around 40 days after the initial injection on day 0 are expected to be higher compared to SARS-CoV-2 N-Fc fusion proteins without adjuvant. These data strongly support the selection of Montanide® ISA 720 as the lead adjuvant candidate for this vaccine development program.

[0153] The kinetic response, i.e., the duration of the response, to dose levels ranging from 1 μg to 100 μg after the first, second, and third doses is expected to show an increase in anti-SARS-CoV-2 nucleocapsid protein antibody titers at all dose levels for at least 56 days after vaccination.

[0154] SARS-CoV-2 N-Fc fusion protein for use as a booster vaccine In several instances, the exemplary SARS-CoV-2 N-Fc fusion proteins of this disclosure, e.g., SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, or SEQ ID NO: 20, can be used as booster vaccines. Administration of these fusion proteins to subjects already possessing low but measurable antibody levels against the SARS-CoV-2 antigen enhances their antiviral protection by amplifying their antibody titers. The N-protein (nucleocapsid) fragment variants are synthesized to maximize antigenicity, and the Fc region extends the antigen's residence time. While we do not wish to be bound to a specific mechanism, it is thought that during longer in vivo residence times, the naturally occurring glycosylated human Fc fragments assist in binding to the Fc(γ) receptor on antigen-presenting cells (APCs), resulting in increased presentation of the SARS-CoV-2 nucleocapsid analog antigen to T cells and / or B cells (as shown in Figure 11), and a strong immune response to the SARS-CoV-2 nucleocapsid antigen is expected. Specifically, APCs internalize the SARS-CoV-2 nucleocapsid antigen via the Fc(γ) receptor, process the nucleocapsid fragment, and present it to CD4+ Th cells, which then promote ("assist") B cell activation and anti-SARS-CoV-2 nucleocapsid IgG (i.e., Ab) production. Antigen-presenting cells may be dendritic cells (DCs), monocytes, or macrophages that can internalize molecules of the SARS-CoV-2 N-Fc fusion protein, such as SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, or SEQ ID NO: 20, via N-Fc receptor-mediated phagocytosis (e.g., via the Fc region of the SARS-CoV-2 N-Fc fusion protein that binds to the Fc(γ) receptor of immune cells, as shown in Figure 11). For example, Fc-mediated uptake of the SARS-CoV-2 N-Fc fusion protein by a subset of DCs (e.g., cDC2S) promotes the development of anti-SARS-CoV-2 T helper 2 (Th2) cells via the secretion of IL-10 and IL-33. Anti-SARS-CoV-2 Th2 cells activate anti-SARS-CoV-2 B cells, for example, by cross-linking antigen receptors so that B cells can attract Th2 cells.Uptake via the B cell antigen receptor (BCR) involves the binding of the SARS-CoV-2 RNABD to the SARS-CoV-2 N-Fc fusion protein molecule, which then transports the SARS-CoV-2 antigen to an intracellular site where it is degraded and returned to the B cell surface as a peptide bound to an MHC class II molecule. This peptide MHC class II complex is recognized by SARS-CoV-2-specific helper T cells, stimulating them to proliferate and produce proteins that differentiate their progeny into B cells that secrete anti-SARS-CoV-2 antibodies. The SARS-CoV-2 N-Fc fusion proteins, such as SEQ ID NOs. 15, 16, 19, or 20, may, due to the presence of the Fc fragment, allow for prolonged direct exposure of the SARS-CoV-2 RNABD (nucleocapsid) fragment to antigen-producing cells. Furthermore, as previously described, the glycosylated Fc fragments in the SARS-CoV-2 N-Fc fusion protein of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, or SEQ ID NO: 20 are expected to induce a very strong immune response directed towards the therapeutic or antigenic portion of the fusion protein. The combination of these properties significantly increases the amount of antiviral antibodies while decreasing the amount of antigen required to produce the necessary immune response.

[0155] In several embodiments, therapies including treatment of patients with the SARS-CoV-2 N-Fc fusion protein of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, or SEQ ID NO: 20 or a pharmaceutical composition thereof can be administered as a booster vaccine to recovered COVID-19 patients who are already antibody-positive to SARS-CoV-2, as a means of amplifying their antibody titers and affinity, thereby providing these treated patients with sufficient immunity to prevent infection by the SARS-CoV-2 virus and / or severe symptoms associated with infection when they subsequently encounter the virus. Furthermore, therapies including treatment of patients with the SARS-CoV-2 N-Fc fusion protein of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, or SEQ ID NO: 20 or a pharmaceutical composition thereof can be administered as a booster vaccine to subjects previously immunized with a vaccine against the SARS-CoV-2 virus, as a means of specifically amplifying antibody titers and affinity to the SARS-CoV-2 N-Fc fusion protein nucleocapsid. Such therapies are important when vaccines are not 100% effective and / or when induced antibody titers decrease over time. In several cases, the SARS-CoV-2 N-Fc fusion protein or its pharmaceutical composition, such as SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, or SEQ ID NO: 20, can be administered to patients by subcutaneous (sc) or intramuscular (im) injection because the subcutaneous or intramuscular space has a high concentration of dendritic cells (DCs), making the sc or im injection site more likely to induce a strong antibody response.

[0156] Production of Fc fusion proteins In several embodiments, the fusion protein can be expressed by cells as described in more detail in the Examples section.

[0157] Expression and Purification SARS-CoV-2 N-Fc fusion proteins can be expressed by recombination in eukaryotic cells, e.g., mammalian cells or non-mammalian cells. Exemplary mammalian cells used for expression include HEK cells (e.g., HEK293 cells) or CHO cells. CHO cells can be subdivided into various cell lines or subclasses (e.g., CHO DG44, CHO-M, CHO-SE®, and CHO-K1), some of which are genetically engineered for optimal use with specific types of nucleic acid molecules (e.g., vectors containing DNA) or specific cell growth medium compositions, as described in the Examples section. Cells can be transfected with nucleic acid molecules encoding the SARS-CoV-2 N-Fc fusion protein (e.g., vectors) (e.g., if the entire SARS-CoV-2 N-Fc fusion protein is encoded by a single nucleic acid molecule). HEK293 cells can be transfected with vectors encoding SARS-CoV-2 N-Fc fusion proteins, but this process only results in transient expression of the SARS-CoV-2 N-Fc fusion protein for a set period (e.g., 3, 4, 5, 7, 10, 12, 14 days, or more), after which the host cells cease expressing the SARS-CoV-2 N-Fc fusion protein at a recognizable level (i.e., transient transfection). HEK293 cells transiently transfected with nucleic acid sequences encoding SARS-CoV-2 N-Fc fusion proteins often enable more rapid production of recombinant proteins, thereby facilitating the creation and screening of multiple SARS-CoV-2 N-Fc fusion protein candidates. CHO cells can be transfected with vectors that permanently integrate into host cell DNA, resulting in consistent and persistent expression of the SARS-CoV-2 N-Fc fusion protein (i.e., stable transfection) as long as the cells are properly cultured.CHO cells and cell lines stably transfected with nucleic acids encoding SARS-CoV-2 N-Fc fusion proteins often take a long time to develop but offer high protein yields and are often more suitable for the production of low-cost products (e.g., products for use in the veterinary pharmaceutical market). Cells and cell lines can be cultured using standard methods in the art.

[0158] In several examples, the SARS-CoV-2 N-Fc fusion protein can be purified or isolated from cells (e.g., by cell lysis). The SARS-CoV-2 N-Fc fusion protein can be secreted by cells and purified or isolated from the cell culture medium in which the cells have grown. Purification of the SARS-CoV-2 N-Fc fusion protein may include the use of column chromatography (e.g., affinity chromatography) or other separation methods based on differences in size, charge, and / or affinity for specific molecules. Purification of the SARS-CoV-2 N-Fc fusion protein includes selecting or concentrating the protein containing the Fc fragment, for example, using protein A beads or a protein A column to ensure that the protein containing the Fc fragment binds with high affinity to protein A covalently bound to protein A beads at a neutral solution pH. The bound SARS-CoV-2 N-Fc fusion protein is then eluted from the protein A beads by a change in the solution variable (e.g., a decrease in solution pH). Other separation methods, such as ion exchange chromatography and / or gel filtration chromatography, may also be employed as alternatives or additional measures. Purification of SARS-CoV-2 N-Fc fusion proteins may further include filtration or centrifugation of the protein preparation, diafiltration, ultrafiltration, and filtration through porous membranes of various sizes, as well as final formulation with excipients.

[0159] Purified SARS-CoV-2 N-Fc fusion proteins can be characterized for purity, protein yield, structure, and / or activity using various methods, such as absorbance at 280 nm (e.g., to determine protein yield), size exclusion or capillary electrophoresis (e.g., to determine molecular weight, aggregation rate, and / or purity), mass spectrometry (MS) and / or liquid chromatography (LC-MS) (e.g., to determine purity and / or glycosylation), and / or ELISA (e.g., to determine binding to SARS-CoV-2 antibody or ACE2, e.g., the degree of affinity). Exemplary characterization methods are also described in the Examples section.

[0160] The protein yield of SARS-CoV-2 N-Fc fusion protein produced in transiently transfected HEK cells and after protein A purification may be greater than 5 mg / L, 10 mg / L, or 20 mg / L, or more preferably greater than 50 mg / L (e.g., greater than 60 mg / L, greater than 70 mg / L, greater than 80 mg / L, greater than 90 mg / L, greater than 100 mg / L). The % homodimer of SARS-CoV-2 N-Fc fusion protein produced in transiently transfected HEK cells and after protein A purification may be greater than 70% (e.g., greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%). The homodimeric value (calculated as the product of SARS-CoV-2 N-Fc fusion protein yield and % homodimer) of SARS-CoV-2 N-Fc fusion protein produced in transiently transfected HEK cells and after protein A purification may be greater than 50 mg / L (e.g., greater than 60 mg / L, greater than 70 mg / L, greater than 80 mg / L, greater than 90 mg / L, greater than 100 mg / L).

[0161] Pharmaceutical composition and route of administration The amount and concentration of SARS-CoV-2 N-Fc fusion protein in the pharmaceutical composition, as well as the amount of the pharmaceutical composition administered to the subject, may be selected based on clinically relevant factors such as the subject's medically relevant characteristics (e.g., age, weight, sex, other medical conditions), the solubility of the compound in the pharmaceutical composition, the potency and activity of the compound, and the method of administration of the pharmaceutical composition.

[0162] The formulations of this disclosure include those suitable for parenteral administration. As used herein, the terms “parenteral administration” and “administered parenterally” mean modes of administration other than enteral and topical administration, typically by intravenous, intramuscular, or subcutaneous injection.

[0163] Examples of suitable aqueous and non-aqueous carriers usable in the pharmaceutical compositions of this disclosure include water, physiological saline, ethanol, salts, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, injectable organic esters such as ethyl oleate, buffers such as potassium phosphate and / or sodium phosphate, and pH buffers such as hydrochloric acid and / or sodium hydroxide. Appropriate fluidity can be maintained, for example, by the use of coating or emulsifying materials such as lecithin, maintaining the required particle size in the case of dispersions, and by the use of surfactants, such as Tween-like surfactants. In some examples, the pharmaceutical compositions (e.g., those described herein) comprise a Tween-like surfactant, such as polysorbate-20, Tween-20, or Tween-80. In some examples, a pharmaceutical composition (e.g., as described herein) comprises a Tween-like surfactant, such as Tween-80, in a concentration of about 0.001% to about 2%, or about 0.005% to about 0.1%, or about 0.01% to about 0.5%.

[0164] SARS-CoV-2 N-Fc fusion proteins can be administered as a bolus, infusion, or intravenous push, or via syringe injection, pump, pen, needle, or indwelling catheter. SARS-CoV-2 N-Fc fusion proteins can be administered by subcutaneous bolus injection. In several cases, SARS-CoV-2 N-Fc fusion proteins or their pharmaceutical compositions are administered to patients by subcutaneous (sc) or intramuscular (im) injection because the subcutaneous or intramuscular space contains a high concentration of dendritic cells (DCs), making the sc or im injection site more likely to induce a strong antibody response. Delivery methods using refillable or biodegradable devices can also be provided. In recent years, various sustained-release polymer devices have been developed and tested in vivo for the controlled delivery of drugs, including protein-based biopharmaceuticals. Various biocompatible polymers (including hydrogels), including both biodegradable and non-biodegradable polymers, can be used to form implants for the sustained release of compounds at specific target sites. Additional pharmaceutically acceptable ingredients for use in the composition include buffers, salts, stabilizers, diluents, preservatives, antibiotics, isotonic agents, and the like.

[0165] Administration In use, a therapeutically effective amount of SARS-CoV-2 N-Fc fusion protein is administered to a subject in need. Administration of SARS-CoV-2 N-Fc fusion protein induces an immune response in the subject, more specifically to coronavirus infection, more specifically to SARS-CoV-2 or a variant. The immune response is indicated by the absence of observable clinical symptoms or a reduction in the clinical symptoms typically presented by an infected subject, decreased viral shedding, shortened recovery time from infection, and / or shortened duration of infection. In another embodiment, a method is provided for activating immune cells at a site of infection or disease, comprising administering a therapeutically effective amount of SARS-CoV-2 N-Fc fusion protein to a mammal. In yet another embodiment, a method is provided for increasing antibody production in a subject, comprising administering a therapeutically effective amount of SARS-CoV-2 N-Fc fusion protein to a mammal.

[0166] It should be understood that the therapeutic and prophylactic methods described herein are applicable not only to humans but also to dogs, cats, and other companion animals, as well as any suitable warm-blooded animals, including rodents, primates, horses, cattle, sheep, and pigs. These methods may also be applied to clinical research and / or trials.

[0167] As used herein, the terms “effective dose” or “therapeutically effective dose” mean a therapeutic or prophylactic dose of SARS-CoV-2 N-Fc fusion protein that, when administered according to a desired therapeutic regimen, would produce a desired therapeutic or prophylactic effect or response, including the alleviation of some or all of the symptoms of the infection or a reduction in the predisposition to infection, and which would be appropriate to embodiments of this disclosure. Those skilled in the art will recognize that a dose may be considered therapeutically “effective” if, even if the condition is not completely eradicated or prevented, the condition or its symptoms and / or effects are partially improved or alleviated in the subject. The therapeutically effective dose of SARS-CoV-2 N-Fc fusion peptide may vary depending on the size and species of the subject and the mode of administration.

[0168] The actual dose level of SARS-CoV-2 N-Fc fusion protein can be varied to obtain an amount of the active ingredient effective in achieving the desired therapeutic response for a particular subject. The selected dose level depends on various factors, including the activity of the specific fusion protein or its ester, salt, or amide being employed, the route of administration, the time of administration, the excretion rate of the specific compound being employed, the duration of treatment, other drugs, compounds, and / or materials used in combination with the specific fusion protein being employed, the age, sex, weight, condition, health status, and medical history of the subject being treated, and similar factors well known in the medical field. Generally, the appropriate dose of SARS-CoV-2 N-Fc fusion protein is the minimum dose effective in producing a therapeutic effect. Such an effective dose generally depends on the factors mentioned above.

[0169] Immunogenic formulations are provided in various embodiments in unit dosage forms for ease of administration and dose uniformity. “Unit dosage form,” as used herein, refers to physically discrete units suitable as a unit dose for a subject to be treated, each unit containing a predetermined amount of SARS-CoV-2 N-Fc fusion protein calculated to produce a desired therapeutic effect in conjunction with the required pharmaceutical carrier. The specifications of the unit dosage form are determined and directly depend on the inherent properties of the excipients and therapeutic agents, as well as the specific biological effect to be achieved. In one or more embodiments, the formulation is provided as a kit of components for the administration of SARS-CoV-2 N-Fc fusion protein to a subject. In one or more embodiments, a pharmaceutical composition comprising SARS-CoV-2 N-Fc fusion protein dispersed in a suitable carrier is provided in unit dosage forms (e.g., vials). In one or more embodiments, the kit further comprises discrete unit dosage forms (e.g., vials) containing adjuvants and / or other carrier systems for in-situ mixing of SARS-CoV-2 N-Fc fusion protein for administration. In one or more embodiments, the kit comprises one or more emulsifying needles and syringes for mixing immunogenic preparations in situ for administration. In one or more embodiments, the kit comprises one or more administration syringes for administering the prepared immunological composition to a target. In one or more embodiments, the kit further comprises instructions for preparing and / or administering the immunogenic composition.

[0170] In the example following the procedure described above and in the examples described later, it was shown that a 10 μg dose level of SARS-CoV-2 N-Fc fusion protein induced significant anti-nucleocapsid Ab titers in mice 42 days after the first injection on day 0 and the second injection on day 21. Dose levels of 10 μg and 30 μg are expected to be effective in rabbits and non-human primates.

[0171] This disclosure intends to formulate the SARS-CoV-2 N-Fc fusion protein in any of the aforementioned pharmaceutical compositions and formulations. Furthermore, this disclosure intends to administer the protein via any of the aforementioned routes of administration. Those skilled in the art can select an appropriate formulation, dose level, and route of administration based on the condition being treated and the overall health, age, and physique of the patient being treated. [Examples]

[0172] The present technology will be further described below with reference to examples. However, it should be understood that these examples are provided for illustrative purposes only, and what is described therein should not be considered to limit the overall scope of the present technology.

[0173] General examples of the synthesis, purification, and validation of SARS-CoV-2 N-Fc fusion proteins. Example 1: Synthesis and production method of SARS-CoV-2 N-Fc fusion protein in HEK293 cells The SARS-CoV-2 N-Fc fusion protein was synthesized as follows: The target gene sequence was constructed using proprietary software (LakePharma, Belmont, CA) and cloned into a high-expression mammalian vector. HEK293 cells were seeded in a shaking flask 24 hours before transfection and grown in chemically defined serum-free medium. The DNA expression construct encoding the target SARS-CoV-2 N-Fc fusion protein was transiently transfected into a suspension of HEK293 cells using the standard transient transfection procedure (LakePharma, Belmont, CA). After 20 hours, cells were counted to determine viability and the number of viable cells, and titers were measured using ForteBio® Octet® (Pall ForteBio LLC, Fremont, CA). Additional measurements were performed during transient transfection production. Cultures were collected from day 5 onwards.

[0174] Example 2: Purification of SARS-CoV-2 N-Fc fusion protein produced with HEK293 The SARS-CoV-2 N-Fc fusion protein was purified as follows: The supernatant of cell-conditioned medium containing the secreted SARS-CoV-2 N-Fc fusion protein was collected from the HEK293 production test and clarified by centrifugation. The supernatant containing the target SARS-CoV-2 N-Fc fusion protein was passed through a Protein A column, washed with a low pH gradient, and eluted. Subsequently, the eluted fraction containing the target protein was pooled and buffered with 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0 buffer. Final filtration was performed using a 0.2 μm membrane filter. The final protein concentration was calculated from the optical density of the solution at 280 nm. Further purification by ion exchange chromatography (e.g., using anion exchange bead resin or cation exchange bead resin), gel filtration chromatography, or other optional methods was performed as needed.

[0175] Example 3: Confirmation of SARS-CoV-2 N-Fc fusion protein structure using non-reducible and reduced CE-SDS Capillary electrophoresis of sodium dodecyl sulfate (CE-SDS) analysis is performed on a LabChip® GXII (Perkin Elmer, Waltham, MA) using a solution of purified SARS-CoV-2 N-Fc fusion protein dissolved in 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, and pH 7.0 buffer, and the electropherogram is plotted. Under non-reducing conditions, the sample is electrophoresed against a protein standard of known molecular weight (MW), and the elution peak represents the "apparent" MW of the fusion protein homodimer.

[0176] Under reducing conditions (for example, by using β-mercaptoethanol to cleave the disulfide bond of the SARS-CoV-2 N-Fc fusion protein homodimer), the apparent molecular weight (MW) of the obtained SARS-CoV-2 N-Fc fusion protein monomer is compared to half the molecular weight of the SARS-CoV-2 N-Fc fusion protein homodimer as a method to determine if the structural purity of the SARS-CoV-2 N-Fc fusion protein is appropriate.

[0177] Example 4: Identification of SARS-CoV-2 N-Fc fusion protein sequence by LC-MS after removal of glycans. To obtain an accurate estimate of the SARS-CoV-2 N-Fc fusion protein mass by mass spectrometry (MS), the sample is first treated to remove naturally occurring glycans that may interfere with MS analysis. 100 μL of 2.5 mg / mL SARS-CoV-2 N-Fc fusion protein, dissolved in a buffer of 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0, is first buffer-exchanged using a Zeba desalting column (Pierce, ThermoFisher Scientific, Waltham, MA) to a buffer of 0.1 M Tris, pH 8.0 containing 5 mM EDTA. 1.67 μL of PNGase F enzyme (Prozyme N-glycanase) is added to this solution to remove N-linked glycans present in the fusion protein (e.g., glycans attached to the asparagine side chain located at the cNg-N site), and the mixture is incubated overnight at 37°C. Subsequently, analysis of this sample by LC-MS (NovaBioassays, Woburn, MA) yields the molecular weight of the molecule corresponding to the target homodimer without glycans. This mass is further corrected for the deamination of the asparagine side chains to form aspartic acid by the enzymatic treatment used to cleave the glycans from cNg-asparagine, thereby increasing the overall mass of the enzymatically treated homodimer by 2 Da, which corresponds to 1 Da of mass for each chain present in the homodimer. Therefore, the actual molecular weight is obtained by subtracting 2 Da from the measured mass to correct for each enzymatic modification of the SARS-CoV-2 N-Fc fusion protein structure in the analytical sample.

[0178] Example 5: % homodimer of SARS-CoV-2 N-Fc fusion protein by size exclusion chromatography Size exclusion chromatography (SEC-HPLC) of SARS-CoV-2 N-Fc fusion proteins was performed at a wavelength of 280 nm using a Waters 2795HT HPLC (Waters Corporation, Milford, MA) connected to a 2998 photodiode array. Samples of 100 μL or less containing the target SARS-CoV-2 N-Fc fusion protein were injected at a flow rate of 0.2 mL / min into a MAbPac SEC-1, 5 μm, 4 × 300 mm column (ThermoFisher Scientific, Waltham, MA) operating with a mobile phase containing 50 mM sodium phosphate, 300 mM NaCl, and 0.05% w / v sodium azide, pH 6.2. The MAbPac SEC-1 column operates on the principle of molecular size separation. Therefore, larger soluble SARS-CoV-2 N-Fc aggregates (e.g., multimers of SARS-CoV-2 N-Fc fusion protein homodimers) elute with shorter retention times, while non-aggregated homodimers elute with slower retention times. When separating a mixture of homodimers from aggregated multimers by analytical SEC-HPLC, the purity of the SARS-CoV-2 N-Fc fusion protein solution was confirmed as a percentage of non-aggregated homodimers.

[0179] Example 6: In vitro binding affinity of Fc(γ), FcRn, and ACE2 receptor to SARS-CoV-2 N-Fc fusion protein The SARS-CoV-2 N-Fc fusion protein was conjugated to the Fc(γ) receptor at pH 7.4 using an ELISA assay as follows. Human Fc(γ) receptors I, IIa, IIb, III, and FcRn receptors were used as mammalian receptors. The SARS-CoV-2 N-Fc fusion protein was diluted to 10 μg / mL in sodium bicarbonate buffer at pH 9.6 and coated onto a Maxisorp (Nunc) microtiter plate overnight at 4°C. The microplate strip was then washed five times again with PBST (PBS / 0.05% Tween-20) buffer and blocked with Superblock blocking reagent (ThermoFisher). Serial dilutions of biotinylated rhFc(γ) receptors (recombinant human Fc(γ)RI, Fc(γ)RIIa, Fc(γ)RIIb, Fc(γ)RIII, FcRn; R&D Systems) are prepared in PBST / 10% Superblock buffer at 6000 ng / mL to 8.2 ng / mL and added to microplate strips coated with SARS-CoV-2 N-Fc fusion protein at 100 μL / well. Recombinant human ACE2 receptors are purchased from R&D Systems, biotinylated using a known binding procedure, prepared in PBST / 10% Superblock buffer at 6000 ng / mL to 8.2 ng / mL, and added to microplate strips coated with SARS-CoV-2 N-Fc fusion protein at 100 μL / well. Incubate the microtiter plate at room temperature for 1 hour, then wash the microplate strip five times with PBST, and add 100 μL / well of streptavidin-HRP diluted 1:10000 in PBST / 10% Superblock buffer. Incubate for 45 minutes, then wash the microplate strip five times with PBST. Add TMB to remove bound Fc(γ), FcRn, or ACE2 receptor proteins, and stop the ELISA with Boston Bioproducts.This plate is read at 450 nm using an ELISA plate reader, and a binding curve is created by plotting the OD value (proportional to the binding of each rhFc(γ), FcRn, or ACE2 receptor to the SARS-CoV-2 N-Fc fusion protein) against the logarithmic concentration of each rhFc(γ) receptor, FcRn, or ACE2 receptor added to each well using GraphPad Prism software.

[0180] General examples of vivo serological assays for evaluating SARS-CoV-2 N-Fc fusion protein activity in serum Example 7: In vivo quantitative ELISA for evaluating SARS-CoV-2 RBD IgG antibody titers in vaccinated mouse serum. Anti-SP / RBD IgG Ab titers were measured in serum and plasma samples from vaccinated mice containing thermally inactivated serum or plasma using a quantitative SARS-CoV-2 SP / RBD-specific IgG ELISA. In this ELISA method, recombinant SP / RBD immobilized on the plastic wells of a 96-well microtiter ELISA plate was used as a capture antigen that binds to anti-SP / RBD-specific antibodies (IgG) in serum samples during incubation in microplate wells (as shown in Figure 21).

[0181] Test serum samples (simple), diluted 1:100 to 1:500 with sample dilution buffer (SDB; containing a mixture of PBS / 0.05% Tween 20 with 10% Superblock (Thermo)), are added in two or one replicates to wells coated with SP / RBD and incubated for 1 hour. After washing the plate with PBS / 0.05% Tween 20 (PBST) buffer to remove all unbound molecules, HRP-conjugated anti-mouse IgG secondary antibody (diluted 1:40,000) is added as the detection reagent and incubated for 45 minutes. After washing with PBST buffer, HRP enzyme-catalyzed trimethylbenzidine (TMB) reagent is added to each well and incubated for 10-20 minutes. This results in a colorimetric change proportional to the amount of bound HRP-antibody complex. Subsequently, the enzyme-substrate reaction was stopped by adding Stop Reagent (1% H2SO4), and the color intensity (optical density, OD) of each well was measured at a wavelength of 450 nm using a spectrophotometric microplate reader.

[0182] Standard curves were prepared using serial dilutions of purified mouse IgG samples in known quantities (i.e., μg / mL) directly bound to plastic wells (i.e., the wells did not contain bound SP / RBD or serum samples), and in the same manner as described above for serum samples. The amount (i.e., titer) of SP / RBD-specific antibody in each serum sample was expressed in μg / mL units derived from the standard curve using a four-parameter curve-fitting model with appropriate software (e.g., SoftMaxPro or Gen 5).

[0183] Example 8: In vivo general preclinical evaluation of the efficacy of SARS-CoV-2 N-Fc formulations in inducing anti-SP / RBD IgG Ab titer or anti-nucleocapsid protein IgG titer responses in mice. In vivo studies to evaluate the efficacy of SARS-CoV-2 N-Fc fusion protein formulations were conducted in mice as follows.

[0184] BALB / c mice (n=7 / group) (Jackson Laboratories, Bar Harbor, ME) were acclimatized for at least 7 days, then their body weight was measured, and they were subsequently assigned to test groups for administration. Mice were randomly assigned to each test group. Mice were housed in auto-ventilated racks, 5 per cage, and were given ad libitum access to standard irradiated feed and filtered water. Mice were individually tagged with ear tags for identification, and test cages were labeled with animal ID, test name, test group identification, and test records including individual and group administration sheets, blood / serum collection sheets, body weight measurements, and health observation records, in accordance with standard operating procedures (SOPs) and specific test protocols.

[0185] Mice were subcutaneously administered up to three times at a dose of 10 μg / dose, with or without Montanide® ISA 720 (30% / 70% v / v), the SARS-CoV-2 N-Fc fusion protein of SEQ ID NO: 15, synthesized in HEK293 cells transiently transfected according to Example 1. Administrations were performed on days 0, 21, and 46. Mice were observed for immediate reactions 1–3 hours after administration, and then daily for health status. Serum samples for SARS-CoV-2 nucleocapsid IgG Ab titer evaluation were obtained from all mice by submandibular vein puncture to induce non-terminal bleeding on day 0 before the initial immunization, and thereafter on days 14, 21, 28, 35, 42, 55, 70, 83, and 105. The collected blood was allowed to coagulate, and the serum was separated by centrifugation of the microvacutainer tube for antibody analysis by ELISA according to the method described in Example 7 or Example 9. The serum was then aliquoted and frozen.

[0186] Example 9: ELISA for in vitro measurement of IgG antibodies against SARS-CoV-2 nucleocapsid protein (e.g., N-protein) in human serum after treatment with the SARS-CoV-2 N-Fc fusion protein of SEQ ID NO: 15 The SARS-CoV-2 N-Fc fusion protein of SEQ ID NO: 15 was synthesized according to Example 1 and purified according to Example 2. The fusion protein structure was confirmed by non-reducible and reduced CE-SDS according to Example 3, and the sequence of the fusion protein was confirmed by LC-MS after removal of glycans according to Example 4. Following the procedure of Example 8, the SARS-CoV-2 N-Fc fusion protein of SEQ ID NO: 15 was administered by subcutaneous injection to N=7 BALB / c mice (Jackson Laboratories) on days 0, 21, and 46, with or without Montanide® ISA 720 adjuvant.

[0187] The COVID-19 N-Protein Human IgG ELISA Kit (Abcam# ab274339) is an in vitro indirect ELISA for the quantitative measurement of human IgG antibodies against the SARS-CoV-2 nucleocapsid protein (e.g., N protein) in human serum. SARS-CoV-2 N protein was coated onto a standard 96-well plate (12 strips of 8 wells / strip, as shown in Figure 21), combined with the corresponding antibody present in the human serum sample and a positive control to be used as a calibration curve for interpretation. The wells were washed, and mouse IgG secondary antibody (1:40,000 dilution) was added to the mouse sample. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin was pipetted into the wells. The wells were washed again, and TMB substrate solution was added to the wells, developing color proportional to the amount of bound COVID19 N protein human / mouse IgG antibody. The Stop Solution changed the color from blue to yellow, and the color intensity was measured at 450 nm. The positive control was derived from an inactivated serum sample containing human IgG antibodies against the SARS-CoV-2N protein.

[0188] Anti-nucleocapsid protein IgG titers were measured on days 14 and 21 (after one dose), 28 and 42 (after two doses), and 55, 70, 83 and 105 (after three doses) in mice administered with the SARS-CoV-2 N-Fc fusion protein of SEQ ID NO: 15. As shown in Figure 13, the SARS-CoV-2 N-Fc fusion protein of SEQ ID NO: 15 induced significant anti-nucleocapsid protein IgG titers when measured on days 42, 55, 70 and 83. On days 42, 55, 70, and 83, the levels of anti-nucleocapsid protein IgG titers induced by the SARS-CoV-2 N-Fc fusion protein of SEQ ID NO: 15, accompanied by Montanide® ISA 720 adjuvant, were measurably elevated. This indicates that the adjuvant, when delivered with the SARS-CoV-2 N-Fc fusion protein of SEQ ID NO: 15, produces a strong enhancing effect, supporting its dose-saving properties and selection as a clinically important adjuvant.

[0189] General examples of synthesis, purification, and validation of canine insulin-Fc fusion proteins Example 10: Method for synthesizing and producing insulin-Fc fusion protein in HEK293 cells The insulin-Fc fusion protein was synthesized as follows: The target gene sequence was constructed using proprietary software (LakePharma, Belmont, CA) and cloned into a high-expression mammalian vector. HEK293 cells were seeded in a shaking flask 24 hours before transfection and grown in chemically defined serum-free medium. The DNA expression construct encoding the target insulin-Fc fusion protein was transiently transfected into a suspension of HEK293 cells using the standard transient transfection procedure (LakePharma, Belmont, CA). After 20 hours, cells were counted to determine viability and the number of live cells, and titers were measured using ForteBio® Octet® (Pall ForteBio LLC, Fremont, CA). Additional measurements were performed during transient transfection production. Cultures were collected from day 5 onwards.

[0190] Example 11: Method for synthesizing and producing insulin-Fc fusion protein in CHO cells The CHO cell line was originally derived from CHO-K1 (LakePharma, Belmont, CA), in which the endogenous glutamine synthetase (GS) gene was knocked out using recombinant techniques known in the art. A stable expression DNA vector was designed, optimized for CHO expression and GS selection, and incorporated into a high-expression mammalian vector (LakePharma, Belmont, CA). After sequencing of each complete construct, scale-up experiments were initiated. CHO cells adapted to suspension were cultured in a chemically defined medium (CD OptiCHO; Invitrogen, Carlsbad, CA) in a humidified 5% CO2 incubator at 37°C. No serum or other animal-derived products were used in culturing the CHO cells.

[0191] To generate stable CHO cell lines for each insulin-Fc fusion protein (the DNA construct contains the full-length sequence of the insulin-Fc fusion protein), approximately 80 million suspension-adapted CHO cells growing in CD OptiCHO medium during the exponential growth phase were transfected with 80 μg of DNA by electroporation using the MaxCyte® STX® system (MaxCyte, Inc., Gaithersburg, MD). After 24 hours, the transfected cells were counted and placed under selection for stable integration of the insulin-Fc fusion gene. The transfected cells were seeded at a cell density of 0.5 × 10 6 cells / mL in CD OptiCHO selection medium containing 0 - 100 μM methionine sulfoximine (MSX) in a shaker flask and incubated at 37 °C and 5% CO2. During the selection process, the cells were centrifuged and resuspended in fresh selection medium every 2 - 3 days until the CHO stable pool recovered its growth rate and viability. Cell culture growth and titer were monitored.

[0192] The cells grew to 2.5 × 10 6 cells / mL. At the time of collection for cell banking, the viability exceeded 95%. Subsequently, the cells were centrifuged and the cell pellet was resuspended in CD OptiCHO medium supplemented with 7.5% dimethyl sulfoxide (DMSO) at a cell number of 15 × 10 6 cells per mL per vial. The vials were frozen for storage in liquid nitrogen. <​Small-scale production was performed using CHO cells as follows: Cells were scaled up for production at 37°C in CD OptiCHO growth medium containing 100 μM MSX, supplemented with glucose-supplemented CD OptiCHO growth medium every 2-4 days as needed, and amino acids were added for approximately 14-21 days as needed. The supernatant of the cell-conditioned medium taken from the stable pool production test was clarified by centrifugation. This protein was passed through a Protein A (MabSelect, GE Healthcare, Little Chalfont, UK) column pre-equilibriumized with binding buffer. Next, it was passed through a washing buffer until the OD280 value (NanoDrop, Thermo Scientific) was measured to be at or near the background level. The insulin-Fc fusion protein was eluted using a low pH buffer, the eluted fraction was collected, and the OD280 value of each fraction was recorded. The fractions containing the target insulin-Fc fusion protein were pooled and, optionally, further filtered using a 0.2 μM membrane filter.

[0194] This cell line was further subcloned and monocloned at the discretion of the user, and clones expressing high-titer insulin-Fc fusion protein were selected at the discretion of the user, using the limiting dilution method, a method known to those skilled in the art. After obtaining high-titer monoclonal insulin-Fc fusion protein expressing cell lines, insulin-Fc fusion protein production was achieved in growth medium without MSX, or at the discretion of MSX, as described above, to obtain cell culture supernatant containing recombinant insulin-Fc fusion protein produced by CHO. At the discretion of the user, the MSX concentration was increased over time to further enhance selectivity for clones that yielded higher product titers.

[0195] Example 12: Purification of insulin-Fc fusion protein The insulin-Fc fusion protein was purified as follows: The supernatant of cell-conditioned medium containing the secreted insulin-Fc fusion protein was collected from transiently or stably transfected HEK293 manufacturing samples and clarified by centrifugation. The supernatant containing the target insulin-Fc fusion protein was passed through a Protein A column and eluted using a low pH gradient. The eluted fraction containing the target protein was then pooled and buffer-exchanged with 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0 buffer. Final filtration was performed using a 0.2 μm membrane filter. The final protein concentration was calculated from the optical density of the solution at 280 nm. Further purification by optional means, such as ion exchange chromatography (e.g., using anion exchange bead resin or cation exchange bead resin), gel filtration chromatography, or other methods, was performed as needed.

[0196] Example 13: Confirmation of insulin-Fc fusion protein structure using non-reducing and reduced CE-SDS Capillary electrophoresis of purified insulin-Fc fusion protein in a solution of 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, and pH 7.0 buffer was performed using a LabChip® GXII (Perkin Elmer, Waltham, MA), and the electropherogram was plotted. Under non-reducing conditions, the sample was electrophoresed against a known molecular weight (MW) protein standard, and the elution peak represented the "apparent" MW of the insulin-Fc fusion protein homodimer.

[0197] Under reducing conditions (for example, by using β-mercaptoethanol to cleave the disulfide bond of the insulin-Fc fusion protein homodimer), the apparent molecular weight (MW) of the obtained insulin-Fc fusion protein monomer is compared to half the molecular weight of the insulin-Fc fusion protein homodimer as a method to determine if the structural purity of the insulin-Fc fusion protein is appropriate.

[0198] Example 14: Identification of insulin-Fc fusion protein sequences by LC-MS after removal of glycans. To obtain an accurate estimate of the insulin-Fc fusion protein mass by mass spectrometry (MS), the sample was first treated to remove naturally occurring glycans that could interfere with MS analysis. 100 μL of 2.5 mg / mL insulin-Fc fusion protein, dissolved in a buffer of 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0, was first buffer-exchanged using a Zeba desalting column (Pierce, ThermoFisher Scientific, Waltham, MA) to a buffer of 0.1 M Tris, pH 8.0 containing 5 mM EDTA. 1.67 μL of PNGase F enzyme (Prozyme N-glycanase) was added to this solution to remove N-linked glycans present in the insulin-Fc fusion protein (e.g., glycans attached to the asparagine side chain located at the cNg-N site), and the mixture was incubated overnight at 37°C. Subsequently, analysis of this sample by LC-MS (NovaBioassays, Woburn, MA) yielded the molecular weight of the molecule corresponding to the target homodimer without glycans. This mass was further corrected for the deamination of the asparagine side chains to form aspartic acid by the enzymatic treatment used to cleave the glycans from cNg-asparagine. In doing so, the enzymatically treated homodimer increased by 2 Da overall, which corresponds to a mass of 1 Da for each chain present in the homodimer. Therefore, the actual molecular weight was obtained by subtracting 2 Da from the measured mass to correct for the enzymatic modification of the insulin-Fc fusion protein structure in the analytical sample.

[0199] Example 15: Size exclusion chromatography of insulin-Fc fusion protein to obtain % homodimer Size exclusion chromatography (SEC-HPLC) of insulin-Fc fusion proteins was performed at a wavelength of 280 nm using a Waters 2795HT HPLC (Waters Corporation, Milford, MA) connected to a 2998 photodiode array. Samples of 100 μL or less containing the target insulin-Fc fusion protein were injected at a flow rate of 0.2 mL / min into a MAbPac SEC-1, 5 μm, 4 × 300 mm column (ThermoFisher Scientific, Waltham, MA) operating with a mobile phase containing 50 mM sodium phosphate, 300 mM NaCl, and 0.05% w / v sodium azide, pH 6.2. The MAbPac SEC-1 column operates on the principle of molecular size separation. Therefore, larger soluble insulin-Fc aggregates (e.g., multimers of insulin-Fc fusion protein homodimers) elute with faster retention times, while non-aggregated homodimers elute with slower retention times. When separating a mixture of homodimers from aggregated macromeric homodimers by analytical SEC-HPLC, the purity of the insulin-Fc fusion protein solution was confirmed as the percentage of non-aggregated homodimers.

[0200] Example 16: In vitro Fc(γ) receptor I binding affinity assay for insulin-Fc fusion protein The insulin-Fc fusion protein was conjugated to the Fc(γ) receptor at pH 7.4 using an ELISA assay as described below. Since canine Fc(γ) receptor I was not commercially available, human Fc(γ) receptor I (i.e., rhFc(γ) receptor I) was used as a surrogate mammalian receptor. The insulin-Fc compound was diluted to 10 μg / mL in sodium bicarbonate buffer at pH 9.6 and coated onto Maxisorp (Nunc) microtiter plates overnight at 4°C. The microplate strips were then washed five times with PBST (PBS / 0.05% Tween-20) buffer and blocked with Superblock blocking reagent (ThermoFisher). Serial dilutions of biotinylated rhFc(γ) receptor I (recombinant human Fc(γ)RI; R&D Systems) were prepared in PBST / 10% Superblock buffer at concentrations of 6000 ng / mL to 8.2 ng / mL and added to microplate strips coated with insulin-Fc fusion protein at a concentration of 100 μL / well. Microtiter plates were incubated at room temperature for 1 hour, after which the microplate strips were washed five times with PBST. Then, 100 μL / well of streptavidin-HRP diluted 1:10000 in PBST / 10% Superblock buffer was added. After incubation for 45 minutes, the microplate strips were washed again five times with PBST. The bound Fc(γ) receptor I protein was removed by adding TMB, and the ELISA was stopped using ELISA stop reagent (Boston Bioproducts). This plate was read at 450 nm using an ELISA plate reader, and a binding curve was created by plotting the OD value (proportional to the binding of rhFc(γ) receptor I to insulin-Fc fusion protein) against the logarithmic concentration of rhFc(γ) receptor I added to each well using GraphPad Prism software.

[0201] Example 17: In vivo pharmacokinetics (PD) after regular administration of insulin-Fc fusion protein in client-owned dogs. The living insulin-Fc fusion homodimer constructs were synthesized according to Example 10 or Example 11, purified according to Example 12, and their effect on fasting blood glucose levels was evaluated as follows.

[0202] Protocol 1 is an open-label, self-controlled, single-arm, demonstrative efficacy trial treating client-owned dogs diagnosed with diabetes with insulin-Fc fusion protein. The drug's effectiveness is evaluated by comparing glycemic control (based on clinical signs, fructosamine levels, and interstitial glucose concentration using a continuous glucose monitoring unit (CGMS)) during standard insulin therapy (1 week) versus treatment with escalating doses of insulin-Fc fusion protein over 8 weeks. The dose is started at 0.1 mg / kg subcutaneously and then increased weekly to a maximum of 0.5 mg / kg based on CGMS results and clinical signs. Table 2 shows the timeline for the Protocol 1 trial used in some dogs.

[0203] [Table 2]

[0204] Protocol 2 is an open-label, self-controlled, single-arm, demonstrative efficacy trial treating client-owned dogs diagnosed with diabetes with insulin-Fc fusion protein. The drug's effectiveness is evaluated by comparing glycemic control (based on clinical signs, fructosamine levels, and interstitial glucose concentration using a continuous glucose monitoring unit (CGMS)) during standard insulin therapy (1 week) versus treatment with escalating doses of insulin-Fc fusion protein over 5 weeks. The dose is started at 0.1 mg / kg and administered subcutaneously, then increased weekly to a maximum of 0.5 mg / kg based on CGMS results and clinical signs. Table 3 shows the timeline of the Protocol 2 trial used in some dogs. [Table 3]

[0205] After completion of any of the above protocols, an optional "home use" evaluation of up to one year is available. During this stage, the veterinarian treats each dog on a case-by-case basis, similar to other patients using conventional insulin. Clinical signs and interstitial glucose concentration using a continuous glucose monitoring unit (CGMS) are monitored at this stage of the study. In addition, blood samples are taken as frequently as possible to assess fructosamine levels, blood chemistry, and blood cell counts, and to test for the presence of anti-drug and anti-insulin antibodies.

[0206] Example 18: Measurement of in vivo anti-insulin antibody (AIA) titers after periodic administration of insulin-Fc fusion protein in dogs - Protocol 1 Maxisorp ELISA plates (Nunc) were coated overnight at 4°C with purified RHI diluted to 30 μg / mL in coating buffer (pH=9.6 Carbonate-Biocarbonate buffer). The plates were then washed five times with PBST (PBS + 0.05% Tween 20) and blocked with SuperBlock blocking solution (ThermoFisher) for at least 1 hour (or overnight). To calculate AIA in canine IgG units, strips were directly coated overnight at 4°C with 1:2 serial dilutions of canine IgG (Jackson Immunoresearch) in pH=9.6 Carbonate-Biocarbonate (Carb-Biocarb) coating buffer at concentrations ranging from 300 to 4.69 ng / mL, and used to create a 7-point pseudo-standard curve. Standard strip plates were also washed and blocked with SuperBlock blocking solution for at least 1 hour (or overnight).

[0207] Test serum samples were diluted in PBST / SB / 20%HS sample dilution buffer (PBS + 0.1% Tween 20 + 10% SuperBlock + 20% horse serum) at a ratio of ≥1:100 (usually tested at 1:200) and added in two replicates to 100 μL / well of RHI coated strips. Two canine IgG coated standard strips were also added to each plate and filled with 100 μL / well of PBST / SB (PBS + 0.1% Tween 20 + 10% SuperBlock) buffer. Plates were incubated at room temperature for 1 hour. After incubation, plates were washed five times with PBST. For AIA detection, HRP-conjugated goat anti-canine IgG F(ab')2 (Jackson Immunoresearch), which cross-reacts with canine IgG, was diluted 1:10,000 in PBST / SB and added to both the sample well and standard well at a ratio of 100 μL / well. Incubation was performed in the dark at room temperature for 45 minutes. The plates were washed five times with PBST, 100 μL / well of TMB substrate (Invitrogen) was added, and the plates were allowed to develop color in the dark at room temperature for 15-20 minutes. Then, 100 μL / well of ELISA Stop Solution (Boston Bioproducts) was added to stop the color development, and the absorbance was read at 450 nm using a SpectraMax plate reader within 30 minutes. The anti-drug antibody concentration was determined by interpolating the OD value using a 4-PL pseudo-standard curve with SoftMax Pro software.

[0208] Example 19: Measurement of in vivo anti-insulin antibody (AIA) titers after periodic administration of insulin-Fc fusion protein in dogs - Protocol 2 The general procedure is as follows: Serum containing the labeled antigen is incubated overnight with and without cold insulin. Antibody-bound labeled antigen is precipitated in a 96-well plate using Protein A / G Sepharose, with each test serum in two replicates. The 96-well plate is washed to remove unbound labeled antigen. Each well is counted using a 96-well plate beta counter. The results are expressed as an index that corrects the Δcpm (counts per minute) of the test serum for the Δcpm of the positive and negative control serums in the specific assay.

[0209] The specific procedure involves preparing two buffers as follows: Buffer 1 (150 mM NaCl, 20 mM Tris-HCl, 1% BSA, 0.15% Tween-20, 0.1% sodium azide, pH 7.4) and Buffer 2 (same as Buffer 1 except that 0.1% BSA is used instead of 1% BSA). Remove fibrin clots from each serum sample by rotating and settling as needed. Next, prepare the radiolabeled insulin stock solution by dissolving 10 μCi of 125I-insulin powder in 1 mL of 5% BSA in PBS. Prepare a "hot" insulin antigen solution using 3040 μL of Buffer 1 and 160 μL of the radiolabeled insulin stock solution. Prepare a "cold inhibitor" insulin antigen solution using 2784 μL of Buffer 1, 160 μL of the radiolabeled insulin stock solution, and 256 μL of Humulin® solution (Eli Lilly, IN). All solutions should be kept on ice before use. Mix 6 μL of each serum sample with 30 μL of "hot" insulin antigen solution in a PCR tube, and mix 6 μL of each serum sample with 30 μL of "cold inhibitor" insulin antigen solution. Incubate the resulting mixtures overnight at 4°C. Then, add 150 μL of Buffer 1 to each well, incubate overnight at room temperature under an aluminum foil cover, and then coat the plates with BSA by washing and removing the wash buffer. The Protein A / G Sepharose mixture is prepared in two parts. Prepare the Protein A Sepharose solution to a concentration of 62.5 vol% with Buffer 1. Prepare the Protein G Sepharose solution to a concentration of 40 vol% with Buffer 1. Finally, prepare the Protein A / G Sepharose mixture by mixing the Protein A Sepharose solution and the Protein G Sepharose solution in a 4:1 ratio (final concentration: 50% Protein A Sepharose / 8% Protein G Sepharose). To perform the assay, add 50 μL of Protein A / G Sepharose mixture and 30 μL of overnight incubated serum solution to each well in two replicates.The plate is mixed on a plate shaker at 4°C for 45 minutes, then washed seven times with 200 μL of washing buffer per well using a Millipore plate washing device, and dried in a 37°C incubator for 15 minutes. 50 μL of scintillation cocktail (Microscint-20) is added to each well, the plate is counted using a 96-well plate counter, and the cpm of each well is determined.

[0210] The doctrine of equality In the claims, articles such as “a,” “an,” and “the” may mean one or more unless otherwise indicated or otherwise obvious from the context. A claim or statement containing “or” among one or more members of a group is deemed satisfied if one or more or all of the group members are present in, adopted or otherwise relevant to a given product or method, unless otherwise indicated or otherwise obvious from the context. This disclosure includes embodiments in which just one member of the group is present in, adopted or otherwise relevant to a given product or method. This disclosure includes embodiments in which one or more or all of the group members are present in, adopted or otherwise relevant to a given product or method.

[0211] Furthermore, this disclosure encompasses all variations, combinations, and permutations where one or more limitations, elements, terms, and descriptive terms from one or more of the enumerated claims are introduced in another claim. For example, a claim dependent on another claim may be modified to include one or more limitations found in other claims dependent on the same basic claim. Where elements are presented as a list, for example in the form of hemp-Marcush groups, each subgroup of the elements is also disclosed, and any element may be excluded from that group. In general, where this disclosure or an aspect of this disclosure is said to comprise certain elements and / or features, it should be understood that certain embodiments of this disclosure or an aspect of this disclosure comprise, or are essentially comprised of, such elements and / or features. For simplicity, these embodiments are not specifically shown in this specification in these terms. Also, note that the terms “comprise” and “include” are intended to be open and their use may allow for the inclusion of additional elements or processes. Where a scope is given, endpoints are included. Furthermore, unless otherwise indicated, or unless otherwise obvious from the context and the understanding of those skilled in the art, values ​​expressed as a range may, in different embodiments of this disclosure, assume any specific value or subrange within the range described, up to one-tenth of the lower limit unit of the range, unless the context explicitly indicates otherwise.

[0212] Further advantages of various embodiments of the present technology will become apparent to those skilled in the art upon consideration of the disclosures herein and the following examples. It will be understood that the various embodiments described herein are not necessarily mutually exclusive unless otherwise indicated herein. For example, features described or depicted in one embodiment may, but not necessarily, be included in other embodiments. Accordingly, the present invention encompasses various combinations and / or integrations of the specific embodiments described herein.

[0213] This specification also uses numerical ranges to quantify certain parameters relating to various embodiments of the invention. Where numerical ranges are provided, it should be understood that such ranges are to be interpreted as providing literal support for claims that specify only the lower limit of a range, and for claims that specify only the upper limit of a range. For example, the disclosed numerical range of about 10 to about 100 provides literal support for claims that specify “greater than about 10” (no upper limit) and claims that specify “less than about 100” (no lower limit).

Claims

1. It comprises a nucleocapsid domain and an Fc fragment, The nucleocapsid domain and the Fc fragment are linked by a peptide linker. The nucleocapsid domain has the following sequence: RSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAE (Sequence ID 8) A fusion protein comprising [the specified element].

2. The Fc fragment has the following sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 1) The fusion protein according to claim 1, comprising:

3. The linker has the following arrangement: SGGGSGGGS (Sequence No. 14) The fusion protein according to claim 1, comprising:

4. The following array: RSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAESGGGSGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 15) The fusion protein according to claim 1, comprising:

5. The fusion protein according to claim 1, which is a homodimer.

6. The fusion protein according to claim 1, wherein the Fc fragment is glycosylated.

7. An immunogenic composition comprising the fusion protein described in claim 1 and a pharmaceutically acceptable carrier.

8. The immunogenic composition according to claim 7, further comprising an adjuvant.

9. The immunogenic composition according to claim 8, wherein the adjuvant is Montanide™ ISA-720.

10. The immunogenic composition according to claim 8, wherein the fusion protein is emulsified with the adjuvant.

11. A method for increasing antibody production against an antigenic substance in a subject, A method comprising administering a therapeutically effective amount of the fusion protein described in claim 1 to the subject.

12. The method according to claim 11, wherein the subject has a measurable antibody titer against the antigenic substance before administration of the fusion protein.

13. The method according to claim 11, wherein the subject is antibody-naive before administration of the fusion protein.

14. The method according to claim 11, wherein the fusion protein is administered by injection.

15. The method according to claim 11, wherein the fusion protein is administered subcutaneously or intramuscularly.

16. The method according to claim 11, wherein the fusion protein is administered in combination with an adjuvant.

17. The method according to claim 16, further comprising pre-mixing the fusion protein and the adjuvant before administration.

18. The aforementioned premixing emulsifies the adjuvant and the fusion protein to form an emulsion. The method according to claim 17, comprising administering the emulsion to the subject.

19. A method for inducing an immune response to viral infection in a subject, A method comprising administering a therapeutically effective amount of the fusion protein described in claim 1 to the subject.

20. The method according to claim 19, wherein the subject has an antibody titer that can be measured against the viral infection before administration of the fusion protein.

21. The method according to claim 19, wherein the subject is antibody-naive before administration of the fusion protein.

22. The method according to claim 19, wherein the fusion protein is administered by injection.

23. The method according to claim 22, wherein the fusion protein is administered subcutaneously or intramuscularly.

24. The method according to claim 19, wherein the fusion protein is administered in combination with an adjuvant.

25. The method according to claim 24, further comprising pre-mixing the fusion protein and the adjuvant before administration.

26. The aforementioned premixing emulsifies the adjuvant and the fusion protein to form an emulsion. The method according to claim 25, comprising administering the emulsion to the subject.

27. A method for producing the fusion protein described in claim 1, The method comprises transiently transfecting HEK293 cells with a nucleic acid encoding the aforementioned fusion protein. Transfected HEK293 cells express the fusion protein, A method wherein the yield of the purified or isolated fusion protein is higher than 100 mg / L in any of the expression systems.

28. Cells manipulated to express the fusion protein described in claim 1.

29. cDNA encoding the fusion protein described in claim 1.

30. A fusion protein or a pharmaceutical composition thereof comprising the sequence of Sequence ID No. 15 for use in the treatment and / or prevention of SARS-CoV-2 virus infection.

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