TW202334198A - Vaccine compositions against sars-cov-2 omicron ba.4/ba.5 to prevent infection and treat long-haul covid - Google Patents

Abstract

The present disclosure is directed to amino acid sequences from SARS-CoV2 S1-RBD Omicron variants BA.4/BA.5 protein (SEQ ID NO: 11) and N, M and S2 derived Th and CTL epitope peptides (SEQ ID Nos: 17-22) and an idealized pathogen derived artificial Th epitope peptide (SEQ ID NO: 23) to offer effective prevention and treatment of long-haul COVID with specificities against SARS-CoV2 Omicron variants BA.4/BA.5. The disclosed vaccine compositions utilize amino acid sequences for the design and manufacture of optimal SARS-CoV-2 antigenic proteins, Th/CTL peptide immunogen constructs, CHO- derived S1-RBD Omicron variants BA.4/BA.5-scFc protein, and compositions thereof, as vaccines for prevention and treatment of long-haul COVID.

Description

Vaccine composition against SARS-CoV-2 OMICRON BA.4/BA.5 for preventing infection and treating long-term COVID-19

The present disclosure relates to vaccine compositions against SARS-CoV-2 Omicron BA.4/BA.5 variants for preventing infection and treating long-term COVID.

SARS is the abbreviation created in 2003 for severe acute respiratory syndrome, also known as COVID, and is the abbreviation created in 2020 for the coronavirus infectious disease. The disease may initially have few or no symptoms, or it may progress to fever, cough, shortness of breath, muscle aches and fatigue. Complications may include pneumonia and acute respiratory distress syndrome. SARS-CoV-2, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), refers to the strain of coronavirus first discovered in Wuhan, China, that causes the 2019 coronavirus infectious disease (COVID-19).

The SARS-CoV-2 Omicron lineage has swept the world, starting with the original Wuhan strain, with rapid succession of dominant subvariants, from BA.1, BA.2, and now BA.4/BA.5, which account for 10% of SARS infections More than 90% of cases have overwhelming advantages in transmissibility and neutralizing antibody escape. The most recent publications of the inventor group (Wang, CY et al 2022a and b) are attached together with the literature citations included herein for easy background reference.

Although an additional third dose of the mRNA SARS vaccine can compensate for the reduction in serum neutralizing antibodies caused by Omicron (BA.1) (20-30-fold reduction), when compared with the original Wuhan strain, as well as hospitalization rates and severe disease (80 -90% protection), it is less effective at protecting against mild and asymptomatic infections (40-50% protection). Even after the fourth dose (and the second booster for adults 18 years and older), breakthrough infections, identified as high viral loads, are common.

Omicron BA.1 has severe mutations compared with the Wuhan original strain of SARS-CoV-2, including more than 35 amino acid changes in the S protein. Related to 2 mutations in the S-1 receptor binding domain (S1-RBD, residues 319-541), 12 mutations are shared between BA.1 and BA.2, with an additional 3 mutations each in BA.1 and BA.2 and 4 unique mutations, which give BA.2 greater immune evasion capabilities. BA.4 and BA.5 have the same spike protein. They differ from BA.2 in that they have additional mutations in the wild-type amino acids at positions 69-70del, L452R, F486V, and Q493 within the spike protein, giving them a higher immune evasion ability than BA.2. BA.2 exhibits 1.3-1.5 times greater transmissibility and 1.3-fold immune evasion than BA.1, consistent with the finding that BA.1 immune sera neutralize BA.2 with a low titer by a factor of 1.3 to 1.4. Moreover, BA.2 can cause reinfection after BA.1. BA.4/BA.5 are more transmissible and resistant to immunity and monoclonal antibodies of BA.1/BA.2.

In doubly vaccinated adults, a booster (third dose) against BA.4/BA.5 induced significantly lower neutralizing titers than against BA.1/BA.2. These suggest that booster vaccination or BA.1/BA.2 infection may not achieve sufficient immunity to protect people against BA.4/BA.5, and reinfection is common.

The development of (variant-specific) vaccines with newer compositions has been strongly advocated. There remains an urgent need to develop vaccine components to prevent individuals from becoming infected with SARS-CoV-2 Omicron BA.4/BA.5 in order to control the outbreak and reduce the resulting suffering, including long-term COVID-19 and death.

This disclosure relates to vaccine compositions against SARS-CoV-2 Omicron BA.4/BA.5 for preventing infection and treating SARS (COVID) patients. More specifically, the vaccine composition uses a fusion protein produced in CHO cells as the B cell immunogen, which contains an S-RBD BA.4/BA.5 at the N-terminus with a modified hub of human IgG region is covalently linked to the Fc fragment (CH2 and CH3 regions) (Figure 1). The vaccine ingredients incorporate hybrid site-directed SARS-CoV2 Th/CTL epitope peptides to provide optimal T cell immunity to the vaccinated.

In summary, the disclosed vaccine composition utilizes the amino acid sequence of the SARS-CoV-2 Omicron BA.4/BA.5 protein to design and manufacture antigenicity derived from the SARS-CoV-2 M, N and S2 proteins. Th/CTL epitope peptides (e.g., SEQ ID NO: 17-22), CHO-derived S1-RBD Omicron BA.4/BA.5-sFc fusion protein (e.g., SEQ ID NO: 11) and formulations thereof , as a vaccine to avoid and treat COVID caused by SARS-CoV2 Omicron BA.4/BA.5.

The present disclosure relates to vaccine compositions against SARS-CoV-2 Omicron variants with specificity for Omicron BA.4/BA.5 to prevent infection and treat those suffering from SARS (i.e., COVID). More specifically, the vaccine composition uses as its B cell immunogen a fusion protein produced in CHO cells that contains the S1-RBD BA.4/BA.5 protein at the N-terminus with the hinge region and Fc fragment (CH2 and CH3 regions) human IgG. Site-directed SARS-CoV2 Th/CTL epitope peptides have also been designed and synthesized for incorporation into vaccine formulations to provide optimal T cell immunity to recipients. The disclosed vaccine system uses amino acid sequences to design and manufacture SARS-CoV2 M, N and S2-protein-derived Th/CTL epitope peptides (Sequence ID: 17-22) and T helper cells (Th) Antigenic epitopes derived from pathogen proteins (e.g., SEQ ID NO: 23), CHO-derived S1-RBD Omicron BA.4/BA.5 sFc fusion proteins (SEQ ID NO: 11), and preparations thereof As a vaccine against SARS (i.e. COVID) caused by the SARS-CoV 2 Omicron variant.

Various aspects of the disclosed invention are discussed in greater detail below.

generally

The section headings used in this article are for organizational purposes only and should not be construed as limiting the subject matter described. All references, or portions of references, cited in this application are expressly incorporated by reference in their entirety for any purpose.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The singular terms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Thus, the phrase "comprising A or B" means including A or B, or both A and B. It is also understood that all amino acid sizes and all molecular weight or molecular weight values given for polypeptides are approximations and descriptions are provided. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosed methods, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, this manual (including explanation of terms) shall prevail. Furthermore, the materials, methods, and examples disclosed herein are illustrative only and not limiting.

SARS is the abbreviation of Severe Acute Respiratory Syndrome, also known as COVID, and is the abbreviation of Corona Virus Infectious Disease. The disease may initially have few symptoms or may progress to fever, cough, shortness of breath, muscle pain and tiredness. Complications may include pneumonia and acute respiratory distress syndrome. SARS-CoV-2, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), refers to the coronavirus strain that causes coronavirus disease 2019 (COVID-19) first discovered in Wuhan, China.

for prevention SARS-COV-2 OMICRON VARIANTS BA.4/BA.5 infectious protein / win Peptide vaccine compositions.

The disclosed vaccine composition relates to a protein/peptide vaccine composition for the prevention of infection with SARS-CoV-2 Omicron Variants BA.4/BA.5.

1. Based on S1 Designer proteins for receptor binding domains

Most vaccines currently in clinical trials only target the full-length S protein to induce neutralizing antibody responses. The induced T cell response will be limited compared to the response produced by natural SARS-CoV-2 infection. The S1-RBD region is a key component of SARS-CoV-2. It is required for cell attachment and represents the major neutralizing domain of the highly similar SARS-CoV virus described in 2003, providing full-length There is a safety margin that cannot be achieved with the S antigen, when the antibodies produced by the vaccine actually help the virus infect more cells than it can infect on its own. In this case, the antibodies bind to the virus and help it enter cells more easily than the virus alone, causing more severe disease than in unvaccinated people.

Due to the clear advantages of using shorter receptor binding domain (S1-RBD) focused B cell vaccine components, protein/peptide vaccine compositions include designer proteins based on the S1-RBD, also known as S1-RBD-sFc Fusion proteins with specific Omicron specificity, such as BA.4/BA.5. As mentioned above, S1-RBD-sFc is a recombinant protein fused by the S1-RBD of SARS-CoV-2 and the single-chain fragment crystallized region (sFc) of human IgG. In addition, engineered Fc has been used as a solution for many therapeutic antibodies to minimize nonspecific binding, increase solubility, yield, thermal stability, and in vivo half-life.

In some embodiments, the vaccine composition contains the S1-RBD-sFc fusion protein of SEQ ID NOs: 10 or 11. Each of these S1-RBD-sFc proteins contains a respective S1-RBD protein (Sequence Identification Number: 1 or 2), which corresponds to amino acid residues 331-530 of the full-length S protein of SARS-CoV-2 via IgG The mutated hub region of (SEQ ID NO: 4 or 5) is fused to a single-chain Fc peptide (SEQ ID NO: 7-9).

In some embodiments, cysteine C) residues at positions 61 and 195 of the S1-RBD sequence of SEQ ID NO: 10 or 11 and Figures 3A and 3B are mutated to alanine (A) residues, ( Residues 61 and 195 of S-RBD correspond to residues 391 and 525 of the full-length S protein of the original Wuhan strain sequence identification number: 13). The C61A and C195A mutations were introduced into the S1-RBD sequence to avoid mismatching of disulfide bonds during recombinant protein expression. The amino acid sequence of S1-RBD Omicron BA.4/BA.5 representing the Omicron BA.4/BA.5 strain fused to a single-chain Fc peptide (S-RBD Omicron BA.4/BA.5-sFc) is: Serial ID: 11.

The amount of designer protein based on the S1 receptor binding region in the vaccine composition can vary depending on the need or application. The vaccine composition may include about 1 µg to about 1000 µg of the designer protein based on the S1 receptor binding region. In some embodiments, the vaccine composition contains about 10 µg to about 200 µg of a designer protein based on the S1 receptor binding region.

2.Th/CTL win peptide

Neutralization responses targeting the S protein alone are unlikely to provide durable protection against SARS-CoV-2 and its emerging variants harboring mutated B cell epitopes. A durable cellular response can enhance the initial neutralizing response (via memory B cell activation) and provide a longer duration of immunity as antibody titers wane. Recent studies have shown that within 2-3 months, >90% of SARS-CoV-2-infected individuals have a rapid decline in IgG responses to S (Long, Q.X., et al., 2020). In contrast, SARS-specific memory T cells have been shown to persist for 11–17 years following the 2003 SARS outbreak (Ng, O.W., et al., 2016; and Le Bert, N., et al., 2020 ). S protein is a key antigen that triggers humoral immunity and mainly contains CD4+ epitopes (Braun, J., et al., 2020). Additional antigens are needed to enhance/enhance the cellular immune response to clear SARS-CoV-2 infection. The vast majority of reported CD8+ T cell epitopes in SARS-CoV-2 proteins are located in the ORF1ab, N, M and ORF3a regions; only 3 are located in S, and only 1 CD8+ epitope is located in S1-RBD (Ferretti , A.P., et al., 2020). The smaller M and N structural proteins were recognized by T cells from patients who successfully controlled the infection. In a study of nearly 3,000 people in the UK, it was found that people with higher numbers of T cells were more protected against SARS-CoV-2 than those with lower T-cell responses, suggesting that T-cell immunity may Play a key role in avoiding COVID-19 (Wyllie, D., et al., 2020).

To provide immunogens that elicit T cell responses, Th/CTL epitopes from highly conserved sequences of the S, N, and M proteins of SARS-CoV and SARS-CoV-2 were identified. These Th/CTL peptides are shown in Tables 3 and 4 . Each selected peptide contains a Th or CTL epitope, is pre-validated for MHC I or II binding, and exhibits good manufacturability characteristics (optimal length for high-quality synthesis). They should also best demonstrate the intrinsic ability to stimulate PBMCs in ordinary individuals. These Th/CTL peptides have undergone extensive screening, identification, validation and design, and are further modified by adding a Lys-Lys-Lys tail to the N-terminus of each peptide to improve the solubility of the peptide and enrich the potential for vaccine formulations. Positive charge. The design and sequences of the five final peptides and their respective HLA alleles are shown in Table 3 .

In order to enhance the immune response, the proprietary peptide UBITh®1a (SEQ ID NO: 23) can be added as a catalyst to the peptide mixture of the vaccine composition. UBITh®1a is a proprietary synthetic peptide with original framework sequences derived from the measles virus fusion protein (MVF). The sequence was further modified to display a palindromic profile within the sequence, allowing for the accommodation of multiple MHC class II binding motifs within the short 19 amino acid peptide. A Lys-Lys-Lys sequence was also added to the N-terminus of this artificial Th peptide to increase its positive charge, thus facilitating the subsequent binding of the peptide to highly negatively charged CpG oligonucleotide molecules through "charge neutralization" "Formation of immunostimulatory complexes. In previous studies, the linkage of UBITh®1a to the target "functional B epitope peptide" derived from self-protein made the self-peptide immunogenic, thus disrupting immune tolerance (Wang, C.Y., et al. , 2017). The Th epitope of UBITh®1 exhibits this stimulatory activity whether covalently linked to the target peptide or as a free charged peptide administered together with other designed target peptides that interact with CpG1 Charge-neutralizing effects combine to trigger site-directed B or CTL reactions. Such immunostimulatory complexes have been shown to enhance weak or moderate responses accompanying target immunogens (e.g., WO2020/132275A1).

CpG1 is engineered to bring together rationally designed immunogens via "charge neutralization" to allow for the generation of balanced B cell (induction of neutralizing antibodies) and Th/CTL responses in the vaccinated host. In addition, activation of TLR-9 signaling by CpG is known to promote IgA production and favor Th1 immune responses. UBITh®1 peptide was included as one of the Th peptides due to its "cluster" properties to further enhance the antiviral activity of SARS-CoV-2-derived Th and CTL epitope peptides. The amino acid sequence of UBITh®1 is SEQ ID NO: 23. The nucleic acid sequence of CpG1 is SEQ ID NO: 26.

In view of the above, the protein/peptide vaccine composition may comprise one or more Th/CTL peptides. Th/CTL peptides can include:

a. Peptide derived from SARS-CoV-2M protein (for example, sequence identification number: 21);

b. Peptide derived from SARS-CoV-2N protein (for example, sequence identification number: 19);

c. Peptides derived from SARS-Cov-2S protein (for example, sequence identification numbers: 17, 18, 20, 22); and/or

d. Artificial Th epitopes derived from pathogen proteins (eg, SEQ ID NO: 23).

Vaccine compositions may contain one or more Th/CTL peptides. In certain embodiments, vaccine compositions include a mixture of more than one Th/CTL peptide. When present in a mixture, each Th/CTL peptide may be present in any amount or ratio compared to the other peptide or peptides. For example, the Th/CTL peptides can be mixed in equimolar, equal weight amounts, or the amount of each peptide in the mixture can be different from the amount of other peptides in the mixture. If more than two Th/CTL peptides are present in the mixture, the amount of peptide may be the same as or different from any other peptide in the mixture.

The amount of Th/CTL peptide present in the vaccine composition can vary depending on the need or application. The vaccine composition may contain a total amount of Th/CTL peptide between about 0.1 μg and about 100 μg. In some embodiments, the vaccine composition contains a total of about 1 μg to about 50 μg of Th/CTL peptides. In certain embodiments, the vaccine composition includes a mixture of SEQ ID NO: 17-22. These Th/CTL peptides can be mixed in equimolar amounts, equal weights, or the amount of each peptide in the mixture can be different from the amount of other peptides in the mixture. In certain embodiments, these Th/CTL peptides are mixed in equal weight amounts in the vaccine composition.

The presence of Th and CTL epitopes in pharmaceutical/vaccine formulations triggers an immune response in the treated subject by initiating antigen-specific T cell activation, which is associated with protection from SARS-CoV-2 infection. In addition, formulations containing carefully selected endogenous Th epitopes and/or CTL epitopes on SARS-CoV-2 proteins can generate broad cell-mediated immunity, which also makes these formulations effective in treating and protecting Subjects with different genetic makeup.

In pharmaceutical compositions containing one or more endogenous SARS-CoV-2 Th/CTL epitope peptides, the S1-RBDOmicronBA.4/BA.5-sFc protein brings the peptides into close contact with each other, thereby making the epitope consist of Antigens are seen and processed by B cells, macrophages, dendritic cells, etc. These cells process the antigens and present them to surfaces for contact with B cells to produce antibodies, while T cells trigger further T cell responses to help mediate the killing of virus-infected cells. The endogenous SARS-CoV-2 CTL epitope peptide contains a Lys-Lys-Lys (KKK) tail at the N-terminus.

The endogenous SARS-CoVTh/CTL epitope peptides of SEQ ID NO: 17-22 and 23 are particularly useful when used in pharmaceutical compositions formulated with CpG oligonucleotides (ODN) into immunostimulatory complexes , because the cationic KKK tail is able to interact with CpGODN through electrostatic association. Use of endogenous SARS-CoV-2 Th epitopes in peptide immunogen constructs enhances the immunogenicity of S1-RBDOmicronBA.4/BA.5B cell epitope peptides, thereby promoting specificity and high efficacy following infection Generation of high-valent antibodies against optimized S1-RBDB cell epitope peptides based on screening and selection based on design principles.

In some embodiments, the pharmaceutical composition includes one or more S1-RBDOmicronBA.4/BA.5sFc fusion proteins (SEQ ID NO: 10, 11 or any combination thereof) and one or more S1-RBDOmicronBA.4/BA.5sFc fusion proteins containing endogenous SARS-CoV-2Th /CTL epitope peptide (SEQ ID NO: 17-22 and 23, or any combination thereof).

3. Excipients

Vaccine compositions may also contain pharmaceutically acceptable excipients.

As used herein, the term "excipient" or "excipient" refers to any component of the vaccine composition that is not (a) a designer protein based on the S1-receptor binding region or (b) a Th/CTL peptide. Examples of excipients include carriers, adjuvants, antioxidants, binders, buffers, fillers, chelating agents, colorants, diluents, disintegrants, emulsifiers, surfactants, solvents, fillers, gums Coagulants, pH buffers, preservatives, solubilizers, stabilizers, etc. Therefore, the vaccine composition may contain pharmaceuticals.

The vaccine composition may contain one or more adjuvants, which function to accelerate, prolong or enhance the immune response to the API without having any specific antigenic effect. Adjuvants may include oils, oil emulsions, aluminum salts, calcium salts, immunostimulatory complexes, bacterial and viral derivatives, virions, carbohydrates, cytokines, polymeric particles. In certain embodiments, the adjuvant can be selected from the group consisting of CpG oligonucleotides, alum (e.g., potassium aluminum phosphate), aluminum phosphate (e.g., ADJU-PHOS®), aluminum hydroxide (e.g., ALHYDROGEL®), calcium phosphate, incomplete Freund's Adjuvant (IFA), Freund's Complete Adjuvant, MF59, Adjuvant 65, Lipovant, ISCOM, liposyn, saponin, squalene, L121, EMULSIGEN®, EmulsIL-6n®, Monophosphoryl Lipid A (MPL) , Quil A, QS21, MONTANIDE® ISA 35, ISA 50V, ISA 50V2, ISA 51, ISA 206, ISA 720, liposomes, phospholipids, peptidoglycan, lipopolysaccharide (LPS), ASO1, ASO2, ASO3, ASO4, AF03 , lipophilic phospholipids (lipid A), gamma inulin, algammulin, dextran, dextran, glucomannan, galactomannan, fructans (levans), xylans (xylans), dimethyl Dimethyldioctadecylammonium bromide (DDA), as well as other additives and emulsifiers.

In some embodiments, the vaccine composition includes ADJU-PHOS® (aluminum phosphate), MONTANIDE™ ISA 51 (an oil adjuvant composition consisting of vegetable oil and mannitol oleate used to produce a water-in-oil emulsion), TWEEN® 80 (also known as: Polysorbate 80 or Polyoxyethylene (20) sorbitan monooleate), CpG oligonucleotides and/or or any combination thereof. In other embodiments, the pharmaceutical composition is a water-in-oil-in-water (i.e., w/o/w) emulsion with EMULSIGEN or EMULSIGEN D as an adjuvant.

In certain embodiments, multi-epitope protein/peptide vaccine compositions contain ADJU-PHOS® (aluminum phosphate) as an adjuvant to improve immune response. Aluminum phosphate acts as a Th2-directed adjuvant through the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome pathway. In addition, it has pro-phagocytic and storage effects, has a long safety record, and is able to improve immune responses to target proteins in many vaccine formulations.

The vaccine composition may contain pH adjusters and/or buffers, such as hydrochloric acid, phosphoric acid, citric acid, acetic acid, histidine, histidine HCl·H ₂ O, lactic acid, tromethamine, gluconic acid, asparagine Acid, glutamic acid, tartaric acid, succinic acid, malic acid, fumaric acid, alpha-ketoglutarate and arginine HCl.

Vaccine compositions may contain surfactants and emulsifiers, such as polyoxyethylene sorbitan fatty acid ester (Polysorbate, TWEEN®), polyoxyethylene 15 hydroxy stearate (Macrogol 15 hydroxy stearate, SOLUTOL HS15®), Polyoxyethylene castor oil derivatives (CREMOPHOR® EL, ELP, RH 40), Polyoxyethylene stearates (MYRJ®), Sorbitan fatty acid ester (Sorbitan fatty acid ester) acid esters) (SPAN®), Polyoxyethylene alkyl ethers (BRIJ®) and Polyoxyethylene nonylphenol ether (NONOXYNOL®).

Vaccine compositions may include carriers, solvents, or osmotic maintainers such as water, alcohols, and saline solutions (eg, sodium chloride).

Vaccine compositions may contain preservatives such as alkyl/aryl alcohols (e.g., benzyl alcohol, chlorobutanol, 2-ethoxyethanol), amino aryl acid esters (e.g., methyl, Ethyl, propyl butyl parabens and combinations), alkyl/aryl acids (e.g., benzoic acid, sorbic acid), biguanides (e.g., chlorhexidine), aromatic ethers (e.g., phenol , 3-cresol, 2-phenoxyethanol), organic mercury (such as thimerosal, phenylmercurate salts).

4. formula

Vaccine compositions can be formulated as immediate release or sustained release formulations. Additionally, vaccine compositions can be formulated to induce systemic or local mucosal immunity via immunogen entrapment and co-administration with microparticles. Such delivery systems are readily identifiable by those of ordinary skill in the art.

Vaccine compositions may be prepared as injectables, or as liquid solutions or suspensions. A liquid carrier containing the vaccine composition can also be prepared prior to injection. Vaccine compositions may be administered by any suitable mode of application, eg, i.d., i.p., i.m., intranasally, orally, subcutaneously, etc., and in any suitable delivery device. In certain embodiments, vaccine compositions are formulated for subcutaneous, intradermal, or intramuscular administration. Vaccine compositions may also be formulated for other modes of administration, including oral and intranasal application.

Vaccine compositions may also be formulated in suitable dosage unit form.

In some embodiments, the vaccine composition contains about 1 μg to about 1,000 μg of API (eg, a designer protein based on the S1 receptor binding region and/or one or more Th/CTL peptides). The effective dose of a vaccine composition can vary based on many different factors, including the mode of administration, the target site, the physiological state of the subject, whether the subject is human or animal, other drugs administered, and whether the treatment is prophylactic or therapeutic sexual. Typically, the subject is a human, but non-human mammals can also be treated. When delivered in multiple doses, the vaccine composition can be conveniently divided into appropriate amounts/dosage unit forms. The dosage administered will depend on the age, weight and general health of the subject, as is well known in the therapeutic field.

In some embodiments, the vaccine composition includes a designer protein based on the S1 receptor binding region and one or more Th/CT winning L peptides in a formulation with additives and/or excipients. In certain embodiments, the vaccine composition includes a designer protein based on the S1 receptor binding region and more than one Th/CTL peptide in a formulation with additives and/or excipients. Vaccine compositions containing more than one Th/CTL peptide mixture may synergistically enhance the immune efficacy of the composition. Vaccine compositions containing designer proteins based on the S1 receptor binding region and more than one Th/CTL peptide in formulations with additives and/or excipients are compared with compositions containing only the designer protein or one Th/CTL peptide In comparison, due to the broad MHC class II coverage, it can be more effective in a larger genetic population, thus providing a better immune response to the vaccine composition.

When the vaccine composition includes a designer protein based on the S1 receptor binding region and one or more Th/CTL peptides as an API, the relative amounts of the designer protein and Th/CTL peptides may be present in any amount or ratio with each other. For example, the designed protein and Th/CTL peptide can be mixed in equimolar amounts and equal weights, or the amounts of the designed protein and Th/CTL peptide can be different. In addition, if more than one Th/CTL peptide is present in the composition, the amounts of the designed protein and each Th/CTL peptide can be the same or different from each other. In some embodiments, a molar or weight amount of the designer protein is present in the composition in an amount greater than the Th/CTL peptide. In other embodiments, the molar or weight amount of the designer protein is present in the composition in an amount less than the Th/CTL peptide. The ratio (wt:wt) of designer protein to Th/CTL peptide can vary depending on the need or application. In some cases, the ratio (w:w) of the designed protein to Th/CTL peptide can be 70:30, 80:20, or 90:10. In specific embodiments, the ratio (w:w) of the designed peptide to Th/CTL peptide is 90:10, 88:12 or 85:15, etc. In a specific embodiment, the ratio (w:w) of the designed protein to Th/CTL peptide is 88:12.

In some embodiments, the vaccine composition comprises one or more S1 receptor binding domain-based designer proteins of SEQ ID NO: 10, 11. In other embodiments, the vaccine composition includes one or more Th/CTL peptides. In some embodiments, the vaccine composition comprises an S1 receptor binding domain-based designer protein from one of SEQ ID NOs: 10, 11 in combination with a Th/CTL peptide of SEQ ID NOs: 17-22 and 23.

In some embodiments, the vaccine composition includes one of the S1-receptor binding region-based design proteins of SEQ ID NO: 10 and 11 and one of the Th/CTL peptides of SEQ ID NO: 17-22 and 23. or a variety of more adjuvants and/or excipients. In various embodiments, the vaccine composition includes one or more designed proteins based on the S1 receptor binding region of SEQ ID NO: 10 and 11 and Th/CTL peptides of SEQ ID NO: 17-22 and 23, wherein Th/ CTL peptides exist in an equal weight ratio to each other. The combined weight ratio (w:w) of one or more designed proteins based on the S1 receptor binding region of Sequence ID: 10 and 11 and Th/CTL peptides is 88: 12. Tables 7-9 provide specific embodiments of vaccine compositions containing 20 μg/mL, 40 μg/mL, 60 μg/mL and 200 μg/mL respectively, based on S1-RBD Omicron BA.4/BA.5- The total weight of the sfc protein (SEQ ID NO: 11) and one of the Th/CTL peptides SEQ ID NO: 17-22 and 23.

5. method

The present disclosure also relates to methods of making and using vaccine compositions and formulations thereof.

a. Methods for manufacturing designed proteins and Th/CTL peptides based on the S1 receptor binding region.

The disclosed designed protein based on the S1-receptor binding region can be produced according to the method described in Examples 2 and 3. In addition, the disclosed Th/CTL peptides can be produced according to the method described in Example 1.

b. Methods of manufacturing vaccine compositions.

The disclosed vaccine compositions can be prepared according to the methods described in Examples 5 and 6 and their compounding procedures.

c. Methods of using vaccine compositions.

In preventive applications, the disclosed protein/peptide vaccine compositions can be administered to subjects susceptible to or at risk of infection with SARS-CoV-2 and its related variants, resulting in viral elimination or risk reduction or mitigation of COVID. severity, or delaying the onset of disease.

The amount of vaccine composition sufficient to accomplish preventive treatment is defined as the prophylactically effective dose. The disclosed protein/peptide vaccine compositions can be administered to a subject in one or more doses to generate a sufficient immune response to prevent SARS-CoV-2 infection. Typically, the immune response is monitored and the dose is repeated if the immune response begins to wane.

Vaccine compositions can be formulated as immediate release or sustained release formulations. Additionally, vaccine compositions can be formulated to induce systemic or local mucosal immunity via immunogen entrapment and co-administration with microparticles. Such delivery systems are readily identifiable by those of ordinary skill in the art. Vaccine compositions can be prepared as injectables, or as liquid solutions or suspensions. Liquid vehicles containing the vaccine composition can also be prepared prior to injection. Vaccine compositions may be administered by any suitable mode of application, eg, i.d., i.p., i.m., intranasally, orally, subcutaneously, etc., and in any suitable delivery device. In certain embodiments, vaccine compositions are formulated for subcutaneous, intradermal, or intramuscular administration. Vaccine compositions may also be formulated for other modes of administration, including oral and intranasal application.

The dosage of the vaccine composition will vary depending on the subject and the particular mode of administration. The required dosage will vary depending on many factors known to those of ordinary skill in the art, including, but not limited to, the type and size of the subject. Dosage can range from 1 μg to 1,000 μg based on the total weight of the design protein and Th/CTL peptide. The ratio (weight:weight) of designer protein to Th/CTL peptide can vary depending on the need or application. In some cases, the ratio (w:w) of designer protein to Th/CTL peptide can be 70:30, 80:20, or 90:10. In specific embodiments, the ratio (w:w) of the designed protein to Th/CTL peptide is 95:5, 90:10, 88:12 or 85:15, etc. In a specific embodiment, the ratio (w:w) of the designed protein to Th/CTL peptide is 88:12. In specific embodiments, the vaccine compositions comprise the components shown in Tables 7-9.

The vaccine composition can be administered in a single dose or in multiple doses over a period of time.

Vaccine compositions can be administered according to specific dosage schedules. Effective doses can be extrapolated from dose-response curves obtained in animal models. In some embodiments, the vaccine composition is provided to the subject in a single administration. In other embodiments, the vaccine composition is provided to the subject in multiple administrations (two or more). When provided as multiple doses, the duration between doses may vary depending on the application or need. In some embodiments, the subject is provided with a first dose of the vaccine composition and a second dose is provided about 1 week to about 12 weeks after the first dose. In certain embodiments, the second dose is administered about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about Dosing at or about 11 weeks. In a specific embodiment, the second dose is administered approximately 4 weeks after the first dose.

A booster dose of the vaccine composition can be administered to the subject after the initial vaccination regimen to increase immunity to SARS-CoV-2. In some embodiments, a booster dose of the vaccine composition is administered to the subject about 6 months to about 10 years after the initial vaccination regimen. In certain embodiments, the booster dose of the vaccine composition is about 3 months, about 6 months, about 1 year, about 2 years, about 3 years, about 4 years after the initial vaccination regimen or the last booster dose. , administered at about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years.

Specific embodiments of the present invention include, but are not limited to, the following embodiments.

1. A fusion protein, comprising an Fc fragment of an IgG molecule and a biologically active molecule, wherein the Fc fragment is a single chain Fc (single chain Fc, sFc), wherein the Fc fragment includes a hinge region, Wherein the hub region is mutated and does not form a disulfide bond, wherein the hub region includes an amino acid sequence selected from the group consisting of sequence identification numbers: 3 to 5, wherein the biologically active molecule is from SARS- A receptor binding domain (RBD) of the S protein (S1-RBD) of CoV2 (sequence identification number: 1 or 2), one of which is the Wuhan strain (Wuhan), the sequence identification number is: 1, one of which is Omicron BA The .4/BA.5 variant has sequence identification number: 2.

2. The fusion protein of claim 1, wherein the fusion protein is selected from the group consisting of sequence identification numbers: 10 and 11.

3. The fusion protein of claim 1, wherein the hub region includes an amino acid sequence of sequence identification number: 4.

4. A pharmaceutical composition, including the fusion protein of claim 1 and a pharmaceutically acceptable carrier or excipient.

5. A method for producing the fusion protein of claim 1, comprising: a) Provide a biologically active molecule, wherein the biologically active molecule is a receptor binding domain (RBD) (Sequence Identification Number: 1) of the S protein (S-RBD) from SARS-CoV2 Wuhan or its Omicron BA.4/ One of the BA.5 variants, wherein the receptor binding domain (RBD) of the S protein (S-RBD) is sequence identification number: 2, b) Provide an Fc fragment of an IgG molecule, wherein the Fc fragment includes a hub region, wherein the hub region is mutated by substitution and/or deletion of a cysteine residue to form a mutated Fc, and the The mutated Fc does not form a disulfide bond, wherein the hub region includes an amino acid sequence selected from the group consisting of Sequence Identification Number: 3-5, and c) Binding the biologically active molecule to the mutated Fc via the hub region.

6. A fusion protein selected from the group consisting of S1-RBD Omicron BA.4/BA.5 variant-sFc with sequence identification number: 11.

7. A composition comprising the fusion protein of claim 6.

8. The composition of claim 7, further comprising a Th/CTL peptide, wherein the Th/CTL peptide is derived from SARS-CoV-2 M, N or S protein, a pathogen protein, or any combination thereof , wherein the Th/CTL peptide is selected from the group consisting of sequence identification numbers: 17-23 and any combination thereof.

9. The composition of claim 8, wherein the Th/CTL peptide is selected from the group consisting of sequence identification numbers: 17-23 and any combination thereof.

10. A COVID vaccine composition comprising: a). An S-RBD Omicron BA.4/BA.5 variant protein, selected from the group with sequence identification number: 11; b). A Th/CTL peptide, selected from the group consisting of free sequence identification numbers: 17-23 and any combination thereof; c). A pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient is an adjuvant, buffer, surfactant, emulsifier, pH adjuster, saline solution, preservative, solvent, or any combination thereof.

11. For the COVID vaccine composition of claim 10, the Th/CTL peptide in (b) is selected from the group consisting of sequence identification numbers: 17-23 and any combination thereof.

12. For example, the COVID vaccine composition of claim 11, wherein the pharmaceutically acceptable excipient is a CpG1 oligonucleotide, ALUM (aluminum phosphate or aluminum hydroxide), histidine, histidine HCl·H ₂ O, arginine HCl, TWEEN 80 (polyoxyethylene (20) sorbitan monooleate), hydrochloric acid, sodium chloride and 2-phenoxyethanol in water ( 2-phenoxyethanol).

13. For example, the COVID vaccine composition of claim 12, wherein the pharmaceutically acceptable excipient is CpG1 (sequence identification number: 26).

14. A method of preventing COVID in an individual, comprising administering to the individual a pharmaceutically effective amount of the vaccine composition of claim 10.

15. A method of preventing COVID in an individual, comprising administering to the individual a pharmaceutically effective amount of the vaccine composition of claim 11.

16. A method of generating antibodies against the SARS-CoV-2 Omicron BA.4/BA.5 variant, comprising administering a pharmaceutically effective amount of the vaccine composition of claim 10 to an individual.

17. A method of generating antibodies against the SARS-CoV-2 Omicron BA.4/BA.5 variant, comprising administering a pharmaceutically effective amount of the vaccine composition of claim 11 to an individual.

18. A COVID vaccine composition comprising an ingredient in any of Tables 7-9 in the amounts indicated.

19. A cell strain transfected with a cDNA sequence encoding the fusion protein of claim 6 (sequence identification number: 16).

20. The cell line of claim 19, which is a Chinese Hamster Ovary (CHO) cell line.

21. For example, the cell strain of claim 19, wherein the cDNA sequence is selected from the group consisting of sequence identification number: 16.

Example 1

Synthesis of SARS-CoV2 related peptides.

SARS-CoV-2-related Th and CTL peptides serve as immunogens for vaccine development, can be synthesized in small-scale quantities useful for serological testing, laboratory experiments and field studies, and can be used in pharmaceutical compositions It is synthesized in large-scale (kilogram) quantities for commercial production of the substance. A large repertoire of SARS-CoV2-related Th/CTL epitope peptides ranging from approximately 9 to 40 amino acids in length was designed and selected as peptide immunogenic constructs for use in vaccine formulations.

Tables 3 and 4 provide the sequences of Th/CTL peptides (SEQ ID NO: 17-22) derived from SARS-CoV-2 M, N and S proteins with known MHC binding activity as designer peptides (e.g., For better formulation, having KKK as a linker at the N-terminus to increase its positive charge) was used for inclusion in the final SARS-CoV2 vaccine formulation. For T cell activation in the vaccine composition, an ideal artificial Th epitope peptide (SEQ ID NO: 23) is also used as a catalyst.

Use F-moc chemistry to synthesize all peptides that can be used in immunogen studies or related serology tests on a small scale using Applied BioSystems Models 430A, 431 and/or 433 peptide synthesizers. Each peptide is produced by independent synthesis on a solid support, accompanied by N-terminal F-moc protection and trifunctional amino acid side chain protecting groups. After synthesis, the peptides were cleaved from the solid support, followed by removal of side chain protecting groups with 90% trifluoroacetic acid (TFA). The synthesized peptide preparation was evaluated by Matrix-Assisted Laser Desorption/Ionization-Time-Of-Flight (MALDI-TOF) mass spectrometry to confirm the correct molecular weight. and amino acid content. Each synthesized peptide was evaluated by reverse phase HPLC (RP-HPLC) to confirm the synthesis profile and concentration of the preparation. Although the synthesis process is tightly controlled, including step-by-step monitoring of coupling efficiency, peptide analogs are generated due to unexpected events during the extension cycle, including amino acid insertions, deletions, substitutions, and premature terminations. Therefore, synthetic preparations often contain multiple peptide analogs, albeit in small amounts, together with the target peptide.

Despite the inclusion of such unintended peptide analogs, the resulting peptide preparations are suitable for immunological applications and as peptide immunogens. In general, such peptide analogs are often as effective as the purified peptides, as long as a discernible QC program is developed to monitor both the manufacturing and evaluation processes to ensure that the final product using these peptides is reproducible and effective. sex. On a custom-made automatic peptide synthesizer UBI2003, large-scale peptide synthesis of hundreds to thousands of kilograms was carried out on a scale of 15 mmole to 150 mmole.

For active ingredients in final pharmaceutical compositions for clinical trials or commercial use, peptide immunogenic constructs are purified by preparative RP-HPLC under a shallow elution gradient and analyzed by MALDI-TOF mass spectrometry, amine Basic acid analysis and RP-HPLC were used to characterize purity and identity.

Example 2

Design, plasmid construction, and performance of S-RBD fusion protein in CHO cells.

1. Design of cDNA sequences

Optimized cDNA sequences encoding SARS-CoV2-RBD Wuhan Sequence ID: 12) and SARS-CoV2-RBD Omicron BA.4/BA.5 (Sequence ID: 13) for CHO cell expression. In order to produce S-RBD Wuhan-scFc (DNA sequence identification number: 15) and SARS-CoV2-RBD Omicron variant BA.4/BA.5-sFc (DNA sequence identification number: 16) fusion proteins, the encoding SARS- The nucleic acid sequences of S-RBD Wuhan and Omicron _BA.4/BA.5 (aa331-530) of CoV-2 (DNA sequence identification numbers: 12 and 13) are fused to the single strand of immunoglobulin Fc (DNA sequence identification number: 14), along with the plasmid map shown in Figure 2. Plasmids carrying the genes for the respective S-RBD Omicron _BA.4/BA.5 -sFc proteins will be transfected into the CHO cell system and produce the respective S-RBD-sFc fusion proteins (amino acid sequence identification number: 11; DNA Serial ID: 16).

Since no disulfide bonds are formed in the hub region, large proteins fused to sFc will not limit the performance of the corresponding S-RBD protein. The single-chain Fc structure also has the advantage of being purified via a "Protein A Binding and Elution" purification process.

2. Plastid constructs and protein expression

a. plastid construct

To express the S-RBD-sFc fusion protein, cDNA encoding each of these target protein lipids is generated in a suitable cell line. The N-terminus of the cDNA fragment is added with a leader signal sequence for protein secretion, while the C-terminus can be connected to a single-stranded Fc (sFc). The cDNA fragment was inserted into the pND expression vector, which contains the neomycin resistance gene for screening and the dhfr gene for gene amplification. The vector and cDNA fragment were digested with PacI/EcoRV restriction enzymes and then ligated to generate four expression vectors, each corresponding to pS-RBD Omicron BA.4/BA.5-sFc.

b. host cell strain

CHO-S™ cell line (Gibco, A1134601) is a stable aneuploidy cell line established from the ovaries of adult Chinese hamsters. The host cell line CHO-S™ is adapted to serum-free suspension growth and is compatible with FREESTYLE™ MAX reagent to achieve high transfection efficiency. CHO-S cells were cultured in DYNAMIS™ medium (Gibco, Cat. 0010057DG) supplemented with 8 mM glutamine supplement (Life Technologies, Cat. 25030081) and anti-clumping agent (Gibco, Cat. 0010057DG). Cat. A26175-01).

ExpiCHO-S™ cell line (Gibco, Cat. A29127) is a clonal derivative of the CHO-S cell line. ExpiCHO-S™ cells are adapted to high-density suspension culture in ExpiCHO™ Performance Medium (Gibco, Cat. A29100) without any supplementation. Cells were maintained in a 37°C incubator with a humidified atmosphere of 8% _CO2 .

c. instantaneous performance (transient expression)

For transient expression, expression vectors were individually transfected into ExpiCHO-S cells using the EXPIFECTAMINE™ CHO Kit (Gibco, Cat. A29129). On day 1 post-transfection, add EXPIFECTAMINE™ CHO Enhancer with the first feed and transfer cells from a 37°C incubator with a humidified atmosphere of 8% _CO to a humidified atmosphere with 5% _CO of a 32°C incubator. Afterwards, a second feed was added on day 5 post-transfection, and cells were harvested 12-14 days post-transfection. After harvesting the cell culture, the supernatant was clarified by centrifugation and 0.22-µm filtration. Recombinant proteins containing single-chain Fc and His-tags were purified by Protein A chromatography (Gibco, Cat. 101006) and Ni-NTA chromatography (Invitrogen, Cat. R90101) respectively.

d. Stable transfection and cell screening

The expression vector was transfected into CHO-S cells using FreeStyle MAX reagent (Gibco, Cat. 16447500), followed by anti-agglutination reagent containing 8 mM L-glutamine diluted 1:100, puromycin. (puromycin) (InvovoGen, Cat. ant-pr-1) and MTX (Sigma, Cat. M8407) were cultured in selected DYNAMIS™ medium. After 2 rounds of selection stages, four stable pools (1A, 1B, 2A, 2B) were obtained. In addition, cell colonies were plated in semi-solid CloneMedia (Molecular Devices, Cat. K8700), and detection antibodies were added for colony screening and single-cell selection via the high-throughput system ClonePixTM2 (CP2). separation. Colonies selected via CP2 were screened without selection using a 14-day glucose simple fed-batch culture in DYNAMIS™ medium with 8mM glutamine and anti-agglutination reagent. After screening, single-cell isolation of selected colonies with high yield was performed by limiting dilution and monoclonality was confirmed by imaging using CloneSelect Imager (Molecular Devices).

e. Simple feeding batch culture

A simple fed batch culture was used to confirm the productivity of CHO-S cells expressing recombinant proteins. Inoculate CHO-S cells in a 125-mL shake flask at ³ Cells were cultured in a 37°C incubator with a humidified atmosphere of 8% _CO2 . Add 4 g/L glucose on days 3 and 5, and add 6 g/L glucose on day 7. Cultures were collected daily to confirm cell density, viability, and productivity until cell viability dropped below 50% or day 14 of culture was reached.

f. gene transcript (transcript) accuracy

The accuracy of gene transcription by CHO-S expressing cells was confirmed by RT-PCR. Briefly, cells were isolated using the PURELINK™ RNA Mini Kit (Invitrogen Cat. 12183018A). Afterwards, the first strand cDNA (first strand cDNA) was reverse transcribed from the total RNA using the Maxima H Minus first strand cDNA synthesis kit (Thermo Cat. K1652). The cDNA of the recombinant protein was purified and ligated into the yT&A vector (Yeastern Biotech Co., Ltd Cat.YC203). Finally, the cDNA sequence was confirmed by DNA sequencing.

g. Express cell stability

Cells were seeded at 1~2 x ¹⁰⁵ cells/mL and cultured in a medium without selective reagent for 60 passages. During this period, once the cell density of the culture reaches 1.0 x 10 ⁶ cells/mL or higher, the cell culture is passaged again at a cell density of 1 to 2 x 10 ⁵ cells/mL. After 60 passages in culture, cell performance and productivity were compared with cells freshly thawed from LMCB using glucose simple fed batch culture. The standard for the stability of product productivity in cells is that the potency is greater than 70% after 60 generations of culture.

Example 3

f Purification and biochemical characterization of the fusion protein.

1. sFc Purification of fusion protein

All sFc fusion proteins were purified by Protein A-sepharose chromatography from the culture medium containing the harvested cell cultures. The sFc fusion protein is captured via a Protein A affinity column. After washing and elution, the pH of the protein solution was adjusted to 3.5. The protein solution was then neutralized to pH 6.0 by the addition of 1 M Tris base buffer, pH 10.8. The purity of the fusion protein was confirmed by polyacrylamide gel electrophoresis. Protein concentration is measured based on UV absorption at a wavelength of 280 nm.

2. Include S1-RBD Omicron Variants BA.4/BA.5-sFc Fusion proteins are used for COVID Avoidance and treatment of immunogenic S1-RBD-sFc Description of the biochemical properties of the fusion protein

S-RBD Omicron BA.4/BA.5-sFc proteins were prepared and purified according to the method described in Example 2 above for use in designing representative immunogen fusion proteins in vaccine formulations with high precision for immunization. Originality assessment.

After purification of the sFc fusion protein, the purity of the protein was confirmed by SDS-PAGE under non-reducing and reducing conditions using Coomassie blue staining). Figure 5 is an image showing a highly purified preparation of S1-RBD-sFc protein under non-reducing conditions (lane 2) and reducing conditions (lane 3).

Purified proteins were characterized by mass spectrometry and glycosylation analysis.

a. S-RBD-sFc - LC mass analysis and glycosylation analysis

i. Glycosylation

Glycoproteins can have two types of glycosylation linkage: N-linked glycosylation and O-linked glycosylation. N-linked glycosylation usually occurs on asparagine (Asn) residues within a sequence: Asn-Xaa-Ser/Thr, where Xaa is any amino acid residue except Pro, and the carbohydrate moiety Attach to protein via NH ₂ on the side chain of asparagine. O-linked glycosylation utilizes side chain OH groups of trace or threonine residues.

The glycosylation site of S1-RBD-sFc was studied by trypsin digestion, followed by LC-MS and MS/MS, which showed that the arginine residue at amino acid position 13 (N13) of S1-RBD-sFc It has an N-linked glycosylation site on the base and an O-linked glycosylation site on the serine residues at amino acid positions 211 (S211) and 224 (S224).

ii. N-glycosylation

The N-linked glycan structure of S1-RBD-sFc was analyzed by mass spectrometry (MS) spectroscopy. Briefly, PNGase F was used to release N-oligosaccharides from purified proteins. Then, the N-glycan part was further labeled with 2-aminobenzamide (2-AB) to enhance the glycan signal in mass spectrometry analysis.

iii. O-glycosylation

The O-linked glycans of S1-RBD-sFc were studied by trypsin digestion followed by mass spectrometry. After trypsin digestion, peaks containing O-linked glycans were collected and their molecular weights confirmed by mass spectrometry.

iv. LC mass spectrometry analysis

Purified S1-RBD-sFc protein was characterized by LC mass spectrometry analysis. The theoretical molecular weight of the S1-RBD-sFc protein based on its amino acid sequence is 48,347.04 Da. The mass spectrum profile of S1-RBD-sFc protein has a main peak at 49,984.51 Da. The difference between the theoretical molecular weight and the observed weight via LC mass spectrometry analysis was 1,637.47 Da, suggesting that the purified S1-RBD-sFc protein contains N- and/or O-glycans.

Figure 4 illustrates the general manufacturing process of drug substance ( DS) S1-RBDVoC-sFc. The process begins with a Working Cell Bank (WCB) to seed cells and scale up the culture in a 2000 L fed-batch bioreactor. After the cell culture process, the unprocessed bulk solution is collected and clarified through sterile filtration to produce a clarified bulk solution. In order to purify the drug matrix, the stock solution was subjected to protein A affinity chromatography, depth filtration (Depth Filtration) and ion-exchange (IEX) chromatography, and then tangential flow filtration (Tangential Flow Filtration, TFF) to buffer exchange. to achieve formulated DS. To avoid contamination by foreign viruses, the clarified stock solution also undergoes a process with solvent detergent treatment, acid inactivation in protein A chromatography, and nano-filtration. Finally, the formulated S1-RBDVoCs-sFc DS concentrate was produced after sterile filtration.

Example 4

Target SARS-CoV2 Designer proteins / win Composite process for manufacturing peptides .

Figure 7 illustrates the composite process for the manufacture of a designed multitope COVID vaccine targeting VoCs of SARS-CoV2. In order to produce a vaccine product, the process is to successively add peptide, CpG1, alum (Alum) adjuvant and protein components to the solution. First, Th/CTL peptide is added to WFI and then CpG1 is added to the peptide solution to form a peptide/CpG1 complex. Thereafter, protein buffer, alum and NaCl were added to the solution containing the peptide/CpG1/alum/NaCl complex. Finally, the S1-RBDVoCs-sFc protein solution is added, mixed well, and the protein concentration, pH, and other buffer conditions are adjusted to achieve the final vaccine product.

Example 5

Designing protein/peptide COVID vaccine products versus placebo.

COVID vaccines are designed to activate both humoral and cellular responses for use in preclinical, Phase 1, 2 and 3 clinical trials or in expanded catch-up vaccinations. For the SARS-CoV-2 immunogen, the COVID vaccine product combines a CHO-expressed S1-RBD-sFc fusion protein (Wuhan strain or Omicron BA.4/BA.5 variant) with a synthetic T helper (Th) and A mixture of cytotoxic T lymphocyte (CTL) epitope peptides selected from immunodominant M-peptides known to bind to human major histocompatibility complex (MHC) I and II , S2 and N areas. The preparation of vaccine products consists of compounding, filtration, mixing and filling operations. Before adding the subunit protein S1-RBD-sFc, filter the individual components of the vaccine through a 0.22 micron membrane filter, including peptide solution (2 µg/mL), CpG1, and a proprietary oligonucleotide ( oligonucleotide, ODN), solution (2 µg/mL), containing 40 mM histidine, 500 mM arginine and 0.6% Tween 80 in 10X protein buffer, 20% sodium chloride stock solution. After each component is added sequentially, the S1-RBD-sFc fusion protein and peptide are formulated together with the above components to form a protein-peptide complex, which is then adsorbed on aluminum phosphate (Adju-Phos®) adjuvant. The final step is to add water for injection containing 2-phenoxyethanol preservative solution to bring the final drug product to 200 μg/mL. Store the completed vaccine product at 2 to 8°C.

The placebo used in all experiments was sterile 0.9% normal saline.

Example 6

used for COVID Vaccine B cell or T Complementary methods for the assessment of cellular immunogenicity.

1. according to ELISA resistance S1-RBDWT combine IgG antibody.

A 96-well ELISA plate was coated with 2 µg/mL recombinant S1-RBDWT-His protein antigen (100 µL/well in coating buffer, 0.1 M sodium carbonate, pH 9.6) and incubated overnight at room temperature (16 to 18 hours). One hundred μL/well of serially diluted serum samples (dilutions from 1:20, 1:1,000, 1:10,000, and 1:100,000, a total of 4 dilutions) were added in 2 replicates, and the plate was incubated at 37°C for 1 hour. . The plate was washed six times with 250 μL of wash buffer (PBS-0.05% Tween 20, pH 7.4). Bound antibodies were probed with HRP-rProtein A/G for 30 minutes at 37°C, followed by six washes. Finally, add 100 μL/well of TMB (3,3',5,5'-tetramethylbenzidine (3, 3',5,5'-tetramethylbenzidine)) and incubated in the dark at 37°C for 15 minutes, and the reaction was stopped by adding 100 μL/well of H ₂ SO ₄ , 1.0 M. The color of the developed samples was measured on an ELISA reader (Molecular Device, VersaMax). Use the UBI® EIA potency calculator to calculate the relative potency. Anti-S1-RBD antibody levels were expressed as Log10 of one endpoint dilution of one test sample (SoftMax Pro 6.5, Quadratic fitting curve, cut-off value 0.248).

2. according to ELISA Of RBWT combine to ACE2 of inhibition.

96-well ELISA plates were coated with 2 µg/mL ACE2-ECD-Fc antigen (100 µL/well in coating buffer, 0.1 M sodium carbonate, pH 9.6) and incubated overnight at 4°C (16 to 18 hours). Wash the plate six times with wash buffer (25x solution of phosphate buffered saline with 0.05% Tween 20, pH 7.0-7.4, 250 μL/well/wash) using an automated microplate cleaner. . Additional binding sites were blocked with 200 μL/well of blocking solution (5 N HCl, sucrose, Triton X-100, casein, and Trizma base). Immune serum or a five-fold dilution of a positive control group (diluted in a buffered saline solution containing carrier protein and preservatives) was mixed with a 1:100 dilution of RBDWT-HRP conjugate (horseradish peroxidation). Mix the horseradish peroxidase-conjugated recombinant protein S1-RBD-His (horseradish peroxidase-conjugated recombinant protein S1-RBD-His), incubate at 25±2°C for 30±2 minutes, wash, and add TMB matrix (3,3',5,5'-tetramethylbenzidine(3,3',5,5'-tetramethylbenzidine) diluted in citrate buffer containing hydrogen peroxide). The reaction was stopped by stopping the solution (diluted sulfuric acid, H ₂ SO ₄ , solution, 1.0 M) and the absorbance of each well at 450 nm was read within 10 minutes using a microplate reader (VersaMax). Calibration standards used for quantitation ranged from 0.16 to 2.5 μg/mL. Samples with potency values below 0.16 μg/mL were defined as half the detection limit. Samples with titers above 2.5 μg/mL were further diluted for reanalysis.

3. based on CPE live virus neutralization assay for SARS-CoV-2 Virus-neutralizing antibody titers of wild-type versus variants.

Neutralizing antibody titers by CPE-based live virus neutralization assay using wild type (SARS-CoV-2-Taiwan-CDC#4, Wuhan) and Delta variant (SARS-CoV-2-Taiwan-CDC# 1144, B.1.617.2) Challenge Vero-E6 cells were measured, which was performed in a BSL-3 laboratory at Academia Sinica, Taiwan. Vero-E6 (ATCC® CRL-1586) cells were cultured in DMEM (Hyclone) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1x penicillin-streptomycin solution (Thermo) in a humidified atmosphere with ₅ % CO Cultured at 37°C. A 96-well microtiter plate was seeded at 1.2×10 ⁴ cells/100 μL/well. Incubate the plate overnight at 37°C in a _CO2 incubator. The next day, test sera were heated at 56°C for 30 minutes to inactivate complement and then diluted in DMEM (supplemented with 2% FBS and 1x penicillin/streptomycin). Serial 2-fold dilutions of serum were performed on the dilutions. Fifty μL of diluted serum was mixed with one volume of virus (100 TCID50) and incubated at 37°C for 1 hour. After removing the overnight culture medium, 100 μL of serum-virus mixture was inoculated in triplicates onto a confluent monolayer of Vero-E6 cells in a 96-well plate. After culturing for 4 days at 37°C with 5% _CO2 , cells were fixed with 10% formaldehyde and stained with 0.5% crystal violet staining solution for 20 minutes at room temperature. Individual holes were scored with a binary outcome of 'infected' or 'not infected' in terms of CPE. The confirmation of SARS-CoV-2 virus-specific neutralizing titer is based on the principle of VNT ₅₀ titer (≥50% reduction in virus-induced cytopathic effects) to measure neutralization against SARS-CoV-2 virus. and antibody titer. The virus-neutralizing titer of serum was defined as the reciprocal of the highest serum dilution at which a 50% reduction in cytopathic effect was observed, and the results were calculated by the method of Reed and Muench.

4. pseudovirus (pesudovirus) Target of inspection Omicron BA.1/BA.2/BA.5 of neutralizing potency.

Neutralizing antibody titers were measured by neutralization assay using HEK-293T-ACE2 cells challenged with SARS-CoV-2 pseudovirus variants. This study was performed in a BSL2 laboratory of the RNAi core of the Biomedical Translation Research Center (BioTReC), Academia Sinica. Human embryonic kidney (HEK-293T/17; ATCC® CRL-11268 ^TM ) cells were obtained from the American Type Culture Collection (ATCC). Cells were cultured in DMEM (Gibco) supplemented with 10% fetal calf serum (Hyclone) and 100 U/mL penicillin-streptomycin solution (Gibco), and then incubated at 37°C in a humidified atmosphere with ₅ % CO culture in. HEK-293T-ACE2 cells are generated by transduction of VSV-G pseudotyped lentivirus carrying the human ACE2 gene. To generate SARS-CoV-2 pseudovirus, TransIT-LT1 transfection reagent (Mirus Bio) was used to generate a C-terminal truncated wild-type Wuhan-Hu-1 strain SARS-CoV-2 spike protein. The plasmid (pcDNA3.1-nCoV-SΔ18) was co-transfected into HEK-293T/17 cells with packaging and reporter plasmids (pCMVΔ8.91 and pLAS2w.FLuc.Ppuro, respectively) (BioTReC, Academia Sinica). Site-directed mutagenesis was used to generate Omicron BA.1, BA.2 and BA.4/BA.5 variants by changing nucleotides in the Wuhan-Hu-1 reference strain. For the BA.1 variant, the mutations in the spike protein are A67V, Δ69-70, T95I, G142D/Δ143-145, Δ211/L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F. For the BA.2 variant, the mutations in the spike protein are T19I, L24S, Δ25-27, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K. For the BA.4/5 variant, the mutations in the spike protein are T19I, L24S, Δ25-27, Δ69-70, G142D, V213G, G339D, S371F, S373P, S375F, T,376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, L486V, Q493, Q498R, N501Y, Y505H, D614G, H655Y, N679K, N764K, D796Y, N856K and Q954H, & L969K.

Different SARS-CoV-2 pseudoviruses were generated by delivering the indicated plasmids into HEK-293T/17 cells using TransITR-LT1 transfection reagent (Mirus Bio). After 72 hours of transfection, cell debris was removed by centrifugation at 4,000 xg for 10 minutes, and the supernatant was collected, filtered (0.45 μm, Pall Corporation) and frozen at -80°C until use. HEK-293-hACE2 cells (1x104 cells/well) were inoculated into 96-well white isoplates and cultured overnight. Test sera were heated at 56°C for 30 minutes to inactivate complement and diluted in culture medium (DMEM supplemented with 1% FBS and 100 U/ml penicillin/streptomycin), and then serial 2-fold dilutions were performed for a total of 8 dilution. Before adding to the plate with cells, 25 μL of diluted serum was mixed with an equal volume of pseudovirus (1,000 TU) and incubated at 37°C for 1 hour. After 1 hour of incubation, 50 μL of the mixture was added to the plate with cells containing 50 μL of DMEM culture per well at the indicated dilution factor. During the next 16 h of culture, 50 μL of fresh medium (supplemented with Replace the culture medium with 10% FBS and 100 U/ml penicillin/streptomycin in DMEM. After 72 hours of infection, cells were lysed and relative light units (RLU) were measured using the Bright-Glo ^™ Luciferase Assay System (Promega). Luciferase activity was detected by Tecan i-control (Infinite 500). The percent inhibition was calculated as the ratio of RLU reduction in the presence of diluted serum to the RLU value of the virus-only control group, calculated as follows: (RLU ^{control group} - RLU ^serum ) / RLU ^{control group} . The 50% protective titer (NT50 titer) was confirmed by the Reed and Muench method.

5. according to ELISPOT Of T Cellular response.

Human peripheral blood mononuclear cells (PBMCs) were used in the detection of T cell responses. For the third dose series extension study, the Human IFN-γ/IL-4 FluoroSpot ^PLUS Kit (MABTECH) performed the ELISpot assay. Aliquots of 250,000 PBMCs were inoculated into each well and inoculated with 10 μg/mL (each stimulus) RBD-WT+Th/CTL, Th/CTL or Th/CTL pool without UBITh1a (CoV2 peptide) Stimulate and incubate in culture _medium alone for 24 hours at 37°C with 5% CO as a negative control for each plate. Analyzes were performed according to manufacturer's guidelines. Pot-forming units (SFU) per million cells were calculated by subtracting negative control holes.

6. intracellular factor staining (Intracellular cytokine staining, ICS) .

CD4+ and CD8+ T cell responses were assessed using intracellular cytokine staining and flow cytometry. PBMCs were cultured in culture medium with S1-RBD-His recombinant protein plus Th/CTL peptide pool, only Th/CTL peptide pool, CoV2 peptide, PMA + Inonmycin (as positive control group) or alone in culture medium as negative control group. Control group, 6 hours at 37°C with 5% _CO2 . After stimulation, cells were washed and stained with viability dye for 20 minutes at room temperature, followed by surface staining for 20 minutes at room temperature. Cells were fixed and permeabilized using BD cytofix/cytoperm kit (Catalog # 554714) at room temperature. permeabilization for 20 minutes, followed by intracellular staining at room temperature for 20 minutes. CD4+ T cell responses were assessed using intracellular cytokine staining for IFN-γ, IL-2, and IL-4. CD8+ T cell responses were assessed using intracellular cytokine staining for IFN-γ, IL-2, CD107a, and Granzyme B. After staining, cells were analyzed in a FACSCanto II flow cytometer (BD Biosciences) using BD FACSDiva software.

7. Statistics.

For the Phase II extension booster vaccination study. Immunogenicity results for geometric mean titer (GMT) are presented as 95% confidence intervals. Statistical analysis was performed using SAS® Version 9.4 (SAS Institute, Cary, NC, USA) or the Wilcoxon sign rank test. Spearman correlation is used to evaluate the monotonic relationship between non-normally distributed data sets. For the Phase II primary 2-dose series, the trial was designed with a sample size that meets the minimum safety requirement of 3,000 study participants in the vaccine group of healthy adults, as recommended by the U.S. FDA and WHO.

Example 7

High-precision designed vaccines against SARS-CoV-2 infection containing respective S1-RBD fusion proteins, including S1-RBD Omicron BA.4/BA.5 variants.

1. General design.

An effective immune response against viral infection depends on both humoral and cellular immunity. More specifically, the potential to design preventive vaccines with high precision would employ engineered immunogens, peptides or proteins that (1) bind to B cells via viral proteins involving the virus to its receptor on target cells. Induction of neutralizing antibodies through the use of epitopes; (2) cellular responses against invading viral antigens including primary and memory B cell and CD8+ T cell responses through the use of endogenous Th and CTL epitopes Induced active pharmaceutical ingredients. Such vaccines can be formulated with adjuvants such as ALHYDROGEL, ADJUPHOS, MONTANIDE ISA, CpG and other excipients to enhance the immunogenicity of high-precision designed immunogens.

2. use CHO cell expression S-RBD-sFc Protein (sequence identification number: 10 Amino acid sequence and sequence identification number: 15 The nucleic acid sequence) is B A representative design of cellular immunogens COVID-19 vaccine UB-612 .

This protein is designed and prepared to express the receptor binding domain (RBD) on the SARS CoV-2 Spike (S) protein with a very carbohydrate structure within the RBD to induce during immunization High affinity neutralizing antibodies. Vaccines can also use designed peptides to incorporate a mixture of endogenous SARS-CoV-2 Th and CTL epitope peptides (SEQ ID NO: 17-22, 23) that can promote host-specific Th cell-mediated immunity. Promote virus-specific primary and memory B cell and CTL responses to SARS-CoV-2 to avoid SARS-CoV-2 infection. An effective vaccine requires priming memory T cells and B cells for rapid recall upon viral infection/challenge.

In order to improve the effectiveness of the disclosed designed immunogens, two representative adjuvant formulations of ALUM (ALHYDROGEL/CpG, ADJU-PHOS®/CpG and MONTANIDE™ ISA/CpG) were used to induce the best Anti-SARS-CoV-2 immune response.

Alum (ADJUPHOS and ALHYDROGEL) is generally accepted as an adjuvant for human vaccines. This adjuvant induces a Th2 response by enhancing the attraction and uptake of the designed immunogen to antigen-presenting cells (APCs). MONTANIDE™ ISA 51 is an oil that forms an emulsion when mixed with aqueous designed peptide/protein immunogens to elicit an effective immune response against SARS-CoV-2. CpGs oligonucleotides are TLR9 agonists that improve antigen presentation and induction of vaccine-specific cellular and humoral responses. Typically, negatively charged CpG molecules are combined with positively charged designer immunogens to form immunostimulatory complexes suitable for antigen presentation to further enhance immune responses.

Weak or inappropriate antibody presentation compared to vaccines with more complex immunogenic content using inactivated virus lysates or other less characterized immunogens, based on the vaccine compositions as shown in Table 7 The disclosed high-precision designed vaccines (such as UB-612) have the advantage of generating highly specific immune responses. Additionally, there are potential pitfalls in COVID-19 vaccine development related to a mechanism called antibody-dependent enhancement (ADE). Specifically, ADE is a phenomenon in which virus binding to non-neutralizing antibodies enhances its entry into host cells and sometimes its replication. This mechanism leading to increased infectivity and virulence has been observed in mosquito-borne flaviviruses, HIV, and coronaviruses. The disclosed high-precision vaccines are designed to avoid vaccine-induced disease enhancement by monitoring the quality and quantity of antibody responses, as they will determine functional outcomes.

Representative studies discussed in Examples 8 and 9 illustrate methods for designing the disclosed highly accurate SARS-CoV-2 vaccines that are safe and promote the elicitation of antibodies capable of (1) binding to to the S1-RBD-sFc protein expressed in CHO; (2) inhibit the binding of S1 protein to the ACE2 receptor that overexpresses the ACE2 receptor protein fixed on the microwell surface or the cell surface, and (3) in cell-mediated neutralization and experimentally neutralize virus-mediated cytopathic effects.

Example 8

Potent booster and long-lasting immunity demonstrated in the Phase 1/2 trial of UB-612, a SARS-CoV-2 (Wuhan) protein-peptide vaccine.

1. Test procedures and safety

a. Main and additional third dose series of Phase 1 trial

The Phase 1 trial began with a sentinel group of 6 participants receiving the low 10-μg dose, followed by the remaining 14 participants if there were no vaccine-related grade ≥3 adverse reactions. The same procedure was extended to the escalating 30- and 100-μg dose groups. Additional follow-up visits were scheduled for all participants on days 14, 28, 35, 42, 56, 112, and 196. Study participants were scheduled to be visited on days 14 and 84 after addition. Participants who completed the 7-day period were provided with an electronic diary after each injection to record local reactions at the injection site (pain, induration/swelling, rash/redness, itching, and cellulitis) and the resulting symptoms. Systemic reactions (17 different constitutional symptoms). Severity is graded using a 5-point (0 to 4) scale from none to life-threatening. In addition, participants recorded their axillary temperatures every evening starting on the day of vaccination and continuing for 6 days. Safety endpoints included non-suggested AEs reported up to 14 days after the add-on in this interim Phase 1 extension report.

b. Main series of Phase 2 trials

The primary safety endpoint of the Phase 2 trial is to assess the safety and tolerability of all participants receiving the study intervention from Day 1 to Day 57 (28 days after the second dose). Assess vital signs before and after each injection. Participants were observed for 30 minutes after each injection for changes in vital signs or any acute allergic reactions. After each injection, participants were required to record local and systemic AEs in their self-assessment electronic diary for seven days and skin allergic reactions in their electronic diary for fourteen days. Safety endpoints included non-reported AEs from Day 1 to Day 57 reported in this interim Phase 2 report.

2. result

a. Experimental group.

i. Phase 1 main and additional third dose series

Characteristics of participants in the open-label Phase 1 trial including those who received two doses (28 days apart) of 10, 30, or 100 μg of UB-612 in three dose groups (n = 20 each) A 196-day main series in 60 healthy adults (age 20-55 years); with an extended booster vaccination 84 days after the main series, including 10-μg (n = 17), 30-μg ( n = 15) and 100-μg (n = 18) groups, 50 participants were enrolled to receive an additional 100-μg boost between 7.6 and 9.6 months after the second injection. In this interim report, additional participants were followed for 14 days to assess safety and immunogenicity, and then for 84 days post-addition.

ii. Phase 2 main 2-dose series

The Phase 2 trial used a randomized double-blind design. A total of 3,875 participants who received at least one dose of 100 μg vaccine (3,321 received UB-612 and 554 received placebo in a 6:1 ratio) were enrolled and included in the safety population, of which 1,012 participants (vaccine 871 versus placebo 141) were included in the evaluable immunogenicity population. The average age of participants who received UB-612 was 44.9 years (range, 18 to 83 years), while the average age of participants in the placebo group was 44.4 years (range, 19 to 84 years). The ratio of younger adults (18 to 65 years) to older adults (≥65 years) was approximately 80:20 for both the UB-612 and placebo groups. Except for 5 people, all participants were Taiwanese.

b. Reactogenicity and safety.

i. Phase 1 main 2-dose and additional third-dose series

No vaccine-related serious adverse events (SAEs, including grade 3/4 AEs) were recorded in the 196-day main series and up to 14 days post-boost, nor were they recorded in incidence or severity The dose-limited increase. Local and systemic AEs reported within 7 days in all vaccination groups were mild to moderate (Grade 1/2) and transient, with the majority of systemic AEs compared with local reactions. Reactions are less frequent. The incidence of local AEs caused after the first and second vaccinations was similar, with a slight increase after the additional dose. The most common local AE caused after the additional dose was pain at the injection site (60-71% ). The incidence of systemic AEs was similar after each vaccination, with the most common post-boost systemic AE being fatigue (11-33%). The safety profile observed in the primary 2-dose vaccination series and the booster phase was similar.

ii. Phase 2 main 2-dose series

There are no vaccine-related SAEs. Both local and systemic AEs were mild, transient, and self-limited within a few days. Overall, 2546 participants reported local AEs after doses 1 and 2, 2386 (72.0%) in the UB-612 group and 160 (28.9%) in the placebo group. The severity of these local AEs ranged from mild (Grade 1) to moderate (Grade 2), with the most common events being injection site pain and occasional allergic skin reactions among 2246 (67.8%) participants in the vaccine group.

There was no significant difference in the incidence of systemic AEs across age groups between the UB-612 vaccine group and the placebo group (P ＞ 0.05). Systemic AEs were reported in 38.6% of older participants (65-85 years) in the vaccine group, compared with 63.3% in the overall safety group. The most common systemic AE was fatigue/tiredness reported in 1,488 (44.9%) of UB-612-treated participants and was generally mild.

c. Neutralizing antibodies against live SARS-CoV-2 wild-type and Delta variants, and neutralizing antibodies against pseudo-SARS-CoV-2 wild-type and VoC including Alpha, Beta, Gamma and Omicron.

i. Main 2 doses and additional third dose in Phase 1

A booster dose of 100 μg, given 7.6-9.6 months after the second dose, induced robust neutralization against live SARS-CoV-2 wild-type (WT, Wuhan strain) and Delta variants in 100% of participants Antibodies (Figure 8). Boosters resulted in geometric mean 50% virus neutralizing titers (VNT ₅₀ ) of 4643, 3698, and 3992 against WT in the 10-, 30-, and 100-μg UB-612 dose groups, respectively (Figures 8A-8D) , representing 104, 118, and 37-fold increases (geometric mean fold increases, GMFI (GMFI)), respectively, compared to the peak response in the main series (14 days after 2 doses, i.e., day 42), and (b ) GMFI were 465, 216 and 65 respectively, exceeding the pre-addition levels. Compared with a panel of human convalescent sera (HCS) collected ~1 month after the onset of hospitalized COVID-19 cases, the levels of neutralizing antibodies after supplementation were 45.5 times, 36.2 times, and 39.1 times higher respectively. (GMFI). Neutralizing antibody titers in the same live virus test standardized to WHO reference antisera and expressed in international units (IU/mL) were similar (Figures 9A-9D).

Additional doses also induced significantly higher VNT ₅₀ titers against the live Delta variant, reaching 2854, 1646, and 2358 (Fig. 9A), which represent differences between the 10-, 30-, and 100-μg groups relative to the WT strain. Moderate GMFRs of 1.6, 2.4, and 1.7 (i.e., ~63%, ~42%, and ~60% preservation of neutralization strength, respectively).

pVNT ₅₀ observed 14 days after the boost in the 100-μg group (n = 18) was evaluated for cross-reactive neutralizing antibody titers against pseudo-SARS-CoV-2 wild type (WT) and other VoCs containing Omicron , as shown in Figure 9B. Compared to 14,171 for the wild type, the pVNT ₅₀ for WT, Omicron, Alpha, Gamma and Beta were 12,778, 2,325, 9,300, 13,408 and 4,974 respectively. Compared to the WT strain, the GMRFs were 5.5, 1.4, 1.0 and 2.6 respectively (i.e. Maintaining neutralizing strengths of 18.2%, 72.7%, 105% and 38.9% respectively).

Neutralizing antibodies in the primary series were durable in the 100-μg group compared with the lower doses of 10- and 30-μg, associated with the highest increase in VNT ₅₀ against WT observed 14 to 28 days after 2 doses (Figures 8A-8C). The peak neutralizing antibody GMT in the 100-μg group (108 on day 42; 103 on day 56) (Figure 8C) was close to the GMT of 102 in the control human convalescent serum (HCS) group. For the 100 μg dose, the seroconversion rate based on SARS-CoV-2 neutralizing antibody titers was 100% on day 57 of Phase 1 and remained 100% thereafter throughout the monitoring period.

Before the boost (days 255 to 316), none of the 18 participants (0%) in the 100-μg group had VNT ₅₀ titers below the limit of detection (LLOQ), indicating that the induced neutralizing effect was long-lasting period of time. Antibody persistence after 2 doses from the 100-μg arm of the Phase 1 trial for anti-WT neutralizing VNT ₅₀ on days 42 to 196 was calculated using first-order exponential model fitting (SigmaPlot) (r ² = 0.9877, decay rate Constant K _el = -0.0037; t _1/2 = 0.693/K _el ). Neutralizing antibody VNT ₅₀ GMT decreased slowly with t _1/2 at 187 days (Figure 9C).

We also studied the neutralization of Delta versus other VoCs during the Phase 1 main vaccination phase with all serum samples (n = 20) from the main series of the Phase 1 trial in the 100-μg UB-612 dose group (Figure 10 ). The results showed that virus neutralizing activity was retained, especially against the Delta B.1.617.2 variant, for which 63% neutralizing activity (1.6 GMFR) was retained relative to the wild-type Wuhan strain. Significant neutralizing antibodies were also retained against the Alpha (B.1.1.7) variant with 91% retention (GMFR 1.1), the Gamma (P.1) variant with 56% retention (GMFR 1.8), and against Beta B.1.351 is weaker, with 20% retention (GMFR of 5.1).

ii. Main 2 doses in Phase 2

At day 57 (4 weeks after the second dose), across participants of all ages (18 to 85 years), the GMT of anti-S1-RBD titers was 518.8 (Figure 11A) versus the original wild (WT Wuhan) strain The virus neutralizing titer was age-dependent, with _a total VNT of 87.2 (Figure 11B). Younger adults (18 to 65 years old) had a higher VNT ₅₀ of 96.4, which was reproducibly close to that observed in Phase 1 study participants 20 to 55 years old (VNT 50 of 103) (Figure 8C), whereas Older adults (≥65 years) showed a lower VNT ₅₀ of 51.6. An extension study of the Phase 2 trial with an additional third dose was conducted. Across participants of all ages (18 to 85 years) in Phase 2, seroconversion rates at day 57 (or day 56 after dose 1) based on wild-type SARS-CoV-2 neutralizing antibody titers from 88.6% among elders to 96.4% among young adults.

On day 57, significant levels of anti-Delta neutralizing antibodies were observed. A pool of 48 serum samples were randomly selected from vaccine recipients across age groups (n = 39 for younger adults 18–65 years; n = 9 for older adults ≥ 65 years) at two independent laboratories Underwent ad hoc live virus assay analysis (Academia Sinica and California Division of Virology and Rickettsial Diseases). The results were consistent, showing that immune sera could neutralize two key SARS-CoV-2 prototypes with similar VNT ₅₀ : 329 against the Wuhan WT obtained in Taiwan, and 308 against the USA WA 1/2020 ( Figure 12). Compared with the USA WA 1/2020 variant, the VNT ₅₀ estimates for Alpha B.1.1.7 and Delta B.1617.2 are 122 and 222 respectively, representing a reduction of 2.7 times and 1.4 times.

d. Neutralizing antibodies against S1-RBD binding to the ACE2 receptor.

i. Phase 1 main 2 doses and additional third dose series

The ELISA results for functional inhibition (neutralization) of the S1-RBD:ACE2 interaction (Figure 13) are generally consistent with the VNT ₅₀ data (Figure 8). The 100-μg dose group showed the highest neutralizing titer (Figure 13C), with an anti-S1-RBD:ACE2 quantitative neutralizing antibody (qNeuAb) level of 6.4 μg/mL at day 112, compared with the 100-μg dose group from 20 human recovered The 1.4 μg/mL of serum (HCS) increased 4.6 times. After booster vaccination, anti-S1-RBD:ACE2 qNeuAb levels reached 303 to 521 μg/mL, representing a 77- to 168-fold increase over the peak following primary series vaccination; similarly, compared with pre-boost levels, a significant 82-fold increase was observed to a 579-fold increase (Figures 21A-21C). Therefore, a UB-612 booster dose can elicit a significant immune response in vaccinated subjects, regardless of how low their pre-boost levels were.

Neutralization of S1-RBD:ACE2 binding in the ELISA correlated well with the VNT50 results (Spearman's r = 0.9012) (Figure 13D) thus confirming the anti-WT _VNT50 results by the cytopathic effect (CPE) assay. Effectiveness (Figures 8A-8C). In addition, post-boost anti-S1-RBD:ACE2 qNeuAb levels ranged from 303 to 521 μg/mL (Figures 8A-8C), which were 216 to 372 times higher than human convalescent serum (HCS). This suggests that most antibodies in HCS appear to interact more with the allosteric site (N of S1) than with the orthosteric (RBD) site where the viral S1-RBD interacts with the ACE2 receptor. or C-terminal domain).

e. S1-RBD IgG antibody ELISA reaction.

i. In Phase 1 trial

S1-RBD binding antibodies measured by ELISA (Figure 14) again showed that the 100-μg vaccination group elicited the highest immune response in the 196-day main series, with a GMT of 2,240 on day 42, which far exceeded GMT from 141 of 20 human convalescent sera (HCS). After booster vaccination, anti-S1-RBD GMT peaked at 7,154 to 9,863 in the three dose groups (a 3- to 28-fold increase (GMFIs) from the peak during the main series); again, compared with pre-boost levels, A significant increase of 37 to 378 times.

f. T cell response according to ELISpot.

i. Phase 1 trial

In the main vaccination series of the Phase 1 trial, peripheral blood mononuclear cells (PBMC) were collected from vaccinees for assessment by interferon-γ+ (IFN-γ+)-ELISpot (Figures 15A-15C). The highest antigen-specific response was observed in the 100-μg dose group: 254 spot-forming units (SFU)/10 ⁶ PBMC after stimulation with S1-RBD+Th/CTL peptide pool on day 35, and There were 173 Th/CTL peptide pools after stimulation alone (Figure 15C), proving that the Th/CTL peptide in the UB-612 vaccine is mainly responsible for T cell responses.

On day 196, the IFN-γ+ ELISpot response in the 100-μg dose group remained at ~50% of the peak response and decreased from 254 SFU/ ¹⁰ cells to 121 on restimulation with the RBD+Th/CTL peptide pool. SFU/10 ⁶ cells, or restimulation with Th/CTL peptide pool only from 173 to 86.8. This observation suggests that the T cell response elicited by the UB-612 vaccine persisted for at least 6 months after two doses of the vaccine. This is consistent with the persistence of neutralizing antibodies mentioned earlier (Figure 8C).

ii. Phase 2 trial

In the phase 2 trial, a strong IFN-γ+-ELISpot response was also observed on day 57: the geometric mean, with S1-RBD+Th/CTL restimulation was 370 (SFU/10 ⁶ cells), with Th /CTL re-stimulation was 322, and the Th/CTL peptide pool without UBITh1a was 181 (Figure 15D), which were both much higher than the counterparts in the placebo group (p<0.0001). Responses to IL-4 were much lower compared to IFN-γ: 13.6, 7.5, and 5.4, respectively (Figure 15E). The overall ELISpot results indicate that the content of Th/CTL peptide is essential and mainly responsible for the T cell response, while the recombinant protein S1-RBD only plays a minor role. Importantly, the direction of T cell responses is primarily Th1-directed. UBITh1a acts as a catalyst as usual, triggering the Th1 response through a pool of virus-specific Th/CTL peptides.

g. CD4+ versus CD8+ T cell responses based on intracellular cytokine staining (ICS).

i. Phase 2 Trial

T cell responses were assessed by intracellular cytokine staining (ICS) (Figure 16). Significant increases in IFN-γ and IL-2-producing CD4+ and CD8+ cells were observed across the three peptide restimulation groups; and, consistent with the ELISpot findings (Figures 15D-15E), lower IL-4 production was detected of CD4+ T cells, confirming the Th1 advantage of the T cell response.

After restimulation with S1-RBD+Th/CTL, Th/CTL, and Th/CTL pools without UBITh1a, CD8+ T cells expressing cytotoxicity markers CD107a and granzyme B were observed, accounting for 3.5% of circulating CD8+ T cells. , 2.1% and 1.8%. Overall, UB-612 elicited Th1-directed immunity with a robust CD8+ cytotoxic T cell response that would facilitate clearance of viral infection, and restimulation results indicated the inclusion of non-spiny nucleocapsid (N) and membrane (M) Th/CTL peptide of structural protein is the main factor responsible for T cell immunity.

3. Conclusion

No vaccine-related serious adverse events (SAEs) were recorded. The most common AEs are injection site pain and fatigue, most of which are mild and transient. In both trials, UB-612 elicited neutralizing antibody titers similar to those in a panel of human convalescent sera. The most striking findings are: persistent virus-neutralizing antibodies and broad T-cell immunity against SARS-CoV2 VoCs including Delta and Omicron, as well as strong catch-up with high cross-reactive neutralizing titers against Delta and Omicron variants Memory immunity.

UB-612 has a favorable safety profile, effective boosting against VoC, and durable B and broad T cell immunity, and is worthy of further development for primary immunity and heterologous boosting of other COVID-19 vaccines.

Of particular note are the five precisely designed T cell epitopes representing Th and CTL epitopes from the Sarbecovirus regions of the N, M and S2 proteins. These epitope peptides are highly conserved across all variants of interest, including Delta and Omicron, and are promiscuous epitopes that allow induction of memory recall, T cell priming and effector functions in a broad range of populations. Therefore, in addition to the effective anti-Delta and anti-Omicron effects of the additional third dose of UB-612, long-lasting and powerful T cell immunity is effective against all VoCs including Omicron. Since currently approved COVID vaccines do not recognize non-spiny M and N proteins, the UB-612 vaccine is well-positioned to protect against new variants of concern such as Delta and Omicron, which require large-scale field trials to evaluate.

Example 9

The UB-612 phase 2 booster vaccine provides better protection against Omicron infection than those with the "Spike" only COVID vaccine.

As with the results for the Phase 1 booster vaccine against Delta and Omicron BA.1 variants shown in Example 8, the Phase 2 booster results confirm that UB-612 can serve as a universal (Pan Sabic virus) vaccine to Protect against Omicron variants and other emerging variants.

In addition to spine S1-RBD (restricted ACE2 receptor-binding domain) as an immunogen to activate B cells (long-lasting neutralizing antibodies), UB-612 has multiple structural differences between spine S2 and non-spine (nucleocapsid N vs. membrane M). The protein is rich in five sequence conserved and mixed Th/CTL epitopes, which are used to promote more comprehensive T cell (helper and cytotoxic) memory immunity.

Due to the uniqueness of the vaccine design, UB-612 was included on the agenda of the White House Next Generation COVID-19 Vaccine Summit on July 26, along with the Pfizer BNT and Moderna vaccines, to showcase the Pioneer vaccine platform. UB-612 booster vaccination can induce effective, broadly recognized and durable B cell (neutralizing antibodies) and T cell (helper and cytotoxic) memory immunity, which can simulate infection with any SARS-CoV-2 variant.

1. Comparison of the UB-612 vaccine with COVID vaccines that employ other platform technologies and use only the “ Spike ” protein as the immunogen .

The recent approval of the use of the bivalent mRNA vaccine (Modena and Pfizer) as a fourth dose (second booster dose) in the UK, US and Taiwan has aroused concern and skepticism. Therefore, amid concerns about the use of upcoming imported bivalent mRNA vaccines, now is the time to compare the performance of current vaccines in terms of the extent of virus-neutralizing antibodies against Omicron variants and the strength of T-cell immunity.

a. Pseudovirus neutralizing activity

UB against the BA.1/BA.2/BA.5 variants was measured by a “pseudovirus neutralization assay” after a booster dose (third dose, homologous booster) of each vaccine in uninfected subjects -612 immune serum (50% geometric mean GMT, i.e. pVNT ₅₀ /ID ₅₀ titer) showed higher than Moderna (mRNA-1273), Pfizer (BNT162b2) and NVX-CoV2373; and much higher than MVC-COV1901, AZD1222 , CoronaVac and BBIBIP vaccines (Table 10). Taking the most infectious BA.5 pVNT50 as an example, the neutralizing titers were reported to be 854 for UB-612, 582 for NVX-CoV2373, 378 for Moderna mRNA-1273, 360 for Pfizer BNT162b2, and 75 for CoronaVac. AZD1222 is 43. These data show that the UB-612 vaccine achieves the design concept of synergistic B cell and T cell immunity, and that the third dose (the first booster dose) has been able to basically neutralize the Omicron BA.5 variant, which is currently facing Taiwan A powerful, dominant SARS-CoV-2 variant. The UB-612 booster dose performed better than the AZ vaccine.

It is important to note that pseudovirus detection is based on an artificial (pseudo)virus with only a spike protein on its crown, while clinical isolates of live viruses are actual isolates that contain spike and non-spike proteins on the main body of the virus. All currently licensed vaccines designed using only spine immunogens are unable to recognize the bulk structure of the virus's non-spike proteins.

Non-Spike proteins also mutate during the evolution of the virus (Table 12). The antibodies produced by the current Spike-only vaccine can neither homing nor induce B and T cell memory immunity to recognize non-Spike proteins. . Therefore, for those vaccines that only have spike proteins, there will be data inconsistencies between pseudovirus and live virus assays, unless the vaccine antigen is designed to account for both spike and non-spike proteins. The inconsistency is verified below.

b. Live virus neutralizing activity

After a booster dose of each vaccine (third dose, homologous booster) was administered to uninfected persons, UB was evaluated by the "live virus neutralization test" (50% geometric mean GMT, i.e., VNT ₅₀ / FRNT ₅₀ titer). -612 (value of 670) showed higher GMT potency than other vaccines (values of 46 to 106) (mRNA-1273, BNT162b2 and AZD1222 vaccines), representing 6 to 12 times higher titer intensity (Table 10) .

Comparing live virus and artificial pseudovirus neutralization test methods (Tables 10 and 11), only the UB-612 vaccine showed consistency between methods, and the virus neutralization intensity of UB-612 was better than that of any virus test method. All other EUL listed vaccines.

There are data differences between pseudovirus and live virus assays for these brands of vaccines. Note, taking the anti-BA.1 pVNT50 as an example (Table 10), the _pVNT50 between UB-612 (value 1,196-2,325) and mRNA-1273/BNT162b2 (value 945-1,116) (pseudovirus neutralization assay) The difference is small; while the difference in the live virus test against BA.1 VNT50 is even larger, about 6-12 times (Table 11).

2.UB-612 Behind the superior virus neutralizing activity.

The superiority of UB-612 to other vaccines in terms of pseudovirus and live virus neutralization potency can be attributed to its recognition of targets on spike and non-spike proteins (conserved and promiscuous Th/CTL antigen determination on S2, M and N proteins). position), to generate a compelling and widely recognized comprehensive T cell immune memory, which should strengthen the B cell immune response and T cell immunity with cross-neutralizing antibodies against BA1, BA.2 to BA.5, in a synergistic manner reaction.

Top-up vaccinations (third doses) of branded vaccines have exposed their weaknesses against the live BA.1 virus, not to mention BA.2 and BA.5. Judging from the fact that it is difficult for the current mRNA vaccine to produce a VNT ₅₀ peak titer of >100 against the BA.1 live virus after the additional dose, it can be predicted that the vaccine will be further weakened in neutralizing the BA.2 and BA.5 live viruses.

3. spine Mutation sites on protein and non-spike proteins.

In addition to the more than 30 mutations on the Spike protein, as shown in Table 12, there are also mutation sites on the non-spike proteins (E, M and N) of the BA.1, BA.2 and BA.5 variants. These mutation sites The point that Echinacea-only vaccines fail to recognize is that they are inherently deficient in promoting more adequate T-cell immunity. And, in contrast, by targeting the smaller S1-RBD protein fragment of spike protein and designing T immunogens with conserved and promiscuous T epitopes, UB-612-induced immunity is better than that encountered by other vaccines. Fewer viral mutants are resistant, making Omicron evasion less likely, as UB-612 vaccine-induced immunity can behave more closely like infection-induced immunity.

The above observations also show that: 1) when comparing the virus neutralization efficacy of different vaccine platforms, only live virus test detection is truly reliable; 2) artificial pseudovirus detection is beneficial in those that focus on Spike (Spike-focused). Comparison among vaccines.

4. A potential pan- Sabak virus vaccine.

It is particularly noteworthy that non-spine structural proteins such as the mantle (E), membrane (M) and nucleocapsid (N) are crucially involved in the induction of host cell interferon response and T cell memory. Therefore, the profound T cell memory immunity evoked by the UB-612 vaccine can play a key role in the long-term control of SARS-CoV-2 infection. Therefore, UB-612 as a booster agent may potentially benefit infected patients the most in preventing reinfection.

Unlike other vaccines that use spike (S) protein as the only B and T immunogen, UB-612's composition includes the immunogen S1-RBD to trigger B cells to produce neutralizing antibodies, and five conserved, immutable hybrids Antigenic epitopes (S2x3, N and M proteins) were used as T immunogens (Table 13). The unique and rational vaccine design makes UB-612 a possible pan-Sabavirus vaccine.

5. Powerful T cell immunity that prevents immune evasion .

Because vaccines can generate strong T cell immune responses to conserved, non-mutable epitopes, they can prevent immune escape. It is of great interest to study the T cell immune responses generated by different forms of vaccines. As described below, UB-612, as a booster (third dose, homologous boost), induces a much higher degree of T cell immunity (SFU/10 ⁶ PBMC cells) than either the mRNA vaccine (BNT162b2) or the adenovirus DNA vaccine ( ChAdOx1 or AZD1222).

The SFU units before and after the booster dose were 38/45 for 3 doses of ChAd/ChAd/ChAd and 28/82 for 3 doses of BNT/BNT/BNT, which were lower than those observed in the phase 2 extension study. To 261/374 SFU of UB-612 supplement. These results are consistent with the fact that UB-612 has strong neutralizing titers against BA.1 live virus, whereas BNT162b2 has weaker potencies with ADZ1222 (Table 11). In general, strong T-cell immunity is also known to be critical for protecting people against severe disease and for the long-term success of vaccines.

6.UB-612 Effectiveness of vaccines against infection.

While the extent to which the vaccine is effective against infection in the real world is unknown, the strong blockade of ACE2:RBDWT interaction by UB-612 was associated with virus neutralization of VNT ₅₀ (live virus WT vs. Delta) and pVNT ₅₀ (pseudovirus BA.1 ), inferring that the clinical efficacy against COVID-19 is considerable. In fact, using models of S protein binding activity and neutralizing antibodies, the clinical efficacy of 2 doses of primary immunization with UB-612 against Wuhan/Delta is predicted to be 70-80%, while follow-up vaccination may lead to 70-80% clinical efficacy against ancestral Wuhan/Delta or Efficacy against symptomatic COVID-19 caused by the Omicron strain is 95%.

7. Ongoing investigation of the effectiveness of UB -612 in Taiwan and the United States.

It should be noted that among the 1,480 subjects who received two or three doses of the UB-612 vaccine (Phase 2 trial in Taiwan), there were no cases of infection as of the end of the designed Phase 2 clinical study in March this year. report.

In addition, as Omicron infections rapidly escalated in Taiwan in May this year, telephone interviews are being conducted with participants in the Phase 2 clinical trial, as well as those who received two or three doses of the UB-612 vaccine against Omicron during the unprecedented outbreak in Taiwan. Initial vaccine protection is estimated to be greater than 95% in participants (the trial is ongoing).

Additionally, Phase 3 clinical efficacy in protecting against infection with circulating subvariants including primary Omicron B5 will await results from an ongoing Phase 3 trial comparing UB-612 to an approved vaccine under homologous versus heterologous boost [ClinicalTrials .gov ID: NCT05293665].

Use the mRNA bivalent vaccine as the fourth dose. Currently, Moderna’s bivalent vaccine mRNA-1273.214 (original spine plus Omicron BA.1 spine), which is the fourth dose (second booster shot), recently received Emergency Use Authorization (Emergency Use Authorization). It is reported that the pseudovirus neutralizing titer (pVNT50) against BA.5 is 727, which is 50-60% higher than the original mRNA-1273 vaccine (fourth dose) and 90% higher than the third dose of mRNA-1273 vaccine (Table 10); none of them exceeded 2 times. This small increase in pVNT50 has been shown not to result in an increase in vaccine efficacy. Another bivalent vaccine containing BA.5 also received emergency use authorization on September 6; the approval was based on only an eight-mouse study.

Unfortunately, Moderna did not submit key "live virus neutralizing efficacy" data at the US FDA review meeting on June 28. At present, there is only VNT ₅₀ data for the live virus against BA.1. It is reported that the VNT ₅₀ of the original mRNA-1273 after the third dose is 81.0 (Table 11); while the anti-BA after the fourth dose of the bivalent vaccine mRNA-1273.214. 5The VNT ₅₀ for live viruses may be lower.

According to the overall estimate of anti-pseudovirus pVNT50 based on the data in Table 10, compared with anti-BA.1/anti-BA.2, the neutralizing efficacy against BA.1 is 1.3 times; the neutralizing efficacy against BA.5 is within 2-3 times. . If you do not wear a mask, wash your hands frequently, and maintain appropriate social distance, you are likely to be reinfected or have a breakthrough infection with the Omicron BA.5 variant. While comprehensive vaccinations and booster shots are needed to prevent infection, the question remains whether people are getting both the vaccine and the dosage regimen right.

8. Prevent remote COVID potential benefits.

Finally, regardless of vaccination status or mixed immunity, each reinfection increases the risk of death, hospitalization, and other health harms, including the burden of long COVID, and immunity with currently approved vaccines has limited benefit in mitigating long COVID.

Since long-range COVID was found to be associated with a decline in IFNγ-producing CD8+ T cells, in preventing long-range COVID, it is hoped that a system that can induce strong and long-lasting T cell immunity to clear residual systemic infection (persistent viral reservoir) vaccine platform, for which UB-612 may play a positive role.

Example 10

Prevention and treatment SARS-CoV2 Infections, clear viral infections and treat remotely COVID Pansabico (Pan-Sarbeco) Vaccine development.

Starting from the original Wuhan strain, the SARS-CoV-2 Omicron strain has evolved in rapid succession with dominant subvariants, from BA.1, BA.2 to the current BA.4/BA.5, accounting for 90% of SARS infection cases. More than %, it has overwhelming advantages in transmissibility and neutralizing antibody escape, sweeping the world.

Omicron BA.1 has undergone severe mutations from the original SARS-CoV-2 Wuhan strain, including more than 35 amino acid changes in the S protein. Compared with the 2 mutations related to Delta of the S-1 receptor binding domain (S1-RBD, residues 319-541), BA.1 and BA.2 have a total of 12 mutations, each of BA.1 and BA.2 There are 3 and 4 unique ones, which respectively give BA.2 higher immune evasion capabilities. BA.4 and BA.5 have the same spike protein. They differ from BA.2 by having additional mutations at 69-70del, L452R, F486V and the wild-type amino acid at position Q493 within the spike protein (Table 12), which contributes to their comparison with BA.2 Higher levels of immune evasion. BA.2 showed 1.3 to 1.5 times higher transmission rate and 1.3 times immune evasion than BA.1, which is consistent with the results of BA.1 immune serum neutralizing BA.2 with a lower titer of 1.3 to 1.4 times, BA.2 reinfection may occur after BA.1. BA.4/BA.5 are more transmissible and resistant to BA.1/BA.2 immunity and monoclonal antibodies.

In addition, after additional injections in UB-612 vaccine recipients, the ability of immune serum to neutralize live Wuhan virus was still low, and its titer was reduced (50% geometric mean GMT, i.e., VNT50/FRNT50 titer) by 10 to 50 times. range (GMFR = ~10 to 50), as shown in Table 10, this falls short of the goal of developing a Pansabiko vaccine.

The development of compositionally updated (variant specific) vaccines is strongly advocated to address this urgent need to prevent individuals from becoming infected with SARS-CoV-2 Omicron BA.4/BA.5 to control the outbreak and reduce the resulting suffering, including remote COVID-19 with death.

The disclosed high-precision designer vaccines (such as UB-612, UniCoVac-2, UniCoVac-3) prepared according to the vaccine compositions shown in Tables 7 to 9 have fewer features than the use of inactivated virus lysates or other features The immunogen content of the immunogen has the advantage of generating a highly specific immune response compared to the presentation of weak or inappropriate antibodies in more complex vaccines. Additionally, there are potential pitfalls in COVID-19 vaccine development related to a mechanism called antibody-dependent enhancement (ADE). Specifically, ADE is a phenomenon in which binding of a virus to non-neutralizing antibodies enhances its entry into host cells and sometimes its replication. This mechanism results in increased infectivity and virulence and has been observed in mosquito-borne flaviviruses, HIV, and coronaviruses. The disclosed high-precision vaccine composition, which uses only S1-RBD-sFc protein as the B-cell immunogen, is designed to avoid vaccine-induced disease enhancement by monitoring the quality and quantity of antibody responses, as they will determine functional outcomes. .

In addition to the advantages of UB-612 using the "spike" protein as a vaccine target compared to other COVID vaccines using different platforms, as discussed extensively in Example 9, our monovalent UniCoVac 2 and bivalent UniCoVac 3 will have variable The additional advantage of updated vaccines with body-specific (Omicron BA.4/BA.5) ingredients to meet the urgent need for COVID vaccines. UniCoVac 2 will complement the existing UB-612 monovalent vaccine by providing a highly complementary S1-RBD-Omicron BA.4/BA.5 variant-specific sFc using S1-RBD WuHan-sFc derived from the WuHan sequence of the original strain The protein serves as a B cell immunogen, thereby inducing neutralizing antibodies that can effectively neutralize the currently prevalent BA.4/BA.5 variants.

The combination vaccine prepared according to Table 9 uses S1-RBD Wuhan-sFc (SEQ ID NO: 10) and S1-RBD Omicron-BA.4/BA.5-sFc protein (SEQ ID NO: 11) as B cell immunogens. Allows the generation of complementary neutralizing antibodies against the broad spectrum of RBD of SARS-CoV2. Such a broad range of neutralizing antibodies, coupled with the long-lasting immune memory provided by conservative T cell immunity, is achieved by adding SARS-CoV Th/CTL peptide (Sequence ID: 17-22) and idealized artificial Th peptide ( SEQ ID NO: 23) As a catalyst for T cell priming, an optimal pan-Sabacovirus vaccine composition can be developed that (1) is safe due to the high precision and sub-unit nature of the vaccine platform; ( 2) Can promote the stimulation of antibodies that bind to the S1-RBD-sFc protein expressed by CHO, covering from the original Wuhan strain to the latest Omicron BA4./BA.5 strain, and neutralize virus-mediated viruses in cell-mediated neutralization experiments induced cytopathological effects; (3) can produce Th1-biased T cell immunity, activate IFN-γ-producing Th cells after mucosal contact, and immediately defend against invading SARS-CoV-2 variants; (4) can produce Viral antigen (M.M and S2)-specific CD8+ cells lyse cells to eliminate virus-infected cells; and (5) provide long-lasting immunity due to far enhanced immune memory to achieve the ideal Pan Sabah The ultimate goal of a viral vaccine is to prevent individuals from becoming infected with SARS-CoV-2 Omicron BA.4/BA.5, control SARS-COV2 outbreaks, and reduce the resulting suffering, including remote COVID and death.

sheet

Table 1

Amino acid sequence of S1-RBD-scFc fusion protein of SARS-CoV2 Omicron BA.4/BA.5 serial identification number sequence Type 1 NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDL C FTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATV C GPKKS S protein RBD-Wuhan 2 nitnlcpfDevfnatrfasvyawnrkrisncvadysvlynFaPfFAfkcygvsptklndl c ftnvyadsfvirgNevSqiapgqtgniadynyklpddftgcviawnsnKldskvGgnynyRyrlfrksnlkpferdisteiyqagNKpcNgvAgVncyfplQsyGfRptygvgHqpyrvvv lsfellhapatv c gpkks S protein RBD-Omicron BA.4/BA.5 (B.1.1.529.4/ B.1.1.529.5, South Africa) 3 EPKSCDKTHTCPPCP Wild-type hub region from human IgG1 4 EPKS S DKTHT S PP S P Mutated hub region from human IgG1 5 EPKS X DKTHT X PP X P Mutated hub region from human IgG1 6 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY N STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG Fc peptide (wild type) 7 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY H STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG Fc peptide Mut. Glycos. (N->H) 8 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY A STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG Fc peptide Mut. Glycos. (N->A) 9 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY VMHEALHNHYTQKSLSLSPG Fc peptideMut. Glycos. (N->X) X = N,H,A 10 NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDL C FTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATV C GPKKS EPKSSDKTHTSPPSP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY H STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG S-RBD-Wuhan-sFc fusion protein 11 nitnlcpfDevfnatrfasvyawnrkrisncvadysvlynFaPfFAfkcygvsptklndl c ftnvyadsfvirgNevSqiapgqtgniadynyklpddftgcviawnsnKldskvGgnynyRyrlfrksnlkpferdisteiyqagNKpcNgvAgVncyfplQsyGfRptygvgHqpyrvvv lsfellhapatv c gpkks EPKSSDKTHTSPPSP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY H STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG S-RBD-Omicron BA.4/BA.5- sFc fusion protein

Table 2

Nucleic acid sequences of SARS-CoV2 Wuhan, Omicron BA.4/BA.5 S1-RBD protein and S1-RBDs-scFc fusion protein serial identification number sequence Type 12 AACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGGTTCGCCTCCGTGTACGCCTGGAACAGGAAGGATCTCCAACTGCGTGGCCGACTACTCCGTGCTGTACAACTCCGCCTCCTTCTCCACCTTCAAGTGCTACGGCGTGTCCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACTCCTTCGTGATCAGGGGCGACGAGGTGAGGCAGATCGCCCCCGGCCAGACCGGCAAGA TCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACTCCAACAACCTGGACTCCAAGGTGGGCGGCAACTACAACTACCTGTACAGGCTGTTCAGGAAGTCCAACCTGAAGCCCTTCGAGAGGGACATCTCCACCGAGATCTACCAGGCCGGCTCCACCCCCTGCAACGGCGTGGAGGGCTTCAACTGCTACTTCCCCCTGCAGTCCTACGGCTTCCAGCCCACCAACGGCGTGGGCTACCAG CCCTACAGGGTGGTGGTGCTGTCCTTCGAGCTGCTGCACGCCCCCGCCACCGTGTGCGGCCCCAAGAAGTCC S protein RBD (Wuhan) 13 AACATCACCAACCTGTGCCCCTTCGACGAGGTGTTCAACGCCACCAGGTTCGCCTCCGTGTACGCCTGGAACAGGAAGAGGATCTCCAACTGCGTGGCCGACTACTCCGTGCTGTACAACTTCGCCCCCTTCTTCGCCTTCAAGTGCTACGGCGTGTCCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACTCCTTCGTGATCAGGGGCAACGAGGTGTCGCAGATCGCCCCCGGCCAGACCGGCAACAT CGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACTCCAACAAGCTGGACTCCAAGGTGGGCGGCAACTACAACTACAGGTACAGGCTGTTCAGGAAGTCCAACCTGAAGCCCTTCGAGAGGGACATCTCCACCGAGATCTACCAGGCCGGCAACAAGCCCTGCAACGGCGTGGCCGGCGTCAACTGCTACTTCCCCCTGCAGTCCTACGGCTTCAGGCCCACCTACGGCGTGGGCCACCAG CCCTACAGGGTGGTGGTGCTGTCCTTCGAGCTGCTGCACGCCCCCGCCACCGTGTGCGGCCCCAAGAAGTCC S protein RBD-Omicron BA.4/BA.5 (B.1.1.529.4, B.1.1.529.5, South Africa) 14 GCCCCCGAGCTGCTGGGCGGCCCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCTCCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAGTACCACTCCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGG AGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCCGCCCCCATCGAGAAGACCATCTCCAAGGCCAAGGGCCAGCCCAGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACTCCGACGGCTCCTTCTCCTGTACTCCAA GCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCCCCCGGC Fc peptide Mut. Glyco. (N->H) 15 AACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGGTTCGCCTCCGTGTACGCCTGGAACAGGAAGAGGATCTCCAACTGCGTGGCCGACTACTCCGTGCTGTACAACTCCGCCTCCTTCTCCACCTTCAAGTGCTACGGCGTGTCCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACTCCTTCGTGATCAGGGGCGACGAGGTGAGGCAGATCGCCCCCGGCCAGACCGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACTCCAACAACCTGGACTCCAAGGTGGGCGGCAACTACAACTACCTGTACAGGCTGTTCAGGAAGTCCAACCTGAAGCCCTTCGAGAGGGACATCTCCACCGAGATCTACCAGGCCGGCTCCACCCCCTGCAACGGCGTGGAGGGCTTCAACTGCTACTTCCCCCTGCAGTCCTACGGCTTCCAGCCCACCAACGGCGTGGGCTACCAGCCCTACAGGGTGGTGGTGCTGTCCTTCGAGCTGCTGCACGCCCCCGCCACCGTGTGCGGCCCCAAGAAGTCC GAGCCCAAGTCCTCCGACAAGACCCACACCTCCCCCCCCTCCCCC GCCCCCGAGCTGCTGGGCGGCCCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCTCCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAGTAC CAC TCCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCCGCCCCCATCGAGAAGACCATCTCCAAGGCCAAGGGCCAGCCCAGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCCCCCGGC S-RBD-Wuhan sFc fusion protein 16 AACATCACCAACCTGTGCCCCTTCGACGAGGTGTTCAACGCCACCAGGTTCGCCTCCGTGTACGCCTGGAACAGGAAGAGGATCTCCAACTGCGTGGCCGACTACTCCGTGCTGTACAACTTCGCCCCCTTCTTCGCCTTCAAGTGCTACGGCGTGTCCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACTCCTTCGTGATCAGGGGCAACGAGGTGTCGCAGATCGCCCCCGGCCAGACCGGCAACATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACTCCAACAAGCTGGACTCCAAGGTGGGCGGCAACTACAACTACAGGTACAGGCTGTTCAGGAAGTCCAACCTGAAGCCCTTCGAGAGGGACATCTCCACCGAGATCTACCAGGCCGGCAACAAGCCCTGCAACGGCGTGGCCGGCGTCAACTGCTACTTCCCCCTGCAGTCCTACGGCTTCAGGCCCACCTACGGCGTGGGCCACCAGCCCTACAGGGTGGTGGTGCTGTCCTTCGAGCTGCTGCACGCCCCCGCCACCGTGTGCGGCCCCAAGAAGTCC GAGCCCAAGTCCTCCGACAAGACCCACACCTCCCCCCCCTCCCCC GCCCCCGAGCTGCTGGGCGGCCCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCTCCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAGTAC CAC TCCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCCGCCCCCATCGAGAAGACCATCTCCAAGGCCAAGGGCCAGCCCAGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCCCCCGGC S-RBD-Omicron BA.4/BA.5-sFc fusion protein

table 3

Selection of peptides containing SARS-CoV-2 Th/CTL epitopes with known MHC I/II binding for high-precision SARS-CoV-2 vaccine design SEQ ID NO Location Type of epitope amino acid sequence MHC I MHC II 17 S _957-984 Th/CTL KKK- QALNTLVKQLSSNFGAI S SVLNDILSRL QALNTLVKQLSSNFGAI (HLA RB1*04:01) SVLNDILSR (HLA-A*11:01; 43.38% coverage) HLA-A*11:01 HLA-DRB1*04:01 18 S _891-917 Th KKK- GAALQIPFAMQMA YRFNGIGV TQNVLY GAALQIPFAMQMAYRF (HLA-DRA*01:01; HLA-DRB1*07:01) MA YRFNGIGV TQNVLY (HLA-DRB1*04:01) HLA-DRA*01:01 HLA-DRB1*07:01 HLA-DRB1*04:01 19 N _305-331 Th/CTL KKK- AQFAPSASAFFGMSRIGMEVTPSGTWL AQFAPSASAFFGMSRIGM (HLA-B*40:01) MEVTPSGTWL (HLA-B*40:01; 77.23% coverage) HLA-B*40:01 II 20 S _996-1028 Th/CTL KKK- LITGRLQSLQTYVTQ QLIRAAEIRASANLAATK LITGRLQSL (HLA-A2) QLIRAAEIRASANLAATK (HLA-DRB1*04:01) RLQSLQTYV (HLA-A*02:01, 69.63% coverage) VQIDR LITGR (HLA-A*31:01; 80.62% coverage) HLA-A2 HLA-A*02:01 HLA-A*31:01 HLA-DRB1*04:01 twenty one M _89-111 Th/CTL KKK- GLMWLSYF I ASFRLFARTRSMWS GLMWLSYFI (HLA-A*02:01) 100% coverage of MHC I IASFRLFARTRSMWS (MHC II) 65% coverage of MHC II HLA-A*02:01 II Bold : MHC I, Bottom : MHC II

Table 4

Conserved Th/CTL epitopes on membrane (M), nucleocapsid (N) and spike-2 (S2) proteins of SARS-CoV-2 variants of concern (VoC) used in T-cell vaccines against COVID Serial ID: 21 Serial ID: 19 Serial ID: 17 Serial ID: 18 Serial ID: 20 Wild types & VoCs M protein KKK-SARS-CoV2 M _101-156 CTL epitope N protein KKK-SARS-CoV2 N _305-331 Th/CTL epitopes S2 protein ^b,c KKK-SARS-CoV2 S _957-984 (Th/CTL epitopes) S2 Protein KKK-SARS-CoV2 S _891-917 (Th epitope) S2 Protein KKK-SARS-CoV2 S _996-1028 (Th/CTL epitope) Wuhan (original) KKK-GLMWLSYFIASFRLFARTRSMWS KKK-AQFAPSASAFFGMSRIGMEVTPSGTWL KKK-QALNTLVKQLSSNFGAISSVLNDILSRL KKK-GAALQIPFAMQMAYRFNGIGVTQNVLY KKK-LITGRLQSLQTVVTQLIRAAEIRASANLAATK Alpha, Beta, & Gamma KKK-GLMWLSYFIASFRLFARTRSMWS KKK-AQFAPSASAFFGMSRIGMEVTPSGTWL KKK-QALNTLVKQLSSNFGAISSVLNDILSRL KKK-GAALQIPFAMQMAYRFNGIGVTQNVLY KKK-LITGRLQSLQTVVTQLIRAAEIRASANLAATK Delta KKK-GLMWLSYFIASFRLFARTRSMWS KKK-AQFAPSASAFFGMSRIGMEVTPSGTWL KKK-QALNTLVKQLSSNFGAISSVLNDILSRL KKK-GAALQIPFAMQMAYRFNGIGVTQNVLY KKK-LITGRLQSLQTVVTQLIRAAEIRASANLAATK Omicron ^c (BA.1) KKK-GLMWLSYFIASFRLFARTRSMWS KKK-AQFAPSASAFFGMSRIGMEVTPSGTWL KKK-QALNTLVKQLSS K FGAISSVLNDI F SRL KKK-GAALQIPFAMQMAYRFN GIGVTQNVLY KKK-LITGRLQSLQTVVTQLIRAAEIRASANLAATK Omicron ^c (BA.2/BA.4/ BA.5) KKK-GLMWLSYFIASFRLFARTRSMWS KKK-AQFAPSASAFFGMSRIGMEVTPSGTWL KKK-QALNTLVKQLSS K FGAISSVLNDI L SRL (Serial ID: 22) KKK-GAALQIPFAMQMAYRFN GIGVTQNVLY KKK-LITGRLQSLQTVVTQLIRAAEIRASANLAATK The presence of ^a T cell epitope is critical for inducing B and T cell memory responses to viral antigens. SARS-CoV-2 CTL and Th epitopes verified by HLA binding and T cell function assays are highly conserved between SARS-CoV-2 and SARS-CoV-1 viruses, with smaller ones observed only in S957-984 Differences between variants. Wuhan wild-type peptides (M, N and S2x3) are used to precisely design the UB-612 vaccine against COVID-19. An HLA binding assay was used to identify T cell epitopes on SARS-CoV-1 (2003), which was used to determine the corresponding T cell epitopes on SARS-CoV-2 (2019) by sequence alignment. ^bExcept for N969K (on BA.1 to BA.5) and L981F (on BA.1) in the S957-984 peptide on the S2 spike protein, the other four designed epitope peptides of the UB-612 vaccine are not present. aa residues that overlap with reported mutation sites on spinin, M, and N proteins (Table 12). ^c In S957-984, there are minor sequence differences between Omicron BA.1 and BA.2/BA.4/BA.5, marked in bold.

table 5

Amino acid sequences of pathogen protein-derived Th epitopes including idealized artificial Th epitopes used to design SARS CoV immunogenic structures serial identification number describe sequence twenty three KKK-MvF5 Th (UBITh®1) KKK-ISITEIKGVIVHRIETILF

Table 6

Examples of optional heterologous spacers and CpG oligonucleotides serial identification number describe sequence / composition N/A naturally occurring amino acids Naturally occurring amino acids include: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamic acid, glycine, histidine, isoleucine , leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine N/A Gly-Gly -GG- N/A Epsilon-N Lysine ε-K twenty four Epsilon-N Lysine-KKK ε-K-KKK 25 KKK-Epsilon-N lysine KKK-ε-K 26 CpG1 5' TCg TCg TTT TgT CgT TTT gTC gTT TTg TCg TT 3' (completely phosphorothioated)

Table 7

UB-612 (Wuhan) 200 μg/mL composition serial identification number describe UnitQ'ty /mL Function Quality level 10 S1-RBD-sFc(Wuhan) 176 μg B-immunogen (GMP) ¹ 17 Th/CTL peptide 4 μg T-immunogen (GMP) ¹ 18 4 μg 19 4 μg 20 4 μg twenty one 4 μg twenty three 4 μg 26 CpG1 4 μg Adjuvant GMP -- ADJU-PHOS 1.6 mg Adjuvant GMP -- Histidine 4.0mM protein buffer Ph Eur, JP, USP -- Histidine HCl•H ₂ O 6.0mM protein buffer Ph Eur, BP, JP -- Arginine HCl 50.0mM protein buffer Ph Eur, BP, JP, USP -- TWEEN 80 0.06% (v/v) Surfactants/emulsifiers Ph Eur, JP, NF -- hydrochloric acid qs to pH 5.9~6.0 pH adjuster Ph Eur, BP, JP, NF -- sodium chloride 9 mg Osmotic pressure maintaining agent Ph Eur, BP, USP -- 2-phenoxyethanol 0.5% (v/v) Preservatives USP-NF -- water for injection (qsto) 1.0mL Solvent Ph USP ^1Materials used in Phase 2 and Phase 2/3 clinical trials will be manufactured in accordance with cGMP

Table 8

UniCoVac 2 unit price (Omicron BA.4/BA.5) 40 μg/mL composition serial identification number describe UnitQ'ty /mL Function level 11 S1-RBD-sFc (Omicron) 35.2 μg B-immunogen (GMP) ¹ twenty two Th/CTL peptide 0.8 μg T-immunogen (GMP) ¹ 18 0.8 μg 19 0.8 μg 20 0.8 μg twenty one 0.8 μg twenty three 4.0 μg 26 CpG1 120 μg Adjuvant (GMP) ¹ -- Alhydrol gel 1.2 mg Adjuvant GMP -- Histidine 4.0mM protein buffer Ph Eur, JP, USP -- Histidine HCl•H ₂ O 6.0mM protein buffer Ph Eur, BP, JP -- Arginine HCl 50.0mM protein buffer Ph Eur, BP, JP, USP -- TWEEN 80 0.06% (v/v) Surfactants/emulsifiers Ph Eur, JP, NF -- hydrochloric acid qs to pH 5.9~6.0 pH adjuster Ph Eur, BP, JP, NF -- sodium chloride 9 mg Osmotic pressure maintaining agent Ph Eur, BP, USP -- 2-phenoxyethanol 0.5% (v/v) Preservatives USP-NF -- water for injection (qsto) 1.0mL Solvent Ph USP ^1Materials used in Phase 2 and Phase 2/3 clinical trials will be manufactured in accordance with cGMP

Table 9

Composition of UniCoVac 3 bivalent (Wuhan + Omicron BA.4/BA.5) 40 μg/mL serial identification number describe UnitQ'ty /mL Function level 10+11 (1:1) S1-RBD-sFc(Wuhan+Omicron BA.4/BA.5) 35.2 μg B-immunogen (GMP) ¹ twenty two Th/CTL peptide 0.8 μg T-immunogen (GMP) ¹ 18 0.8 μg 19 0.8 μg 20 0.8 μg twenty one 0.8 μg twenty three 0.8 μg 26 CpG1 120 μg Adjuvant GMP -- Alhydrol gel 1.2mg Adjuvant GMP -- Histidine 4.0mM protein buffer Ph Eur, JP, USP -- Histidine HCl•H ₂ O 6.0mM protein buffer Ph Eur, BP, JP -- Arginine HCl 50.0mM protein buffer Ph Eur, BP, JP, USP -- TWEEN 80 0.06% (v/v) Surfactants/emulsifiers Ph Eur, JP, NF -- hydrochloric acid qs to pH 5.9~6.0 pH adjuster Ph Eur, BP, JP, NF -- sodium chloride 9 mg Osmotic pressure maintaining agent Ph Eur, BP, USP -- 2-phenoxyethanol 0.5% (v/v) Preservatives USP-NF water for injection (qsto) 1.0mL Solvent Ph USP ^1Materials used in Phase 2 and Phase 2/3 clinical trials will be manufactured in accordance with cGMP

Table 10. Pseudovirus neutralization assay (pVNT ₅₀ /ID ₅₀ ) Vaccine s ^a (same-boosting dose) ^a Participant (No.) NeuAb assay (method/unit) WT (GMT) ^b Omicrons BA.1/BA.2/BA.5 (GMT) ^b WT/Omicrons BA.1/BA.2/BA.5 (GMFR) UB-612 (Ph-1) (n = 45) PNA/pVNT ₅₀ 12,778 2,325/ND/ND 5.5/ND/ND UB-612 (Ph-2) ^c (n = 41) PNA/pVNT ₅₀ 6,254 1,196/985/ND 5.2/6.3/ND UB-612 (Ph-2) ^c (n = 12) PNA/pVNT ₅₀ 11,167 2,314/1,890/854 4.8/5.9/13.0 BNT162b2 (n = 24) PNA/pVNT ₅₀ 6,539 1066/776/ND 6.1/8.4/ND BNT162b2 (n = 19) PNA/pVNT ₅₀ 4,122 1,116/1,113/360 3.7/3.7/11.5 BNT162b2 (n = 27) PNA/ID ₅₀ 5783 900/829/275 6.4/7.0/21.0 NVX-CoV2373 (n = 48) PNA/ID ₅₀ 10,862 1,197/ND/582 9.1/ND/18.7 mRNA-1273 (n = 16) PNA/ID ₅₀ 4,679 945/780/378 5.0/5.9/12.4 MVC-COV1901 (n = 15) PNA/ID ₅₀ 1,280-640 160-80/ND/ND 8.7/ND/ND CoronaVac (n = 40) PNA/pVNT ₅₀ 632 122/122/75 5.2/5.2/8.4 AZD1222 (n = 41) PNA/pVNT ₅₀ 516 89/76/43 5.8/6.8/12 BBIBIP-CorV (n = 75) PNA/pVNT ₅₀ 295 15/ND/ND 20.1/ND/ND Abbreviations: PNA = pseudotyped virus neutralization assay; GMT = geometric mean titer; GMFR = geometric mean fold reduction relative to WT; WT = wild-type strain of SARS-CoV-2; Omicrons = Omicron subvariant BA. 1/BA.2/BA.5; ND = Not Confirmed. pVNT ₅₀ & ID ₅₀ = 50% neutralizing GMT pseudovirus assay. ^aVaccines reporting a homologous booster (third dose) vaccine. ^b GMT for WT measured 14 or 28 days after the third dose. ^c UB-612 - Pseudovirus assay in sera from a subset of participants when Omicron infection was sequentially dominated by BA.2 and BA.5 subvariants (Phase 2 add-on extension study).

Table 11 Live virus neutralization assay (VNT ₅₀ /FRNT ₅₀ /ID ₅₀ ) Vaccinea (same ^- boosting dose) ^a Participant (No.) NeuAb analysis (method/unit) WT (GMT) ^b Omicrons BA.1/BA.2 (GMT) ^b WT/Omicrons BA.1/BA.2 (GMFR) UB-612 (n = 15) MNA/VNT ₅₀ 6,159 670/485 9.2/12.7 mRNA-1273 (n = 20) FRNT/ID ₅₀ 1659 81.0/ND 20.5/ND BNT162b2 (n = 20) FRNT/ID ₅₀ 640 46.2/ND 13.3/ND BNT162b2 (n = 30) PRNT/VNT ₅₀ 673 106/ND 6.3/ND AZD1222 (n = 41) FRNT/FRNT ₅₀ 723 57.0/ND 12.7/ND Abbreviations: MNA = microneutralization assay; PRNT = plaque reduction neutralization test; FRNT = focal reduction neutralization test; GMT = geometric mean titer; GMFR = geometric mean fold reduction relative to WT; WT = SARS-CoV- Wild-type strain of 2; Omicrons = Omicron subvariant BA.1/BA.2; ND = not confirmed. pVNT ₅₀ & ID ₅₀ = 50% neutralized GMT, by live virus detection; VNT ₅₀ , ID ₅₀ and FRNT ₅₀ = 50% neutralized GMT, by live virus detection. ^aVaccines reporting a homologous booster (third dose) vaccine. b GMT for WT measured 14 or 28 days after the third additional dose.

Table 12 Mutation sites on SARS-CoV-2 spine (S) and non-envelope (E) , membrane (M) and nucleocapsid (N) proteins VoC ^b, Spiny (S1-RBD residues based on 319-541) E M N Delta T19R, G142D, Δ156-157, R158G, L452R , T478K , D614G, P681R , & D950N T9I I82T D63G, R203M, & D377Y Omicron (BA.1) A67V, Δ69-70, T95I, G142D , Δ143, Y144del, Δ145, Δ211, L212I, +214EPE, G339D, R346K, S371L, S373P , S375F , K417N , N440K , G446S, S477N , T47 8K, E484A , Q493R , G496S , Q498R , N501Y , Y505H , T547K , D614G , H655Y , N679K , N764K , D796Y , N856K , and Q954H , L969K , & L981F T9I D3G, Q19E , & A63T P13L , Δ31-33, R203K , & G204R Omicron (BA.2) T19I, L24S, Δ25-27, G142D , V213G, G339D, S371F, S373P , S375F , T,376A, D405N, R408S, K417N , N440K , S477N, T478K , E484A , Q493R , Q498R , N501Y , Y505H , D614G , H655Y , N679K , N764K , D796Y , N856K , Q954H , & L969K T9I Q19E & A63T P13L , Δ31-33, R203K , G204R , & S413R Omicron (BA. 4) T19I, L24S, Δ25-27, Δ69-70, G142D , V213G, G339D, S371F, S373P , S375F , T,376A, D405N, R408S, K417N , N440K , L452R, S477N , T478K , E484A , L486V, Q493, Q498R , N501Y , Y505H , D614G , H655Y , N679K , N764K , D796Y , N856K , and Q954H , & L969K T9I Q19E & A63T P13L , Δ31-33, P151S, R203K , G204R , & S413R Omicron (BA.5) T19I, L24S, Δ25-27, Δ69-70, G142D , V213G, G339D, S371F, S373P , S375F , T,376A, D405N, R408S, K417N , N440K , L452R, S477N , T478K , E484A , L486V, Q493, Q498R , N501Y , Y505H , D614G , H655Y , N679K , N764K , D796Y , N856K , and Q954H , & L969K T9I D3N, Q19E , & A63T P13L , Δ31-33, R203K , G204R , & S413R ^aReported mutation sites of spine, E, M and N proteins. ^b Omicron BA.4 and BA.5 have the same mutation site spectrum on the spike protein, and are more related to BA.2 than to BA.1. In BA.2, BA.4 and BA.5, the differences between the mutation sites of S, E, M and N proteins are marked in red. ^c . In addition to N969K (on BA.1 to BA.5) and L981F (on BA.1) in the S _957-984 peptide on S2 spike protein, the other four designed epitope peptides of UB-612 vaccine (Table 1B below) There are no aa-residues that overlap with the reporter mutation sites on the Spinin, M, and N proteins.

sequence serial identification number describe sequence 1 S protein RBD-Wuhan NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDL C FTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATV C GPKKS 2 S protein RBD-Omicron BA.4/BA.5 (B.1.1.529.4/ B.1.1.529.5, South Africa) nitnlcpfDevfnatrfasvyawnrkrisncvadysvlynFaPfFAfkcygvsptklndl c ftnvyadsfvirgNevSqiapgqtgniadynyklpddftgcviawnsnKldskvGgnynyRyrlfrksnlkpferdisteiyqagNKpcNgvAgVncyfplQsyGfRptygvgHqpyrvvv lsfellhapatv c gpkks 3 Wild-type hub region from human IgG1 EPKSCDKTHTCPPCP 4 Mutated hub region from human IgG1 EPKS S DKTHT S PP S P 5 Mutated hub region from human IgG1 EPKS X DKTHT X PP X P X : Ser, Gly, Thr, Ala, Val, Leu, Ile, Met, and/or missing. Underlined residues represent mutation sites relative to the sequence of wild-type IgG. 6 Fc peptide (Wild type) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY N STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG 7 Fc peptide Mut. Glycos. (N->H) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY H STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG 8 Fc peptide Mut. Glycos. (N->A) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY A STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG 9 Fc peptide Mut. Glycos. (N->X) X = N,H,A APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY VMHEALHNHYTQKSLSLSPG 10 S-RBD-Wuhan -sFc fusion protein NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDL C FTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATV C GPKKS EPKSSDKTHTSPPSP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY H STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG 11 S-RBD-Omicron BA.4/BA.5- sFc fusion protein nitnlcpfDevfnatrfasvyawnrkrisncvadysvlynFaPfFAfkcygvsptklndl c ftnvyadsfvirgNevSqiapgqtgniadynyklpddftgcviawnsnKldskvGgnynyRyrlfrksnlkpferdisteiyqagNKpcNgvAgVncyfplQsyGfRptygvgHqpyrvvv lsfellhapatv c gpkks EPKSSDKTHTSPPSP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY H STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 12 S protein RBD (Wuhan) AACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGGTTCGCCTCCGTGTACGCCTGGAACAGGAAGGATCTCCAACTGCGTGGCCGACTACTCCGTGCTGTACAACTCCGCCTCCTTCTCCACCTTCAAGTGCTACGGCGTGTCCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACTCCTTCGTGATCAGGGGCGACGAGGTGAGGCAGATCGCCCCCGGCCAGACCGGCAAGA TCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACTCCAACAACCTGGACTCCAAGGTGGGCGGCAACTACAACTACCTGTACAGGCTGTTCAGGAAGTCCAACCTGAAGCCCTTCGAGAGGGACATCTCCACCGAGATCTACCAGGCCGGCTCCACCCCCTGCAACGGCGTGGAGGGCTTCAACTGCTACTTCCCCCTGCAGTCCTACGGCTTCCAGCCCACCAACGGCGTGGGCTACCAG CCCTACAGGGTGGTGGTGCTGTCCTTCGAGCTGCTGCACGCCCCCGCCACCGTGTGCGGCCCCAAGAAGTCC 13 S protein RBD-Omicron BA.4/BA.5 (B.1.1.529.4, B.1.1.529.5, South Africa) AACATCACCAACCTGTGCCCCTTCGACGAGGTGTTCAACGCCACCAGGTTCGCCTCCGTGTACGCCTGGAACAGGAAGAGGATCTCCAACTGCGTGGCCGACTACTCCGTGCTGTACAACTTCGCCCCCTTCTTCGCCTTCAAGTGCTACGGCGTGTCCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACTCCTTCGTGATCAGGGGCAACGAGGTGTCGCAGATCGCCCCCGGCCAGACCGGCAACAT CGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACTCCAACAAGCTGGACTCCAAGGTGGGCGGCAACTACAACTACAGGTACAGGCTGTTCAGGAAGTCCAACCTGAAGCCCTTCGAGAGGGACATCTCCACCGAGATCTACCAGGCCGGCAACAAGCCCTGCAACGGCGTGGCCGGCGTCAACTGCTACTTCCCCCTGCAGTCCTACGGCTTCAGGCCCACCTACGGCGTGGGCCACCAG CCCTACAGGGTGGTGGTGCTGTCCTTCGAGCTGCTGCACGCCCCCGCCACCGTGTGCGGCCCCAAGAAGTCC 14 Fc peptide Mut. Glyco. (N->H) GCCCCCGAGCTGCTGGGCGGCCCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCTCCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAGTACCACTCCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGG AGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCCGCCCCCATCGAGAAGACCATCTCCAAGGCCAAGGGCCAGCCCAGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACTCCGACGGCTCCTTCTCCTGTACTCCAA GCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCCCCCGGC 15 S-RBD-Wuhan sFc fusion protein AACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCAGGTTCGCCTCCGTGTACGCCTGGAACAGGAAGGATCTCCAACTGCGTGGCCGACTACTCCGTGCTGTACAACTCCGCCTCCTTCTCCACCTTCAAGTGCTACGGCGTGTCCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACTCCTTCGTGATCAGGGGCGACGAGGTGAGGCAGATCGCCCCCGGCCAGACCGGCAAGA TCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACTCCAACAACCTGGACTCCAAGGTGGGCGGCAACTACAACTACCTGTACAGGCTGTTCAGGAAGTCCAACCTGAAGCCCTTCGAGAGGGACATCTCCACCGAGATCTACCAGGCCGGCTCCACCCCCTGCAACGGCGTGGAGGGCTTCAACTGCTACTTCCCCCTGCAGTCCTACGGCTTCCAGCCCACCAACGGCGTGGGCTACCAG CCCTACAGGGTGGTGGTGCTGTCCTTCGAGCTGCTGCACGCCCCCGCCACCGTGTGCGGCCCCAAGAAGTCC GAGCCCAAGTCCTCCGACAAGACCCACACCTCCCCCCCCTCCCCCCGCCCCCGAGCTGCTGGGCGGCCCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCTCCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAGTAC CAC TCCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCCGCCCCCATCGAGAAGACCATCTCCAAGGCCAAGGGCCAGCCCAGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCC AGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACTCCGACGGCTCCTTCTCCTGTACTCCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCCCCCGGC 16 S-RBD-Omicron BA.4/BA.5-sFc fusion protein AACATCACCAACCTGTGCCCCTTCGACGAGGTGTTCAACGCCACCAGGTTCGCCTCCGTGTACGCCTGGAACAGGAAGAGGATCTCCAACTGCGTGGCCGACTACTCCGTGCTGTACAACTTCGCCCCCTTCTTCGCCTTCAAGTGCTACGGCGTGTCCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACTCCTTCGTGATCAGGGGCAACGAGGTGTCGCAGATCGCCCCCGGCCAGACCGGCAACAT CGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACTCCAACAAGCTGGACTCCAAGGTGGGCGGCAACTACAACTACAGGTACAGGCTGTTCAGGAAGTCCAACCTGAAGCCCTTCGAGAGGGACATCTCCACCGAGATCTACCAGGCCGGCAACAAGCCCTGCAACGGCGTGGCCGGCGTCAACTGCTACTTCCCCCTGCAGTCCTACGGCTTCAGGCCCACCTACGGCGTGGGCCACCAG CCCTACAGGGTGGTGGTGCTGTCCTTCGAGCTGCTGCACGCCCCCGCCACCGTGTGCGGCCCCAAGAAGTCC GAGCCCAAGTCCTCCGACAAGACCCACACCTCCCCCCCCTCCCCCGCCCCCGAGCTGCTGGGCGGCCCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCTCCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAGTAC CAC TCCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCCGCCCCCATCGAGAAGACCATCTCCAAGGCCAAGGGCCAGCCCAGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCC AGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACTCCGACGGCTCCTTCTCCTGTACTCCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCCCCCGGC 17 KKK-SARS-CoV2 S _957-984 (Th/CTL peptide) KKK-QALNTLVKQLSSNFGAISSVLNDILSRL ​ 18 KKK-SARS-CoV2 S _891-917 (Th peptide) KKK-GAALQIPFAMQMAYRFNGIGVTQNVLY ​ 19 KKK-SARS-CoV2 N _305-331 (Th/CTL peptide) KKK-AQFAPSASAFFGMSRIGMEVTPSGTWL ​ 20 KKK-SARS-CoV2 S _996-1028 (Th/CTL peptide) KKK-LITGRLQSLQTYVTQQLIRAAEIRASANLAATK ​ twenty one KKK-SARS-CoV2 M _89-111 (Th/CTL peptide) KKK-GLMWLSYFIASFRLFARTRSMWS ​ twenty two KKK-SARS-CoV2 S _957-984 (Omicron BA.2/BA.4/ BA.5 Th/CTL peptide) KKK-QALNTLVKQLSS K FGAISSVLNDI L SRL twenty three KKK-MvF5 Th (UBITh®1) KKK-ISITEIKGVIVHRIETILF

without

Figure 1 , illustrates the design of a single-chain fusion protein according to various embodiments of the present disclosure. Specifically, this figure illustrates the general structure of a fusion protein that includes an S-RBD Omicron BA.4/BA.5 (SEQ ID NO: 2) at the N-terminus, which interacts with the hub region of human IgG ( SEQ ID NO: 4) and the Fc fragment (CH2 and CH3 regions) (SEQ ID NO: 7 or 8) are covalently linked. Figure 2 , schematically illustrates the overall view of the pZD/S-RBD Omicron BA.4/BA.5-sFc plasmid. According to one embodiment of the present invention, the pZD/S-RBD Omicron BA.4/BA.5-sFc plasmid encodes the S-RBD Omicron BA.4/BA.5-sFc fusion protein. Figure 3A illustrates the amino acid sequence, structure and function of S1-RBD Omicron BA.4/BA.5-sFc (SEQ ID NO: 11). Figure 3B includes the pairing of six disulfide bonds in the S1-RBD Omicron BA.4/BA.5-sFc fusion protein. Figure 4. Schematic illustration illustrating the general manufacturing process of drug matrix (DS) S1-RBD Omicron BA.4/BA.5-sFc (SEQ ID NO: 11). The process begins with a working cell bank (WCB) to seed cells and scale up the culture in a 2000 L fed-batch bioreactor. After the cell culture procedure, the unprocessed stock solution was collected and clarified by sterile filtration to produce a clear stock solution. To purify the drug matrix (DS), the drug substance is passed through protein A affinity chromatography, depth filtration and ion exchange (IEX) chromatography, followed by tangential flow filtration (TFF) for buffer exchange to achieve the formulated drug matrix. To avoid exogenous virus contamination, the clarified stock solution must be treated with solvent detergent, protein A chromatographic acid inactivation, and nanofiltration. Finally, the formulated S1-RBD Omicron BA.4/BA.5-sFc DS concentrate is produced after sterile filtration. Figure 5 , schematically illustrates the biochemical characterization by SDS-PAGE of a representative engineered S1- RBD -scFc protein of the invention in both non-reduced and reduced forms. Picture 6 . Schematic diagram illustrating the composition of the protein/peptide vaccine disclosed herein. The vaccine composition contains an S1-RBD Omicron BA.4/BA.5-sFc fusion protein (SEQ ID NO: 11) as the main B cell immunogen, and five synthetic Th/CTL peptides (SEQ ID NO: 17-22) are class I and class II MHC molecules, derived from SARS-CoV-2 Omicron BA.4/BA.5 M, N and S2 proteins, and UBITh®1a peptide (sequence identification number: 23) as T Catalyst for cell initiation. These ingredients are mixed with CpG1 (SEQ ID NO: 26), which binds to a positively charged peptide via bipolar interactions and also acts as an adjuvant, and is then combined with an alum adjuvant to form the vaccine composition. Figure 7 schematically illustrates the composite process for the manufacture of the designed COVID vaccine against SARS-CoV2 Omicron BA.4/BA.5 (or named UniCoVac Omicron ). To produce the vaccine composition, the peptide, CpG1, alum adjuvant, and finally the protein component are added sequentially. Specifically, the designed Th/CTL peptide was added to WFI, and then CpG1 was added to the mixture to form the peptide/CpG1 complex. Thereafter, protein buffer, alum and sodium chloride were added to the solution now containing peptide/CpG1/alum/sodium chloride. Finally, the S1-RBD Omicron BA.4/BA.5-sFc protein solution was added to the solution mixture to achieve the final vaccine composition. Figures 8A to 8D . Graph showing virus neutralizing titers (VNT50) against live SARS-CoV-2 wild type after an initial 2 doses of vaccine and a third booster dose in a Phase 1 trial. In the UB-612 primary 2-dose vaccine series of the 196-day Phase 1 trial, 50 of the 60 participants enrolled in the 10-μg, 30-μg, and 100-μg dose groups (n = 20 per group) Participants were enrolled in the extension study and received a third booster dose at 100-μg (n = 17 for the 10-μg dose; n = 15 for the 30-μg dose; n = 18 for the 100-μg dose group). Virus-neutralizing antibody geometric mean titers (GMT, 95% CI) that inhibited 50% of live SARS-CoV-2 wild-type (WT, Wuhan strain) were measured and expressed as 10-μg (Figure 8A ) , 30-μg ( Panel 8B ) VNT 50 for the 100-μg (Panel 8C ) dose group _. Figure 8D illustrates the 100-μg dose group. For study participants in the three dose groups, VNT ₅₀ data were recorded on Day 0 (before the first dose), Day 14 (14 days after the first dose). ), day 28 (1 month after the first dose; before the second dose), day 42 (14 days after the second dose), day 56 (1 month after the second dose), Day 112 (3 months after the 2nd dose), Day 196 (6 months after the 2nd dose), Days 255 to 316 (before the 3rd dose, before the booster dose) and Days 269 to 330 (after the booster dose) 14 days). Valence for individual participants is shown by circles. The horizontal dashed line represents the lower limit of quantification (LLOQ). HCS: Human convalescent serum samples in the control group (n = 20). Figures 9A to 9C . Chart showing effective neutralizing titers against SARS-CoV2 wild-type, Delta, Omicron, and other variants of concern generated by a third dose of UB-612 in a Phase 1 trial. A 196-day phase 1 trial was conducted with a main 2-dose series (days 0 and 28) with an extended booster 3rd dose of 100 mcg administered on a mean day 286 (days 255-316). Figure 9A provides the VNT ₅₀ titers observed for participants in the 100-μg group 14 days post-boost. The VNT ₅₀ titers observed 14 days after the boost reached 3992 for live SARS-CoV-2 wild type (WT) and 2358 for live Delta. Similar high levels of anti-WT and anti-Delta VNT ₅₀ were observed in the lower 30- and 10-μg dose groups. Figure 9B provides the observed pVNT ₅₀ titers against pseudo-SARS-CoV-2 wild type (WT) and against pseudo-SARS-CoV-2 variants including Omicron 14 days after the spike. Figure 9C provides antibody persistence after 2 doses (Phase 1 trial): anti-WT neutralizing VNT ₅₀ efficacy based on first-order exponential model fitting (SigmaPlot) from days 42-196 The valency decays slowly, with a half-life of 187 days (R ² = 0.9877; decay rate constant K _el = -0.0037; half-life = 0.693/K _el ). This figure shows that virus-neutralizing antibodies are persistent in the presence of WT live virus. Picture 10 . Bar graph showing virus-neutralizing pVNT ₅₀ titers observed against different SARS-CoV-2 variants in the main series of the Phase 1 trial at the 100 μg UB-612 dose group. In the main 2-dose vaccination series of the Phase 1 trial, in which vaccinees received two 100 μg doses of UB-612, twenty of the day 56 immune serum samples (n = 20) were selected to measure response to concern. Comparative neutralizing antibody activity of variants (VoCs). pVNT ₅₀ titers were assessed by pseudovirus-luciferase assay (microneutralization of viable virus in vitro). The research was conducted in the BSL2 laboratory of the RNAi Core Facility of Academia Sinica. Figures 11A and 11B . Graph showing anti-S1-RBD IgG antibodies and virus neutralizing responses to the wild-type Wuhan strain of SARS-CoV-2. Figure 11A provides mean ELISA -based GMT of anti-S1-RBD IgG responses across age groups at days 1, 29, and 57 in a Phase 2 study of 100 μg of UB-612 (n = 871, all; n = 731 for 18-65 years old; n = 140 for 65-85 years old). The error bars represent 95% CI, and the dashed line represents the limit of the ELISA assay. Figure 11B provides the GMT of the 50% virus neutralizing response (VNT ₅₀ ) to SARS -CoV-2-TCDC#4 (Wuhan wild type) virus at day 1 and day 57 across age groups. GMTs values are measured by microneutralization CPE assay. Error bars represent 95% CI and dashed lines indicate limits of microneutralization assay. In younger adults 18-65 years, the VNT50 of 96.4 was largely reproducible, as seen on Day 56 of the Phase 1 trial in vaccine recipients (20-55 years) at the 100 μg vaccine dose, where VNT ₅₀ is estimated to be 103 (Figure 8C ) . Figures 12A and 12B . Graph showing neutralizing antibody titers ( _VNT50 ) against SARS-CoV-2 variants in the primary 2-dose series of the Phase 2 trial. Figure 12A provides measurements of 50% virus neutralizing titers ( _VNT50 ) against live SARS-CoV-2 virus variants in immune sera from 48 (n = 39 to 18- Younger adults 65 years of age; n = 9 for older adults ≥65 years of age) were randomly selected among vaccinees who received two doses of UB-612 vaccine in the Phase 2 trial. Live wild-type Wuhan SARS-CoV-2-TCDC#4 and US WA 1/2020, as well as two VoCs listed by WHO (B.1.1.7 and B.1.617.2 lineages) were used for CPE detection. VNT ₅₀ values are marked at the top of each column and arranged with 95% confidence intervals (CI) shown as horizontal bars. Figure 12B provides the fold change (reduction) of VNT ₅₀ for each variant compared to wild type, Wuhan vs. US WA 1/2020 by two-sample t-test (** p < 0.01; **** p < 0.0001 ). The 2.7-fold and 1.4-fold reductions also represent 37% and 72% retention of neutralizing titers relative to the two Wuhan wild types isolated from the two different geographical locations where CPE assays were performed. Academia Sinica: Academia Sinica, Taiwan; CDPH: California Department of Public Health (CDPH), United States. Figures 13A to 13D . Graph showing inhibitory potency against S1-RBD:ACE2 binding after the main 2 doses of vaccination and the additional 3rd dose according to ELISA. ELISA-based S1-RBD:ACE2 binding titers were measured in the main 2-dose vaccine series of a 196-day Phase 1 trial (60 participants) and in an extension study with an additional third dose. Participants in the 10-μg (Figure 13A ) , 30-μg (Figure 13B), and 100-μg (Figure 13C ) dose groups ( n = 20 per dose group ) received two allocated vaccine doses separated by 28 days and an additional third dose of 100 μg was administered to 50 participants over 6 months (n = 17 for the 10-μg dose, n = 15 for the 30-μg dose, and n = 100-μg dose = 18 groups). Serum samples were collected at designated time points to measure the inhibitory potency on S1-RBD binding to ACE2 by ELISA. The horizontal dashed line represents the lower limit of quantification (LLOQ). Figure 13D illustrates the good correlation between S1-RBD: ACE2 binding inhibition and _VNT50 . Data are plotted for all primary/boost vaccinated participants (10-, 30-, and 100-μg dose groups). Data points for participants on Day 0 were excluded from the correlation analysis. Correlation was analyzed by non-parametric Spearman correlation method. Figures 14A to 14D . Graph showing anti-S1-RBD IgG binding titers by ELISA after primary 2 doses of vaccination and an additional third dose. ELISA-based anti-S1-RBD antibody binding titers were measured in the main 2-dose vaccine series of the 196-day Phase 1 trial (60 participants) and in an extension study with an additional third dose. Participants in the 10-μg (Figure 14A ) , 30-μg (Figure 14B), and 100-μg (Figure 14C ) dose groups ( n = 20 per dose group ) received two doses 28 days apart. vaccine dose, and a booster third dose of 100 μg was given to 50 participants over 6 months (n = 17 for the 10-μg dose, n = 15 for the 30-μg dose, and n = 100-μg dose = 18 groups). Serum samples were collected at indicated time points for measurement of anti-S1-RBD antibody binding by ELISA, expressed as geometric mean titer GMT with 95% CI. The horizontal dashed line represents the lower limit of quantification (LLOQ). Figure 14D illustrates the good correlation between anti-S1- RBD antibody binding and _VNT50 . Data are plotted for all primary/boost vaccinated participants (10-, 30-, and 100-μg dose groups). Data points for Day 0 participants were excluded from correlation analyses. Correlation was analyzed by non-parametric Spearman correlation method. Figures 15A to 15E . Graph showing the long-lasting, robust Th1-dominated cellular response induced by UB-612 as measured by IFN-γ and IL-4 ELISpot after restimulation of PBMCs with designated peptide antigens. In a 196-day Phase 1 trial, two doses of UB-612 were administered on days 0 and 28, administered by IFN-γ ELISpot at 10-μg (Figure 15A ) , 30-μg ( Figure 15B ) Vaccine-induced T cell responses were measured on PBMC cells from young adults (20 to 55 years old) in 100-μg (Figure 15C ) dose groups (n = 20 each). In a phase 2 pilot study, participants (young adults, >18 to <65 years) received two doses of 100 μg of UB-612 (n = 88) or saline placebo (n = 12) via IFN-γ ELISpot ( Figure 15D ) and IL-4 ELISpot ( Figure 15E ) T cell responses in PBMC of vaccine recipients restimulated with designed antigenic proteins/peptides on day 57. Shown are spots per 1×10 ⁶ PBMC after stimulation with S1-RBD+Th/CTL peptide pool, Th/CTL peptide pool, or CoV2 T peptide (Th/CTL peptide pool without UBITh1a) Forming units (SFU) produce IFN-γ and IL-4. Statistical analysis was performed using two-sample t-test (**** p<0.0001). Figures 16A -16C . Graph showing UB-612-induced Th1-dominated T cells as measured by IFN-γ and IL-4 intracellular staining (ICS) after restimulation of PBMCs with designed peptide antigens in the Phase 2 main 2-dose vaccination series. Cellular response (CD4 vs. CD8). In the Phase 2 trial, study participants (young adults >18 to 65 years of age) received 2 doses (28 days apart) of UB-612 (n = 88) or saline placebo (n = 12) at 100 mcg. Their PBMCs harvested on days 1 and 57 (4 weeks after the second injection) were restimulated with designed antigenic proteins/peptides, and T cell responses were assessed by intracellular staining (ICS). Indicator cells were produced The frequency of CD4+ and CD8+ T cells of factors affects the S1-RBD+Th/CTL peptide pool (Figure 16A ) , the Th/CTL peptide pool ( Figure 16B ) and the CoV2 T peptide (Th/ CTL peptide pool Responses to stimulation ( Figure 16C ) without UBITh1a). Statistical analysis was performed using Mann-Whitney t test. (* p<0.05; ** p<0.01;***p<0.001; **** p<0.0001) . [Citations] 1. Braun, J., Loyal, L., Frentsch, M., et al. SARS-CoV-2-reactive T cells in healthy donors and patients with COVID-19. Nature. 2020;587( 7833):270-274. 2. Ferretti, AP, Kula, T., Wang, Y., et al. Unbiased Screens Show CD8(+) T Cells of COVID-19 Patients Recognize Shared Epitopes in SARS-CoV-2 that Largely Reside outside the Spike Protein. Immunity. 2020;53(5):1095-1107 e3. 3. Le Bert, N., Tan, AT, Kunasegaran, K., et al. SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls. Nature. 2020;584(7821):457-462. 4. Long, QX, Tang, XJ, Shi, QL, et al. Clinical and immunological assessment of asymptomatic SARS-CoV-2 infections. Nat Med. 2020;26(8):1200-1204. 5. Ng, OW, Chia, A., Tan, AT, et al. Memory T cell responses targeting the SARS coronavirus persist up to 11 years post-infection. Vaccine. 2016;34(17):2008-2014. 6. Wang, CY, Wang, PN, Chiu, MJ, et al. UB-311, a novel UBITh® amyloid beta peptide vaccine for mild Alzheimer's disease. Alzheimers Dement (NY). 2017;3(2):262-272. 7. Wang, CY Artificial promiscuous t helper cell epitopes as immune stimulators for synthetic peptide immunogens. WO2020/132275A1. International Publication date on June 25th, 2020 (Priority Data: 62/782,253 on December 19th, 2018). 8. Wang, CY, Lin, F., Ding, S., et al. Designer peptides and proteins for the detection, prevention and treatment of coronavirus disease, 2019 (COVID 19). WO2021/168305A1. International Publication date on August 26th, 2021 (Priority Data: 62/978,596 on February 19th, 2020; 62/990,382 on March 16th, 2020, 63/027,290 on May 19th 2020; and 63/118,596 on November 25th, 2020). 9. Wang, CY, Hwang, KP, Kuo, HK, et al. A multitope SARS-CoV-2 vaccine provides long-lasting B cell and T cell immunity against Delta and Omicron variants. 2022a. J. Clinical Invest. 2022:312(10):e157707. https://doi.org/10.1172/JCI157707 and https://www.jci.org/articles/view/157707/sd /1 . 10. Wang, CY, Peng, WJ et al. UB-612 Multitope Vaccine targeting both Spike and Non-Spike Proteins of SARS-CoV-2 Provides Broad and Durable Immune Responses. 2022b. MedRxiv https://www. medrxiv.org/conten(https://www.medrxiv.org/content/10.1101/2022.08.26.22279232v1) . 11. Wyllie, D., Mulchandani, R., Jones, HE, et al. SARS-CoV-2 responsive T cell numbers are associated with protection from COVID-19: A prospective cohort study in keyworkers. medRxiv. 2020:2020.11.02.20222778.

TW202334198A_111137163_SEQL.xml

Claims (21)

A fusion protein comprising an Fc fragment of an IgG molecule and a biologically active molecule, wherein the Fc fragment is a single chain Fc (single chain Fc, sFc), wherein the Fc fragment includes a hinge region, wherein the The hub region is mutated and does not form a disulfide bond, wherein the hub region includes an amino acid sequence selected from the group consisting of sequence identification numbers: 3 to 5, wherein the biologically active molecule is from SARS-CoV2 A receptor binding domain (RBD) of the S protein (S1-RBD) (sequence identification number: 1 or 2), one of which is the Wuhan strain (Wuhan), one of which is the sequence identification number: 1, one of which is Omicron BA.4 The /BA.5 variant is Sequence ID: 2. Such as the fusion protein of claim 1, wherein the fusion protein is selected from the group consisting of sequence identification numbers: 10 and 11. Such as the fusion protein of claim 1, wherein the hub region includes an amino acid sequence of sequence identification number: 4. A pharmaceutical composition including the fusion protein of claim 1 and a pharmaceutically acceptable carrier or excipient. A method of producing a fusion protein as claimed in claim 1, comprising: a) Provide a biologically active molecule, wherein the biologically active molecule is a receptor binding domain (RBD) (Sequence Identification Number: 1) of the S protein (S-RBD) from SARS-CoV2 Wuhan or its Omicron BA.4/ One of the BA.5 variants, wherein the receptor binding domain (RBD) of the S protein (S-RBD) is sequence identification number: 2, b) providing an Fc fragment of an IgG molecule, wherein the Fc fragment includes a hub region, wherein the hub region is mutated by substitution and/or deletion of a cysteine residue to form a mutated Fc, and the The mutated Fc does not form a disulfide bond, wherein the hub region includes an amino acid sequence selected from the group consisting of Sequence Identification Number: 3-5, and c) Binding the biologically active molecule to the mutated Fc via the hub region. A fusion protein is selected from the group consisting of S1-RBD Omicron BA.4/BA.5 variant-sFc with sequence identification number: 11. A composition including the fusion protein of claim 6. The composition of claim 7 further includes a Th/CTL peptide, wherein the Th/CTL peptide is derived from SARS-CoV-2 M, N or S protein, a pathogen protein, or any combination thereof, wherein The Th/CTL peptide is selected from the group consisting of sequence identification numbers: 17-23 and any combination thereof. Such as the composition of claim 8, wherein the Th/CTL peptide is selected from the group consisting of sequence identification numbers: 17-23 and any combination thereof. A COVID vaccine composition including: a). An S-RBD Omicron BA.4/BA.5 variant protein, selected from the group with sequence identification number: 11; b). A Th/CTL peptide, selected from the group consisting of free sequence identification numbers: 17-23 and any combination thereof; c). A pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient is an adjuvant, buffer, surfactant, emulsifier, pH adjuster, saline solution, preservative, solvent, or any combination thereof. For example, the COVID vaccine composition of claim 10, wherein the Th/CTL peptide in (b) is selected from the group consisting of sequence identification numbers: 17-23 and any combination thereof. For example, the COVID vaccine composition of claim 11, wherein the pharmaceutically acceptable excipient is a CpG1 oligonucleotide, ALUM (aluminum phosphate or aluminum hydroxide), histidine, histidine HCl·H ₂ O , Arginine HCl, TWEEN 80 (polyoxyethylene (20) sorbitan monooleate), hydrochloric acid, sodium chloride and 2-phenoxyethanol (2- phenoxyethanol). For example, the COVID vaccine composition of claim 12, wherein the pharmaceutically acceptable excipient is CpG1 (sequence identification number: 26). A method of preventing COVID in an individual comprising administering to the individual a pharmaceutically effective amount of the vaccine composition of claim 10. A method of preventing COVID in an individual comprising administering to the individual a pharmaceutically effective amount of the vaccine composition of claim 11. A method of generating antibodies against the SARS-CoV-2 Omicron BA.4/BA.5 variant, comprising administering a pharmaceutically effective amount of the vaccine composition of claim 10 to an individual. A method of generating antibodies against the SARS-CoV-2 Omicron BA.4/BA.5 variant, comprising administering a pharmaceutically effective amount of the vaccine composition of claim 11 to an individual. A COVID vaccine composition including an ingredient in any of Tables 7-9 in the amounts indicated. A cell strain is transfected with a cDNA sequence encoding the fusion protein of claim 6 (SEQ ID NO: 16). For example, the cell line of claim 19 is a Chinese Hamster Ovary (CHO) cell line. Such as the cell strain of claim 19, wherein the cDNA sequence is selected from the group consisting of sequence identification number: 16.