WO2023023466A1

WO2023023466A1 - Sars-cov-2 multitope peptide/protein vaccine for the prevention and treatment of coronavirus disease, 2019 (covid-19)

Info

Publication number: WO2023023466A1
Application number: PCT/US2022/074904
Authority: WO
Inventors: Farshad Guirakhoo; Chang Yi Wang; Thomas P. Monath; Mei Mei HU
Original assignee: Vaxxinity, Inc.
Priority date: 2021-08-14
Filing date: 2022-08-12
Publication date: 2023-02-23
Also published as: TW202320845A

Abstract

The present disclosure is directed to high-precision, site-directed peptide immunogen constructs for the prevention of infection by SARS-CoV-2; receptor-based antiviral therapies for the treatment of the disease in infected patients; and designer protein vaccine containing S1-RBD-sFc. The disclosed invention utilizes amino acid sequences from SARS-CoV-2 proteins as well as human receptors for the design and manufacture of optimal SARS-CoV-2 antigenic peptides, peptide immunogen constructs, CHO-derived protein immunogen constructs, long-acting CHO-derived ACE2 proteins, and formulations thereof, as diagnostics, vaccines, and antiviral therapies for the detection, prevention, and treatment of COVID-19.

Description

SARS-CoV-2 MULTITOPE PEPTIDE/PROTEIN VACCINE FOR THE PREVENTION

AND TREATMENT OF CORONAVIRUS DISEASE, 2019 (COVID-19)

FIELD OF THE INVENTION

The present disclosure relates to a Coronavirus Disease, 2019 (COVID-19) multitope peptide/protein vaccine for the prevention, and treatment of COVID- 19, caused by the virus SARS-CoV-2. The disclosed invention utilizes viral and host-receptor amino acid sequences for the manufacture of optimal SARS-CoV-2 antigenic peptides, peptide immunogen constructs, CHO-derived protein immunogen constructs, long-acting CHO-derived ACE2 proteins, and formulations thereof, as vaccines, and antiviral therapies for the prevention, and treatment of COVID-19.

BACKGROUND OF THE INVENTION

In December 2019, a zoonotic coronavirus crossed species to infect human populations for the third time in recent decades. The disease caused by the virus, SARS-CoV-2, has been officially named by the World Health Organization (WHO) as “COVID- 19” for Coronavirus Disease, 2019, as the illness was first detected at the end of 2019. The virus SARS-CoV-2 was first identified in Wuhan, China and affected people exposed to a seafood wholesale market where other live animals were also sold. The virus SARS-CoV-2 is transmitted human-to- human and causes a severe respiratory disease similar to outbreaks caused by two other pathogenic human respiratory coronaviruses (i.e., severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV)).

Coronaviruses (family Coronaviridae, order Nidovirales) are large, enveloped, positive-stranded RNA viruses with a typical crown-like appearance (website: en.wikipedia.org/wiki/Coronavirus). Their viral genomes (26 to 32 kb) are some of the largest known among all RNA viruses. Coronaviruses are classified into four subgroups (Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus), initially based on antigenic relationships of the spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. The Betacoronavirus subgroup includes HCoV-OC43, HCoV- HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2. Genetic recombination readily occurs between members of the same and of different subgroups providing opportunity for increased genetic diversity.

Zhu, N., et al., 2020, identified and characterized SARS-CoV-2 and sequenced the viral genome from clinical specimens (bronchoalveolar-lavage fluid) and human airway epithelial cells virus isolates. The sequences were found to have 86.9% nucleotide sequence identity to a previously published bat SARS-like CoV genome (bat-SL-CoVZC45, MG772933.1). Additional articles (Chen, Y, et al., 2020 and Perlman, S., 2020) further characterize the genome structure, replication, and pathogenesis of emerging coronaviruses, including SARS- CoV, MERS-CoV, and SARS-CoV-2. A schematic diagram of the SARS-CoV-2 structure is shown in Figure 1. The viral surface proteins (S, E, and M proteins) are embedded in a lipid bilayer envelope produced by the host cell and the single stranded positive-sense viral RNAis associated with the nucleocapsid protein (N protein). Unlike other betacoronaviruses, SARS- CoV-2 does not possess a hemagglutinin esterase glycoprotein.

SARS-CoV-2 can be propagated in the same cells used for growing SARS-CoV and MERS-CoV. However, SARS-CoV-2 grows better in primary human airway epithelial cells, whereas both SARS-CoV and MERS-CoV infect intrapulmonary epithelial cells more than cells of the upper airways. In addition, transmission of SARS-CoV and MERS-CoV occurs primarily from patients demonstrating known signs and symptoms of the illness, whereas SARS-CoV-2 can be transmitted from asymptomatic patients or patients with mild or nonspecific signs. These differences likely contribute to the faster and more wide-spread transmission of SARS-CoV-2 compared to SARS-CoV and MERS-CoV.

It has been reported that SARS-CoV-2 uses the cellular receptor hACE2 (human angiotensin-converting enzyme 2) for cell entry, which is the same receptor used by SARS- CoV and different from the CD26 receptor used by MERS-CoV (Zhou, P., et al, 2020 and Lei, C , 2020). Accordingly, it has been suggested that transmission of SARS-CoV-2 is expected only after signs of lower respiratory tract disease have developed.

SARS-CoV mutated over the 2002-2004 epidemic to better bind to its cellular receptor and to optimize replication in human cells, which enhanced its virulence. Adaptation readily occurs because coronaviruses have error-prone RNA-dependent RNA polymerases, making mutations and recombination events frequent. By contrast, MERS has not been found to have mutated significantly to enhance human infectivity since it was detected in 2012. It is likely that SARS-CoV-2 will behave more like SARS-CoV and will further adapt to the human host, with enhanced binding to hACE2.

Following the SARS-CoV and MERS-CoV epidemics, great efforts were devoted to the development of new antiviral agents that target coronavirus proteases, polymerases, MTases, and entry proteins. However, none of them has been shown to be efficacious in clinical trials (Chan, JFW, et al., 2013; Cheng, KW, et al., 2015; Wang, Y, et al., 2015). Plasma and antibodies obtained from the convalescent patients have been used, out of the emergency situations, to treat patients with severe clinical symptoms (Mair-Jenkins, J., et al., 2015). In addition, various vaccine strategies targeting SARS-CoV and MERS-CoV, such as inactivated viruses, live-attenuated viruses, viral vector-based vaccines, subunit vaccines, recombinant proteins, and DNA vaccines, have been developed but have only been evaluated in animals so far (Graham, RL, et al., 2013; de Wit, E., et al., 2016).

Since there is no effective therapy or vaccine in face of the tragic outbreaks of CO VID- 19, the best current measures to reduce transmission of the virus, and to avoid unnecessary social panic resulting in huge economic losses, are to control the source of infection through (1) early detection by RT-PCR assays, (2) case reporting and quarantining of those in contact with the confirmed positive individuals with strict adherence to universal precautions in health care settings, (3) supportive treatments, and (4) timely publishing epidemic information. Individuals can also help reduce the transmission of SARS-CoV-2 through good personal hygiene, using a fitted mask, and avoiding crowded places.

There is an urgent need for the development of (a) serological assays for effective and rapid detection and surveillance of SARS-CoV-2, (b) vaccines to prevent non-infected individuals from contracting SARS-CoV-2, and (c) antiviral therapies to effectively treat individuals infected with SARS-CoV-2, in order to control the outbreak and reduce the resulting sufferings, including death.

References:

The following documents that are cited in this application as well as additional references cited therein are hereby incorporated by reference in their entireties as if fully disclosed herein.

1. AHMED, S.F., et al., “Preliminary identification of potential vaccine targets for 2019- nCoV based on SARS-CoV immunological studies.” DOI: 10.1101/2020.02.03.' 933226 (2020)

2. ARENDSE, L.B., et al., “Novel therapeutic approaches targeting the Renin-Angiotensin system and associated peptides in hypertension and heart failure.” Pharmacol. Rev., 71, 539-570 (2019)

3. BLUMBERG, R.S., et al., “Receptor specific transepithelialus transport of therapeutics.” US Patent Nos. 6,030,613 (2000), 6,086,875 (2000), and 6,485,726 (2002) . BLUMBERG, R.S., et al., “Central airway administration for systemic delivery of therapeutics.” WO 03/077834 (2002) and US Patent Publication US2003-0235536A1 (2003)

5. BRAUN, J., et al., “SARS-CoV-2-reactive T cells in healthy donors and patients with COVID-19.” Nature, 587, 270-274 (2020).

6. CAPON, D.J., et al., “Designing CD4 immunoadhesins for AIDS therapy.” Nature, 337:525 (1989)

7. CAPON, D.J., et al., “Hybrid immunoglobulins.” US Patent No. 5,116,964 (1992)

8. CHAN, J.F.W., et al., “Broad-spectrum antivirals for the emerging Middle East respiratory syndrome coronavirus.” J. Infect., 67(6):606- 616 (2013)

9. CHANG, J.C.C., et al., “Adjuvant activity of incomplete Freund’s adjuvant.” Advanced Drug Delivery Reviews, 32(3): 173-186 (1998)

10. CHEN, Y, et al., “Emerging coronaviruses: Genome structure, replication, and pathogenesis.” J Med Virol. DOI: 10.1002/jmv.25681 (2020)

11. CHENG, K.W., et al., “Thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of Middle East respiratory syndrome coronavirus.” Antiviral Res., 115: 9-16 (2015)

12. DE WIT, E., et al., “SARS and MERS: recent insights into emerging coronaviruses.” Nat. Rev. Microbiol., 14(8): 523-534 (2016)

13. FERRETTI, A.P., 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) doi: 10.1016/j.immuni.2020.10.006.

14. FIELDS, G.B., et al., Chapter 3 in Synthetic Peptides: A User’s Guide, ed. Grant, W.H. Freeman & Co., New York, NY, p.77 (1992)

15. GOEBL, N.A., et al., “Neonatal Fc Receptor Mediates Internalization of Fc in Transfected Human Endothelial Cells.” Mol. Biol. Cell, 19(12):5490-5505 (2008)

16. GRAHAM, R.L., et al., “A decade after SARS: strategies for controlling emerging coronaviruses.” Nat. Rev. Microbiol., 11 (12): 836- 848 (2013)

17. JUNGHANS, R.P, et al., “The protection receptor for IgG catabolism is the beta2- microglobulin-containing neonatal intestinal transport receptor.” Proc. Natl. Acad. Sci. USA, 93(11):5512-5516 (1996)

18. LE BERT, N., et al., “SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls.” Nature, 584, 457-462 (2020). 19. LEI, C., et al., “Potent neutralization of 2019 novel coronavirus by recombinant ACE2- Ig.” DOI: 10.1101/2020.02.01.929976 (2020)

20. LIU, H., et al., “Fc Engineering for Developing Therapeutic Bispecific Antibodies and Novel Scaffolds.” Frontiers in Immunology. , 8, 38 (2017).

21. LONG, Q.-X., et al., “Clinical and immunological assessment of asymptomatic SARS- CoV-2 infections.” Nat. Med. 26, 1200-1204 (2020).

22. MAIR- JENKINS, J., et al., “The effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis.” J. Infect. Dis., 211 (1): 80-90 (2015)

23. NG, O.-W., et al., “Memory T cell responses targeting the SARS coronavims persist up to 11 years post-infection.” Vaccine, 34, 2008-2014 (2016).

24. OSBORN, B.L., et al., “Pharmacokinetic and pharmacodynamic studies of a human serum albumin-interferon-alpha fusion protein in cynomolgus monkeys.” J. Pharmacol. Exp. Then, 303(2):540-8 (2002)

25. PERLMAN, S., “Another decade, another coronavirus.” N. Engl. J. Med., DOI: 10.1056/NEJMe2001126 (2020)

26. SHUBIN, Z., et al., “An HIV Envelope gpl20-Fc Fusion Protein Elicits Effector Antibody Responses in Rhesus Macaques.” Clin. Vaccine Immunol., 24, (2017).

27. SUI, J., et al. “Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to SI protein that blocks receptor association.” Proc. Natl. Acad. Sci. USA, 101, 2536-2541 (2004).

28. WANG, C.Y., et al., “UB-311, a novel UBITh(®) amyloid P peptide vaccine for mild Alzheimer’s disease.” Alzheimer ’s Dement., 3, 262-272 (2017).

29. WANG, C.Y., “Artificial promiscuous T helper cell epitopes as immune stimulators for synthetic peptide immunogens.” PCT Publication No. WO 2020/132275A1 (2020).

30. WANG, Y, et al., “Coronavirus nspl0/nspl6 methyltransferase can be targeted by nsplO- derived peptide in vitro and in vivo to reduce replication and pathogenesis.” J. Virol., 89(16):8416-8427 (2015)

31. WIKIPEDIA, The free encyclopedia, “Coronavirus” available at website: en.wikipedia.org/wiki/Coronavirus (accessed February 17, 2020).

32. WYLLIE, D., et al., “SARS-CoV-2 responsive T cell numbers are associated with protection from COVID-19: A prospective cohort study in keyworkers.” medRxiv 2020.11.02.20222778 (2020) doi: 10.1101/2020.11.02.20222778. 33. ZHAO, B., et al., “Immunization With Fc-Based Recombinant Epstein-Barr Virus gp350 Elicits Potent Neutralizing Humoral Immune Response in a BALB/c Mice Model.” Front. Immunol., 9, 932 (2018).

34. ZHOU, P, et al., “Discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin.” DOI: 10.1101/2020.01.22.914952 (2020)

35. ZHU, N., et al., “A novel coronavirus from patients with pneumonia in China, 2019.” N.

Engl. J. Med., DOI: 10.1056/NEJMoa2001017 (2020)

36. Giandhari, J. et al. Early transmission of SARS-CoV-2 in South Africa: An epidemiological and phylogenetic report, Int J Infect Dis. 2021 Feb; 103:234-241

37. Tegally, H., Wilkinson, E., Giovanetti, M. et al. Detection of a SARS-CoV-2 variant of concern in South Africa. Nature 592, 438-443 (2021).

38. PCT International Patent Application No: PCT/US2021/018855, filed February 19, 2021.

SUMMARY OF THE INVENTION

The present disclosure is directed to a SARS-CoV-2 multitope peptide/protein-based vaccine for the effective prevention, and treatment of COVID-19 containing Sl-RBD-sFc. The invention utilizes amino acid sequences from SARS-CoV-2 proteins as well as human receptors for the design and manufacture of optimal SARS-CoV-2 antigenic peptides, peptide immunogen constructs, CHO-derived protein immunogen constructs, long-acting CHO- derived ACE2 proteins, and formulations thereof, as diagnostics, vaccines, and antiviral therapies for the detection, prevention, and treatment of COVID-19.

More specifically, the present invention relates to a systematic approach to develop designer protein vaccine containing Sl-RBD-sFc (e.g., SEQ ID NOs: 235 and 236); utilizing bioinformatics including SARS-CoV-2 viral and receptor amino acid sequences for the design and manufacture of SARS-CoV-2 antigenic peptides, peptide immunogen constructs, and long acting ACE2 receptor proteins and formulations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Schematic diagram showing the structure of SARS-CoV-2. The viral surface proteins (spike, envelope, and membrane) are embedded in a lipid bilayer envelope derived from the host cell. Unlike other betacoronaviruses, SARS-CoV-2 does not possess a hemagglutinin esterase glycoprotein. The single stranded positive-sense viral RNA is associated with the nucleocapsid protein. Figure 2. A representative design of SARS-CoV-2 S-RBD (i.e., Receptor Binding Domain from the Spike protein) derived B cell epitope peptide immunogen constructs comprising constrained loop A, B, and C, respectively, based on an adapted 3D structure of ACE2 and SARS-CoV binding complex (image acquired through the Protein Data Bank (PDB) entry: 2AJF)(SEQ ID NOs: 28, 26, 25 and 23, respectively).

Figure 3. Alignment of M protein sequences from SARS-CoV-2, SARS-CoV, and MERS-CoV. An asterisk (*) represents an identical amino acid for the position, a colon (:) represents conserved substitution, a period (.) represents semi-conserved substitution, and an underline (_) represents an antigenic peptide )(SEQ ID NOs: 1, 2, and 3, respectively).

Figure 4. Alignment of N protein sequences from SARS-CoV-2, SARS-CoV and MERS- CoV )(SEQ ID NOs: 6, 7, and 8, respectively). An asterisk (*) represents identical amino acid for the position, a colon (:) represents conserved substitution, a period (.) represents semiconserved substitution, an underline ( ) represents an antigenic peptide, a dashed line (— ) represents a CTL epitope, and a dotted line (...) represents a Th epitope.

Figures 5A-5C. Alignment of S protein sequences from SARS-CoV-2, SARS-CoV and MERS-CoV. An asterisk (*) represents identical amino acid for the position, a colon (:) represents conserved substitution, a period (.) represents semi-conserved substitution, an underline (_) represents an antigenic peptide, a dashed line (— ) represents a CTL epitope, a dotted line (...) represents a Th epitope, and a box (□) represents a B cell epitope )(SEQ ID NOs: 20, 21, and 22, respectively).

Figures 6A-6D. Illustrates the design of a single chain fusion protein according to various embodiments of the present disclosure. Fig. 6A illustrates the structure of a fusion protein comprising an S-RBD at the N-terminus that is covalently linked to a hinge region and Fc fragment (CH2 and CH3 domains) of human IgG. Fig. 6B illustrates a fusion protein comprising an S-RBD from SARS-CoV-2 at the N-terminus that is covalently linked through a linker to a hinge region and Fc fragment (CH2 and CH3 domains) of human IgG. Fig. 6C illustrates a fusion protein comprising an ACE2-ECD (i.e., extra-cellular domain of ACE2) at the N- terminus that is covalently linked to a hinge region and Fc fragment (CH2 and CH3 domains) of human IgG. Fig. 6D illustrates a fusion protein comprising an ACE2-ECD at the N-terminus that is covalently linked through a linker to a hinge region and Fc fragment (CH2 and CH3 domains) of human IgG. Figure 7. Illustrates a map of pZD/S-RBD-sFc plasmid. The pZD/S-RBD -sFc plasmid encodes an S-RBD-sFc fusion protein according to an embodiment of the present invention.

Figure 8. Illustrates a map of pZD/hACE2-sFc plasmid. The pZD/hACE2-sFc plasmid encodes an ACE2-sFc fusion protein according to an embodiment of the present invention.

Figure 9. Illustrates the design and identification of antigenic peptides from SARS-CoV-2 N (Nucleocapsid) protein. A schematic of the full-length N protein is shown at the top and the designer peptide antigens disclosed herein are shown below.

Figure 10. Illustrates the design and identification of antigenic peptides from SARS-CoV-2 S (Spike) protein. A schematic of the full-length S protein is shown at the top and the designer peptide antigens disclosed herein are shown below.

Figure 11. Illustrates the design and identification of antigenic peptides from SARS-CoV-2 M (Membrane) protein. A schematic of the full-length M protein is shown at the top and the designer peptide antigens disclosed herein are shown below.

Figure 12. Illustrates the design and identification of antigenic peptides from SARS-CoV-2 E (Envelope) protein. A schematic of the full-length E protein is shown at the top and the designer peptide antigens disclosed herein are shown below.

Figure 13. Illustrates the design and identification of antigenic peptides from SARS-CoV-2 ORF9b protein. A schematic of the full-length ORF9b protein is shown at the top and the designer peptide antigens disclosed herein are shown below.

Figure 14. Illustrates the reactivities with identified antigenic peptides from various regions derived from SARS-CoV-2 N (Nucleocapsid) protein by serum antibodies obtained from representative COVID-19 patients.

Figure 15. Illustrates the mapping of antigenic regions from SAR.S-CoV-2 S (Spike) protein by serum antibodies from representative COVID-19 patients.

Figure 16. Illustrates the sites of four antigenic peptides on the SARS-CoV-2 S (Spike) protein by a 3D structure.

Figure 17. Illustrates the antigenic regions from SARS-CoV-2 E (Envelope) protein by serum antibodies from representative COVID-19 patients.

Figure 18. Illustrates of the antigenic regions from SARS-CoV-2 M (Membrane) protein by serum antibodies from representative COVID-19 patients. Figure 19. Illustrates of the antigenic regions from SARS-CoV-2 ORF9b protein by serum antibodies from representative COVID-19 patients.

Figure 20. Illustrates the assessment of immunogenicity associated with varying forms of designer proteins by ELISA using SI protein coated plates.

Figure 21. Illustrates assessment of neutralizing antibody titers by an Sl-RBD and ACE2 Binding inhibition assay using two separate methods, Method A and Method B.

Figure 22 shows detailed procedure of a cell-based Sl-RBD and ACE2 binding inhibition assay is illustrated in detail in Figure 22.

Figure 23A-23B. Illustrates the amino acid sequence, structure, and function of Sl-RBD-sFc. Fig. 23A provides the sequence of Sl-RBD-sFc and identifies the N-linked glycosylation site (*), the O-linked glycosylation site (+) the Asn-to-His mutation (underlined residue), and the disulfide bonds (connected lines)(SEQ ID NO: 235). Fig. 23B summarizes the disulfide bonding in the Sl-RBD-sFc fusion protein.

Figure 24. A schematic illustrating the components of a multitope protein/peptide vaccine disclosed herein. The vaccine composition contains an Sl-RBD-sFc fusion protein for the B cell epitopes, five synthetic Th/CTL peptides for class I and II MHC molecules derived from SARS-CoV-2 S, M, and N proteins, and the UBITh®la peptide. These components are mixed with CpGl which binds to the positively (designed) charged peptides by dipolar interactions and also serves as an adjuvant, which is then bound to ADJU-PHOS® adjuvant to constitute the multitope vaccine drug product.

Figure 25. Depicts the immunogenicity of UB-612 and VACCINE CANDIDATE A in Cynomolgus Macaques

Figure 26. Depicts a study of the pre- SARS-Cov-2 challenge neutralizing Abs at Days 0, 14, 28 and 50 of UB-612 and VACCINE CANDIDATE A.

Figure 27. Depicts a study of variant neutralization: Comparison WT vs SA Viruses with UB- 612 and VACCINE CANDIDATE A.

Figure 28. Depicts a study of variant neutralization: Comparison WT vs SA Viruses at Day 50 with UB-612 and VACCINE CANDIDATE A.

Figure 29. Depitcs a study of viral loads in BAL, nasal and rectal swabs for VACCINE CANDIDATE A post SARS-Cov-2 Challenge Figure 30A-30C. Depicts Loop 1, Loop 3 and Loop 5 of the N-terminal domain (NTD) of SARS-CoV-2 (Figure 30A); the sequence of Loop 3 (N3) and Loop 5 (N5) (SEQ ID NO: 20) (Figure 30B), and the loop conformation of ligand free vs antibody contact (Figure 30C)

Figure 31. Depicts a compilation of SARS-CoV-2 spike mutations in humans and animals

Figure 32. Depicts the NT50 values for COVID-19 convalescent plasma measured at 1.3 months and 6.2 months as well as plasma from mRNA- Vaccines.

Figure 33. Alignment of S protein sequences from SARS-CoV-2 parent (SEQ ID NO: 20) and SARS-CoV-2 South Africa, Beta Variant (S.501Y.V2) (SEQ ID NO: 371).

Figure 34. Alignment of Sl-RBD-sFc of UB612 (SARS-CoV-2) parent (SEQ ID NO: 235) and the Sl-RBD-sFc of VACCINE CANDIDATE A (SARS-CoV-2 South Africa, Beta Variant (S.501Y.V2)(SEQ ID NO: 371)).

Figure 35. Depicts the neutralization of (SARS-CoV-2 WT) and (SARS-CoV-2 Delta) at day 50 after treatment with VACCINE CANDIDATE A.

Figure 36. Depicts N-linked glycan structures of S-RBD-sFc.

Figure 37. Depicts O-linked glycan structures of S-RBD-sFc.

Figure 38. Depicts N-linked glycan structures of ACE2-ECD-sFc.

Figure 39. Depicts O-linked glycan structures of ACE2-ECD-sFc.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is directed to a SARS-CoV-2 multitope peptide/protein-based vaccine for the effective prevention, and treatment of COVID- 19 containing S 1 -RBD-sFc. The invention utilizes amino acid sequences from SARS-CoV-2 proteins as well as human receptors for the design and manufacture of optimal SARS-CoV-2 antigenic peptides, peptide immunogen constructs, CHO-derived protein immunogen constructs, long-acting CHO- derived ACE2 proteins, and formulations thereof, as diagnostics, vaccines, and antiviral therapies for the detection, prevention, and treatment of COVID-19.

Each aspect of the disclosed invention is discussed in further detail below.

General

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

Unless otherwise explained, 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 context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Hence, the phrase “comprising A or B” means including A, or B, or A and B. It is further to be understood that all amino acid sizes, and all molecular weight or molecular mass values, given for polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosed method, 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, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

The term “SARS-CoV-2”, as used herein, refers to the 2019 novel coronavirus strain that was first identified in Wuhan, China and affected people exposed to a seafood wholesale market where other live animals were also sold. SARS-CoV-2 is also known as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is the cause of the coronavirus disease 2019 (CO VID-ID).

The term “COVID- 19”, as used herein, refers to the human infectious disease caused by the SARS-CoV-2 viral strain. COVID-19 was initially known as SARS-CoV-2 acute respiratory disease. The disease may initially present with few or no symptoms, or may develop into fever, coughing, shortness of breath, pain in the muscles and tiredness. Complications may include pneumonia and acute respiratory distress syndrome.

A. RECEPTOR-BASED ANTIVIRAL THERAPIES FOR THE TREATMENT OF COVID-19 IN INFECTED PATIENTS

One aspect of the invention relates to receptor-based antiviral therapies for the treatment of COVID-19 in infected patients. The present disclosure is directed to novel fusion proteins comprising a bioactive molecule and portions of an immunoglobulin molecule. Various aspects of the present disclosure relate to fusion proteins, compositions thereof, and methods for making and using the disclosed fusion proteins. The disclosed fusion proteins are useful for extending the serum half-life of bioactive molecules in an organism.

The following is a detailed description provided to aid those skilled in the art in practicing the present invention. Those of ordinary skill in the art would understand that modifications or variations of the embodiments expressly described herein, which do not depart from the spirit or scope of the information contained herein, are encompassed by the present disclosure. The terminology used in the description is for describing particular embodiments only and is not intended to be limiting of the invention. The section headings used below are for organizational purposes only and are not to be construed as limiting the subject matter described.

1. Fusion Protein

As used herein, “fusion protein” or a “fusion polypeptide” is a hybrid protein or polypeptide comprising at least two proteins or peptides linked together in a manner not normally found in nature.

One aspect of the present disclosure is directed to a fusion protein comprising an immunoglobulin (Ig) Fc fragment and a bioactive molecule. The bioactive molecule that is incorporated into the disclosed fusion protein has improved biological properties compared to the same bioactive molecule that is either not-fused or incorporated into a fusion protein described in the prior art (e.g., fusion proteins containing a two chain Fc region). For example, the bioactive molecule incorporated into the disclosed fusion protein has a longer serum halflife compared to its non-fused counterpart. Additionally, the disclosed fusion protein maintains full biological activity of the bioactive molecule without any functional decrease, which is an improvement over the fusion proteins of the prior art that have a decrease in activity due to steric hindrance from a two chain Fc region.

The fusion proteins of the present disclosure provide significant biological advantages to bioactive molecules compared to non-fused bioactive molecules and bioactive molecules incorporated into fusion proteins described in the prior art.

The disclosed fusion protein can have any of the following formulae (also shown in Figures 6A-6D):

(B)-(Hinge)-(C_H2-C_H3) or

(C_H2-C_H3)-(Hinge)-(B) or

(B)-(L)_m-(Hinge)-(C_H2-C_H3) or

(CH2-C_H3)-(Hinge)-(L)_m-(B) wherein

“B” is a bioactive molecule;

“Hinge” is a hinge region of an IgG molecule;

“CH2-CH3” is the CH2 and CH3 constant region domains of an IgG heavy chain;

“L” is an optional linker; and

“m” may be an any integer or 0.

The various portions/fragments of the fusion protein are discussed further below. a. Fc Region and Fc Fragment

The fusion protein of the present disclosure contains an Fc fragment from an immunoglobulin (Ig) molecule.

As used below, “Fc region” refers to a portion of an immunoglobulin located in the c- terminus of the heavy chain constant region. The Fc region is the portion of the immunoglobulin that interacts with a cell surface receptor (an Fc receptor) and other proteins of the complement system to assist in activating the immune system. In IgG, IgA and IgD isotypes, the Fc region contains two heavy chain domains (CH2 and CH3 domains). In IgM and IgE isotypes, the Fc region contains three heavy chain constant domains (CH2 to CH4 domains). Although the boundaries of the Fc portion may vary, the human IgG heavy chain Fc portion is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index.

In certain embodiments, the fusion protein comprises a CH2-CH3 domain, which is an FcRn binding fragment, that can be recycled into circulation again. Fusion proteins having this domain demonstrate an increase in the in vivo half-life of the fusion proteins.

As used herein, “Fc fragment” refers to the portion of the fusion protein that corresponds to an Fc region of an immunoglobulin molecule from any isotype. In some embodiments, the Fc fragment comprises the Fc region of IgG. In specific embodiments, the Fc fragment comprises the full-length region of the Fc region of IgGl . In some embodiments, the Fc fragment refers to the full-length Fc region of an immunoglobulin molecule, as characterized and described in the art. In other embodiments, the Fc fragment includes a portion or fragment of the full-length Fc region, such as a portion of a heavy chain domain (e.g., CH2 domain, CH3 domain, etc.) and/or a hinge region typically found in the Fc region. For example, the Fc fragment of can comprise all or part of the CH2 domain and/or all or part of the CH3 domain. In some embodiments, the Fc fragment includes a functional analogue of the full-length Fc region or portion thereof.

As used herein, “functional analogue” refers to a variant of an amino acid sequence or nucleic acid sequence, which retains substantially the same functional characteristics (binding recognition, binding affinity, etc.) as the original sequence. Examples of functional analogues include sequences that are similar to an original sequence but contain a conservative substitution in an amino acid position; a change in overall charge; a covalent attachment to another moiety; or small additions, insertions, deletions or conservative substitutions and/or any combination thereof. Functional analogues of the Fc fragment can be synthetically produced by any method known in the art. For example, a functional analogue can be produced by modifying a known amino acid sequence by the addition, deletion, and/or substitution of an amino acid by site-directed mutation. In some embodiments, functional analogues have an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95% 96%, 97%, 98%, or 99% identical to a given sequence. Percent identity between two sequences is determined by standard alignment algorithms such as ClustalOmega when the two sequences are in best alignment according to the alignment algorithm.

The immunoglobulin molecule can be obtained or derived from any animal (e.g., human, cows, goats, swine, mice, rabbits, hamsters, rats, guinea pigs). Additionally, the Fc fragment of the immunoglobulin can be obtained or derived from any isotype (e.g., IgA, IgD, IgE, IgG, or IgM) or subclass within an isotype (IgGl, IgG2, IgG3, and IgG4). In some embodiments, the Fc fragment is obtained or derived from IgG and, in particular embodiments, the Fc fragment is obtained or derived from human IgG, including humanized IgG.

The Fc fragment can be obtained or produced by any method known in the art. For example, the Fc fragment can be isolated and purified from an animal, recombinantly expressed, or synthetically produced. In some embodiments, the Fc fragment is encoded in a nucleic acid molecule (e.g., DNA or RNA) and isolated from a cell, germ line, cDNA library, or phage library.

The Fc region and/or Fc fragment can include a hinge region found in some immunoglobulin isotypes (IgA, IgD, and IgG). In certain embodiments, the Fc fragment is modified by mutating the hinge region so that it does not contain any Cys and cannot form disulfide bonds. The hinge region is discussed further below.

The Fc fragment of the disclosed fusion protein is preferably a single chain Fc. As used herein, “single chain Fc” (of “sFc”) means that the Fc fragment is modified in such a manner that prevents it from forming a dimer (e.g., by chemical modification or mutation addition, deletion, or substation of an amino acid).

In certain embodiments, the Fc fragment of the fusion protein is derived from human IgGl, which can include the wild-type human IgGl amino acid sequence or variations thereof. In some embodiments, the Fc fragment of the fusion protein contains an Asn (N) amino acid that serves as an N-glycosylation site at amino acid position 297 of the native human IgGl molecule (based on the European numbering system for IgGl, as discussed in U.S. Patent No. 7,501,494), which corresponds to residue 67 in the Fc fragment (SEQ ID NO: 231), shown in Table 11. In other embodiments, the N-glycosylation site in the Fc fragment is removed by mutating the Asn (N) residue with His (H) (SEQ ID NO: 232) or Ala (A) (SEQ ID NO: 233) (Table 11). An Fc fragment containing a variable position at the N-glycosylation site is shown as SEQ ID NO: 234 in Table 11.

In some embodiments, the CH3-CH2 domain of the Fc fragment has an amino acid sequence corresponding to the wild-type sequence (disclosed in SEQ ID NO: 231). In certain embodiments, the CH3-CH2 domain of the Fc fragment has the amino acid sequence of SEQ ID NO: 232, where the N-glycosylation site is removed by mutating the Asn (N) residue with His (H). In certain embodiments, the CH3-CH2 domain of the Fc fragment has the amino acid sequence of SEQ ID NO: 233, where the N-glycosylation site is removed by mutating the Asn (N) residue with Ala (A). b. Hinge Region

The disclosed fusion protein can include a hinge region found in some immunoglobulin isotypes (IgA, IgD, and IgG). The hinge region separates the Fc region from the Fab region, and adds flexibility to the molecule, and can link two heavy chains via disulfide bonds. Formation of a dimer, comprising two CH2-CH3 domains, is required for the functions provided by intact Fc regions. Interchain disulfide bonds between cysteines in the wild-type hinge region help hold the two chains of the Fc molecules together to create a functional unit.

In certain embodiments, the hinge region is be derived from IgG, preferably IgGl . The hinge region can be a full-length or a modified (truncated) hinge region.

In specific embodiments, the hinge region contains a modification that prevents the fusion protein from forming a disulfide bond with another fusion protein or an immunoglobulin molecule. In specific embodiments, the hinge region is modified by mutating and/or deleting one or more cysteine amino acids to prevent the formation of a disulfide bond. The N-terminus or C-terminus of the full-length hinge region may be deleted to form a truncated hinge region. In order to avoid the formation of disulfide bonds, the cysteine (Cys) in the hinge region can be substituted with a non-Cys amino acid or deleted. In specific embodiments, the Cys of hinge region may be substituted with Ser, Gly, Ala, Thr, Leu, He, Met or Vai. Examples of wild-type and mutated hinge regions from IgGl to IgG4 include the amino acid sequences shown in Table 9 (SEQ ID NOs: 166-187). Disulfide bonds cannot be formed between two hinge regions that contain mutated sequences. The IgGl hinge region was modified to accommodate various mutated hinge regions with sequences shown in Table 10 (SEQ ID NOs: 188-225). c. Linker

The fusion protein may have the bioactive molecule linked to the N-terminus of the Fc fragment. Alternatively, the fusion protein may have the bioactive molecule linked to the C- terminus of the Fc fragment. The linkage is a covalent bond, and preferably a peptide bond.

In the present invention, one or more bioactive molecule may be directly linked to the C-terminus or N-terminus of the Fc fragment. Preferably, the bioactive molecule(s) can be directly linked to the hinge of the Fc fragment.

Additionally, the fusion protein may optionally comprise at least one linker. Thus, the bioactive molecule may not be directly linked to the Fc fragment. The linker may intervene between the bioactive molecule and the Fc fragment. The linker can be linked to the N- terminus of the Fc fragment or the C-terminus of the Fc fragment.

In one embodiment, the linker includes amino acids. The linker may include 1-5 amino acids. d. Bioactive Molecule

As used herein, the term “biologically active molecule” refers to proteins, or portions of proteins, derived either from proteins of SARS-CoV-2 or host-receptors involved in viral entry into a cell. Examples of biologically active molecules include the spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins from 2019-CoV, the human receptor ACE2 (hACE2), and/or fragments thereof.

In one embodiment, the biologically active molecule is the S protein of SARS-CoV-2 (SEQ ID NO: 20). In certain embodiments, the biologically active molecule is the receptor binding domain (RBD) of the S protein (S-RBD or Sl-RBD) of SARS-CoV-2 (SEQ ID NO: 226), which corresponds to amino acid residues 331-530 of the full-length S protein. In certain embodiments, the cysteine (C) residues at positions 61 and 195 of the S-RBD sequence of SEQ ID NO: 226 are mutated to alanine (A) residues, as shown in SEQ ID NO: 227 (residues 61 and 195 of S-RBD correspond to residues 391 and 525 of the full-length S protein of SEQ ID NO: 20). The mutated S-RBD sequence is also referred to as S-RBDa in this disclosure. The C61A and C195A mutations in the S-RBD sequence are introduced to avoid a mismatch of disulfide bond formation in the recombinant protein expression. Exemplary formulations of VACCINE CANDIDATE A can be found in Tables 33-39

In another embodiment, the biologically active molecule is the S protein of SARS-CoV- 2 SA, beta variant (Figures 33 and 34). In certain embodiments, the biologically active molecule is the receptor binding domain (RBD) of the S protein (S-RBD or Sl-RBD) of SARS- CoV-2 SA, beta variant (Figures 33 and 34), which corresponds to amino acid residues 331- 530 of the full-length S protein. In certain embodiments, the cysteine (C) residues at positions 61 and 195 of the S-RBD sequence of SEQ ID NO: Figures 33 and 34 are mutated to alanine (A) residues, as shown in SEQ ID NO: Figures 33 and 34 (residues 61 and 195 of S-RBD correspond to residues 391 and 525 of the full-length S protein of SEQ ID NO: Figures 33 and 34). A particular embodiment using the S protein of SARS-CoV-2 SA, beta variant is referred to herein as VACCINE CANDIDATE B. Exemplary formulations of VACCINE CANDIDATE B can be found in Tables 36-38

In another embodiment, the biologically active molecule includes both the S protein of SARS-CoV-2 (SEQ ID NO: 20) and the S protein of SARS-CoV-2 SA, beta variant (Figures 33 and 34). In certain embodiments, the biologically active molecule is the receptor binding domain (RBD) of the S protein (S-RBD or Sl-RBD) of SARS-CoV-2 (SEQ ID NO: 20), and the RDB of the S protein of SARS-CoV-2 SA, beta variant (Figures 33 and 34). A particular embodiment using both the S protein of SARS-CoV-2 (SEQ ID NO: 20) and the S protein of SARS-CoV-2 SA, beta variant is referred to herein as VACCINE CANDIDATE B-bivalent or VACCINE CANDIDATE C - BIVALENT. Exemplary formulations of VACCINE CANDIDATE C - BIVALENT can be found in Tables 39-41

In another embodiment, the biologically active molecule is the human receptor ACE2 (hACE2) (SEQ ID NO: 228). In certain embodiments, the biologically active molecule is the extracellular domain (ECD) of hACE2 (1IACE2ECD) (SEQ ID NO: 229), which corresponds to amino acid residues 1-740 of the full-length hACE2 protein. In some embodiments, the histidine (H) residues at positions 374 and 378 in the IIACE2ECD sequence of SEQ ID NO: 229 are mutated to asparagine (N) residues, as shown in SEQ ID NO: 230 (also referred to as ACE2NECD in this disclosure). The H374N and H378N mutations are introduced to abolish the peptidase activity of hACE2.

2. Compositions

In certain embodiments, the present invention relates to compositions, including pharmaceutical compositions, comprising the fusion protein and a pharmaceutically acceptable carrier, adjuvant, and/or other excipients such as diluents, additives, stabilizing agents, preservatives, solubilizing agents, buffers, and the like. Pharmaceutical compositions can be prepared by mixing the fusion protein with optional pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers include solvents, dispersion media, isotonic agents and the like. Examples of carriers include water, saline solutions or other buffers (such as phosphate, citrate buffers), oil, alcohol, proteins (such as serum albumin, gelatin), carbohydrates (such as monosaccharides, di saccharides, and other carbohydrates including glucose, sucrose, trehalose, mannose, mannitol, sorbitol or dextrins), gel, lipids, liposomes, stabilizers, preservatives, antioxidants including ascorbic acid and methionine, chelating agents such as EDTA; salt forming counter-ions such as sodium; nonionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG), or combinations thereof.

Pharmaceutical compositions can contain one or more adjuvant that act(s) to accelerate, prolong, or enhance the immune response to the fusion protein without having any specific antigenic effect itself. Adjuvants used in the pharmaceutical composition can include oils, oil emulsions, aluminum salts, calcium salts, immune stimulating complexes, bacterial and viral derivatives, virosomes, carbohydrates, cytokines, polymeric microparticles. In certain embodiments, the adjuvant can be selected from alum (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, lipopolysaccahrides (LPS), ASO1, ASO2, ASO3, ASO4, AF03, lipophilic phospholipid (lipid A), gamma inulin, algammulin, glucans, dextrans, glucomannans, galactomannans, levans, xylans, dimethyl dioctadecyl ammonium bromide (DDA), as well as the other adjuvants and emulsifiers.

In some embodiments, the pharmaceutical composition contains MONTANIDE™ ISA 51 (an oil adjuvant composition comprised of vegetable oil and mannide oleate for production of water-in-oil emulsions), TWEEN® 80 (also known as: Polysorbate 80 or Polyoxyethylene (20) sorbitan monooleate), a CpG oligonucleotide, and/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 the adjuvant.

Pharmaceutical compositions can also include pharmaceutically acceptable additives or excipients. For example, pharmaceutical compositions can contain antioxidants, binders, buffers, bulking agents, carriers, chelating agents, coloring agents, diluents, disintegrants, emulsifying agents, fillers, gelling agents, pH buffering agents, preservatives, solubilizing agents, stabilizers, and the like.

Pharmaceutical compositions can be formulated as immediate release or for sustained release formulations. Additionally, the pharmaceutical compositions can be formulated for induction of systemic, or localized mucosal, immunity through immunogen entrapment and coadministration with microparticles. Such delivery systems are readily determined by one of ordinary skill in the art.

Pharmaceutical compositions can be prepared as injectables, either as liquid solutions or suspensions. Liquid vehicles containing the S-RBD peptide immunogen construct can also be prepared prior to injection. The pharmaceutical composition can be administered by any suitable mode of application, for example, i.d., i.v., i.p., i.m., intranasally, orally, subcutaneously, etc. and in any suitable delivery device. In certain embodiments, the pharmaceutical composition is formulated for subcutaneous, intradermal, or intramuscular administration. Pharmaceutical compositions suitable for other modes of administration can also be prepared, including oral and intranasal applications.

Pharmaceutical compositions can also be formulated in a suitable dosage unit form. In some embodiments, the pharmaceutical composition contains from about 0.1 pg to about 1 mg of the fusion protein per kg body weight. Effective doses of the pharmaceutical compositions vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human but nonhuman mammals including transgenic mammals can also be treated. When delivered in multiple doses, the pharmaceutical compositions may be conveniently divided into an appropriate amount per dosage unit form. The administered dosage will depend on the age, weight and general health of the subject as is well known in the therapeutic arts.

In some embodiments, the pharmaceutical composition contains more than one fusion protein. A pharmaceutical composition containing a mixture of more than one fusion protein to allow for synergistic enhancement of the immunoefficacy of the fusion proteins. Pharmaceutical compositions containing more than one fusion protein can be more effective in a larger genetic population due to a broad MHC class II coverage thus provide an improved immune response to the fusion protein.

The pharmaceutical compositions can also contain more than one active compound. For example, the formulation can contain one or more fusion protein and/or one or more additional beneficial compound(s). The active ingredients can be combined with the carrier in any convenient and practical manner, e.g., by admixture, solution, suspension, emulsification, encapsulation, absorption and the like, and can be made in formulations such as powder (including lyophilized powder), suspensions that are suitable for inj ections, infusion, or the like. Sustained-release preparations can also be prepared. In certain embodiments, the pharmaceutical composition contains the fusion protein for human use. The pharmaceutical compositions can be prepared in an appropriate buffer including, but not limited to, citrate, phosphate, Tris, BIS-Tris, etc. at an appropriate pH and can also contain excipients such as sugars (50 mM to 500 mM of sucrose, trehalose, mannitol, or mixtures thereof), surfactants (e.g., 0.025% - 0.5% of TWEEN 20 or TWEEN 80), and/or other reagents. The formulation can be prepared to contain various amounts of fusion protein. In general, formulations for administration to a subject contain between about 0.1 ug/mL to about 200 pg/mL. In certain embodiments, the formulations can contain between about 0.5 pg/mL to about 50 pg/mL; between about 1.0 pg/mL to about 50 pg/mL; between about 1 pg/mL to about 25 pg/mL; or between about 10 pg/mL to about 25 pg/mL of fusion protein. In specific embodiments, the formulations contain about 1.0 pg/mL, about 5.0 pg/mL, about 10.0 pg/mL, or about 25.0 pg/mL of fusion protein.

3. Methods

Another aspect of the present invention relates to methods for making and using a fusion protein and compositions thereof. a. Producing the Fusion Protein

In some embodiments, the method for making the fusion protein comprises (i) providing a bioactive molecule and an Fc fragment comprising a hinge region, (ii) modifying the hinge region to prevent it from forming a disulfide bond, and (iii) linking the bioactive molecule directly or indirectly to the sFc through the mutated hinge region to form the fusion protein, hybrid, conjugate, or composition thereof. The present disclosure also provides a method for purifying the fusion protein, comprising (i) providing a fusion protein, and (ii) purifying the fusion protein by Protein A or Protein G-based chromatography media.

The fusion protein may alternatively be expressed by well-known molecular biology techniques. Any standard manual on molecular cloning technology provides detailed protocols to produce the fusion protein of the invention by expression of recombinant DNA and RNA. To construct a gene expressing a fusion protein of this invention, the amino acid sequence is reverse translated into a nucleic acid sequence, preferably using optimized codons for the organism in which the gene will be expressed. Next, a gene encoding the peptide or protein is made, typically by synthesizing overlapping oligonucleotides which encode the fusion protein and necessary regulatory elements. The synthetic gene is assembled and inserted into the desired expression vector. The synthetic nucleic acid sequences encompassed by this invention include those which encode the fusion protein of the invention, and nucleic acid constructs characterized by changes in the non-coding sequences that do not alter the biological activity of the molecule encoded thereby. The synthetic gene is inserted into a suitable cloning vector and recombinants are obtained and characterized. The fusion protein is expressed under conditions appropriate for the selected expression system and host. The fusion protein is purified by an affinity column of Protein A or Protein G (e.g., SOFTMAX®, ACROSEP®, SERA-MAG®, or SEPHAROSE®).

The fusion protein of the present invention can be produced in mammalian cells, lower eukaryotes, or prokaryotes. Examples of mammalian cells include monkey COS cells, CHO cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.

The invention also provides a method for producing a single chain Fc (sFc) region of an immunoglobulin G, comprising mutating, substituting, or deleting the Cys in a hinge region of Fc of IgG. In one embodiment, the Cys is substituted with Ser, Gly, The, Ala, Vai, Leu, He, or Met. In another embodiment, the Cys is deleted. In an additional embodiment, a fragment of the hinge is deleted.

The invention further provides a method for producing a fusion protein comprising: (a) providing a bioactive molecule and an IgGFc fragment comprising a hinge region, (b) mutating the hinge region by amino acid substitution and/or deletion to form a mutated Fc without disulfide bond formation, and (c) combining the bioactive molecule and the mutated Fc. b. Using the Fusion Protein

Pharmaceutical compositions containing the fusion proteins can be formulated as immediate release or for sustained release formulations. Additionally, the pharmaceutical compositions can be formulated for induction of systemic, or localized mucosal, immunity through immunogen entrapment and co-administration with microparticles. Such delivery systems are readily determined by one of ordinary skill in the art.

The fusion protein of the invention can be administered intravenously, subcutaneously, intra-muscularly, or via any mucosal surface, e.g., orally, sublingually, buccally, sublingually, nasally, rectally, vaginally, or via pulmonary route. In certain embodiments, the pharmaceutical composition is formulated for subcutaneous, intradermal, or intramuscular administration. Pharmaceutical compositions suitable for other modes of administration can also be prepared, including oral and intranasal applications.

The dose of the fusion protein of the invention will vary depending upon the subject and the particular mode of administration. The dosage required will vary according to a number of factors known to those skilled in the art, including, but not limited to, the fusion protein, the species of the subject and the size of the subject. Dosage may range from 0.1 to 100,000 pg/kg body weight. In certain embodiments, the dosage is between about 0.1 pg to about 1 mg of the fusion protein per kg body weight. The fusion protein can be administered in a single dose, in multiple doses throughout a 24-hour period, or by continuous infusion. The fusion protein can be administered continuously or at specific schedule. The effective doses may be extrapolated from dose-response curves obtained from animal models.

B. MULTITOPE PROTEIN/PEPTIDE VACCINE COMPOSITION FOR THE PREVENTION OF INFECTION BY SARS-COV-2

Aspect of the invention relates to multitope protein/peptide vaccine compositions for the prevention of infection by SARS-CoV-2. The multitope protein/peptide vaccine composition disclosed herein is also referred to as “VACCINE CANDIDATE A,” “VACCINE CANDIDATE B,” and “VACCINE CANDIDATE C - BIVALENT.”

1. Sl-Receptor-Binding Region-Based Designer Protein

Most of the vaccines currently in clinical trials only target the full-length S protein to induce a neutralizing antibody response. The induction of T cell responses would be limited compared to responses generated by natural multigenic SARS-CoV-2 infections. The Sl-RBD region is a critical component of SARS-CoV-2. It is required for cell attachment and represents the principal neutralizing domain of the virus of the highly similar SARS-CoV, providing a margin of safety not achievable with a full-length S antigen and eliminating the possibility of the potentially deadly side effects that led to withdrawal of an otherwise effective inactivated RSV vaccine. Accordingly, the monoclonal antibodies for the treatment of newly diagnosed COVID-19, approved through FDA Emergency Use Authorization (Lilly's neutralizing antibody bamlanivimab, LY-CoV555 and REGN-COV2 antibody cocktail), are all directed to Sl-RBD.

Due to the clear advantages of a strong Sl-RBD vaccine component, the multitope protein/peptide vaccine composition comprises the SI -receptor-binding region-based designer protein described in Part A above. As described above, Sl-RBD-sFc is a recombinant protein made through a fusion of Sl-RBD of SARS-CoV-2 to a single chain fragment crystallizable region (sFc) of a human IgGl. Genetic fusion of a vaccine antigen to a Fc fragment has been shown to promote antibody induction and neutralizing activity against HIV gpl20 in rhesus macaques or Epstein Barr virus gp350 in BALB/c mice (Shubin, Z., et al., 2017; and Zhao, B., et al., 2018). Moreover, engineered Fc has been used in many therapeutic antibodies as a solution to minimized non-specific binding, increase solubility, yield, thermostability, and in vivo half-life (Liu, H., et al., 2017).

In some embodiments, the vaccine composition contains Sl-RBD-sFc fusion protein of SEQ ID NO: 235. The Sl-RBD-sFc protein (SEQ ID NO: 235) contains the Sl-RBD peptide (SEQ ID NO: 226), which corresponds to amino acid residues 331-530 of the full-length S protein of SARS-CoV-2, fused to the single chain Fc peptide (SEQ ID NO: 232) through a mutated hinge region from IgG (SEQ ID NO: 188).

In some embodiments, the cysteine (C) residues at positions 61 and 195 of the S-RBD sequence of SEQ ID NO: 226 are mutated to alanine (A) residues, as shown in SEQ ID NO: 227 (residues 61 and 195 of S-RBD correspond to residues 391 and 525 of the full-length S protein of SEQ ID NO: 20). The mutated S-RBD sequence is also referred to as S-RBDa in this disclosure. The C61A and C195A mutations in the S-RBD sequence are introduced to avoid a mismatch of disulfide bond formation in the recombinant protein expression. The amino acid sequence of the S-RBDa fused to the single chain Fc peptide (S-RBDa-sFc) is SEQ ID NO: 236.

The amount of the SI -receptor-binding region-based designer protein in the vaccine composition can vary depending on the need or application. The vaccine composition can contain between about 1 pg to about 1,000 pg of the SI -receptor-binding region-based designer protein. In some embodiments, the vaccine composition contains between about 10 pg to about 200 pg of the SI -receptor-binding region-based designer protein. 2. Th/CTL Peptides

A neutralizing response against the S protein alone is unlikely to provide lasting protection against SARS-CoV-2 and its emerging variants with mutated B-cell epitopes. A long-lasting cellular response could augment the initial neutralizing response (through memory B cell activation) and provide much greater duration of immunity as antibody titers wane. Recent studies have demonstrated that IgG response to S declined rapidly in >90% of SARS- CoV-2 infected individuals within 2-3 months (Long, Q.-X., et al., 2020). In contrast, memory T cells to SARS have been shown to endure 11-17 years after 2003 SARS outbreak (Ng., O - W., et al., 2016; and Le Bert, N., et al., 2020). The S protein is a critical antigen for elicitation of humoral immunity which mostly contains CD4+ epitopes (Braun, J., et al., 2020). Other antigens are needed to raise/augment cellular immune responses to clear SARS-CoV-2 infection. The vast majority of reported CD8+ T cell epitopes in SARS-CoV-2 proteins are located in ORF lab, N, M, and ORF3a regions; only 3 are in S, with only 1 CD8+ epitope being located in the Sl-RBD (Ferretti, A.P., et al., 2020). The smaller M and N structural proteins are recognized by T cells of patients who successfully controlled their infection. In a study of nearly 3,000 people in the UK, it was found that individuals with higher numbers of T cells were more protected against SARS-CoV-2 compared to those with low T cell responses, suggesting that T cell immunity may play a critical role in preventing COVID-19 (Wyllie, D., et al., 2020).

To provide immunogens to elicit T cell responses, Th/CTL epitopes from highly conserved sequences derived from S, N, and M proteins of SARS-CoV and SARS-CoV-2 (e.g., Ahmed, S.F., et al., 2020/0 were identified after extensive literature search. These Th/CTL peptides are shown in Tables 4 and 5. Several peptides within these regions were selected and subject to further designs. Each selected peptide contains Th or CTL epitopes with prior validation of MHC I or II binding and exhibits good manufacturability characteristics (optimal length and amenability for high quality synthesis). These rationally designed Th/CTL peptides were further modified by addition of a Lys-Lys-Lys tail to each respective peptide’s N-terminus to improve peptide solubility and enrich positive charge for use in vaccine formulation. The designs and sequences of the five final peptides and their respective HLA alleles are shown in Table 28

To enhance the immune response, a proprietary peptide UBITh®la (SEQ ID NO: 66) can be added to the peptide mixture of the vaccine composition. UBITh®la is a proprietary synthetic peptide with an original framework sequence derived from the measles virus fusion protein (MVF). This sequence was further modified to exhibit a palindromic profile within the sequence to allow accommodation of multiple MHC class II binding motifs within this short peptide of 19 amino acids. A Lys-Lys-Lys sequence was added to the N terminus of this artificial Th peptide as well to increase its positive charge thus facilitating the peptide’s subsequent binding to the highly negatively charged CpG oligonucleotide molecule to form immunostimulatory complexes through “charge neutralization”. In previous studies, attachment of UBITh®la to a target “functional B epitope peptide” derived from a self-protein rendered the self-peptide immunogenic, thus breaking immune tolerance (Wang, C.Y, et al, 2017). The Th epitope of UBITh®l has shown this stimulatory activity whether covalently linked to a target peptide or as a free charged peptide, administered together with other designed target peptides, that are brought together through the “charge neutralization” effect with CpGl, to elicit site-directed B or CTL responses. Such immunostimulatory complexes have been shown to enhance otherwise weak or moderate response of the companion target immunogen (e.g., WO 2020/132275A1). CpGl is designed to bring the rationally designed immunogens together through “charge neutralization” to allow generation of balanced B cells (induction of neutralizing antibodies) and Th/CTL responses in a vaccinated host. In addition, Toll-like receptors (TLRs) play critical roles in the innate immune system by recognizing pathogen- associated molecular patterns derived from a variety of microbes. Activation of Toll-like receptor 9 (TLR-9) signaling by CpG is known to promote IgA production and favor Thl immune response. UBITh®! peptide is incorporated as one of the Th peptides for its “epitope cluster” nature to further enhance the SARS-CoV-2 derived Th and CTL epitope peptides for their antiviral activities. The amino acid sequence of UBITh® 1 is SEQ ID NO: 65 and the sequence of UBITh®la is SEQ ID NO: 66. The nucleic acid sequence of CpGl is SEQ ID NO: 104.

In view of the above, the multitope protein/peptide vaccine composition can contain one or more Th/CTL peptides. The Th/CTL peptides can include: a. peptides derived from the SARS-CoV-2 M protein of SEQ ID NO: 1 (e.g., SEQ ID NO: 361); b. peptides derived from the SARS-CoV-2 N protein of SEQ ID NO: 6 (e.g., SEQ ID NOs: 9-16, 19, 153-160, 165, 347, 350, 351, and 363); c. peptides derived from the SARS-Cov-2 S protein of SEQ ID NO: 20 (e.g., SEQ ID NOs: 35-36, 39-48, 145-152, 161-164, 345-346, 348, 362, 364, and 365); and/or d. artificial Th epitopes derived from pathogen proteins (e g., SEQ ID NOs: 49-100).

The vaccine composition can contain one or more of the Th/CTL peptides. In certain embodiments, the vaccine composition contains a mixture of more than one Th/CTL peptides. When present in a mixture, each Th/CTL peptide can 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 amounts, equal weight amounts, or the amount of each peptide in the mixture can be different than the amount of the other peptide(s) in the mixture. If more than two Th/CTL peptides are present in the mixture, the amount of the peptides can be the same as or different from any of the other peptides in the mixture.

The amount of Th/CTL peptide(s) present in the vaccine composition can vary depending on the need or application. The vaccine composition can contain a total of between about 0.1 pg to about 100 pg of the Th/CTL peptide(s). In some embodiments, the vaccine composition contains a total of between about 1 pg to about 50 pg of the Th/CTL peptide(s).

In certain embodiments, the vaccine composition contains a mixture of SEQ ID NOs: 345, 346, 347, 348, 361, and 66. These Th/CTL peptides can be mixed in equimolar amounts, equal weight amounts, or the amount of each peptide in the mixture can be different than the amount of the other peptide(s) in the mixture. In certain embodiments, these Th/CTL peptides are mixed in equal weight amounts in the vaccine composition.

3. Excipients

The vaccine composition can also contain a pharmaceutically acceptable excipient.

As used herein, the term “excipient” or “excipients” refers to any component in the vaccine composition that is not (a) the S 1 -receptor-binding region-based designer protein or (b) the Th/CTL peptide(s). Examples of excipients include carriers, adjuvants, antioxidants, binders, buffers, bulking agents, chelating agents, coloring agents, diluents, disintegrants, emulsifying agents, surfactants, solvents, fdlers, gelling agents, pH buffering agents, preservatives, solubilizing agents, stabilizers, and the like. Accordingly, the vaccine composition can contain a pharmaceutically effective amount of an active pharmaceutical ingredient (API), such as the SI -receptor-binding region-based designer protein and/or one or more Th/CTL peptides, together with a pharmaceutically acceptable excipient.

The vaccine composition can contain one or more adjuvants that act to accelerate, prolong, or enhance the immune response to the API without having any specific antigenic effect itself. Adjuvants can include oils, oil emulsions, aluminum salts, calcium salts, immune stimulating complexes, bacterial and viral derivatives, virosomes, carbohydrates, cytokines, polymeric microparticles. In certain embodiments, the adjuvant can be selected from a CpG oligonucleotide, alum (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, lipopolysaccahrides (LPS), ASO1, ASO2, ASO3, ASO4, AF03, lipophilic phospholipid (lipid A), gamma inulin, algammulin, glucans, dextrans, glucomannans, galactomannans, levans, xylans, dimethyldioctadecylammonium bromide (DDA), as well as the other adjuvants and emulsifiers.

In some embodiments, the vaccine composition contains ALHYDROGEL® (aluminum hydroxide), MONTANIDE™ ISA 51 (an oil adjuvant composition comprised of vegetable oil and mannide oleate for production of water-in-oil emulsions), TWEEN® 80 (also known as: Polysorbate 80 or Polyoxyethylene (20) sorbitan monooleate), a CpG oligonucleotide, and/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 the adjuvant.

In certain embodiments, the multitope protein/peptide vaccine composition contains ALHYDROGEL® (aluminum hydroxide) and a CpG oligonucleotide as the adjuvant to improve the immune response. In particular embodiments, the CpG oligonucleotide is present in an amount of about 100-2500 pg, of about 500-2000 pg, of about 750-1500 pg; or of about 900-1100 pg. In still other embodiments, the CpG oligonucleotide is present in an amount of about 1000 pg. VACCINE CANDIDATE A, VACCINE CANDIDATE B, and VACCINE CANDIDATE C - BIVALENT are exemplary embodiments using ALHYDROGEL® (aluminum hydroxide) and about 1000 pg of CpG oligonucleotide as adjuvant.

The vaccine composition can contain pH adjusters and/or buffering agents, such as hydrochloric acid, phosphoric acid, citric acid, acetic acid, histidine, histidine HCEHzO, lactic acid, tromethamine, gluconic acid, aspartic acid, glutamic acid, tartaric acid, succinic acid, malic acid, fumaric acid, a-ketoglutaric acid, and arginine HC1.

The vaccine composition can contain surfactants and emulsifiers, such as olyoxyethylene sorbitan fatty acid esters (Polysorbate, TWEEN®), Polyoxyethylene 15 hydroxy stearate (Macrogol 15 hydroxy stearate, SOLUTOLHS15®), Polyoxyethylene castor oil derivatives (CREMOPHOR® EL, ELP, RH 40), Polyoxyethylene stearates (MYRJ®), Sorbitan fatty acid esters (SPAN®), Polyoxyethylene alkyl ethers (BRIJ®), and Polyoxyethylene nonylphenol ether (N0N0XYN0L®).

The vaccine composition can contain carriers, solvents, or osmotic pressure keepers, such as water, alcohols, and saline solutions (e.g., sodium chloride).

The vaccine composition can contain preservatives, such as alkyl/aryl alcohols (e.g., benzyl alcohol, chlorbutanol, 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 mercurials (e.g., thimerosal, phenylmercurate salts).

4. Formulations

The vaccine composition can be formulated as immediate release or for sustained release formulations. Additionally, the vaccine composition can be formulated for induction of systemic, or localized mucosal, immunity through immunogen entrapment and coadministration with microparticles. Such delivery systems are readily determined by one of ordinary skill in the art.

The vaccine composition can be prepared as an injectable, either as a liquid solution or suspension. Liquid vehicles containing the vaccine composition can also be prepared prior to injection. The vaccine composition can be administered by any suitable mode of application, for example, i.d., i.v., i.p., i.m., intranasally, orally, subcutaneously, etc. and in any suitable delivery device. In certain embodiments, the vaccine composition is formulated for subcutaneous, intradermal, or intramuscular administration. The vaccine composition can also be prepared for other modes of administration, including oral and intranasal applications.

The vaccine composition can also be formulated in a suitable dosage unit form. In some embodiments, the vaccine composition contains from about 1 pg to about 1,000 pg of the API (e.g., the SI -receptor-binding region-based designer protein and/or one or more of the Th/CTL peptides). Effective doses of the vaccine composition can vary depending upon many different factors, including means of administration, target site, physiological state of the subject, whether the subject is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the subj ect is a human, but nonhuman mammals can also be treated When delivered in multiple doses, the vaccine composition may be conveniently divided into an appropriate amount per dosage unit form. The administered dosage will depend on the age, weight and general health of the subject as is well known in the therapeutic arts.

In some embodiments, the vaccine composition contains a S 1 -receptor-binding regionbased designer protein and one or more Th/CTL peptides in a formulation with additives and/or excipients. In certain embodiments, the vaccine composition contains a S 1 -receptor-binding region-based designer protein and more than one Th/CTL peptides in a formulation with additives and/or excipients. A vaccine composition containing a mixture of more than one Th/CTL peptides can provide synergistic enhancement of the immunoefficacy of the composition. A vaccine composition containing a S 1 -receptor-binding region-based designer protein and more than one Th/CTL peptides in a formulation with additives and/or excipients can be more effective in a larger genetic population compared to compositions containing only the designer protein or one Th/CTL peptide, due to a broad MHC class II coverage, thus providing an improved immune response to vaccine composition.

When the vaccine composition contains a S 1 -receptor-binding region-based designer protein and one or more Th/CTL peptides as the API, the relative amounts of the designer protein and the Th/CTL peptides can be present in any amount or ratio to each other. For example, the designer protein and the Th/CTL peptide(s) can be mixed in equimolar amounts, equal weight amounts, or the amount of the designer protein and the Th/CTL peptide(s) can be different. In addition, if more than one Th/CTL peptide is present in the composition, the amount of the designer protein and each Th/CTL peptide can be the same as or different from each other. In some embodiments, the molar or weight amount of the designer protein is present in the composition in an amount greater than the Th/CTL peptides. 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 peptides. The ratio (weightweight) of the designer protein to Th/CTL peptide(s) can vary depending on the need or application. In some instances, the ratio (w:w) of the designer peptide to Th/CTL peptide(s) can be 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, or 90: 10. In specific embodiments, the ratio (w:w) of the designer peptide to Th/CTL peptide(s) is 95:5, 94:6, 93:7, 92:8, 91:9, 90: 10, 89: 11, 88: 12, 87: 13, 86: 14, or 85:15. In specific embodiments, the ratio (w:w) of the designer peptide to Th/CTL peptide(s) is 88:12.

In some embodiments, the vaccine composition comprises the S 1 -receptor-binding region-based designer protein of SEQ ID NO: 235. In other embodiments, the vaccine composition comprises one or more Th/CTL peptides. In some embodiments, the vaccine composition comprises the SI -receptor-binding region-based designer protein of SEQ ID NO: 235 in combination with Th/CTL peptides of SEQ ID NOs: 345, 346, 347, 348, 361, and 66. In certain embodiments, the vaccine composition comprises the S 1 -receptor-binding regionbased designer protein of SEQ ID NO: 235, the Th/CTL peptides of SEQ ID NOs: 345, 346, 347, 348, 361, and 66, together with one or more adjuvant and/or excipient. In various embodiments, the vaccine composition comprises SEQ ID NO: 235 together with the Th/CTL peptides of SEQ ID NOs: 345, 346, 347, 348, 361, and 66, where the Th/CTL peptides are present in an equal weight ratio to each other and the ratio (w:w) of SEQ ID NO: 235 to the combined weight of the Th/CTL peptides is 88: 12. Specific embodiments of the vaccine composition containing 20 pg/mL, 60 pg/mL, and 200 pg/mL, based on the total weight of the Sl-RBD-sFC protein (SEQ ID NO: 235) together with the Th/CTL peptides of SEQ ID NOs: 345, 346, 347, 348, 361, and 66 are provided in Tables 33-35, respectively. b. Pharmaceutical compositions

The present disclosure is also directed to pharmaceutical compositions containing the disclosed vaccine composition.

Pharmaceutical compositions can contain carriers and/or other additives in a pharmaceutically acceptable delivery system. Accordingly, pharmaceutical compositions can contain a pharmaceutically effective amount of an SI -receptor-binding region-based designer protein together with pharmaceutically-acceptable carrier, adjuvant, and/or other excipients such as diluents, additives, stabilizing agents, preservatives, solubilizing agents, buffers, and the like.

Pharmaceutical compositions can contain one or more adjuvant that act(s) to accelerate, prolong, or enhance the immune response to the vaccine composition without having any specific antigenic effect itself. Adjuvants used in the pharmaceutical composition can include oils, oil emulsions, aluminum salts, calcium salts, immune stimulating complexes, bacterial and viral derivatives, virosomes, carbohydrates, cytokines, polymeric microparticles. In certain embodiments, the adjuvant can be selected from alum (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, lipopolysaccahrides (LPS), ASO1, ASO2, ASO3, ASO4, AF03, lipophilic phospholipid (lipid A), gamma inulin, algammulin, glucans, dextrans, glucomannans, galactomannans, levans, xylans, dimethyldioctadecylammonium bromide (DDA), as well as the other adjuvants and emulsifiers.

Pharmaceutical compositions can also be formulated in a suitable dosage unit form. In some embodiments, the pharmaceutical composition contains from about 0.1 pg to about 1 mg of the SI -receptor-binding region-based designer protein per kg body weight. Effective doses of the pharmaceutical compositions vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human but nonhuman mammals including transgenic mammals can also be treated. When delivered in multiple doses, the pharmaceutical compositions may be conveniently divided into an appropriate amount per dosage unit form. The administered dosage will depend on the age, weight, and general health of the subject as is well known in the therapeutic arts.

In some embodiments, the pharmaceutical composition contains more than one Sl- receptor-binding region-based designer proteins. A pharmaceutical composition containing a mixture of more than one S 1 -receptor-binding region-based designer proteins to allow for synergistic enhancement of the immunoefficacy of the constructs. Pharmaceutical compositions containing more than one S 1 -receptor-binding region-based designer protein can be more effective in a larger genetic population due to a broad MHC class II coverage thus provide an improved immune response to the S-RBD peptide immunogen constructs.

In other embodiments, pharmaceutical compositions comprising a peptide composition of, for example, a mixture of the S 1 -receptor-binding region-based designer protein in contact with mineral salts including Alum gel (ALHYDROGEL) or Aluminum phosphate (ADJUPHOS) as adjuvant to form a suspension formulation was used for administration to hosts.

Pharmaceutical compositions containing an SI -receptor-binding region-based designer protein can be used to elicit an immune response and produce antibodies in a host upon administration. c. Pharmaceutical compositions also containing endogenous SARS-CoV-2 Th and CTL epitope peptides

Pharmaceutical compositions containing a S 1 -receptor-binding region-based designer protein can also include an endogenous SARS-CoV-2 T helper epitope peptide and/or CTL epitope peptide separate from (i.e., not covalently linked to) the peptide immunogen construct. The presence of Th and CTL epitopes in pharmaceutical/vaccine formulations prime the immune response in treated subjects by initiating antigen specific T cell activation, which correlates to protection from SARS-CoV-2 infection. Additionally, formulations that include carefully selected endogenous Th epitopes and/or CTL epitopes presented on proteins from SARS-CoV-2 can produce broad cell mediated immunity, which also makes the formulations effective in treating and protecting subjects having diverse genetic makeups.

Including one or more separate peptides containing endogenous SARS-CoV-2 Th epitopes and/or CTL epitopes in a pharmaceutical composition containing S 1 -receptor-binding region-based designer protein brings the peptides in close contact to each other, which allows the epitopes to be seen and processed by antigen presenting B cells, macrophages, dendritic cells, etc. These cells process the antigens and present them to the surface to be in contact with the B cell for antibody generation and T cells to trigger further T cell responses to help mediate killing of the virus infected cells.

In some embodiments, the pharmaceutical composition contains one or more endogenous SARS-CoV-2 Th epitope peptide separate from the S 1 -receptor-binding regionbased designer protein. In certain embodiments, the endogenous SARS-CoV-2 Th epitope peptide is from the N protein or the S protein of SARS-CoV-2. In specific embodiments, the endogenous SARS-CoV-2 Th epitope peptide is selected from the group consisting of SEQ ID NOs: 13, 39-41, and 44 (Table 5), SEQ ID NOs: 161-165 (Table 8), and any combination thereof. The endogenous SARS-CoV-2 Th epitope peptides of SEQ ID NOs: 161-165 (Table 8) correspond to the sequences of SEQ ID NOs: 39, 40, 44, 41, and 13, respectively, but contain a Lys-Lys-Lys (KKK) tail at the N-terminus. The endogenous Th epitopes of SEQ ID NOs: 161-165 are particularly useful when used in a pharmaceutical composition that has been formulated into an immunostimulatory complex with a CpG oligonucleotide (ODN), because the cationic KKK tail is capable of interacting with the CpG ODN through electrostatic association. The use of endogenous SARS-CoV-2 Th epitopes in the peptide immunogen construct can enhance the immunogenicity of the S-RBD B cell epitope peptide to facilitates the production of specific high titer antibodies, upon infection, directed against the optimized S-RBD B cell epitope peptide screened and selected based on design rationales.

In other embodiments, the pharmaceutical composition contains one or more endogenous SARS-CoV-2 CTL epitope peptide separate from the S-RBD peptide immunogen construct. In certain embodiments, the endogenous SARS-CoV-2 CTL epitope peptide is from the N protein or the S protein of SARS-CoV-2. In specific embodiment, the endogenous SARS- CoV-2 CTL epitope peptide is selected from the group consisting of SEQ ID NOs: 9-12, 14- 16, 19, 35-36, 42-43, 45-48 (Table 4), SEQ ID NOs: 145-160 (Table 8), and any combination thereof. The endogenous SARS-CoV-2 CTL epitope peptides of SEQ ID NOs: 145-160 correspond to the sequences of SEQ ID NOs: 45, 42, 46, 36, 48, 43, 47, 35, 12, 11, 10, 14, 19, 9, 16, and 15, respectively, but contain a Lys-Lys-Lys (KKK) tail at the N-terminus. The endogenous CTL epitopes of SEQ ID NOs: 145-160 are particularly useful when used in a pharmaceutical composition that has been formulated into an immunostimulatory complex with a CpG oligonucleotide (ODN), because the cationic KKK tail is capable of interacting with the CpG ODN through electrostatic association. The use of endogenous SARS-CoV-2 CTL epitopes in the peptide immunogen construct can enhance the immunogenicity of the S- RBD B cell epitope peptide to facilitates the production of specific high titer antibodies, upon infection, directed against the optimized S-RBD B cell epitope peptide screened and selected based on design rationales.

In some embodiments, the pharmaceutical composition contains one or more Sl- receptor-binding region-based designer proteins together with one or more separate peptides containing an endogenous SARS-CoV-2 Th epitope peptide (SEQ ID NOs: 13, 39-41, 44, 161- 165, or any combination thereof) and/or an endogenous SARS-CoV-2 CTL epitope peptides (SEQ ID NOs: 9-12, 14-16, 19, 35-36, 42-43, 45-48, 145-160, or any combination thereof).

5. Antibodies

The present disclosure also provides antibodies elicited by the vaccine composition.

The present disclosure provides a vaccine composition comprising a SI -receptorbinding region-based designer protein (e.g., Sl-RBD-sFc of SEQ ID NO: 235) and one or more Th/CTL peptides (e.g., SEQ ID NOs: 345, 346, 347, 348, 361, and 66) in a formulation with additives and/or excipients capable of eliciting high titer neutralizing antibodies against SARS- CoV-2 and inhibiting the binding of S-RBD to its receptor ACE2 with a high responder rate in immunized hosts.

Antibodies elicited by the disclosed vaccine composition are also included in the present disclosure. Such antibodies can be isolated and purified using methods known in the field. Isolated and purified antibodies can be included into pharmaceutical compositions or formulations for the use in preventing and/or treating subjects exposed to SARS-CoV-2.

6. Methods

The present disclosure is also directed to methods for making and using the vaccine composition and formulations thereof. a. Methods for Manufacturing the Sl-Receptor-Binding Region-Based Designer Protein

The disclosed SI -receptor-binding region-based designer protein can be manufactured according to the methods described in Part A(3) above. b. Methods for Using the Vaccine Composition

In prophylactic applications, the disclosed multitope protein/peptide vaccine composition can be administered to a subject susceptible to, or at risk of, becoming infected with SARS-CoV-2, the virus that causes COVID-19 to eliminate or reduce the risk, lessen the severity, or delay the onset of the disease.

The amount of the vaccine composition that is adequate to accomplish prophylactic treatment is defined as a prophylactically-effective dose. The disclosed multitope protein/peptide vaccine composition can be administered to a subject in one or more doses to produce a sufficient immune response in order to prevent an infection by SARS-CoV-2. Typically, the immune response is monitored, and repeated dosages are given if the immune response starts to wane.

The dose of the vaccine composition will vary depending upon the subject and the particular mode of administration. The dosage required will vary according to a number of factors known to those skilled in the art, including, but not limited to the species and size of the subject. The dosage may range from 1 pg to 1,000 pg of the combined weight of the designer protein and the Th/CTL peptides. The dosage can between about 1 pg to about 1 mg, between about 10 pg to about 500 pg, between about 20 pg to 200 pg of the combined weight of the designer protein and the Th/CTL peptides. The dosage, as measured by the combined weight of the designer protein and the Th/CTL peptides is about 10 pg, about 20 pg, about 30 pg, about 40 pg, about 50 pg, about 60 pg, about 70 pg, about 80 pg, about 90 pg, about 100 pg, about 110 pg, about 120 pg, about 130 pg, about 140 pg, about 150 pg, about 160 pg, about 170 pg, about 180 pg, about 190 pg, about 200 pg, about 250 pg, about 300 pg, about 400 pg, about 500 pg, about 600 pg, about 700 pg, about 800 pg, about 900 pg, about 1,000 pg. The ratio (weight weight) of the designer protein to Th/CTL peptide(s) can vary depending on the need or application. In some instances, the ratio (w:w) of the designer protein to Th/CTL peptide(s) can be 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90: 10, 99: 1, or with a fixed amount of the Th/CTL peptides per dose. In specific embodiments, the ratio (w:w) of the designer protein to Th/CTL peptide(s) is 95:5, 94:6, 93:7, 92:8, 91 :9, 90:10, 89:11, 88:12, 87:13, 86: 14, or 85: 15. In specific embodiments, the ratio (w:w) of the designer peptide to Th/CTL peptide(s) is 88: 12. In specific embodiments, the vaccine composition contains the components shown in Tables 29-31.

The vaccine composition can be administered in a single dose, in multiple doses over a period of time. The effective doses may be extrapolated from dose-response curves obtained from animal models. In some embodiments, the vaccine composition is provided to a subject in a single administration. In other embodiments, the vaccine composition is provided to a subject in multiple administrations (two or more). When provided in multiple administrations, the duration between administrations can vary depending on the application or need. In some embodiments, a first dose of the vaccine composition is administered to a subject and a second dose is administered about 1 week to about 12 weeks after the first dose. In certain embodiments, the second dose is administered about 1 week, about 2 weeks, 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 11 weeks, or about 12 weeks after the first administration. In a specific embodiment, the second dose is administered about 4 weeks after the first administration.

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

Various exemplary embodiments also encompass pharmaceutical compositions containing SI -receptor-binding region-based designer proteins. In certain embodiments, the pharmaceutical compositions employ water in oil emulsions and in suspension with mineral salts.

In order for a pharmaceutical composition to be used by a large population, safety becomes another important factor for consideration. Despite there has been use of water-in-oil emulsions in many clinical trials, Alum remains the major adjuvant for use in formulations due to its safety. Alum or its mineral salts Aluminum phosphate (ADJUPHOS) are, therefore, frequently used as adjuvants in preparation for clinical applications. In particular embodiments, the invention encompasses the use of ALHYDROGEL® (aluminum hydroxide) and a CpG oligonucleotide as the adjuvant to improve the immune response. In particular embodiments, the a CpG oligonucleotide is present in an amount of about 100-2500 pg, of about 500-2000 pg, of about 750-1500 pg; or of about 900-1100 pg. In still other embodiments, the a CpG oligonucleotide is present in an amount of about 1000 pg. VACCINE CANDIDATE A, VACCINE CANDIDATE B, and VACCINE CANDIDATE C - BIVALENT are exemplary embodiments using ALHYDROGEL® (aluminum hydroxide) and about 1000 pg of CpG oligonucleotide as adjuvant.

Other adjuvants and immunostimulating agents include 3 De-O-acylated monophosphoryl lipid A (MPL) or 3-DMP, polymeric or monomeric amino acids, such as polyglutamic acid or polylysine. Such adjuvants can be used with or without other specific immunostimulating agents, such as muramyl peptides (e.g., N-acetylmuramyl-L-threonyl-D- isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N- acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(l'-2' dipalmitoyl-sn-glycero-3- hydroxyphosphoryloxy)-ethylamine (MTP-PE), N-acetylglucsaminyl-N-acetylmuramyl-L- Al-D-isoglu-L-Ala-dipalmitoxy propylamide (DTP -DPP) THERAMIDE™), or other bacterial cell wall components. Oil-in-water emulsions include MF59 (see WO 1990/014837 toVanNest, G., et al., which is hereby incorporated by reference in its entirety), containing 5% Squalene, 0.5% TWEEN 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE) formulated into submicron particles using a microfluidizer; SAF, containing 10% Squalene, 0.4% TWEEN 80, 5% pluronic-blocked polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion; and the RIBI™ adjuvant system (RAS) (RIBI ImmunoChem, Hamilton, Mont.) containing 2% squalene, 0.2% TWEEN 80, and one or more bacterial cell wall components selected from the group consisting of monophosphoryllipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox™). Other adjuvants include Complete Freund's Adjuvant (CFA), Incomplete Freund's Adjuvant (IFA), and cytokines, such as interleukins (IL-1, IL-2, and IL-12), macrophage colony stimulating factor (M-CSF), and tumor necrosis factor (TNF-a).

The choice of an adjuvant depends on the stability of the immunogenic formulation containing the adjuvant, the route of administration, the dosing schedule, the efficacy of the adjuvant for the species being immunized, and, in humans, a pharmaceutically acceptable adjuvant is one that has been approved or is approvable for human administration by pertinent regulatory bodies. For example, alum, MPL or Incomplete Freund's adjuvant (Chang, J.C.C., et al., 1998), which is hereby incorporated by reference in its entirety) alone or optionally all combinations thereof are suitable for human administration.

The compositions can include pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, non-immunogenic stabilizers, and the like.

Pharmaceutical compositions can also include large, slowly metabolized macromolecules, such as proteins, polysaccharides like chitosan, polylactic acids, polyglycolic acids and copolymers (e.g., latex functionalized sepharose, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes). Additionally, these carriers can function as immunostimulating agents (i.e., adjuvants).

The pharmaceutical compositions of the present invention can further include a suitable delivery vehicle. Suitable delivery vehicles include, but are not limited to viruses, bacteria, biodegradable microspheres, microparticles, nanoparticles, liposomes, collagen minipellets, and cochleates.

In some embodiments, the pharmaceutical composition is prepared by combining one or more S 1 -receptor-binding region-based designer proteins (SEQ ID NOs: 107-144 or any combination thereof) together with one or more separate peptides containing an endogenous SARS-CoV-2 Th epitope peptides (SEQ ID NOs: 13, 39-41, 44, 161-165, or any combination thereof) and/or an endogenous SARS-CoV-2 CTL epitope peptides (SEQ ID NOs: 9-12, 14-16, 19, 35-36, 42-43, 45-48, 145-160, or any combination thereof) in the form of an immunostimulatory complex containing a CpG ODN. d. Methods of using pharmaceutical compositions

The present disclosure also includes methods of using pharmaceutical compositions containing S 1 -receptor-binding region-based designer proteins.

In certain embodiments, the pharmaceutical compositions containing SI -receptorbinding region-based designer proteins can be used for the prevention and/or treatment of COVID-19.

In some embodiments, the methods comprise administering a pharmaceutical composition comprising a pharmacologically effective amount of an S 1 -receptor-binding region-based designer protein to a host in need thereof. In certain embodiments, the methods comprise administering a pharmaceutical composition comprising a pharmacologically effective amount of an SI -receptor-binding region-based designer protein to a warm-blooded animal (e.g., humans, macaques, guinea pigs, mice, cat, etc.) to elicit highly specific antibodies cross-reactive with the S-RBD site that is around SARS-CoV-2 S480-509 region (SEQ ID NO: 26) within the full-length sequence of S-RBD (SEQ ID NO: 226) or S-RBD sequences from other coronaviruses (e.g., SARS-CoV or MERS-CoV).

In certain embodiments, the pharmaceutical compositions containing SI -receptor- binding region-based designer protein can be used to prevent COVID-19 caused by infection by SARS-CoV-2.

EXAMPLE 1

SYNTHESIS OF S-RBD RELATED PEPTIDES AND PREPARATION OF FORMULATIONS THEREOF a. Synthesis of S-RBD related peptides

Methods for synthesizing SARS-CoV-2 antigenic peptides, endogenous Th and CTL, and S-RBD related peptides that are included in the development of S-RBD peptide immunogen constructs are described. The peptides can be synthesized in small-scale amounts that are useful for serological assays, laboratory pilot studies, and field studies, as well as large- scale (kilogram) amounts, which are useful for industrial/commercial production of pharmaceutical compositions. A large repertoire of S-RBD B cell epitope peptides having sequences with lengths from approximately 6 to 80 amino acids were identified and selected to be the most optimal sequences for peptide immunogen constructs for use in an efficacious S-RBD targeted therapeutic vaccine.

Tables 1 to 3 provide the full-length sequences of SARS-CoV-2 M, N, and S proteins (SEQ ID NOs: 1, 6, and 20, respectively). Tables 1, 3, 11, and 13 also provide the sequences of antigenic peptides derived from SARS-CoV-2 M, N, E, ORF9b, and S proteins (SEQ ID NOs: 4-5, 17-18, 37-38, 4-5, 17-18, 37-38, 226, 227, 250-252, 259, 261, 263, 265, 266, 270, 281, 308, 321, 322, 323, 324, and 328-334) for use as solid phase/immunoadsorbent peptides for use in diagnostic assays for antibody detection. In addition, Tables 3, 11, and 13 provide the sequences of the full-length S-RBD, its fragments or modification thereof (SEQ ID NOs: 226, 227, 23-24, 26-27, 29-34, and 315-319).

Selected S-RBD B cell epitope peptides can be made into S-RBD peptide immunogen constructs by synthetically linking to a carefully designed helper T cell (Th) epitope peptide derived from pathogen proteins, including Measles Virus Fusion protein (MVF), Hepatitis B Surface Antigen protein (HBsAg), influenza, Clostridum tetani, and Epstein-Barr virus (EBV), identified in Table 6 (e.g., SEQ ID NOs: 49-100). The Th epitope peptides can be used either in a single sequence (e.g., SEQ ID NOs: 49-52, 54-57, 59-60, 62-63, 65-66 for MVF and SEQ ID NOs: 67-71, 73-74, 76-78 for HBsAg) or combinatorial library sequences (e.g., SEQ ID NOs: 53, 58, 61, 64 for MvF and SEQ ID NOs: 72 and 75 for HBsAg) to enhance the immunogenicity of their respective S-RBD peptide immunogen constructs. In order to generate memory T cells which would facilitate the recall of B cell or CTL responses of the vaccinated hosts to the SARS-CoV2, SARS-CoV2 derived endogenous Th and CTL epitopes are shown in Tables 2, 3, 4, 5, and 8 (SEQ ID NOs: 9-19, 35-48, 345-351) with known MHC binding activities are also designed as synthetic immunogens (e.g., SEQ ID NOs: 345-351) and synthesized for inclusion in the final SARS-CoV2 vaccine formulations.

Representative S-RBD peptide immunogen constructs selected from hundreds of peptide constructs are identified in Table 8 (SEQ ID NOs: 107-144). All peptides that can be used for immunogenicity studies or related serological tests for detection and/or measurement of anti-S-RBD antibodies can be synthesized on a small-scale using F-moc chemistry by peptide synthesizers of Applied BioSystems Models 430A, 431 and/or 433. Each peptide can be produced by an independent synthesis on a solid-phase support, with F-moc protection at the N-terminus and side chain protecting groups of trifunctional amino acids. After synthesis, the peptides can be cleaved from the solid support and side chain protecting groups can be removed with 90% Trifluoroacetic acid (TFA). Synthetic peptide preparations can be evaluated by Matrix- Assisted Laser Desorption/Ionization-Time-Of-Flight (MALD TOF) Mass Spectrometry to ensure correct amino acid content. Each synthetic peptide can also be evaluated by Reverse Phase HPLC (RP-HPLC) to confirm the synthesis profile and concentration of the preparation. Despite rigorous control of the synthesis process (including stepwise monitoring the coupling efficiency), peptide analogues might also be produced due to unintended events during elongation cycles, including amino acid insertion, deletion, substitution, and premature termination. Thus, synthesized preparations can typically include multiple peptide analogues along with the targeted peptide.

Despite the inclusion of such unintended peptide analogues, the resulting synthesized peptide preparations will nevertheless be suitable for use in immunological applications including immunodiagnosis (as antibody capture antigens) and pharmaceutical compositions (as peptide immunogens). Typically, such peptide analogues, either intentionally designed or generated through synthetic process as a mixture of byproducts, are frequently as effective as a purified preparation of the desired peptide, as long as a discerning QC procedure is developed to monitor both the manufacturing process and the product evaluation process to guarantee the reproducibility and efficacy of the final product employing these peptides. Large scale peptide syntheses in the multi-hundred to kilo gram quantities can be conducted on a customized automated peptide synthesizer UB 12003 or the like at 15 mmole to 150 mmole scale or larger.

For active ingredients used in the final pharmaceutical composition for clinical trials, S-RBD peptide immunogen constructs can be purified by preparative RP-HPLC under a shallow elution gradient and characterized by MALDLTOF mass spectrometry, amino acid analysis and RP-HPLC for purity and identity. b. Preparation of compositions containing S-RBD peptide immunogen constructs

Formulations employing water-in-oil emulsions and in suspension with mineral salts can be prepared. In order for a pharmaceutical composition designed to be used by a large population, safety is another important factor for consideration. Despite the fact that water-in- oil emulsions have been used in humans as pharmaceutical compositions in many clinical trials, Alum remains the maj or adjuvant for use in pharmaceutical composition due to its safety. Alum or its mineral salts ADJUPHOS (Aluminum phosphate) are therefore frequently used as adjuvants in preparation for clinical applications.

Formulations in study groups can contain all types of designer S-RBD peptide immunogen constructs. A multitude of designer S-RBD peptide immunogen constructs can be carefully evaluated in guinea pigs for their relative immunogenicity against the corresponding S-RBD peptide used as the B cell epitope peptide or the full-length RBD polypeptide (SEQ ID NOs: 226, 235, 236, and 255). Epitope mapping and serological cross-reactivities can be analyzed among the varying homologous peptides by ELISA assays using plates coated with the evaluated peptides (e.g., SEQ ID NOs: 23-24, 26-27, 29-34, 315-319, and 335-344).

The S-RBD peptide immunogen constructs at varying amounts can be prepared in a water-in-oil emulsion with Seppic MONTANIDE™ ISA 51 as the approved oil for human use, or mixed with mineral salts ADJUPHOS (Aluminum phosphate) or ALHYDROGEL (Alum). Compositions can be prepared by dissolving the S-RBD peptide immunogen constructs in water at about 20 to 2,000 pg/mL and formulated with MONTANIDE™ ISA 51 into water-in- oil emulsions (1 :1 in volume) or with mineral salts ADJUPHOS or ALHYDROGEL (Alum) (1:1 in volume). The compositions should be kept at room temperature for about 30 min and mixed by vortex for about 10 to 15 seconds prior to immunization. Animals can be immunized with 2 to 3 doses of a specific composition, which are administered at time 0 (prime) and 3 weeks post initial immunization (wpi) (boost), optionally 5 or 6 wpi for a second boost, by intramuscular route. Sera from the immunized animals can then be tested with selected B cell epitope peptide(s) to evaluate the immunogenicity of the various S-RBD peptide immunogen constructs present in the formulation and for the corresponding sera’s cross-reactivity with the S-RBD site of SEQ ID NO: 26 or with the full-length S-RBD sequence (SEQ ID NO: 226). The S-RBD peptide immunogen constructs with potent immunogenicity found in the initial screening in guinea pigs can be further tested in in vitro assays for their corresponding sera’s functional properties. The selected candidate S-RBD peptide immunogen constructs can then be prepared in water-in-oil emulsion, mineral salts, and alum-based formulations for dosing regimens over a specified period as dictated by the immunization protocols.

Only the most promising S-RBD peptide immunogen constructs will be further assessed extensively prior to being incorporated into final formulations in combination with or without the SARS-CoV2 Th/CTL peptide constructs for immunogenicity, duration, toxicity and efficacy studies in GLP guided preclinical studies in preparation for submission of an Investigational New Drug application followed by clinical trials in patients with COVID-19. EXAMPLE 2

SEROLOGICAL ASSAYS AND REAGENTS

Serological assays and reagents for evaluating functional immunogenicity of the S- RBD peptide immunogen constructs and formulations thereof are described in detail below. a. S-RBD or S-RBD B cell epitope peptide-based ELISA tests for immunogenicity and antibody specificity analysis

ELISA assays that can be used to evaluate immune serum samples and/or samples from individuals for the detection of COVID- 19 are described below.

The wells of 96-well plates are coated individually for 1 hour at 37°C with 100 pL of S-RBD (SEQ ID NO: 226) or with S-RBD B cell epitope peptides (e.g., SEQ ID NOs: 23-24, 26-27, and/or 29-34), at 2 pg/mL (unless noted otherwise), in 10 mM NaHCOs buffer, pH 9.5 (unless noted otherwise).

The S-RBD or S-RBD B cell epitope peptide-coated wells are incubated with 250 pL of 3% by weight gelatin in PBS at 37°C for 1 hour to block non-specific protein binding sites, followed by three washes with PBS containing 0.05% by volume TWEEN® 20 and dried. Sera to be analyzed are diluted 1 :20 (unless noted otherwise) with PBS containing 20% by volume normal goat serum, 1% by weight gelatin and 0.05% by volume TWEEN® 20. One hundred microliters (100 pL) of the diluted specimens (e.g., serum, plasma) is added to each of the wells and allowed to react for 60 minutes at 37°C. The wells are then washed six times with 0.05% by volume TWEEN® 20 in PBS in order to remove unbound antibodies. Horseradish peroxidase (HRP)-conjugated species (e.g., guinea pig or rat) specific goat polyclonal anti-IgG antibody or Protein A/G are used as a labeled tracer to bind with the antibody/peptide antigen complex formed in positive wells. One hundred microliters (100 pL) of the HRP-labeled detection reagent, at a pre-titered optimal dilution and in 1% by volume normal goat serum with 0.05% by volume TWEEN® 20 in PBS, is added to each well and incubated at 37°C for another 30 minutes. The wells are washed six times with 0.05% by volume TWEEN® 20 in PBS to remove unbound antibody and reacted with 100 pL of the substrate mixture containing 0.04% by weight 3’, 3’, 5’, 5’-Tetramethylbenzidine (TMB) and 0.12% by volume hydrogen peroxide in sodium citrate buffer for another 15 minutes. This substrate mixture is used to detect the peroxidase label by forming a colored product. Reactions are stopped by the addition of 100 pL of 1 ,0M H2SO4 and absorbance at 450 nm (A450) is determined. For the determination of antibody titers of the vaccinated animals that received the various peptide vaccine formulations, or individuals who are being tested for infection with SARS-CoV-2, 10-fold serial dilutions of sera from 1:100 to 1:10,000 or 4-fold serial dilutions of sera from 1: 100 to 1: 4.19 x 10⁸ are tested, and the titer of a tested serum, expressed as Logio, is calculated by linear regression analysis of the A450 with the cutoff A450 set at 0.5. b. Assessment of antibody reactivity towards Th peptide by Th peptide-based ELISA tests

The wells of 96-well ELISA plates are coated individually for 1 hour at 37°C with 100 pL of Th peptide at 2 pg/mL (unless noted otherwise), in 10 mM NaHCOs buffer, pH 9.5 (unless noted otherwise) in similar ELISA method and performed as described above. For the determination of antibody titers of the vaccinated animals that received the various formulations containing S-RBD peptide immunogen constructs, 10-fold serial dilutions of sera from 1:100 to 1: 10,000 are tested, and the titer of a tested serum, expressed as Logio, is calculated by linear regression analysis of the A450 with the cutoff A450 set at 0.5. c. Immunogenicity Evaluation

Preimmune and immune serum samples from animal subjects are collected according to experimental vaccination protocols and heated at 56°C for 30 minutes to inactivate serum complement factors. Following the administration of the formulations containing the S-RBD peptide immunogen constructs, blood samples can be obtained according to protocols and their immunogenicity against specific target site(s) can be evaluated using the corresponding S-RBD B cell epitope peptide-based ELISA tests. Serially diluted sera can be tested, and positive titers can be expressed as Logio of the reciprocal dilution. Immunogenicity of a particular formulation is assessed for its ability to elicit high titer antibody response directed against the desired epitope specificity within the target antigen and high cross-reactivities with the S-RBD polypeptide, while maintaining a low to negligible antibody reactivity towards the helper T cell epitopes employed to provide enhancement of the desired B cell responses.

EXAMPLE 3

ANIMALS USED IN SAFETY, IMMUNOGENICITY, TOXICITY, AND EFFICACY

STUDIES a. Guinea Pigs: Immunogenicity studies can be conducted in mature, naive, adult male and female Duncan-Hartley guinea pigs (300-350 g/BW). The experiments utilize at least 3 Guinea pigs per group.

Protocols involving Duncan-Hartley guinea pigs (8-12 weeks of age; Covance Research Laboratories, Denver, PA, USA) are performed under approved IACUC applications at a contracted animal facility under UBI sponsorship. b. Cynomolgus macaques:

Immunogenicity and repeated dose toxicity studies in adult male and female monkeys (Macacafascicularis, approximately 3-4 years of age; JOINN Laboratories, Suzhou, China) are conducted under approved IACUC applications at a contracted animal facility under UBI sponsorship.

EXAMPLE 4

ASSESSMENT OF FUNCTIONAL PROPERTIES OF ANTIBODIES ELICITED BY THE S-RBD PEPTIDE IMMUNOGEN CONSTRUCTS AND FORMULATIONS THEREOF

Immune sera or purified anti-S-RBD antibodies produced in guinea pigs can be further tested for their ability to (1) bind to S-RBD peptide and polypeptides having the sequences of SEQ ID NOs: 26, 226, and 227; (2) inhibit binding by S-RBD protein to ACE2 receptor in an ELISA assay and an immunofluorescent ACE2 surface expression binding assay; and (3) neutralize in vitro target cell viral replication. a. Antibody binding assay

The aim of this assay is to demonstrate that the immune sera derived from immunized guinea pigs could recognize S ARS-CoV-2 Spike (S) protein. Specifically, 1 pg/ml recombinant S proteins is used to coat onto 96-well microtiter plates (MaxiSorp NUNC) in 0.1 M carbonate buffer (pH 9.6) at 4°C overnight. After blocking with 2% BSA, serially diluted antisera are added and incubated at 37°C for 1 h with shaking, followed by four washes with PBS containing 0.1% TWEEN 20. Bound antisera are detected with Goat Anti -Guinea pig IgG H&L (HRP) (ABcam, ab6908) at 37°C for 1 h, followed by 4 washes. The substrate, 3, 3,5,5- tetram ethylbenzidine (TMB), is added into each well and incubated at 37°C for 20 minutes. The absorbance at 450 nm is measured by an ELISA plate reader (Molecular Device). b. Antibody neutralization assay

The aim of this assay is to demonstrate if antibodies in the immune sera from animals that have been administered with S-RBD peptide immunogen constructs (SEQ ID NOs: 107- 144) or S-RBD fusion proteins (S-RBD-sFc and S-RBDa-sFc of SEQ ID NOs: 235 and 236, respectively) have neutralizing or receptor binding inhibition properties in the presence of the ACE2 receptor. Specifically, 1 ug/ml recombinant S protein (SEQ ID NO: 20) or S-RBD protein (SEQ ID NO: 226, 227) is used to coat onto 96-well microtiter plates (MaxiSorp NUNC) in 0.1 M carbonate buffer (pH 9.6) at 4°C overnight. After blocking with 2% BSA, serially diluted immune sera are co-incubated with hACE2 at 37°C in S protein or S-RBD polypeptide coated 96 well plate for 1 hour, followed by four washes with PBS containing 0.1% Tween 20. Bound ACE2ECD or ACE2NECD peptides (SEQ ID NO: 229-230) are detected with Goat-anti- HuACE2 Ab (HRP) (R&D System) at 37°C for 1 hour, followed by 4 washes. The substrate, 3,3,5,5-tetramethylbenzidine (TMB), is added in to each well and incubated at 37°C for 20 minutes. The absorbance at 450 nm is measured by an ELISA plate reader (Molecular Device). The signal is in reverse proportion to the neutralization antibody concentration. The neutralization titers would be presented as reciprocal of the serum dilution fold. c. Cell-based neutralization assay (Flow cytometry)

The neutralization assay for SARS-CoV-2 S protein binding to ACE2-expressed cells by immune sera directed against S-RBD (S-RBD peptide immunogen constructs, S-RBD-sFc fusion protein, or S-RBDa-sFc fusion protein) is measured by flow cytometry. Briefly, 10⁶ HEK293/ACE2 cells are detached, collected, and washed with HBSS (Sigma-Aldrich). S protein from SARS-CoV-2 is added to the cells to a final concentration of 1 pg/mL in the presence or absence of serial diluted immune sera, followed by incubation at room temperature for 30 min. Cells are washed with HBSS and incubated with anti-SARS-CoV-2 S protein antibody (HRP) at 1/50 dilution at room temperature for an additional 30 min. After washing, cells are fixed with 1% formaldehyde in PBS and analyzed in a FACSCalibur flow cytometer (BD Biosciences) using CellQuest software. d. Neutralization of SARS-CoV-2 infection

After immune sera derived from guinea pigs immunized with S-RBD peptide immunogen constructs, S-RBD-sFc fusion protein, or S-RBDa-sFc fusion protein demonstrates effectiveness to neutralize hACE2 in in vitro assays, the immune sera will be tested in a SARS- CoV-2 neutralization assay.

Briefly, Vero E6 cells are plated at 5 x 10⁴ cells/well in 96-well tissue culture plates and grow overnight. One hundred microliters (100 pL) of 50% tissue-culture infectious dose of SARS-CoV-2 is mixed with an equal volume of diluted guinea pig immune sera and incubated at 37°C for 1 h. The mixture is added to monolayers of Vero E6 cells. Cytopathic effect (CPE) is recorded on day 3 post-infection. Neutralizing titers representing the dilutions of GP immune sera that completely prevented CPE in 50% of the wells is calculated by Reed-Muench method.

EXAMPLE 5

ASSAYS EMPLOYED IN THE DEVELOPMENT OFACE2-SFC FUSION PROTEIN AS ANTIVIRAL THERAPIES

1. Assays for the hACE2 protein drug development a. Binding assay

The following assay is designed to demonstrate that the hACE2 fusion proteins (ACE2- ECD-sFc, ACE2N-ECD-sFc of SEQ ID NOs: 237-238) can be recognized by its natural ligand (the S protein of SARS-CoV-2) in comparison with ACE2-ECD-Fc. Specifically, 1 pg/ml recombinant S protein (Sino Biological) is used to coat 96-well microtiter plates (MaxiSorp NUNC) in 0.1 M carbonate buffer (pH 9.6) at 4°C overnight. After blocking with 2% BSA, ACE2 protein at a concentration of 0.5 pg/mL is added and incubated at 37°C for 1 h with shaking, followed by four washes with PBS containing 0.1% TWEEN 20. Bound ACE2 proteins are detected with rabbit anti-human ACE2 polyclonal antibody:HRP (My Biosource, CN: MBS7044727) at 37°C for 1 h, followed by 4 washes. The substrate, 3, 3,5,5- tetram ethylbenzidine (TMB), is added into each well and incubated at 37°C for 20 minutes. The absorbance at 450 nm is measured by an ELISA plate reader (Molecular Device). b. Blocking assay

The aim of this assay is to demonstrate if the binding between the S protein and ACE2 can be blocked by the ACE2 fusion proteins (ACE2-ECD-sFc and ACE2N-ECD-sFc of SEQ ID NOs: 237 and 238, respectively) in comparison to ACE2-ECD-Fc. Specifically, 1 pg/ml ACE2 is used to coat on 96-well microtiter plates (MaxiSorp NUNC) in 0.1 M carbonate buffer (pH 9.6) at 4°C overnight. After blocking with 2% BSA, serially diluted recombinant ACE2 proteins are co-incubated with SARS-CoV-2 S protein at 37°C for 1 hour, followed by four washes with PBS containing 0.1% TWEEN 20. Bound S protein is detected with anti-SARS- CoV-2 S antibody (HRP) at 37°C for 1 hour, followed by 4 washes. The substrate, 3, 3,5,5- tetram ethylbenzidine (TMB), is added into each well and incubated at 37°C for 20 minutes. The absorbance at 450 nm is measured by an ELISA plate reader (Molecular Device). The signal is in proportion to the reciprocal of dilution fold of the proteins. c. Cell-based neutralization assay (Flow cytometry)

The neutralization of SARS-CoV-2 S protein binding to ACE2-expressed cells by ACE2 fusion proteins (ACE2-ECD-sFc and ACE2N-ECD-sFc of SEQ ID NOs: 237 and 238, respectively) is measured by flow cytometry. Briefly, 10⁶ HEK293/ACE2 cells are detached, collected, and washed with HBSS (Sigma- Aldrich). The SARS-CoV-2 S protein is added to the cells to a final concentration of 1 pg/mL in the presence or absence of serial diluted the ACE2 recombinant proteins, followed by incubation at room temperature for 30 min. Cells are washed with HBSS and incubated with Anti-SARS-CoV-2 S Ab (HRP) at 1/50 dilution at room temperature for an additional 30 min. After washing, cells are fixed with 1% formaldehyde in PBS and analyzed in a FACSCalibur flow cytometer (BD Biosciences) using CellQuest software. d. Affinity determination by SPR assay

S-RBD-Fc is immobilized on a CM5 sensor chip as shown in the instruction manual of Capture kit (GE, BR100839) with an SPR instrument (GE, Biacore X100). For a reaction cycle, a constant level of recombinant protein is initially captured onto the sensor chip. Sequentially, the samples (ACE2-ECD-sFc or ACE2N-ECD-sFc) are flowed at various concentrations in each cycle through the chip for association followed by flowing running buffer through for dissociation. Finally, the chip is regenerated with regeneration buffer for next reaction cycle. For data analysis, the binding patterns (or sensorgrams) from at least five reaction cycles are analyzed with BIAevaluation software to acquire affinity parameters such as KD, Ka and kd.

EXAMPLE 6

DESIGN, PLASMID CONSTRUCTION, AND PROTEIN EXPRESSION OF S-RBD FUSION PROTEINS IN CHO CELLS

1. Design of the cDNA sequence The cDNA sequence of the S protein from SARS-CoV-2 (SEQ ID NO: 239) is optimized for CHO cell expression. This nucleic acid encodes the S protein shown as SEQ ID NO: 20. The receptor binding domain (RBD) of the S protein was identified by aligning with the S protein sequence of SARS-CoV (SEQ ID NO: 21) with the corresponding sequence from SARS-CoV-2 (SEQ ID NO: 20). The S-RBD polypeptide from SARS-CoV-2 (aa331-530) (peptide SEQ ID NO: 226; DNA SEQ ID NO: 240) corresponds with the S-RBD sequence of SARS-CoV, which was proved to be the binding domain binding to hACE2 with high affinity.

To develop a pharmaceutical composition to protect individuals from COVID-19 infection, the RBD of the S protein is an important target for inducing the antibodies to neutralize SARS-CoV-2 after immunization. To produce the S-RBD-Fc fusion protein (DNA SEQ ID NO: 246), the nucleic acid sequence encoding S-RBD (aa331-530) of SARS-CoV-2 (DNA SEQ ID NO: 240) is fused to the N-terminus of the single chain of the immunoglobulin Fc (DNA SEQ ID NO: 245), as shown in Figure 6A and the plasmid map shown in Figure 7. To avoid mismatch of the non-critical disulfide bond formation in the S-RBD fusion protein in CHO expression system, Cys391 replaced by Ala391 and Cys525 replaced by Ala525 in the S- RBD polypeptide (amino acid SEQ ID NO: 227; DNA SEQ ID NO: 241) to produce the S- RBDa-sFc fusion protein (amino acid SEQ ID NO: 236; DNA SEQ ID NO: 247).

To develop the neutralizing intervention by virus inhibition as passive immunization, human angiotensin converting enzyme II (ACE2 accession NP_001358344, amino acid SEQ ID NO: 228; DNA SEQ ID NO: 242), which acts as the receptor of SARS-CoV-2 to mediate virus entrance, is the key target to block the S protein. In a previous study (Sui J., et al. 2004), the binding affinity is 1.70E-9 that corresponds to potent mAb for neutralization. Administration of high dose ACE2 should be safe enough for treatment of coronavirus infected patients since some of the ACE2 clinical trial for hypertension treatment demonstrated the safety profile with very high dose administration (Arendse, L.B. et al. 2019).

The extra-cellular domain of ACE2 (amino acid SEQ ID NO: 229; DNA SEQ ID NO:

243) is fused with single chain immunoglobulin Fc (amino acid SEQ ID NO: 232; DNA SEQ ID NO: 245) to produce the S-ACE2ECD-FC fusion protein (DNA SEQ ID NO: 248), as shown in Figure 6C and the plasmid map shown in Figure 8. To reduce the safety uncertainty, a fusion protein can be produced that abolishes peptidase activity in the ACE2ECD fusion protein in CHO expression system. Specifically, His374 is replaced by Asn374 and His378 is replaced by Asn378 in zinc binding domain of ACE2 (amino acid SEQ ID NO: 230; DNA SEQ ID NO:

244) to produce the ACE2NECD fusion protein (amino acid SEQ ID NO: 238; DNA SEQ ID NO: 249). Since no disulfide bonds form in the hinge region, the large protein fusion with sFc would not constrain the binding to S protein to achieve the most potent neutralization effect. The structure of single chain Fc also has the advantage to be purified by protein A binding and elution in purification process. Other disulfide bond forming with Cys345-Cys370, Cys388- Cys441 and Cys489-Cys497 still reserved in the sequence design to maintain the conformation binding to ACE2.

2. Plasmid construction and protein expression a. Plasmid construction

To express the S-RBD-Fc and S-RBDa-Fc fusion proteins, the cDNA sequences encoding these proteins can be produced in an appropriate cell line. The N-terminus of the cDNA fragment can be added a leader signal sequence for protein secretion, and the C-terminus can be linked to single-chain Fc (sFc) or a His-tag following a thrombin cleavage sequence. The cDNA fragments can be inserted into the pND expression vector, which contains a neomycin-resi stance gene for selection and a dhfr gene for gene amplification. The vector and the cDNA fragments are digested with PacI/EcoRV restriction enzymes, and then ligated to yield four expression vectors, pS-RBD, pS-RBD-sFc, pS-RBDa, and pS-RBDa-sFc.

To express the ACE2ECD and ACE2NECD fusion proteins, the cDNA sequences encoding these proteins can be produced in an appropriate cell line. The C-terminus of the cDNA fragment can be linked to single-chain Fc or a His-tag following a thrombin cleavage sequence. The cDNA fragments can be inserted into pND expression vector to yield four expression vectors, pACE2-ECD, pACE2-ECD-sFc, pACE2N-ECD, pACE2N-ECD-sFc. b. Host cell line

CHO-S™ cell line (Gibco, A1134601) is a stable aneuploid cell line established from the ovary of an adult Chinese hamster. The host cell line CHO-S™ are adapted to serum-free suspension growth and compatible with FREESTYLE™ MAX Reagent for high transfection efficiency. CHO-S cells are cultured in DYNAMIS™ Medium (Gibco, Cat. A26175-01) supplemented with 8 mM Glutamine supplement (Life Technologies, Cat. 25030081) and antidumping agent (Gibco, Cat. 0010057DG).

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™ Expression Medium (Gibco, Cat. A29100) without any supplementation. The cells are maintained in a 37° C incubator with a humidified atmosphere of 8% CO2. c. Transient expression

For transient expression, the expression vectors are individually transfected into ExpiCHO-S cells using EXPIFECTAMINE™ CHO Kit (Gibco, Cat. A29129). On day 1 posttransfection, EXPIFECTAMINE™ CHO Enhancer and first feed is added, and the cells are transferred from a37°C incubator with a humidified atmosphere of 8% CO2 to a32°C incubator with a humidified atmosphere of 5% CO2. Then, the second feed is added on day 5 posttransfection, and the cell culture is harvested after 12-14 days post-transfection. After the cell culture is harvested, the supernatant is clarified by centrifugation and 0.22-pm filtration. The recombinant proteins containing single-chain Fc and His-tag are purified by protein A chromatography (Gibco, Cat. 101006) and Ni-NTA chromatography (Invitrogen, Cat. R90101), respectively. d. Stable transfection and cell selection

The expression vector is transfected into CHO-S cells using FreeStyle MAX reagent (Gibco, Cat. 16447500) and then incubation with selection DYNAMIS™ medium, containing 8 mM L-Glutamine, anti-clumping agent at 1: 100 dilution, puromycin (InvovoGen, Cat. ant- pr-1), and MTX (Sigma, Cat. M8407). After 2 rounds of selection phase, four stable pools (1 A, IB, 2A, 2B) are obtained. Furthermore, the cell clones are plated in semi-solid CloneMedia (Molecular Devices, Cat. K8700) and simultaneously added detection antibody for clone screening and single cell isolation by high throughput system ClonePixTM2 (CP2). The clones picked by CP2 are screened by using a 14-day glucose simple fed-batch culture in DYNAMIS™ Medium with 8 mM Glutamine and anti-clumping agent without selections. After screening, single cell isolation of the clones with high yield are performed by limiting dilution, and the monoclonality is confirmed by imaging using CloneSelect Imager (Molecular Devices). e. Simple fed-batch culture

A simple fed-batch culture is used to determine the productivity of CHO-S cells expressing the recombinant proteins. CHO-S cells are seeded at 3 x 10⁵ cells/mL with 30 mL DYNAMIS medium supplemented, 8 mM Glutamine and anti -clumping agent at 1 : 100 dilution in 125-mL shaker flasks. The cells are incubated in a 37°C incubator with a humidified atmosphere of 8% CO2. 4 g/L of glucose are added on day 3 and 5, and 6 g/L of glucose are added on day 7. The cultures are collected daily to determine the cell density, viability, and productivity until the cell viability dropped below 50% or day 14 of culture is reached. f. Accuracy of gene transcript

The accuracy of the gene transcription by the CHO-S expressing cells is confirmed by RT-PCR. Briefly, total RNA of the cells is isolated using PURELINK™ RNA Mini Kit (Invitrogen Cat. 12183018A). Then, the first strand cDNA is reverse transcribed from total RNA using Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Cat. K1652). The cDNA of the recombinant proteins is purified and ligated into yT&A Vector (Yeastem Biotech Co., Ltd Cat.YC203). Finally, the cDNA sequence is confirmed by DNA sequencing. g. Stability of the expressing cells

The cells are seeded at 1~2 x 10⁵ cells/mL and cultured in a medium without selection reagents for 60 generations. Once the cell density of the cultures reached 1.0 x 10⁶ cells/mL or more during this period, the cultures are passaged at the cell density at 1~2 x 10⁵ cells/mL again. After cultivation for 60 generations, the cell performance and productivity are compared to the cells which had just been thawed from the LMCB using glucose simple fed-batch culture. The criterion of stability of product productivity in cells is titer greater than 70% after cultivation for 60 generations.

EXAMPLE 7

PURIFICATION AND BIOCHEMICAL CHARACTERIZATION OF sFc FUSION PROTEINSAND HIS-TAGGED PROTEINS

1. Purification of sFc Fusion proteins

All sFc fusion proteins are purified by protein A-sepharose chromatography from the harvested cell culture conditioned medium. The sFc fusion proteins were captured by a Protein A affinity column. After washing and eluting, the pH of protein solution is 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 is determined by polyacrylamide gel electrophoresis. The protein concentration was measured according to the UV absorbance at a wavelength of 280 nm.

2. His-Tagged proteins

Conditioned medium is mixed with Ni-NTA resin to purify fusion proteins according to manufacturer’s manual. His-tagged proteins are eluted in the elution containing 50 mmol L-l NaEfcPC , 300 mmol L-l NaCl, and 250 mmol L-l imidazole, at pH 8.0. The eluted solution is concentrated using Amicon YM-5 and then passed through a Sephadex G-75 column to get rid of impurities and a Sephadex G-25 column to remove salts; then collected protein solution is lyophilized. The purity of the His-Tagged proteins is determined by polyacrylamide gel electrophoresis. The protein concentration is measured according to the UV absorbance at a wavelength of 280 nm.

The purified proteins are further characterized by mass spectrometry analysis and glycosylation analysis.

3. Sequence and Structure of Sl-RBD-sFc

The sequence and structure of Sl-RBD-sFc fusion protein (SEQ ID NO: 235) is shown in Figure 23. Sl-RBD-sFc protein is a glycoprotein consisting of one N-linked glycan (Asnl3) and two O-linked glycans (Ser211 and Ser224). The shaded portion (aal - aa200) represents the Sl-RBD portion of SARS-CoV-2 (SEQ ID NO: 226), the boxed portion (aa201 - aa215) represents the mutated hinge region (SEQ ID NO: 188), and the unshaded/unboxed portion (aa216 - aa431) represents the sFc fragment of an IgGl (SEQ ID NO: 232). The substitution of His297 for Asn297 (EU-index numbering) in single chain Fc of IgGl, (i.e., His282 in SEQ ID NO: 235 shown in Figure 23 A) is indicated by underline. The molecular mass of Sl-RBD- sFc protein is about 50 kDa and contains 431 amino acid residues including 12 cysteine residues (Cys6, Cys31, Cys49, Cys61, Cysl02, Cysl50, Cysl58, Cysl95, Cys246, Cys306, Cys352 and Cys410), forming 6 pairs of disulfide bonds (Cys6-Cys31, Cys49-Cysl02, Cys61- Cysl95, Cysl50-Cysl58, Cys246-Cys306 and Cys352- Cys410), which are shown as connecting lines in Figure 23A. A summary of the disulfide bonding of Sl-RBD-sFc is shown in Figure 263B. N-linked and O-linked glycan structures of S-RBD-sFc and ACE2-ECD-sFc are shown in Figures 36-39.

There is one N-glycosylation site Asnl3 on the RBD domain and two O-glycosylation sites Ser211 and Ser224 on a sFc fragment. The N-glycosylation site is shown with an asterisk (*) and the two O-glycosylation sites are shown with a plus (+) above the residues shown in Figure 23A. Glycosylation of an IgG Fc fragment on a conserved asparagine residue, Asn297 (EU-index numbering), is an essential factor for the Fc-mediated effector functions such as complement dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). The Fc fragment in Sl-RBD-sFc is designed for purification by protein A affinity chromatography. In addition, the glycosylation site at Asn297 of the heavy chain was removed through mutation to His (N297H - EU numbering, N282H in the Sl-RBD-sFc protein) to prevent the depletion of target hACE2 through Fc-mediated effector functions.

EXAMPLE 8

DESIGN AND IDENTIFICATION OF ANTIGENIC PEPTIDES FROM SARS-CoV-2 NUCLEOCAPSID (N), SPIKE(S), MEMBRANE (M), ENVELOPE (E), AND OPEN READING FRAME 9b (ORF9b) PROTEINS FOR USE AS IMMUNO AD SORBENT IN IMMUNOASSAYS

1. Peptide antigens from the N, S, M, E, and ORF9b proteins

Over 25 carefully designed peptides derived from the SARS-CoV-2 nucleocapsid (N) protein (SEQ ID NO: 6, Table 2) were synthesized for identification of antigenic peptides suitable for use in the preparation of SARS-CoV-2 antigen mixture as immunoadsorbent in various immunoassays for detection of antibodies in infected individuals. The amino acid sequences of the antigenic peptides are shown in Table 13 (SEQ ID NOs: 253 to 278) and the relative position of the peptides within the full-length N protein is shown in Figure 9.

Over 50 carefully designed peptides with sequences derived from the SARS-CoV-2 spike (S) protein (SEQ ID NO: 20, Table 3) were synthesized for identification of antigenic peptides suitable for use in the preparation of SARS-CoV-2 antigen mixture as immunoadsorbent in various immunoassays for detection of antibodies in infected individuals. The amino acid sequences of the antigenic peptides are shown in Table 13 (SEQ ID NOs: 279 to 327) and the relative position of the peptides within the full-length S protein is shown in Figure 10.

Three carefully designed peptides with sequences derived from the exposed regions of SARS-CoV-2 membrane (M) protein (SEQ ID NO: 1, Table 1) were synthesized for identification of antigenic peptides suitable for use in the preparation of SARS-CoV-2 antigen mixture as immunoadsorbent in various immunoassays for detection of antibodies in infected individuals. The amino acid sequences of the antigenic peptides are shown in Tables 1 and 13 (SEQ ID NOs: 4, 5, 250, and 251) and the relative position of the peptides within the full-length M protein is shown in Figure 11. Eight carefully designed peptides with sequences derived from two small SARS-CoV- 2 proteins, being the envelope (E) and ORF9b were synthesized for identification of antigenic peptides suitable for use in the preparation of SARS-CoV-2 antigen mixture as immunoadsorbent in various immunoassays for detection of antibodies in infected individuals. The amino acid sequences of the antigenic peptides are shown in Table 13 (SEQ ID NOs: 252 for the E protein and SEQ ID NOs: 328-334 for the ORF9b protein). The relative position of the peptides within the full-length E protein and ORF9b protein is shown in Figures 12 and 13, respectively.

2. Evaluation of peptide antigens as immunoadsorbent in ELISA

A panel of 10 representative sera from COVID-19 patients, confirmed by both clinical diagnosis and PCR testing, was used for assessment of the relative antigenicity of the peptide antigens.

Figure 14 shows that highly antigenic regions were identified within the N protein that included (a) amino acids 109 to 195 covering part of the N-terminal domain (NTD) and extended to the linker region with SR rich motif (SEQ ID NOs: 259, 261, 263, and 265); (b) amino acids 213 to 266 (SEQ ID NOs: 269 and 270); and (c) amino acids 355-419 (SEQ ID NO: 18) located at the C-terminus covering the NLS and IDR regions.

Figure 15 shows that highly antigenic regions were identified within the S protein that included (a) amino acids 534 to 588 (SEQ ID NO: 281) covering the region right next to the RBM; (b) amino acids 785 to 839 (SEQ ID NO: 37 and 38) covering the FP region of the S2 subunit; (c) amino acids from 928 to 1015 (SEQ ID NO: 308) covering the HR1 region of the S2 subunit; and (d) amino acids 1104 to 1183 (SEQ ID NOs: 321- 324) covering part of the HR2 region of the S2 subunit. Figure 16 shows the localization of four antigenic sites (SEQ ID NOs: 38, 281, 308, and 322) in the 3D structure of the S protein. Two antigenic peptides (SEQ ID NOs: 288 and 38) are exposed as globular domains on the surface of the S protein, as shown on the left panel. One antigenic site (SEQ ID NO: 308) is within the elongated helical loop, as shown on the right panel. A fourth antigenic peptide (SEQ ID No: 322) is located around the C-terminal domain is shown in the left and right panel.

Figures 16-19 show that weak antigenic regions were identified from the E protein (SEQ ID NO: 251), M protein (SEQ ID NO: 5), and ORF9b protein (SEQ ID NO: 27), respectively.

Mixtures of antigenic peptides from N, S, and M regions can be formulated as solid phase immunoadsorbent with optimal binding by antibodies from individuals infected by SARS-CoV-2. The mixture of antigenic peptides from the N, S, and M proteins can be used for a sensitive and specific immunoassay for detection of antibodies to SARS-CoV-2 and for sero-surveillance of SARS-CoV-2 infection.

EXAMPLE 9

HIGH PRECISION DESIGNER VACCINE AGAINST SARS-CoV-2 INFECTION CONTAINING A Sl-RBD FUSION PROTEIN

1. General design

An effective immune response against viral infections depends on both humoral and cellular immunity. More specifically, the potential of a high precision designer preventative vaccine would employ designer immunogens, either peptides or proteins, as active pharmaceutical ingredients for (1) induction of neutralizing antibodies through the employment of B cell epitopes on the viral protein that is involved in the binding of the virus to its receptor on the target cell; (2) induction of cellular responses, including primary and memory B cell and CD8⁺ T cell responses, against invading viral antigens through the employment of endogenous Th and CTL epitopes Such vaccines can be formulated with adjuvants such as ADJUPHOS, MONTANIDE ISA, CpG, etc. and other excipients to enhance the immunogenicity of the high-precision designer immunogens.

A representative designer COVID-19 vaccine employs CHO cell expressed S-RBD-sFc protein (amino acid sequence of SEQ ID NO: 235 and nucleic acid sequence of SEQ ID NO: 246). This protein was designed and prepared to present the receptor binding domain (RBD) on the SARS CoV-2 Spike (S) protein with the very carbohydrate structure within the RBD to induce high affinity neutralizing antibodies upon immunization. The vaccine can also employ a mixture of designer peptides incorporating endogenous SARS-CoV-2 Th and CTL epitopes capable of promoting host specific Th cell mediated immunity to facilitate the viral-specific primary and memory B cell and CTL responses towards the SARS-CoV-2, for the prevention of SARS-CoV-2 infection. An effective vaccine needs to prime the memory T cells and B cells to allow rapid recall upon viral infection/challenge.

To improve the effectiveness of the disclosed designer immunogens, two representative adjuvant formulations are employed (ADJU-PHOS®/CpG and MONTANIDE™ IS A/CpG) for induction of optimal anti-SARS-CoV-2 immune responses.

ADJUPHOS is generally accepted as an adjuvant for human vaccines. This adjuvant induces a Th2 response by improving the attraction and uptake of designer immunogens by antigen presenting cells (APCs). MONTANIDE™ ISA 51 is an oil which forms an emulsion when mixed with the water phase designer peptide/protein immunogens to elicit potent immune responses to SARS-CoV-2. CpGs Oligonucleotides are TLR-9 agonists that improve antigen presentation and the induction of vaccine-specific cellular and humoral responses. In general, the negative charged CpG molecule is combined with positively charged designer immunogens to form immunostimulatory complexes amenable for antigen presentation to further enhance the immune responses.

The disclosed high precision designer vaccine has the advantage of producing highly specific immune responses compared to weak or inappropriate antibody presentation of vaccines with a more complicated immunogen content employing inactivated viral lysate or other less characterized immunogens. In addition, there are potential pitfalls in COVID- 19 vaccine development that are related to a mechanism named 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 also its replication. This mechanism leads to both increased infectivity and virulence has been observed with mosquito- borne flaviviruses, HIV, and coronaviruses. The disclosed high precision vaccine is designed to avoid vaccine-induced disease enhancement by monitoring the quality and quantity of the antibody responses as they would dictate functional outcomes.

Representative studies discussed below set forth the approach in designing the disclosed high precision SARS-CoV-2 vaccine that can facilitate the elicitation of antibodies that can (1) bind to the CHO-expressed Sl-RBD-sFc protein; (2) inhibit the binding of SI protein to the ACE2 receptor that is immobilized on a microwell surface or on a cell surface overly expressing ACE2 receptor protein, and (3) neutralize viral mediated cytopathic effect in a cell mediated neutralization assay.

An immunization schedule of the varying forms of Sl-RBD-sFc designer proteins (SEQ ID NOs: 235, 236, and 355) in guinea pigs is shown in Table 28 for assessment of antibodies to S protein through a S protein antibody binding assay.

2. SI protein antibody binding assay (immunogenicity)

Varying forms of Sl-RBD proteins, including Sl-RBD-sFc, Sl-RBDa-sFc, and SI- RBD-Fc, for each group in the amount of lOOpg were mixed with ISA51 to prepare a w/o emulsion. These formulations were immunized into guinea pigs (n=5 per group) intramuscularly using the immunization schedule shown in Table 28. Briefly, guinea pigs were given a primary immunization of 100 pg per dose followed by a boost of 50 pg per dose at 3 weeks with individual serums collected at 0, 3, and 5 weeks post initial immunization (WPI). The collected serum samples were tested for immunogenicity by an SI -coated ELISA with detailed procedure an illustrated in Figure 20.

The functional properties of the antibodies elicited by these three protein immunogens were evaluated for their ability to inhibit the binding of S 1 -RBD to its surface receptor ACE-2 to prevent entry of the virus into target cells. Two functional assays were established, including (1) an ELISA to assess the direct inhibition of SI -RBD binding to ACE-2 ECD-sFc coated plate by such SI binding antibodies; and (2) a cell-based S1-RBD-ACE2 binding inhibition assay. These functional assays are described further below.

3. ELISA-based assays to determine Sl-RBD binding inhibition to ACE2

The detailed procedure for two separate ELISA-based Sl-RBD / ACE2 binding inhibition assays are illustrated in Figure 21.

In Method A, the ELISA plates are coated with ACE2 (e.g., ACE2 ECD-sFc) and 100 pL of antisera from an animal immunized with S-RBDa-sFc is mixed and incubated with Sl- RBD-His prior to adding the mixture to the ELISA plate. The amount of Sl-RBD-His binding/inhibition can be detected using a HRP conjugated anti-His antibody.

In Method B, the ELISA plates are coated with ACE2 (e.g., ACE2 ECD-sFc) and 100 pL of antisera from an animal immunized with S-RBDa-sFc is mixed and incubated with Sl- RBD-His-HRP prior to adding the mixture to the ELISA plate. The amount of Sl-RBD-His- HRP binding/inhibition can be detected directly.

4.

5. Cell-based assay to determine Sl-RBD binding inhibition to ACE2

The detailed procedure of a cell-based Sl-RBD and ACE2 binding inhibition assay is illustrated in detail in Figure 22. Specifically, ACE-2 over-expressed HEK293 cells were used as the target cells for such binding. Immune sera obtained from guinea pigs immunized with various forms of fusion proteins of Sl-RBD (Sl-RBD-sFc, Sl-RBDa-sFc, and S-RBD-Fc) were mixed and incubated with Sl-RBD-His protein followed by FITC conjugated detection antibody which is an anti-His-FITC. In this FITC traced ACE2 / Sl-RBD binding system, the presence of immune sera collected from guinea pigs immunized with varying forms of S-RBD- sFc, S-RBDa-sFc, or S-RBD-Fc were tested for their respective binding inhibition capabilities.

6. In vitro neutralization assay

Serum samples collected from animals immunized with S-RBD-sFc, S-RBDa-Fc, and S-RBD-Fc were inactivated at 56°C for 0.5h and serially diluted with cell culture medium in two-fold steps. The diluted sera were mixed with either a CNI strain virus, performed in KeXin laboratory in Beijing or a Taiwan strain virus performed independently in Taipei, suspension of 100 TCID50 in 96-well plates at a ratio of 1 : 1, followed by 2 hours incubation at 36.5°C in a 5% CO2 incubator. Vero cells (1-2 x 10⁴ cells) were then added to the serum-virus mixture, and the plates were incubated for 5 days at 36.5°C in a 5% CO2 incubator. The cytopathic effect (CPE) of each well was recorded under microscope, and the neutralizing titer was calculated by the dilution number of 50% protective condition.

As shown in Table 29 immune sera from guinea pigs after single immunization was collected at 3 wpi and submitted for test by KeXin laboratory in Beijing for this in vitro neutralization test. The pre-bleeds (0 wpi) and other control sera were found to be less than 8 by titer. Immune sera from immunogens with designer protein S-RBD-sFc demonstrated the best titer (1 :>256) while the immune sera from Sl-RBDa-sFc and Sl-RBD-Fc were in the range of 128 and 192, respectively. This in vitro neutralization assay that detects the ability to inhibit virus induced CPE further illustrated the functional efficacy of the tested immune sera to prevent SARS-CoV-2 infection.

Another independent testing for these immune sera was conducted at Nangang, Taipei as shown in Table 29. Immune sera collected from guinea pigs after prime and booster shots with blood collected at 0, 3, and 5 wpi were performed by this CPE based in vitro neutralization assay. In this second site testing, highly reproducible results were obtained for the 0 and 3 wpi immune sera with neutralizing titers measured between 128 and 256, while the titers of the immune sera from these designer proteins were around 4,096 and 8,192, about 15 to 30-fold higher than the immune sera upon single administration. The pre-bleeds and other control sera were found to be less than 8 or 4 depending on the respective laboratory scoring system. Immune sera from constructs with designer protein Sl-RBD-sFc demonstrated best titer (1:>256) while the other immune sera were in the range of 128 and 192 as observed in the Beijing laboratory. Thus, at least more than 2-fold in neutralizing titers was found when using the Sl-RBD-sFc as the designer immunogen than the other two designer proteins Sl-RBD-Fc or Sl-RBDa-sFc. The confirmation by this in vitro neutralization assay in two independent laboratories for ability of these designer protein induced antibodies to inhibit virus induced CPE further illustrated the functional efficacy of these immune sera, thus the utility of these high precision designer proteins as immunogens in vaccine formulations for the prevention of SARS-CoV-2 infection.

The neutralizing titers in sera from guinea pigs immunized with Sl-RBD-sFc were compared against those in convalescent sera of COVID-19 patients. Using the S1-RBD:ACE2 binding inhibition ELISA (also termed as qNeu ELISA), the responses in guinea pigs were compared against those in convalescent sera from Taiwanese COVID-19 patients after discharge from hospitalization.

EXAMPLE 10

MANUFACTURING OF THE MULTITOPE PROTEIN/PEPTIDE VACCINE COMPOSITION FOR THE PREVENTION OF INFECTION BY SARS-COV-2

Different formulations of the vaccine composition were prepared and evaluated in a pre-formulation characterization study to test their suitability for vaccine administration. In a forced degradation study, S-RBD-sFc was shown to be sensitive to heat, light exposure, and agitation but not sensitive to freezing and thawing cycles. The conditions considered sensitive to S-RBD-sFc were used for selecting the appropriate pH and excipients suitable for vaccine administration. Heat and UV Exposure

The isoelectric point (pl) value of S-RBD-sFc is between 7.3 to 8.4 so formulations were prepared with pH ranging from 5.7 to 7.0. In general, as the formulation pH moves away from the isoelectric point (pl), the solutions become clearer because protein solubility increases accordingly.

Size exclusion chromatography was used to determine whether the pH of the formulation had an effect on either heat-induced protein aggregation or UV-induced impurities. In this study, solutions containing S-RBD-sFc with pH ranging from 5.7 to 7.0, using a histidine buffer, were prepared and were either incubated at 35 °C for 24 hours or subjected to UV light for 24 hours. Size exclusion chromatography was used to determine the amount of S-RBD- sFc was present as well as several high molecular weight (HMW) impurities. The results from this study are shown in Table 30. Specifically, the results showed that pH had no obvious effect on heat-induced protein aggregation. The results also showed that, after UV exposure for 24 hours, S-RBD-sFc formed fewer high molecular weight impurities as the pH decreases, particularly from pH 5.7 to 6.4.

Based on this study, the final formulation was selected following the evaluation of prototype formulations at stressed conditions at the target pH of 5.9 using 10 mM histidine and the formulation pH specification limits of pH 5.4 and pH 6.4.

2. Surfactant - Agitation

Based on a forced degradation study, S-RBD-sFc was found to be sensitive to agitation stress and prone to form visible particles during agitation. Surfactants are often used to reduce the protein adsorption at the solid-liquid and liquid-air interface, which might lead to protein destabilization. Thus, a study was performed to determine if polysorbate 80 is capable of reducing or preventing precipitation of S-RBD-sFc after agitation.

In this study, three separate solutions containing approximately 2 mg/mL of S-RBD- sFc were agitated at 1,200 RPM at 25 °C for 67 hours. The first solution contained 0.03% (w/v) polysorbate 80, the second solution contained 0.06% (w/v) polysorbate 80, and the third solution was a control without any polysorbate 80. In this study, the results showed that 0.06% (w/v) polysorbate 80 efficiently mitigates precipitation of S-RBD-sFc after agitation (data not shown). Therefore, the presence of 0.06% (w/v) polysorbate 80 was determined to improve stability and reduce precipitation of S-RBD-sFc in the formulation.

3. Protein Buffers

Additives, such as arginine-HCl, sucrose, and glycerol are frequently used as a protectant in the formulation development of proteins.

In this study, solutions containing S-RBD-sFc together with varying amounts of as arginine-HCl (25 mMto 100 mM), sucrose (25 mM to 100 mM), or glycerol (5% to 15%) were incubated at 50 °C for 1 hour. Size exclusion chromatography was used to determine the amount of S-RBD-sFc was present as well as several high molecular weight (HMW) impurities. The results from this study are shown in Table 30. Specifically, the results indicated that the addition of arginine-HCl, sucrose, or glycerol were able to lower heat-induced aggregation. These results were further confirmed by measuring the turbidity (OD600) of samples incubated at 40 °C for 45 min. Consistent with the size exclusion chromatography results, the addition of arginine-HCl, sucrose, or glycerol efficiently reduced the turbidity of samples (data not shown).

The effect of arginine-HCl, sucrose, or glycerol under UV stress on S-RBD-sFc solutions at pH 5.9 was also evaluated. Size exclusion chromatography results indicated that the addition of arginine-HCl slightly increased light-induced aggregation, but sucrose and glycerol did not have any significant impact on aggregation (Table 30).

4. Summary

A summary of the results obtained in the formulation screening studies is provided in Table 31

EXAMPLE 11

PRODUCTION OF THE Sl-RBD-sFc PROTEIN FOR USE IN THE MULTITOPE PROTEIN/PEPTIDE VACCINE COMPOSITION FOR THE PREVENTION OF

INFECTION BY SARS-COV-2

The fed-batch production development for a small pilot scale batch (15L) and large- scale batch (100L) were carried out as described below.

1. Pilot Batch

a. Fed-Batch Cell Culture Upstream Process

The fed-batch production development at pilot scale was carried out in a 15-L Finesse bioreactor with an initial working volume 9 L. HYPERFORMA™ 15 L bioreactor is a glass vessel bioreactor equipped with HYPERFORMA™ G3Lab Controller and TruFlow gas mass flow controller (MFC). The equipped impeller is a pitched blade impeller, and the sparger is a drilled pipe sparger with 0.8 mm diameter holes for aeration. The 15-L bioreactor parameters were as follows: a. Medium: DYNAMIS + 1 g/kg dextran sulfate + 1.17 g/kg glutamine b. Initial Cell Density: 0.3E6 vc/mL c. Temperature: 37 °C; TS to 32 °C on D5 d. pH: pH 7.0 ± 0.3; base: 1 M Na₂CO₃; acid: C0₂ e. Dissolved Oxygen: Setpoint 50% f. Feeding Strategy: 83% EX-CELL® ACF CHO Medium + 17% EX-CELL® 325 PF CHO Medium supplemented with 50 g/kg glucose and 20 g/kg yeast extract.

D3 - D7: 3% daily; D8 - D12: 4% daily (total feeding ratio: 35% w/w) g. Glucose Control: D3 - D13: add 2 g/kg glucose (stock 300 g/kg) when [Glue] < 2 g/L h. Harvest Criteria: Cell viability < 60% or on D 14

In brief, DYNAMIS™ AGT™ Medium (Thermo Fisher Scientific, A2617502) supplemented with L-Glutamine and dextran sulfate was used for both seed train expansion and production process. Bolus nutrient feed to the bioreactor was started on run day 3 (D3). The nutrient feed was formulated by blending 83% EX-CELL® ACF CHO Medium (Merck, C9098) with 17% EX-CELL® 325 PF CHO Medium (Merck, 24340C). Daily monitoring of cell number, cell viability, concentration of the metabolites (glucose, lactate, glutamine, glutamate and ammonia), osmolality, pH, pCO₂ and pO₂ were performed on BioProfile FLEX Analyzer (Nova Biomedical). The harvest criteria were the cell viability below 60% or on production day 14 (DI 4).

On the day of harvest, the cell culture fluid was clarified by C0HC depth filter (Merck, MC0HC05FS1) followed by 0.22 pm capsule filtration. The harvested cell culture fluid (HCCF) was transferred to the Protein Purification Lab for downstream processing immediately.

In this process, the peak VCD was approximately 14E+06 vc/mL on day 7 and the cell viability was able to sustain > 90% till the end of production. The productivity of Sl-RBD-sFc was 1.6 g/L on day 14. b. Harvest

Millistak+ POD C0HC 0.55 m² and Opticap XL 5 Capsule were applied to harvest materials. The filter was flushed with 100 L/m² of purified water at a flux rate of 600 LMH. The flush rate was 5 L/min and flush time was at least 10 minutes. Blow down was performed to drain off purified water from the POD filter before running filtrate (10 psi for at least 10 minutes). Run harvest cell culture fluid (HCCF) with 500 L/min, which was equal to 54.5 LMH. The first 1.4 L retentate was abandoned and the rest of retentate was collected. During the whole operation, the pressure was monitored and should not exceed 30 psi. The pre- clarification and post-clarification turbidities were 1343 NTU and 12.9 NTU, respectively, and the pre-clarification and post-clarification titers were 1.66 g/L and 1.50 g/L, respectively. Upstream product yields were high (1.5 g/L). c. Downstream Purification Process Development

Briefly, the harvested cell culture fluid (HCCF) was first treated with 1% TWEEN 80 (Merck, 8.17061) and 0.3% TNBP (Merck, 1.00002) and held for 1 hour without agitation at ambient temperature (23 ±4 °C) for sol vent/ detergent virus inactivation. The sol vent/ detergent treated HCCF was purified using a Protein A affinity chromatography column (MabSelectSuRe LX resin, Cytiva Life Sciences, 17-5474-03). The eluate from the Protein A column was neutralized to pH 6.0 immediately by 1 M Tris base solution (Merck, 1.08386). The neutralized protein solution was filtered by two types of depth filter, C0HC (23 cm², Merck Millipore, MC0HC23CL3) and X0SP (23 cm², Merck Millipore, MX0SP23CL3) to remove precipitates and impurities. The clarified protein solution was further purified by a cation exchange chromatography column (NUVIA™ HR-S media, Bio-Rad, 156-0515). The protein concentration was adjusted to 5 mg/ml, and the protein solution was subjected to viral filtration (PLANOVATM 20NNano filter, Asahi Kasei, 20NZ-001). The filtrate from the nano filtration was buffer exchanged into formulation buffer by using tangential flow filtration (TANGENX™ SIUS™ PDn TFF Cassette, Repligen, PP030MP1L). After the buffer exchange, TWEEN 80 was then added to the formulated protein solution at a final concentration of 0.06% (w/v) followed by a 0.22 pm filtration, the formulated product was stored at 2-8 °C and protected from light exposure. d. Process Yields, 15L Pilot Lot

The yield of each step was as follows: a. Solvent detergent virus inactivation, protein A chromatography, neutralization and depth filtration: 11.30 g (83.1% yield). b. Cation exchange chromatograph: 10.96 g (96.7% yield). c. Nano-filtration, formulation by diafiltration and 0.2 pg filtration: 10.50 g (99.7% yield). The overall recovery was 80.3% yield.

2, Large Scale Batch (TOOL)

Aclinical batch of S-RBD-sFc (100L) was manufactured from the clonal Research Cell Bank. The changes were made only at the drug substance level without changes in final composition. The raw materials and the process parameters were not changed, only the batch size is scaled up. No significant differences are observed between both lots. The impact of the changes in manufacturing process for S-RBD-sFc drug substance between the pilot batch and the large-scale batch were assessed by a comparability study.

To assess the comparability between drug substance batches from the 15L scale process and drug substance from the 100L scale process, the analytical data of release data generated by characterizations and data of forced degradation study were compared and evaluated.

The S-RBD-sFc lots produced by the 15L scale and 100L scale manufacturing processes all met release specifications set in the respective specifications. All tested lots showed lot-to-lot consistency with similar levels of size variants and impurity, similar distribution of charge variants and comparable potency.

The results of the characterization study demonstrated comparability and consistency in the protein and carbohydrate structures, post translational modifications, purity/impurity, heterogeneity and biological activity of S-RBD-sFc lots produced by the 15L scale or 100L scale manufacturing process. In addition, the forced degradation study showed that the degradation pathways and the sensitivity to specific degradation conditions were similar and comparable for the tested lots manufactured by different process.

Overall, the results demonstrated the comparability of S-RBD-sFc lots between those produced by 15L scale and 100L scale with respect to the results obtained from release testing, forced degradation studies and additional characterizations.

EXAMPLE 12

A MULTITOPE PROTEIN/PEPTIDE VACCINE COMPOSITION FOR THE PREVENTION OF INFECTION BY SARS-COV-2

The initial immunogenicity assessment in guinea pigs established the humoral immunogenicity of our RBD-based protein and allowed selection of Sl-RBD-sFc (SEQ ID NO: 235) as the main immunogenic B cell component for a vaccine against SARS-CoV-2.

The presence of T cell epitopes is important for the induction of B cell memory response against viral antigens. SARS-CoV-2 CTL and Th epitopes, validated by MHC binding and T cell functional assays, that are conserved between SARS-CoV-2 and SARS-CoV-1 (2003) viruses are employed in the design of the high precision SARS-CoV-2 vaccine against CO VID- 19. Identification of T cell epitopes on SARS-CoV-1 (2003), determined using MHC -binding assays, were used to determine corresponding T cell epitopes in SARS-CoV-2 (2019) by sequence alignment (see Figures 3, 4, and 5A-5C and Table 32). CTL epitopes that are incorporated in the design of the disclosed high precision designer SARS-CoV-2 vaccine were identified in a similar manner. The Th and CTL epitopes that are incorporated in SARS-CoV- 2 vaccine design have been validated by MHC Class II binding and T cell stimulation as shown in Table 28. Specific multitope protein/peptide vaccine compositions for the prevention of infection by SARS-CoV-2 containing 20 pg/mL, 60 pg/mL, and 200 pg/mL (combined weight of the Sl-RBD-sFc fusion protein and the Th/CTL peptides) are shown in Tables 29 to 31.

1. Immunogenicity Study in Rats

In a set of experiments conducted in rats, a proprietary mixture of Th/CTL peptides (SEQ ID NOs: 345, 346, 348, 348, 361, and 66) were added to the Sl-RBS-sFc (SEQ ID NO: 235) B cell component for further assessment of optimal formulations and adjuvants and establishment of the cellular immunity components of the vaccine. This vaccine composition was utilized in the following studies. a. Humoral Immunogenicity Testing in Rats

The guinea pig experiments described in Example 13 were tested with three protein candidates with a single dosing regimen with a prime (100 pg or 200 pg) and a boost (50 pg or 100 pg) using ISA 50 as an adjuvant, allowing for a rigorous comparison of the respective candidate constructs. In this set of experiments conducted in rats, varying doses of immunogen and adjuvants were evaluated to allow selection of an optimal adjuvant based on Sl-RBD binding antibody titers and balanced Thl/Th2 responses.

The vaccine composition containing the Sl-RBD-sFc protein with the Th/CTL peptides were combined the candidate vaccine with two different adjuvant systems, (a) ISA51 combined with CpG3 (SEQ ID NO: 106) and (b) ADJU-PHOS® combined with CpGl (SEQ ID NO: 104). These vaccine-adjuvant combinations were administered to rats IM on 0 WPI (prime) and 2 WPI (boost) with a wide dose range of 10 to 300 pg per injection. The animals were bled at 0, 2 (i.e., after 1 dose), 3 and 4 WPI (i.e., 1 and 2 weeks after the 2nd dose) for antibody titer analyses.

Results of binding antibody (BAb) testing at all time points demonstrated that vaccines formulated with both adjuvant systems elicited similar levels of anti Sl-RBD ELISA titers across all doses ranging from 10 to 300 pg, indicative of an excellent immunogenicity of the vaccine formulations even with low quantities of the primary protein immunogen. In addition, a 100-pg dose of Sl-RBD-sFc without the synthetic peptide components stimulated high Sl- RBD binding activity similar to previous guinea pig studies (data not shown). In the S1-RBD:ACE2 binding inhibition ELISA test, doses of 10 and 30 pg induced as strong inhibitory activity as the high doses at 100 and 300 pg at 4 WPI. The most potent inhibitory activity was seen with the lowest dose of Sl-RBD-sFc protein (10 pg) formulated with rationally designed peptides and the ADJU-PHOS®/CpGl adjuvant. In the replicating virus neutralization assay against the Taiwanese SARS-CoV-2 isolate (as discussed above for guinea pig studies), the 4 WPI immune sera induced by the vaccine composition did not show a significant dose-dependent effect. However, low doses of adjuvanted protein, 10 and 30 pg, could neutralize viral infection at VNT50 of >10,240 dilution fold . The rat immune sera at 6 WPI from each vaccinated dose group were assayed, (a) in comparison with a set of convalescent sera of COVID- 19 patients for titers in S1-RBD:ACE2 binding inhibition ELISA, expressed in blocking level of pg/mL ; and (b) by a SARS-CoV-2 CPE assay in Vero-E6 cells, expressed as VNT50. All doses of the vaccine formulations elicited neutralizing titers in rats that are significantly higher than those in convalescent patients by S1-RBD:ACE2 binding ELISA and higher (but not achieving statistical significance due to the spread in the patient data and the low number of animals) by VNT50. b. Cellular Immunogenicity Testing in Rats

To address the issue related to Thl/Th2 response balance, cellular responses in vaccinated rats were evaluated using ELISpot. i. Procedure for Rat Thl/Th2 Balance Study

A total of 12 male Sprague Dawley rats at 8-10 weeks of age (300-350 gm/BW) were purchased from BioLASCO Taiwan Co., Ltd. After a 3-day acclimation, animals were randomly assigned to 4 groups. All procedures on animals were performed in accordance with the regulations and guidelines reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at UBIAsia. The IACUC number is AT-2028. The rats were vaccinated intramuscularly at weeks 0 (prime) and 2 (boost) with different doses ranging from 1 to 100 pg of a vaccine composition containing Sl-RBD-sFc (SEQ ID NO: 235) with five Th/CTL peptides selected from S, M and N proteins of SARS-CoV-2 (SEQ ID NOs: 345, 346, 348, 348, and 361) and a proprietary universal Th peptide UBITh®la (SEQ ID NO: 66) formulated in ADJU-PHOS®/CpGl adjuvant. The immune sera from rats (n = 3 for each dose group) were collected at weeks 0, 2, 3, and 4 for assessment of antigenic activities. Splenocytes were collected at 4 WPI and restimulated in vitro at 2 pg/well either with the Th/CTL peptide pool plus Sl-RBD or with the Th/CTL peptide pool alone. IFN-y, IL-2, and IL-4-secreting splenocytes were determined by ELISpot analysis. Cytokine-secreting cells (SC) per million cells was calculated by subtracting the negative control wells. ii. ELISpot for Measurement of Cellular Responses

Spleens from vaccinated rats at 4 WPI were collected in Lymphocyte-conditioned medium (LCM; RPMI-1640 medium supplemented with 10% FBS and penicillin/streptomycin) and processed into single cell suspensions. Cell pellets were resuspended in 5 mL of RBC lysis buffer for 3 min at room temperature (RT), and RPMI-1640 medium containing penicillin/streptomycin was then added to stop the reaction. After centrifugation, cell pellets resuspended in LCM were used in ELISpot assay. ELISpot assays were performed using the Rat IFN-y ELISpotPLUS kit (MABTECH, Cat. No. : 3220-4APW), Rat IL-4 T cell ELISpot kit (U-CyTech, Cat. No.: CT081) and Rat IL-2 ELISpot Kit (R&D Systems, Cat. No.: XEL502). ELISpot plates precoated with capture antibody were blocked with LCM for at least 30 min at RT. 250,000 rat splenocytes were plated into each well and stimulated with Sl-RBD-His protein plus Th/CTL peptide pool, Sl-RBD-His protein, Th/CTL peptide pool, or each single Th/CTL peptide for 18-24 hrs at 37°C. Cells were stimulated with a final concentration of 1 pg of each protein/peptide per well in LCM. The spots were developed based on manufacturer’s instructions. LCM and ConA were used for negative and positive controls, respectively. Spots were scanned and quantified by AID iSpot reader. Spot-forming unit (SFU) per million cells was calculated by subtracting the negative control wells.

A dose-dependent trend in IFN-y secretion was observed in splenocytes, while little secretion of IL-4 was seen. The results indicated that the vaccine composition was highly immunogenic and induced a Thl-prone cellular immune response as shown by the high ratios of IFN-y/IL-4 or IL-2/IL-4. High ratios of IL-2/IL-4 were also observed in the presence of the Th/CTL peptide pool and for restimulation with individual peptides, which induced little IL-4 secretion. Bars represent the mean SD (n = 3). The secretion of IFN-y or IL-2 was observed to be significantly higher than that of IL-4 in 30 and 100 pg group (*** p < 0.005 using Least Square Mean and paired wise comparison) but they were not statistically different in 1 or 3 pg dose groups.

2. Challenge Studies in Transgenic Mice

The initial challenge study of the vaccine composition was performed in the AAV/hACE2 transduced BALB/c mouse model established by Dr. Tau, Mi-Hua at Academia Sinica in Taiwan; adaptations of this model are also reported by other investigators. a. Animal Procedures for BALB/C Challenge Studies

A total of 12 male BALB/C at 8-10 weeks of age were purchased from BioLASCO Taiwan Co., Ltd. After a 3-day acclimation, animals were randomly assigned to 4 groups. All procedures on animals were performed in accordance with the regulations and guidelines reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at UBI Asia. The IACUC numbers are AT2032 and AT2033.

The mice were vaccinated by IM route at weeks 0 (prime) and 2 (boost) with 3, 9, or 30 pg of the vaccine composition containing Sl-RBD-sFc (SEQ ID NO: 235) together with Th/CTL peptides (SEQ ID NOs: 345, 346, 348, 348, 361, and 66) formulated in ADJU- PHOS®/CpGl adjuvant. The immune sera from mice were collected at weeks 0, 3 and 4 for assessment of immunogenic and functional activities by the assay methods described below.

AAV6/CB-hACE2 and AAV9/CB-hACE2 were produced by AAV core facility in Academia Sinica. BALB/C mice (8-10 weeks old) were anaesthetized by intraperitoneal injection of a mixture of Atropine (0.4 mg/ml)/Ketamine (20 mg/ml)/Xylazine (0.4%). The mice were then intratracheally (IT) injected with 3 x 1011 vg of AAV6/hACE2 in 100 pL saline. To transduce extrapulmonary organs, 1 x 1012 vg of AAV9/hACE2 in 100 pL saline were intraperitoneally injected into the mice.

Two weeks after AAV6/CB-hACE2 and AAV9/CB-hACE2 transduction, the mice were anesthetized and intranasally challenged with 1x104 PFU of the SARS-CoV-2 virus (hCoV- 19/Taiwan/4/2020 TCDC#4 obtained from National Taiwan University, Taipei, Taiwan) in a volume of 100 pL. The mouse challenge experiments were evaluated and approved by the IACUC of Academia Sinica. Surviving mice from the experiments were sacrificed using carbon dioxide, according to the ISCIII IACUC guidelines. All animals were weighed after the SARS-CoV-2 challenge once per day. b. RT-PCR for SARS-CoV-2 RNA Quantification

To measure the RNA levels of SARS-CoV-2, specific primers targeting 26,141 to 26,253 regions in the envelope (E) gene of the SARS-CoV-2 genome were used by Taqman real-time RT-PCR method that described in the previous study (Corman, et al. 2020). Forward primer E-Sarbeco-Fl (5 ’ -AC AGGTACGTTAATAGTTAATAGCGT-3 ’ ; SEQ ID NO: 368) and the reverse primer E-Sarbeco-R2 (5’-ATATTGCAGCAGTACGCACACA-3’; SEQ ID NO: 369), in addition to the probe E-Sarbeco-Pl (5’-FAM- ACACTAGCCATCCTTACTGCGCTTCG-BBQ-3’; SEQ ID NO: 370) were used. A total of 30 pL RNA solution was collected from each sample using RNeasy Mini Kit (QIAGEN, Germany) according to the manufacturer's instructions. 5 pL of RNA sample was added in a total 25 pL mixture using Superscript III one-step RT-PCR system with Platinum Taq Polymerase (Thermo Fisher Scientific, USA). The final reaction mix contained 400 nM forward and reverse primers, 200 nM probe, 1.6 mM of deoxy-ribonucleoside triphosphate (dNTP), 4 mM magnesium sulphate, 50 nM ROX reference dye and 1 pL of enzyme mixture from the kit. The cycling conditions were performed with a one-step PCR protocol: 55°C for 10 min for cDNA synthesis, followed by 3 min at 94°C and 45 amplification cycles at 94°C for 15 sec and 58°C for 30 sec. Data were collected and calculated by Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific, USA). A synthetic 113-bp oligonucleotide fragment was used as a qPCR standard to estimate copy numbers of viral genome. The oligonucleotides were synthesized by Genomics BioSci and Tech Co. Ltd. (Taipei, Taiwan). c. Challenge Study

Groups of 3 mice were vaccinated at study 0 and 2 WPI with the vaccine composition described above containing 3, 9, or 30 pg of protein and formulated with ADJU-PHOS®/CpGl . The mice were infected with adeno-associated virus (AAV) expressing hACE2 at 4 WPI and challenged 2 weeks later with 106 TCID50 of SARS-CoV-2 by the intranasal (IN) route. Efficacy of the vaccine was measured using lung viral loads and body weight measurements. Vaccination with 30 pg of the vaccine composition significantly reduced lung viral loads (~3.5 loglO viral genome copies/pg RNA or ~ 5-fold TCID50/mL of infectious virus) compared to saline group (p <0.05 as measured by paired t test). Vaccination with middle and high doses led to clear reduction in lung pathology. Vaccination with 3 or 9 pg of the vaccine composition reduced live virus detection by cell culture method (TCID50) to below of the level of detection but it did not appear to reduce viral loads significantly when measured by RT-PCR. Similarly, body weight measurements showed a significant difference between the high-dose group and the control group (data not shown). In sum, despite the lack of a statistical power (N=3 mice) in this study, it appears that the highest dose at 30 pg per dose could have had the maximum protective efficacy when one combines the lack of live virus detection and the lack of inflammatory cell infiltrations as well as lack of immunopathology in the lungs altogether.

3. Immunogenicity and Challenge Studies in Rhesus Macaques

Based on an established model using rhesus macaques (RM), an immunization study of the vaccine composition containing Sl-RBD-sFc (SEQ ID NO: 235) together with Th/CTL peptides (SEQ ID NOs: 345, 346, 348, 348, 361, and 66) was performed as described below. a. Immunogenicity Studies in Non-Human Primates

The study was conducted at JOINN Laboratories (Beijing) in rhesus macaques aged approximately 3-6 years. Animals were housed individually in stainless steel cages, an environmentally monitored, and well-ventilated room (conventional grade) maintained at a temperature of 18-26°C and a relative humidity of 40-70%. Animals were quarantined and acclimatized for at least 14 days. The general health of the animals was evaluated and recorded by a veterinarian within three days upon arrival. Detailed clinical observations, body weight, body temperature, electrocardiogram (ECG), hematology, coagulation and clinical chemistry were performed on monkeys. The data were reviewed by a veterinarian before being transferred from the holding colony. Based on pre-experimental body weights obtained on Day -1, all animals were randomly assigned to respective dose groups using a computer-generated randomization procedure. All animals in Groups 1 to 4 were given either control or test article via intramuscular (IM) injection. Doses were administered to the quadriceps injection of one hind limbs. Monkeys were observed at least twice daily (AM and PM) during the study periods for clinical signs which included, but not limited to mortality, morbidity, feces, emesis, and the changes in water and food intake. Animals were bled at regular intervals for the immunogenicity studies described below.

Rhesus macaques (3-6 years old) were divided into four groups and injected intramuscularly with high dose (100 pg/dose), medium dose (30 pg/dose), low dose (10 pg/dose) vaccine and physiological saline, respectively. All grouped animals were immunized at three times (days 0, 28 and 70) before challenged with 106 TCID50/ml SARS-CoV-2 virus by intratracheal routes (performed on day 82). Macaques were euthanized and lung tissues were collected at 7 days post challenge. At days 3, 5, 7 dpi, the throat swabs were collected. Blood samples were collected 0, 14, 28, 35, 42, 70, and 76 days post immunization, and 0, 3, 5, 7 days post challenge for neutralizing antibody test of SARS-CoV-2. Lung tissues were collected at 7 days post challenge and used for RT-PCR assay and histopathological assay. Analysis of lymphocyte subset percent (CD3+, CD4+ and CD8+) and key cytokines (TNF-a, IFN-y, IL-2, IL-4, IL-6) were also performed in collected blood samples on days 0 and 3 post challenge, respectively. b. Immunogenicity and Challenge Studies in Rhesus Macaques

Based on an established model using rhesus macaques (RM), an immunization study of the vaccine composition by IM injection was initiated with RM (N = 4/group) receiving 0, 10, 30, or 100 pg of the composition at 0 and 4 WPI. Immunogenicity measurements indicated that the serum IgG binding to S 1 -RBD was increased over baseline in all animals with binding titers reaching around 3 logs at 5 and 7 WPI. Strong neutralizing antibody responses were induced, with the 30 pg dose being most potent. ELISpot analysis indicated that vaccine composition activated antigen-specific fFN-y-secreting T cells in a dose-dependent manner with T cell responses highest at the 100 pg dose level.

4. Toxicity Study in Preparation for Clinical Trials

To enable clinical trials, the vaccine composition containing Sl-RBD-sFc (SEQ ID NO: 235) together with Th/CTL peptides (SEQ ID NOs: 345, 346, 348, 348, 361, and 66) was tested in a GLP-compliant repeat-dose toxicology study in Sprague-Dawley rats as described below. a. Protocol for Toxicology Studies

A total of 160 rats (80/sex) were randomly assigned to 8 groups based on the body weights obtained on Day -1 (1 days prior to the first dosing, the first dosing day was defined as Day 1), of which 120 rats were assigned to Groups 1, 2, 3 and 4 (15/sex/group) for the toxicity study, and 40 rats to Groups 5, 6, 7 and 8 (5/sex/group) for the satellite study. Rats were treated with saline injection for Groups 1 and 5 as negative control, vaccine composition placebo for Groups 2 and 6 as adjuvant control, and vaccine composition at doses of 100, 300 pg/animal for Groups 3 and 7 as well as Groups 4 and 8, respectively. Rats were treated via intramuscular injection into the one-side hind limbs muscle (quadriceps femoris and gastrocnemius, left side for the first dose and right side for the second dose) at multiple sites once every two weeks for 2 consecutive weeks, total 2 doses (on Days 1 and 15). The dose volume was 0.5 mL/animal. Clinical observations (including injection sites observation), body weight, food consumption, body temperature, ophthalmoscopic examinations, hematology, coagulation, clinical chemistry, urinalysis, T lymphocyte subpopulation, number of T lymphocyte spots secreting fFN-y by peripheral blood mononuclear cells (PBMCs), cytokines, and immunogenicity, neutralizing antibody titer and IgG2b/IgGl ratio analysis were performed during the study. The first 10 animals/sex/group in Groups 1 to 4 were designated for the terminal necropsy after 2 weeks of dosing (Day 18) and the remaining 5 animals/sex/group were designated for the 4-week recovery necropsy after the last dosing (Day 44). All animals in Groups 1 to 4 were given complete necropsy examinations, and then the organ weights, macroscopic and microscopic examinations were evaluated. b. Toxicity Study in Preparation for Clinical Trials

To enable clinical trials, the vaccine composition was tested in a GLP-compliant repeatdose toxicology study in Sprague-Dawley rats. The study included a 300 ug dose, 3 times higher than that of the highest dose intended for clinical use. Although the schedule of 2 injections did not exceed that intended for clinical use, this is acceptable according to the WHO guidelines46. The study was also designed to evaluate the immunogenicity of the vaccine composition. One hundred and sixty (160) rats were randomly divided into 8 groups (80 males and 80 females) of which 40 rats were included in the satellite immunogenicity study. The low- and high dose groups were inoculated with the vaccine composition at 100 pg/animal (0.5 mL) and 300 pg/animal (0.5 mL) respectively; control groups were injected either with saline (0.9% saline) or adjuvant (vaccine composition placebo) at the same dose volume. The first ten animals/sex/group were designated for the terminal necropsy after two weeks of dosing at 2 WPI (Day 18) and the remaining 20 animals/sex/group were designated for the 4-week recovery necropsy after the last dosing at 4 WPI (Day 44). Under the experimental conditions, rats received IM injections into one hind limb muscle (quadriceps fem oris and gastrocnemius, left side for the first dose and right side for the second dose) at multiple sites once every two weeks for 2 consecutive weeks, total 2 doses at 0 and 2 WPI (on Days 1 and 15).

Treatment with the vaccine composition at dose levels of up to 300 pg/animal at weeks 1 and 3 was well tolerated with no signs of systemic toxicity. Neither test article-related mortality nor moribundity was noted throughout the study. No vaccine-related abnormal findings were noted in clinical observations (including injection site observations) throughout the study. Neither erythema nor edema were noted at injection sites, and the Draize score was 0 for all observation time points. Similarly, no vaccine-related changes in body weight, food consumption, body temperature, hematology, chemistries (other than AG ratio), ophthalmoscopic examinations or urinalysis were observed, and no statistically significant changes were noted in CD3+, CD3+CD4+, CD3+CD8+, and the ratio of CD3+CD4+/CD3+CD8. Statistically significant increases were seen in fibrinogen, IFN-y, and IL-6, while decreases in albumin/globulin ratio were observed; these results are consistent with an acute phase response to a vaccine, and all resolved by the end of the recovery period. Histopathological examinations of epididymides, skin, liver, prostate and mammary gland, revealed minimal inflammatory cell infiltrations with no visible lesions or abnormalities.

Immunogenicity of the vaccine composition measured in satellite groups showed that the vaccine was able to induce substantial levels of anti-SARS-CoV-2 Sl-RBD IgG in animals receiving two doses of 100 pg/animal or 300 pg/animal at 2 and 4 WPI (a 14-day interval) (data not shown). The Sl-RBD binding IgG titers rose modestly over time after the boost at 2 WPI (Day 15), which reached around 2.6 loglO and 3.3 loglO in rats immunized with the vaccine composition at 100 pg/animal and 300 pg/animal, respectively, at 6 WPI (Day 44). The findings observed in this study are as expected for a vaccine designed to stimulate immune responses resulting in production of high titers of antibodies. Anti-SARS-CoV-2 Sl-RBD IgG titers, subtype IgG and serum cytokine production by ELISA were performed to determine the Thl/Th2 responses. On analyses of Sl-RBD-specific IgG subclasses, the patterns and induction levels of Th2 -related subclass IgGl anti-SARS-CoV-2 Sl-RBD were comparable to what was observed in total IgG anti-SARS-CoV-2 Sl-RBD. Only slight induction of Thl- related subclass IgG2b anti-SARS-CoV-2 Sl-RBD was detected in rats vaccinated with the vaccine composition at 6 WPI (Day 43). However, the serum cytokine pattern measured by ELISA indicated a Thl/Th2 balanced response (data not shown).

Clinical trials of the vaccine composition have begun in Taiwan. The first study, entitled “Phase 1, Open-Label Study to Evaluate the Safety, Tolerability, and Immunogenicity of UB- 612 Vaccine in Healthy Adult Volunteers”, was initiated in Taiwan in September 2020. This trial includes three dose groups (N=20 per group) of UB-612 (10, 30, or 100 pg) given at days 1 and 29 (2 dose regimen). The primary endpoint is the occurrence of adverse events within seven days of vaccination; secondary endpoints include adverse events during the six-month follow-up period, standard laboratory safety measures, antigen-specific antibody titers, seroconversion rates, T cell responses and increase of neutralizing antibody titers, a.

EXAMPLE 13

DESIGNER LONG-ACTING PROTEIN DRUG ACE2-ECD-sFc GENERATED HIGH ANTIVIRAL EFFECT MEASURED IN A NEUTRALIZING ASSAY FOR INHIBITION OF SARS-COV-2 INDUCED CPE IN VERO CELLS

The coronaviruses SARS-CoV-1 (2003) and SARS-CoV-2 (2019) enter host cells through binding of the viral envelope-anchored spike (S) protein to the receptor angiotensinconverting enzyme 2 (ACE2). Among other unique features of the S protein, SARS-CoV-2 binds to ACE2 with a higher affinity (up to 20-fold) compared to SARS-CoV-1, which corresponds to a rapid human-to-human transmissibility of new infections observed for SARS- CoV-2. As ACE2 plays a crucial role in the spread of SARS-CoV-2, an engineered soluble ACE2-like protein could potentially work as an effective interceptor to block viral invasion, thereby achieving therapeutic purpose while, at the same time, safeguarding the normal physiological function of the membrane-bound ACE2 from being further reduced and damaged.

Using a proprietary technology platform, a unique ACE receptor-based, long-acting fusion protein product of GMP grade can be used to treat COVID-19 of both symptomatic and asymptomatic patients. The technology platform integrates the plasmid construction of extracellular domain of ACE2 (ACE2-ECD) that links to a single chain immunoglobulin Fc fragment (sFc), expression and production in CHO-S cell line of ACE2-sFc fusion protein, and purification and bio-characterization of the protein species. The ACE2-sFc product is under preclinical testing and being planned for a parallel accelerated phase-I safety study with patients confirmed having mild-to-severe SARS-CoV-2 infection upon clinical diagnosis and PCR confirmation.

A diverse array of in vitro bioassays has been performed demonstrating that the fusion protein ACE2-sFc is functionally active. These assays include a SPR-based binding affinity assay, a molecular and cellular recognition by SARS-CoV-2 spike (S) protein, and a neutralization of the S protein- ACE interaction by ACE2-sFc. A proof-of-concept inhibition of SARS-CoV-2 infection has been confirmed on the cellular level. ACE2-sFc, either alone or in synergic combination with anti-IL6R mAb or the currently approved Remdesivir, could be of significant clinical utility for treatment of COVID-19.

A “Single Chain Fc Platform” was employed to produce a potent, long-acting neutralizing protein product ACE2-ECD-sFc (SEQ ID NO: 237). Due to the receptor binding inhibition nature, the ACE2-ECD-sFc protein is anticipated to meet little drug resistance if the coronavirus mutates. Due to the bulky conformation of the bivalent Fc fusion nature, the ACE- ECD-Fc has a faster departure rate (about 10X) when binding to the SI protein compared to the single chain (ACE ECD-sFc protein) indicating that the Fc protein has a 1 OX lower binding affinity when compared to that of the single chain (sFc) fusion protein. Although all three types of ACE-ECD fusion proteins (ACE2 ECD-sFc, ACE2 ECD-Fc, and ACE2 ECD-sFc) all have significant capability to block S 1 binding to ACE-2 coated on an ELISA plate. The ACE2- ECD-sFc has a higher % of blocking inhibition when compared to the other two types. This result indicates that the relative inhibition in viral induced Cytopathic Effect (CPE) on the Vero cells, when tested in two separate laboratories (KeXin Lab in Beijing and Sinica Lab in Taipei) as shown in Table 36, where an equivalent titer of 8, 192 was achieved by 2.4mg/mL of ACE2- ECD-sFc in an assay, would offer a highly effective treatment for patients encountering acute attack by SARS-CoV-2 infection based on the observation that a full protection can be obtained in a primate challenge study with serum titer neutralizing antibodies in the range of around 50. A phase I/II trial will be conducted in mild to severe COVID-19 patients to observe the safety and efficacy of such a long-acting protein drug.

EXAMPLE 14

COMPARISON OF UB-612 AND VACCINE CANDIDATE A

A number of studies are being undertaken to increase the immugonecity of Sl-RBD- sFc vaccine candidates. Three possible candidates include VACCINE CANDIDATE A, VACCINE CANDIDATE B and Vaccine Candidate C - Bivalent.

In UB-612, the CpG (TLR-9 agonist) oligonucleotide concentration was at an excipient level of upto 4 pg (max). In VACCINE CANDIDATE A, the CpG concentration was changed to an adjuvant level of 1000 pg. To allow for the majority of CpG to bind to antigen and aluminum hydroxide adjuvant, aluminum phosphate was changed to aluminum hydroxide which has a higher binding capacity than aluminum phosphate. Increased concentration of CpG also changes the Thl/Th2 balance toward the Thl biased response which is preferred to avoid potential risks of enhanced disease associated with other SARS or RSV which induce a Th2 biased response.

In certain embodiments, the CpG concentration may be about 10-2000 pg for the first dose in a 2 or more dose regimen. The CpG concentration is preferably 1000 pg for the second dose in a 2 or more dose regimen. The concentration of aluminum hydroxide may be about 100 pg to 1200 pg per dose.

VACCINE CANDIDATE A vaccination shows that the response can be broadened and cover the VOC. Vaccine Candidate C - Bivalent vaccination can achieve the same goal as VACCINE CANDIDATE A vaccination. The type of vaccination may have practical considerations as VACCINE CANDIDATE A uses only one RBD while Vaccine Candidate C - Bivalent uses two RBDs (Wuhan and SA).

Figures 25-29 show preliminary testind data for the immugonecity of VACCINE CANDIDATE A as compared to UB-612.

In the challenge study, all vaccines were given at 2 doses of 100 ug 4 weeks apart, except for the single dose group which was given at 50 ug on day 0.

As can be seen from Figure 26, VACCINE CANDIDATE A demonstrates faster onset of NAb production, GMT of 266 two weeks after first dose. VACCINE CANDIDATE A also demonstrates 13-, 7- and 9-folds higher NAbs on D14, D28 (after single dose) and D50, respectively compared to UB-612.

Single half dose of VACCINE CANDIDATE A produced GMT of of 142.

As can be seen from Figure 27, VACCINE CANDIDATE A vaccine demonstrates a 2- fold titer loss against SA (Beta Variant) as compared to UB-612 vaccine which showed a 4- fold titer loss against SA (Beta Variant). The single half dose of VACCINE CANDIDATE A vaccine showed a 14-fold titer loss against SA (Beta Variant).

As can be seen from Figure 28, VACCINE CANDIDATE A produced an 11.6-fold improvement in NAb titers against SA (Beta variant) compared to UB-612 and a 6.3-fold improvement in NAb titers against WT (Wu) compared to UB-612.

Additional challenge protocols for the comparison of VACCINE CANDIDATE A and additional data can be seen in Figure 35 and in Tables 42 to 47.

Table 1

Amino Acid Sequences of Membrane Glycoprotein M from SARS-CoV-2, SARS-CoV, and MERS-CoV

Table 2

Amino Acid Sequences of Nucleocapsid Phosphoprotein N from SARS-CoV-2, SARS-CoV, and MERS-CoV

Table 3

Amino Acid Sequences of Surface Glycoprotein S from SARS-CoV-2, SARS, and MERS

* Peptides are cyclized by cysteine disulfide bonds with the cysteines underlined. The Cysteines/Serines that substitute the amino acids of the SARS-CoV-2 fragments are in italics.

Table 4

SARS-CoV-2 CTL epitopes for use in vaccine design (validated by PBMC binding and stimulation assay through previous SARS-CoV studies)

Adapted from Ahmed, S.F., et al, 2020

Table 5

SARS-CoV-2 Th epitopes for use in vaccine design (validated by PBMC binding and stimulation assay through previous SARS-CoV studies)

Adapted from Ahmed, S.F., et al, 2020

Table 6

Amino Acid Sequences of Pathogen Protein Derived Th Epitopes Including Idealized Artificial Th Epitopes for Employment in the Design of SARS-CoV-2 Peptide Immunogen Constructs

Table 7

Examples of Optional Heterologous Spacers, CpG Oligonucleotides, and RT-PCR Primers/Probes

Table 8

Amino Acid Sequences of SARS-CoV-2 Peptide Immunogen Constructs

KKK-SARS-CoV-2 M89-111 (Th/CTL epitope)

KKK-GLMWLSYFIAS FRLFARTRSMWS

' Peptides are cyclized by cysteine disulfide bonds with the cysteines underlined. The Cysteines/Serines that substitute the amino acids of the SARS-CoV-2 fragments are in italics

Table 9

Wild-Type and Mutated Hinge Regions from IgGl, IgG2, IgG3, and IgG4

X: Ser, Gly, Thr, Ala, Vai, Leu, He, Met, and/or deletion

Table 10

Examples of Amino Acid Sequences of Mutated Hinge Regions Derived from IgGl

¹ Underlined residues represent sites of mutation in relation to the sequence of wild-type IgG Table 11

Amino Acid Sequences of sFC and Fc Fusion Proteins

Table 12

Nucleic Acid Sequences of sFc and Fc Fusion Proteins

Table 13

SARS-CoV-2 antigenic peptides

* The cysteine residues were rep aced by serine that are underlined.

Table 14

Specificity assessment of U Bl SARS-CoV-2 ELISA

Performance Characteristics: Lack of cross-reactivities to other viral infections

Table 15

Specificity Assessment based on data collected from "Non-COVID-19" individuals in the US, Taiwan, and China

Table 16

Sensitivity assessment of anti-SARS-CoV-2 IgG detection with UBI SARS-CoV-2 ELISA

Performance Characteristics: Sensitivity in PCR-confirmed COVID-19 hospitalized patients:

Relative Sensitivity (<10 days post onset of symptoms) = 0/10 = 0%

Relative Sensitivity (>10 days post onset of symptoms) = 23/23 = 100%

Relative Sensitivity (day of discharge from the hospital) = 5/5 = 100%

Overall Sensitivity (All 46 samples) = 36/46 = 78.2%

Accuracy for positive predictive value (for those >10 days post onset of symptoms) = 36/36 = 100% Table 17

Study 1: Performance Characteristics: Sensitivity and Specificity based on COVID-19 samples collected 10 days after onset of symptoms

Relative Sensitivity (>10 days onset of symptoms): 100%

Overall Sensitivity including those at the onset of symptoms (from 45 different individuals): 78.2%

Relative Specificity: 100%

Accuracy for positive predictive value for patients enrolled in the hospital and 10 days post symptom onset = 36/(36+0) = 100%

Accuracy for negative predictive value = 922/(0+922) = 100%

Table 18

Study 2: Anti-SARS-CoV-2 IgG detection using UBI® SARS-CoV-2 ELISA with serum/plasma samples from COVID-19 patients in Taiwan

Table 19

Study 2: Sensitivity assessment with UBI® SARS-CoV-2 ELISA

Relative Sensitivity (<7 days post onset of symptoms) = 1/4 = 25%

Relative Sensitivity (7-14 days post onset of symptoms) = 7/11 = 63.6%

Relative Sensitivity (>14 days post onset of symptoms) = TllTl - 100%

Overall Sensitivity (All 37 samples) = 30/37 = 81.1%

Accuracy for positive predictive value (>14 days post onset of symptoms) = 22/ 22 = 100% Table 20

Positive Agreement by Days Post-Symptom Onset

Table 21

Negative Percent Agreement

Table 22

Summary results of independent evaluation

Table 23

Summary statistics of independent evaluation

Table 24

Immunization schedule of the RBD-sFc designer proteins into Guinea pigs

Table 25

Titers of Neutralizing Antibodies in Immune Sera Assessed by CPE Assay

*CPE assay conducted at Kexin Laboratory in Beijing and Sinica Lab in Taipei independently

Table 26

Size Exclusion Chromatography of S-RBD-sFc (pH from 5.7 to 7.0) at 37 °C for 24 hours

Table 27

Summary of pH and excipient selection of Sl-RBD-sFc

Table 28

Selection of Peptides comprising SARS-CoV-2 Th/CTL epitopes with known MHC I/II binding for high precision SARS-CoV-2 designer vaccine

Bold: MHC I

Underlined: MHC II

Table 29

Composition of UB-612 20 pg/mL

Materials to be used for the Phase 2 and 2/3 clinical trials will be manufactured to cGMP

Table 30

Composition of UB-612 60 pg/mL

¹ Materials to be used for the Phase 2 and 2/3 clinical trials will be manufactured to cGMP

Table 31

Composition of UB-612200 pg/mL

¹ Materials to be used for the Phase 2 and 2/3 clinical trials will be manufactured to cGMP Table 32

Equivalent to Titers of Neutralizing Antibodies in purified ACE2-ECD-sFc by CPE Assay

Table 33

Composition of VACCINE CANDIDATE A 20 pg/mL

Table 34

Composition of VACCINE CANDIDATE A 60 pg/mL

Table 35

Composition of VACCINE CANDIDATE A 200 pg/mL

¹ Materials to be used for the Phase 2 and 2/3 clinical trials will be manufactured to cGMP Table 36

Composition of VACCINE CANDIDATE B 20 pg/mL

Table 37

Composition of VACCINE CANDIDATE B 60 pg/mL

Table 38

Composition of VACCINE CANDIDATE B 200 tig/mL

¹ Materials to be used for the Phase 2 and 2/3 clinical trials will be manufactured to cGMP Table 39

Composition of VACCINE CANDIDATE C - BIVALENT 20 pg/mL

Table 40

Composition of VACCINE CANDIDATE C - BIVALENT 60 pg/mL

Table 41

Composition of VACCINE CANDIDATE C - BIVALENT 200 pg/mL

Table 42 Study for increased CpG: Immunogenicity in Rats

Table 43

Confirmatory NHP Efficacy Study using UB-612 formulation and initial adjuvant improvement in cynomolgus macaque model (UTMB)

Table 44

UB-612 NHP Immunogenicity and non-GLP Tox Study using Adjuvanted 2nd Gen UB-612 vaccine (AFRIMS)

Table 45

Experiment in Rats to determine the optimal concentration of Th/CTL peptides

Table 46

Immunogenicity study of SI NTD N3 and N5 loop peptide constructs

Tables 47 and 48

Challenge Protocols and Experimental Design - NHP Variant Neutralization: Vaccine dosage and comparison of WT and Delta viral strains

Table 49

NHP Variant Neutralization: Vaccine dosage and comparison of WT and Delta viral strains

Titer of 5000 used for graphing samples with tier .4860

Claims

(1) A COVID-19 vaccine composition comprising: a. a S-RBD-sFc protein comprising a receptor binding domain (RBD) of the S protein of SARS-CoV-2 (SEQ ID NO: 20), RDB of the S protein of SARS-CoV-2 SA, beta variant, or both ; b. a Th/CTL peptide selected from the group consisting of SEQ ID NOs: 9-16, 19, 35-36, 39- 100, 145-165, 345-348, 350, 351, 362-365, and any combination thereof; c. ALHYDROGEL (aluminum hydroxide) and a CpG oligonucleotide as adjuvants; d. optionally, one or more a pharmaceutically acceptable excipients.

(2) The COVID-19 vaccine composition according to (1), wherein the S-RBD-sFc protein comprises a receptor binding domain (RBD) of the S proteinof SARS-CoV-2 (SEQ ID NO: 20) and wherein the S-RBD-sFc protein is of SEQ ID NO: 235.

(3) The COVID-19 vaccine composition according to (1), wherein the S-RBD-sFc protein comprises a RBD of the S protein of SARS-CoV-2 SA, beta variant.

(4) The COVID-19 vaccine composition according to (1), wherein the S-RBD-sFc protein comprising both a receptor binding domain (RBD) of the S protein of SARS-CoV-2 (SEQ ID NO: 20), RDB of the S protein of SARS-CoV-2 SA, beta variant.

(5) The COVID-19 vaccine composition according to (1), wherein each of the Th/CTL peptides are present in the mixture in equal weight amounts.

(6) The COVID-19 vaccine composition according to (1), wherein the ration (w:w) of the S- RBD-sFc protein to the total weight of the mixture of Th/CTL peptides is 88:12.

(7) The COVID-19 vaccine composition according to (1), wherein the pharmaceutically acceptable excipient is an adjuvant, buffer, surfactant, emulsifier, pH adjuster, saline solution, preservative, solvent, or any combination thereof.

(8) The COVID-19 vaccine composition according to (1), wherein the pharmaceutically acceptable excipient is selected from the group consisting of a CpG oligonucleotide, ALHYDROGEL (aluminum hydroxide), histidine, histidine HCl’IEO, arginine HC1, TWEEN 80 (polyoxyethylene (20) sorbitan monooleate), hydrochloric acid, sodium chloride, 2- phenoxyethanol, water, and any combination thereof.

(9) The COVID- 19 vaccine composition according to (1), wherein the ALHYDROGEL (aluminum hydroxide) is present in an amount of 1000 pg.

(10) The COVID-19 vaccine composition according to (1), wherein the Th/CTL peptide is a mixture of SEQ ID NOs: 345, 346, 347, 348, 361, and 66, wherein each peptide is present in the mixture in equal weight amounts; the pharmaceutically acceptable excipient is a combination of a CpGl oligonucleotide, ALHYDROGEL (aluminum hydroxide), histidine, histidine HC1*H2O, arginine HC1, TWEEN 80 (polyoxyethylene (20) sorbitan monooleate), hydrochloric acid, sodium chloride, and 2-phenoxyethanol in water.

(11) The COVID-19 vaccine composition according to (10), wherein the total amount of the S-RBD-sFc protein is between about 10 pg to about 200 pg; and the total amount of the Th/CTL peptides is between about 2 pg to about 25 pg.

(12) The COVID-19 vaccine composition according to (10), wherein the total amount of the S-RBD-sFc protein is between about 17.6 pg; and the total amount of the Th/CTL peptides is between about 2.4 pg.

(13) The COVID-19 vaccine composition according to (10), wherein the total amount of the S-RBD-sFc protein o is between about 52.8 pg; and the total amount of the Th/CTL peptides is between about 7.2 pg.

(14) The COVID-19 vaccine composition according to (10), wherein the total amount of the S-RBD-sFc protein is between about 176 pg; and the total amount of the Th/CTL peptides is between about 24 pg.

(15) A method for preventing COVID-19 in a subject comprising administering a pharmaceutically effective amount of the vaccine composition according to (1) to the subject.

(16) The method according to (15), wherein the pharmaceutically effective amount of the vaccine composition is administered to the subject in two doses.

(17) The method according to (15), wherein a first dose of the vaccine composition is administered to the subject and a second dose of the vaccine composition is administered to the subject about 4 weeks after the first dose.

(18) A method for generating antibodies against SARS-CoV-2 comprising administering a pharmaceutically effective amount of the vaccine composition according to (1) to a subject.