WO2023064708A1 - Vaccine compositions against sars-cov-2 variants of concern to prevent infection and treat long-haul covid - Google Patents

Abstract

The present disclosure is directed to amino acid sequences from SARS-CoV-2 S1-RBD variants of concern (VoCs) including Omicron variants BA.4/BA.5 protein and N, M and S2 derived Th and CTL epitope peptides and an idealized pathogen derived artificial Th epitope peptide to offer effective prevention and treatment of long-haul COVID with specificities against SARS-CoV-2 VoCs including SARS-CoV-2 Omicron variants BA.4/BA.5. The disclosed vaccine compositions utilize amino acid sequences for the design and manufacture of optimal SARS-CoV-2 antigenic proteins, Th/CTL peptide immunogen constructs, CHO- derived S1- RBD VoCs-sFc proteins including CHO- derived S1-RBD Omicron variants BA.4/BA.5-sFc protein, and compositions thereof, as vaccines for prevention and treatment of long-haul COVID.

Description

VACCINE COMPOSITIONS AGAINST SARS-COV-2 VARIANTS of CONCERN TO PREVENT INFECTION AND TREAT LONG-HAUL COVID

FIELD OF THE INVENTION

The present disclosure relates to vaccines against SARS-CoV-2 variants of concern (VoCs) including SARS-CoV-2 Omicron BA.4/BA.5 variants to prevent infection and treat long- haul COVID.

BACKGROUND OF THE INVENTION

SARS is the abbreviation created in 2003 for Severe Acute Respiratory Syndrome, which is also known as COVID, the abbreviation created in 2020 for Corona Virus Infectious 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. SARS-CoV-2 is termed as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and refers to the coronavirus strain that was first identified in Wuhan, China which caused the Corona Virus Infectious Disease 2019 (COVID- 19).

The combined effects of SARS-CoV-2 neutralization escape variants, high transmissibility, spread by asymptomatic persons and breakthrough infections due to waning immunity of currently authorized vaccines continue to cost human lives and sap the world’s economy and healthcare system.

The SARS-CoV-2 Omicron lineage has swept the globe from the original Wuhan strain with a rapid succession of dominating subvariants from BA. l, BA.2 and to the current BA.4/BA.5 that makes up more than 90% of SARS infection cases with overriding edges in transmissibility and neutralizing antibody escape. Enclosed are recent publications from the inventors’ group (Wang, CY et al 2022a and b) along with literature citations contained therein for ease of background references.

While a booster 3^rd-dose of mRNA SARS vaccines could compensate Omicron (BA.l)- induced decrease in serum neutralizing antibodies (20- to 30-fold reduction) when compared to the original Wuhan strain, along with rates of hospitalization and severe disease (80-90% protection), it offers less effective protection against mild and asymptomatic infections (40-50% protection). Breakthrough infections identified with high viral loads are common even after the fourth jab (2^nd booster to adults aged 18 and older).

The current vaccines are manufactured with the original virus antigen but antigenic variants (such as Beta, Delta or Omicron) can account for rapid infection cases and is more resistant to neutralization. Individuals infected with the SARS-CoV-2 Variants of Concern (VoCs) (https://en.wikipedia.org/wiki/Variants _of_SARS-CoV-2) can carry many times more virus in their nasal passages than other variants. Amongst these VoCs, the Omicron variant (https://en.wikipedia.org/wiki/SARS-CoV-2_Omicron_varianthas ) has a total of 60 mutations compared to the original Wuhan variant with thirty-two mutations affect the spike protein, the main antigenic target of many vaccines widely administered. Fifteen of those thirty-two mutations are located in the receptor binding domain (RBD).

Omicron BA.l is heavily mutated from the original SARS-CoV-2 Wuhan strain, including more than 35 amino acid changes in Spike protein. Compared to 2 mutations associated with Delta at S-l receptor binding domain (SI -RBD, residues 319-541), BA.l and BA.2 share 12 mutations, with BA.l and BA.2 each having additional 3 and 4 unique ones, respectively, that confers BA.2 a higher immune evasion. BAA and BA.5 have identical spike protein sequence. They differ from BA.2 by having additional mutations at 69-70del, L452R, F486V and wild type amino acid at position Q493 within the spike protein, contributing to their higher degree of immune escape than BA.2. BA.2 exhibits a 1.3- to 1.5 -fold higher transmissibility and a 1.3-fold immune evasion than BA.l, consistent with the finding that BA.l- immune sera neutralizes BA.2 with lower titers by a factor of 1.3 to 1.4 and that BA.2 reinfection can occur after BA. l. BA.4/BA.5 are more transmissible and resistant to BA. l/BA.2-immunity and monoclonal antibodies.

Amongst double-vaccinated adults, the booster (third dose)-induced neutralization titers against BA.4/BA.5 are notably lower than those against BA.1/BA.2. These suggest that booster vaccination or BA.1/BA.2 infection may not achieve sufficient immunity to protect against BA.4/BA.5 while break-through infection or reinfection would be common.

There remains an urgent need for the development of vaccines to prevent non-infected individuals from contracting SARS-CoV-2 VoCs including Beta, Delta and Omicron to control the outbreak and reduce the resulting sufferings, including death. Moreover, development of composition-updated (variant-specific) vaccines has been strongly advocated. There remains an urgent need to develop vaccine compositions to prevent individuals from contracting SARS- CoV-2 Omicron to control the outbreaks and to reduce the resulting sufferings, including long- haul COVID and death.

SUMMARY OF THE INVENTION

The present disclosure is directed to a vaccine composition against SARS-CoV-2 Variants of Concern (VoCs) to prevent infection and treat those with SARS (COVID). More specifically, the vaccine compositions employ as the B cell immunogen a fusion protein produced in CHO cells comprising an S-RBDVoC at the N-terminus that is covalently linked to a modified hinge region and Fc fragment (CH2 and CH3 domains) of human IgG (Figure 1). Promiscuous site-directed SARS-CoV-2 Th/CTL epitope peptides are incorporated in the vaccine compositions to provide optimal T cell immunity to the vaccinees. In summary, The disclosed vaccine compositions utilize amino acid sequences from SARS-CoV-2 proteins of SARS-CoV-2 VoCs for the design and manufacture of SARS-CoV-2 M, N and S2 protein derived antigenic Th/CTL epitope peptides (e.g. SEQ ID NOs: 2-5, 7-12, 14-35), and CHO-derived Sl-RBD VoC-sFc fusion proteins (e.g. SEQ ID NOs:49-53), and formulations thereof, as vaccines for the prevention and treatment of COVID caused by SARS-CoV-2 VoCs.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. illustrates the design of a single chain fusion protein according to various embodiments of the present disclosure. Specifically, this figure illustrates the general structure of a fusion protein comprising an S-RBDvoC at the N-terminus that is covalently linked to a modified hinge region and Fc fragment (CH2 and CH3 domains) of human IgG. In Sl-RBD Omicron -sFc fusion protein, Sl-RBD Omicron at the N-terminus that is covalently linked to a modified hinge region (SEQ ID NO: 39) and Fc fragment (CH2 and CH3 domains) (SEQ ID NO: 46 or 47) of human IgG.

Figure 2. illustrates the general map of pZD/S-RBDVoC-sFc plasmid. The pZD/S-RBDVoC -sFc plasmid encodes the S-RBDVoC-sFc fusion proteins according to embodiments of the present invention.

Figure 3. illustrates the amino acid sequence, structure, and function of Sl-RBDVoC-sFc where the VoC is Beta. Figure 3A provides the sequence of Sl-RBDVoC Beta-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). Figure 3B summarizes the disulfide bonding in the Sl-RBDVoC Beta-sFc fusion protein.

Figure 4. illustrates the amino acid sequence, structure, and function of Sl-RBDVoC Delta-sFc where the VoC is Delta. Figure 4A provides the sequence of Sl-RBDVoC Delta -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). Figure 4B summarizes the disulfide bonding in the Sl-RBDVoC Delta-sFc fusion protein.

Figure 5. illustrates the amino acid sequence, structure, and function of Sl-RBDVoC Omicron-sFc where the VoC is Omicron (B.1.1.529). Figure 5A provides the sequence of Sl-RBDVoC Omicron BI.I.529-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). Figure 5B summarizes the disulfide bonding in the Sl-RBDVoC OmicronBi.i.529-sFc fusion protein.

Figure 6. illustrates the amino acid sequence, structure, and function of Sl-RBD Omicron BA.4/BA.5-SFC. Figure 6A provides the sequence of Sl-RBD Omicron BA.4/BA.5-SFC (SEQ ID NO: 53) 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). Figure 6B summarizes the disulfide bonding in the Sl-RBD Omicron BA.4/BA.5-SFC fusion protein.

Figure 7. illustrates the general manufacturing process of drug substances (DS) Sl-RBDVoC-sFc proteins including Sl-RBD Omicron BA.4/BA.5-SFC protein (SEQ ID NO: 53). The process starts with the Working Cell Bank (WCB) to inoculate the cell seed and expand the culture in 2000 L fed-batch bio-reactor. After the cell-culture process, the unprocessed bulk is collected and clarified by sterile filtration to produce the clarified bulk. To purify the Drug Substance (DS), the bulk is put through the processes of Protein A affinity chromatography, Depth Filtration and Ionexchange (IEX) chromatography followed by Tangential Flow Filtration (TFF) for buffer exchange to arrive at the formulated DS. To avoid the adventitious virus contamination, the clarified bulk is to put through the process with solvent detergent treatment, acid-inactivation in Protein A chromatography and nano-filtration. Finally, the formulated Sl-RBDVoC-sFc DS concentrate is produced after the sterile filtration. Because the Voc includes Omicron BA.4/BA.5, the formulated Sl-RBD Omicron BA.4/BA.5-SFC DS concentrate is also produced using the same manufacturing process mentioned above.

Figure 8. illustrates the biochemical characterization of a representative designer Sl-RBD-sFc protein of the invention by SDS-PAGE in both nonreducing and reducing forms.

Figures 9A and 9B. illustrate the components of the protein/peptide vaccine disclosed herein. Figure 9A illustrates the components of the UB-612 multitope protein-peptide subunit vaccine. The vaccine composition contains an Sl-RBDVoCs-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 VoCs M, N and S2 proteins, and the UBITh®la peptide as a catalyst for T cell activation. 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 Alum adjuvant to constitute the vaccine composition. Figure 9B illustrates the components of the UniCoVac Omicron BA.4/BA.5 subunit vaccine. The vaccine composition contains an Sl-RBD Omicron BA.4/BA.5-SFC fusion protein (SEQ ID NO: 53) as the main B cell immunogen, five synthetic Th/CTL peptides (SEQ ID NOs: 2, 9, 27, 34, and 35) for class I and II MHC molecules derived from SARS-CoV-2 Omicron BA.4/BA.5 M, N and S2 proteins, and the UBITh®la peptide (SEQ ID NO: 36) as a catalyst for T cell activation. These components are mixed with CpGl (SEQ ID NO: 67) which binds to the positively (designed) charged peptides by dipolar interactions and also serves as an adjuvant, which is then bound to Alum adjuvant to constitute the vaccine compositions.

Figures 10A and 10B. illustrate the compounding processes for the manufacturing of Designer COVID Vaccines against VoCs of SARS-CoV-2 including Omicron BA.4/BA.5 of SARS-CoV-2. Figure 10A illustrates the compounding process for the manufacturing of Designer COVID Vaccine against VoCs of SARS-CoV-2. To produce the vaccine composition, sequential addition of peptides, CpGl, alum adjuvant and finally the protein component is carried out. Specifically, the designer Th/CTL peptides are added to WFI, followed by the addition of CpGl in the mixture to form the peptides/CpGl complex. Thereafter, the protein buffer, Alum and NaCl are added to the solution which now contains peptides/CpGl/Alum/NaCl. Finally, the Sl-RBDVoCs- sFc protein solution is added to the solution mixture to arrive at the final vaccine compositions: e.g. UB-612, UB-613 or UB-614 etc. Figure 10B illustrates the compounding process for the manufacturing of Designer COVID Vaccine (or named as Monovalent UniCoVac Omicron) against Omicron BA.4/BA.5 of SARS-CoV-2. To produce the vaccine composition, sequential addition of peptides, CpGl, alum adjuvant and finally the protein component is carried out. Specifically, the designer Th/CTL peptides are added to WFI, followed by the addition of CpGl in the mixture to form the peptides/CpGl complex. Thereafter, the protein buffer, Alum and NaCl are added to the solution which now contains peptides/CpGl/Alum/NaCl. Finally, the Sl- RBD Omicron BA.4/BA.5-SFC protein solution is added to the solution mixture to arrive at the final vaccine compositions.

Figures 11A to 11D. Graphs showing viral-neutralizing titer (VNT50) against live SARS-CoV-2 wild type after the primary 2-dose vaccination and the booster third-dose in the Phase- 1 trial. In the UB-612 primary 2-dose vaccination series of the 196-day phase-1 trial, 60 participants were enrolled for thelO-pg, 30-pg, and 100-pg dose groups (n = 20 each group), of which 50 participants were enrolled for the extension study and received a booster 3^rd-dose at 100 -pg (n = 17 for the 10-pg; n = 15 for the 30-pg, and n = 18 for the 100-pg dose group). The viral - neutralizing antibody geometric mean titers (GMT, 95% CI) that inhibit 50% of live SARS-CoV- 2 wild-type (WT, Wuhan strain) were measured and expressed as VNT50 for the 10-pg (Figure 11A), 30-pg (Figure 11B), and 100-pg (Figure 11C) dose groups. Figure 11D illustrated with the 100-pg dose group, the VNT50 data were recorded on Day 0 (pre-dose 1), Day 14 (14 days post-dose 1), Day 28 (1 mon. post-dose 1; pre-dose 2), Day 42 (14 days post-dose 2), Day 56 (1 mon. post-dose 2), Day 112 (3 mon. post-dose 2), Day 196 (6 mon. post-dose 2), Days 255 to 316 (pre-dose 3, the pre-booster), and Days 269 to 330 (14 days post-booster) for study participants of the three dose groups. The titers for individual participants are shown by the circles. The horizontal dotted lines indicate the lower limit of quantification (LLOQ). HCS: human convalescent serum samples in the control group (n = 20).

Figures 12A to 12C. Graphs showing potent neutralizing titers against SARS-CoV-2 wild-type, Delta, Omicron, and other Variants of Concern produced by UB-612 booster third-dose in the Phase- 1 trial. The primary 2-dose series (Days 0 and 28) of the 196-day phase- 1 trial and the extended booster 3rd dose of 100 pg administered at mean Day 286 (Days 255-316) were conducted. Figure 12A provides the VNT50 titer observed 14 days post-booster in the participants of the 100-pg group. The VNT50 titer observed 14 days post-booster reached 3992 against live SARS-CoV-2 wild type (WT), and at 2358 against live Delta. Similar high anti-WT and ant-Delta VNTso levels were observed for the lower 30- and 10-pg dose groups. Figure 12B provides the pVNT50 titer observed 14 days post-booster against pseudo-SARS-CoV-2 wild type (WT) and against pseudo-SARS-CoV-2 variants including Omicron. Figure 12C provides the antibody persistence after 2 doses (phase 1 trial): The anti-WT neutralizing VNT50 titers decayed slowly with a half-life of 187 days, based on the first-order exponential model fitting (SigmaPlot) over Days 42-196 (R² = 0.9877; the decay rate constant K_ei = -0.0037; half-life = 0.693/Kei). This figure shows that the viral-neutralizing antibodies were long-lasting revealed with the live WT virus.

Figure 13. Bar graph showing viral-neutralizing pVNTso titers against different SARS-CoV-2 variants observed in the primary series of phase-1 trial of the 100-pg UB-612 dose group. In the primary 2-dose vaccination series of phase- 1 trial with vaccinees receiving two UB-612 doses at 100 pg, twenty samples (n = 20) of Day-56 immune sera (28 days after the second dose) were selected for measuring comparative neutralizing antibody activity against Variants of Concerns (VoCs). pVNTso titers were assessed by pseudovirus-luciferase assay (in vitro live virus microneutralization). The study was conducted in BSL2 lab at RNAi core facility in Sinica.

Figures 14A and 14B. Graphs showing Anti-Sl-RBD IgG antibody and viral-neutralizing responses against SARS-CoV-2 wild type Wuhan strain. Figure 14A provides the mean ELISA- based GMTs of anti-Sl-RBD IgG response at Days 1, 29, and 57 across age groups in the phase- 2 study of UB-612 at 100 pg (n = 871 for All; n = 731 for 18-65 years; and n = 140 for 65-85 years). The error bars represent 95%CI, and the dashed lines denote the limit of ELISA assay. Figure 14B provides the GMTs of 50% viral-neutralizing response (VNTso) against SARS-CoV- 2-TCDC#4 (Wuhan wild type) virus at Days 1 and 57 across age groups. The GMTs values were measured by the microneutralization CPE assay. The error bars represent 95%CI, and the dashed lines denote the limit of microneutralization assay. The VNTso of 96.4 in younger adults aged 18-65 years was essentially reproducible as seen at Day 56 in the phase- 1 trial in vaccinees (aged 20-55 years) on the 100-pg vaccine dose where the VNTso was estimated to be 103 (Figure

11C)

Figures 15A and 15B. Graphs showing neutralizing antibody titers (VNTso) against SARS-CoV- 2 variants in the phase-2 trial primary 2-dose series. Figure 15A provides the measurement of 50% viral-neutralization titers (VNTso) against live SARS-CoV-2 virus variants in Day-57 immune sera randomly selected from 48 vaccinees (n = 39 for young adults aged 18-65 years; n = 9 for elderly adults >65 years) who received two UB-612 vaccine doses in Phase-2 trial. Live wild-type Wuhan SARS-CoV-2-TCDC#4 and US WA 1/2020, and two VoCs (B.1.1.7 and B.1.617.2 lineages) listed by WHO, were employed for CPE assays. The VNTso values were marked on top of each column and lined with 95% confidential interval (CI) shown as horizontal bars. Figure 15B provides the fold change (reduction) of VNTso against each of the variants compared with wild types, Wuhan and US WA 1/2020 by the two-sample t-test (** p<0.01; ****pO .0001) The 2.7- and 1.4-fold reductions also stands for 37% and 72% preservation of neutralization titers relative the two Wuhan wild types isolated from two separate geographic locations where CPE assays were performed. Sinica: Academia Sinica, Taiwan; CDPH: California Department of Public Health (CDPH), CA, USA.

Figures 16A to 16D. Graphs showing the inhibition titers against S1-RBD:ACE2 binding by ELISA in the primary 2-dose vaccination and after the booster third-dose. ELISA-based neutralization (inhibition) of S1-RBD:ACE2 binding titers in the primary 2-dose vaccination series of a 196-day phase 1 trial (60 participants) and in the extension study with a booster third- dose were measured. Participants of 10-pg (Figure 16A), 30-pg (Figure 16B), and 100-pg (Figure 16C) dose groups (n = 20 each dose group) received two assigned vaccine doses, 28 days apart, and a booster third dose of 100 pg at a time over 6 months administered to 50 participants (n = 17 for the 10-pg, n = 15 for the 30-pg, and n = 18 for the 100-pg dose groups). Serum samples were collected at the indicated time points for measuring the inhibition titers against Sl-RBD binding to ACE2 by ELISA. The horizontal dotted lines indicate the lower limit of quantification (LLOQ). Figure 16D illustrates the Good correlation between S1-RBD:ACE2 binding inhibition and VNT50. Data are plotted for all prime/boost vaccinated participants (10-, 30- and 100-pg dose groups). Data points for participants at Day 0 are excluded from the correlation analysis. The correlation was analyzed by the Nonparametric Spearman correlation method.

Figures 17A to 17D. Graphs showing anti-Sl-RBD IgG binding titers on ELISA in the primary 2-dose vaccination and after the booster third-dose. ELISA-based anti-Sl-RBD antibody binding titers in the primary 2-dose vaccination series of a 196-day phase 1 trial (60 participants) and in the extension study with a booster third-dose were measured. Participants of 10-pg (Figure 17A), 30-pg (Figure 17B), and 100-pg (Figure 17C) dose groups (n = 20 in each dose group) who had received two assigned vaccine doses, 28 days apart, and a booster third dose of 100 pg at a time over 6 months administered to 50 participants (n = 17 for the 10-pg, n = 15 for the 30- pg, and n = 18 for the 100-pg dose groups). Serum samples were collected at the indicated time points for measuring anti-Sl-RBD antibody binding by ELISA, expressed as geometric mean titer GMT and 95% CI. The horizontal dotted lines indicate the lower limit of quantification (LLOQ). Figure 17D illustrates the good correlation between anti-Sl-RBD antibody binding and VNTso. Data are plotted for all prime/boost vaccinated participants (10-, 30- and 100-pg dose groups). Data points for participants at day 0 are excluded from the correlation analysis. The correlation was analyzed by the Nonparametric Spearman correlation method.

Figures 18A to 18E. Graphs showing the long-lasting, robust Th 1 -predominant cell response induced by UB-612 measured by IFN-y and IL-4 ELISpot after re-stimulation of PBMCs with designer peptide antigens. In the 196-day phase- 1 trial with two UB-612 doses on Days 0 and 28, vaccine-induced T-cell responses were measured by IFN-y ELISpot with PBMC cells from young adults (20 to 55 years) in 10-pg (Figure 18A), 30-pg (Figure 18B), or 100-pg (Figure 18C) dose group (n = 20 each). In the phase-2 trial study, participants (younger adults, >18 to <65 years) received two doses of UB-612 at 100 pg (n = 88) or saline placebo (n = 12), and T- cell responses in PBMCs of vaccinees on Day 57 re-stimulated with designer antigen protein/peptides were measured by IFN-y ELISpot (Figure 18D) and IL-4 ELISpot (Figure 18E). Shown are spot-forming units (SFU) per U I 0⁶ PBMCs producing IFN-y and IL-4 after stimulation with the Sl-RBD+Th/CTL peptide pool, Th/CTL peptide pool, or CoV2 T peptides (Th/CTL peptide pool without UBIThla). Statistical analysis was performed with the use of the two-sample t-test (**** p<0.0001).

Figures 19A-19C. Graphs showing UB-612-induced Th 1 -predominant T-cell responses (CD4 and CD8) measured by IFN-y and IL-4 Intracellular Staining (ICS) after re-stimulation of PBMCs with designer peptide antigens in Phase-2 primary 2-dose vaccination series. In a phase- 2 trial, study participants (younger adults >18 to 65 years receiving 2 doses (28 days apart) of UB-612 at 100 pg (n = 88) or saline placebo (n = 12). Their PBMCs harvested on Days 1 and 57 (4 weeks after the second shot) were re-stimulated with designer antigen protein/peptides to evaluate T cell responses by Intracellular Staining (ICS). Frequencies of CD4⁺ and CD8⁺ T cells that produce indicated cytokines in response to the stimulation of Sl-RBD+Th/CTL peptide pool (Figure 19A), Th/CTL peptide pool (Figure 19B), and CoV2 T peptides (Th/CTL peptide pool without UBIThla) (Figure 19C). Statistics analysis was performed using the Mann-Whitney t test. (* p<0.05; ** p<0.01; ***p<0.001; **** p<0.0001).

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DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is directed to vaccine compositions against SARS-CoV-2 Variants of Concern (VoC) to prevent infection and treat those with SARS (COVID), with specificities for VoCs such as Alpha, Beta, Gamma, Delta and Omicron. More specifically, the vaccine compositions employ as the B cell immunogen a fusion protein produced in CHO cells comprising an Sl-RBDVoC at the N-terminus that is covalently linked to a modifeied hinge region and Fc fragment (CH2 and CH3 domains) of human IgG. Promiscuous site-directed SARS-CoV-2 Th/CTL epitope peptides are incorporated in the vaccine compositions to provide optimal T cell immunity to the vaccinees.

In summary, the disclosed vaccine compositions utilize amino acid sequences from SARS-CoV-2 proteins of SARS-CoV-2 VoCs including SARS-CoV-2 Omicron BA.4/BA.5 for the design and manufacture of SARS-CoV-2 M, N and S2 protein derived antigenic Th/CTL epitope peptides (SEQ ID NOs: 2-5, 7-12, 14-35), and CHO-derived Sl-RBDVoC-sFc fusion proteins (SEQ ID NOs: 49-53) including Sl-RBD Omicron BA.4/BA.5-SFC fusion protein (SEQ ID NO: 53), and formulations thereof, as vaccines against COVID caused by SARS-CoV-2 VoCs including SARS-CoV-2 Omicron BA.4/BA.5. Each aspect of the disclosed invention is discussed in further details 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. 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 (COVID-ID).

SARS is the abbreviation of Severe Acute Respiratory Syndrome, which is also known as COVID, the abbrieviation of Corona Virus Infectious 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. SARS-CoV-2 is termed as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and refers to the coronavirus strain that was first identified in Wuhan, China which caused the coronavirus disease 2019 (COVID-19). PROTEIN/PEPTIDE VACCINE COMPOSITIONS FOR THE PREVENTION OF INFECTION BY SARS-COV-2 VARIANTS OF CONCERN (VOCs) INCLUDING SARS- COV-2 OMICRON VARIANTS BA.4/BA.5

The disclosed vaccine compositions relate to protein/peptide vaccine compositions for the prevention of infection by SARS-CoV-2 VoCs including SARS-CoV-2 Omicron Variants BA.4/BA.5.

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 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 described in 2003, providing a margin of safety not achievable with a full-length S antigen by eliminating the potential side effect such as the antibody dependent enhancing (ADE) effect, when the antibodies generated by the vaccine actually help the virus infect greater numbers of cells than it would have on its own. In this situation, the antibodies bind to the virus and help it more easily get into cells than it would on its own resulting in more severe illness than if the person had been unvaccinated.

Due to the clear advantages of the employment of a shorter receptor binding domain (Sl- RBD) focused B-cell vaccine component, the protein/peptide vaccine compositions comprise the SI -receptor-binding region-based designer protein, also termed as Sl-RBD -sFc fusion protein, with specific variant specificity such as Beta, Delta, Omicron (such as BA.4/BA.5) or bivalent Sl-RBD of Wuhan and Beta. 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. Moreover, engineered Fc has been used in many therapeutic antibodies as a solution to minimize non-specific binding, increase solubility, yield, thermostability, and in vivo half-life

In some embodiments, the vaccine composition contains Sl-RBD-sFc fusion protein of SEQ ID NOs: 49-53. These Sl-RBD-sFc proteins each contains the respective Sl-RBD protein (SEQ ID NOs: 40-44), 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 NOs: 46-48) through a mutated hinge region from IgG (SEQ ID NO: 38 or 39). In some embodiments, as shown in Figures 3-6 the cysteine (C) residues at positions 61 and 195 of the Sl-RBD sequence of SEQ ID NOs: 50- 53 are mutated to alanine (A) residues, (residues 61 and 195 of S-RBD correspond to residues 391 and 525 of the full-length S protein of the original Wuhan strain SEQ ID NO: 13). The C61A and C195A mutations in the Sl-RBD sequence are introduced to avoid a mismatch of disulfide bond formation in the recombinant protein expression. The amino acid sequence of the Sl-RBDVoCs, representing WuHan, Beta, Omicron (e.g. Omicron BA.4/BA.5), Delta strains, fused to the single chain Fc peptide (S- RBDVoCs-sFc) are SEQ ID NOs: 49- 53, wherein S-RBD Omicron BA.4/BA.5-SFC is SEQ ID NO: 53.

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 1000 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 were identified. These Th/CTL peptides are shown in Tables 1-3, 8 and 10. Each selected peptide contains Th or CTL epitopes with prior validation of MHC I or II binding and exhibits good manufacturability characteristics (optimal length amenable for high quality synthesis). They should also demonstrate preferably the intrinsic ability to stimulate PBMCs from regular individuals. These Th/CTL peptides, after extensive screening, identification, validation and designs, 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 charges for use in vaccine formulation. The designs and sequences of the five final peptides and their respective HLA alleles are shown in Table 8.

To enhance the immune response, a proprietary peptide UBITh®la (SEQ ID NO: 36) can be added to the peptide mixture of the vaccine composition as a catalyst. 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, activation of TLR-9 signaling by CpG is known to promote IgA production and favor Thl immune response. UBITh®l 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®l is SEQ ID NO: 36. The nucleic acid sequence of CpGl is SEQ ID NO: 67.

In view of the above, the protein/peptide vaccine compositions can contain one or more Th/CTL peptides. The Th/CTL peptides can include: a. peptides derived from the SARS-CoV-2 M protein (e.g., SEQ ID NOs: 2-5); b. peptides derived from the SARS-CoV-2 N protein (e.g., SEQ ID NOs: 7-12); c. peptides derived from the SARS-Cov-2 S protein (e.g., SEQ ID NOs: 14-35); and/or d. an artificial Th epitope derived from pathogen proteins (e.g., SEQ ID NO: 36)

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 comprises SEQ ID NOs: 22, 27, 9, 34, 2, 35, 23, 36 or any combination thereof. 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.

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 endogenous SARS-CoV-2 Th/CTL epitope peptides in a pharmaceutical composition, the S-RBDVoC-sFc protein including Sl-RBD Omicron BA.4/BA.5 - sFc 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. The endogenous SARS-CoV-2 CTL epitope peptides contain a Lys-Lys-Lys (KKK) tail at the N-terminus.

The endogenous SARS-CoV Th/CTL epitope peptides (e.g. SEQ ID NOs: 2, 9, 22/23, 27, 34, 35, 36) 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 VoC-sFc protein B cell epitope peptides including Sl-RBD Omicron BA.4/BA.5 B cell epitope peptide to facilitate the production of specific high titer antibodies, upon infection, directed against the optimized Sl-RBD B cell epitope peptide screened and selected based on design rationales.

In some embodiments, the pharmaceutical composition contains one or more S-RBDVoC- sFc proteins including Sl-RBD Omicron BA.4/BA.5 sFc fusion protein (SEQ ID NOs: 49, 53 or any combination thereof) together with one or more separate peptides containing an endogenous SARS-CoV-2 Th/CTL epitope peptide (e.g. SEQ ID NOs:2, 9, 22, 23, 27, 34, 35 and 36, or any combination thereof).

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 SI -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, fillers, 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 Sl- 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 (e.g. potassium aluminum phosphate), aluminum phosphate (e.g. ADJU- PHOS®), aluminum hydroxide (e.g. ALHYDROGEL®), calcium phosphate, incomplete Freund’s adjuvant (IF A), 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 ADJU-PHOS® (aluminum phosphate), 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 ADJU-PHOS® (aluminum phosphate) as the adjuvant to improve the immune response. Aluminum phosphate serves as a Th2 oriented adjuvant via the nucleotide binding oligomerization domain (NOD) like receptor protein 3 (NLRP3) inflammasome pathway. Additionally, it has pro-phagocytic and repository effects with a long record of safety and the ability to improve immune responses to target proteins in many vaccine formulations.

The vaccine composition can contain pH adjusters and/or buffering agents, such as hydrochloric acid, phosphoric acid, citric acid, acetic acid, histidine, histidine HCPH2O, 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 Polyoxyethylene sorbitan fatty acid esters (Polysorbate, TWEEN®), Polyoxyethylene 15 hydroxy stearate (Macrogol 15 hydroxy stearate, SOLUTOL HS15®), Polyoxyethylene castor oil derivatives (CREMOPHOR® EL, ELP, RH 40), Polyoxyethylene stearates (MYRJ®), Sorbitan fatty acid esters (SPAN®), Polyoxyethylene alkyl ethers (BRIJ®), and Polyoxyethylene nonylphenol ether (NONOXYNOL®).

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 co-administration 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.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 subject 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 an SI -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 an SI -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 an SI -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 an SI -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 protein to Th/CTL peptide(s) can be 70:30, 80:20, or 90:10. In specific embodiments, the ratio (w:w) of the designer peptide to Th/CTL peptide(s) is 90: 10, 88: 12, or 85: 15 etc. In specific embodiments, the ratio (w:w) of the designer protein to Th/CTL peptide(s) is 88: 12.

In some embodiments, the vaccine composition comprises the SI -receptor-binding region-based designer protein from one of SEQ ID NOs:49 or 51 in combination with Th/CTL peptides of SEQ ID NOs: 2, 9, 22, 27, 34, 35, 36. In certain embodiments, the vaccine composition comprises one of the SI -receptor-binding region-based designer protein of SEQ ID NOs: 49 or 51, the Th/CTL peptides of SEQ ID NOs: 2, 9, 22, 27, 34, 35, 36,. together with one or more adjuvant and/or excipient. In various embodiments, the vaccine composition comprises one or more of the SI -receptor-binding region-based designer proteins of SEQ ID NOs:49 or 51 together with the Th/CTL peptides of SEQ ID NOs: 2, 9, 22, 27, 34, 35, 36, where the Th/CTL peptides are present in an equal-weight ratio to each other and the ratio (w:w) of one or more of the SI -receptor-binding region-based designer proteins of SEQ ID NOs:49 or 51 to the combined weight of the Th/CTL peptides is 88: 12. Specific embodiments of the vaccine composition containing 20 pg/mL, 40 ug/mL, 60 pg/mL, and 200 pg/mL, based on the total weight of one of the Sl-RBD VoC-sFc proteins (SEQ ID NOs:49 or 51) together with the Th/CTL peptides of SEQ ID NOs: 2, 9, 22, 27, 34, 35, 36, are provided in Tables 15-17, respectively. Specific embodiments of the vaccine composition containing 20 pg/mL, 40 ug/mL, 60 pg/mL, and 200 pg/mL, based on the total weight of one of the Sl-RBD-sFc(Wuhan) proteins (SEQ ID NO: 49) and Sl-RBD Omicron B.1.1.529) (SEQ ID NO: 51) together with the Th/CTL peptides of SEQ ID NOs: 2, 9, 22, 27, 34, 35, 36, are provided in Table 18. 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 from one of SEQ ID NO: 53 in combination with Th/CTL peptides of SEQ ID NOs: 2, 9, 27, 34, 35, 36. In certain embodiments, the vaccine composition comprises one of the SI -receptor-binding region-based designer protein of SEQ ID NO: 53, the Th/CTL peptides of SEQ ID NOs: 2, 9, 27, 34, 35, 36, together with one or more adjuvant and/or excipient. In various embodiments, the vaccine composition comprises one or more of the SI -receptor-binding region-based designer proteins of SEQ ID NO: 53 together with the Th/CTL peptides of SEQ ID NOs: 2, 9, 27, 34, 35, 36, where the Th/CTL peptides are present in an equal-weight ratio to each other and the ratio (w:w) of one or more of the SI -receptor-binding region-based designer proteins of SEQ ID NO: 53 to the combined weight of the Th/CTL peptides is 88: 12. Specific embodiments of the vaccine composition containing 20 pg/mL, 40 ug/mL, 60 pg/mL, and 200 pg/mL, based on the total weight of one of the Sl-RBD-sFc(Wuhan) proteins (SEQ ID NO: 49) together with the Th/CTL peptides of SEQ ID NOs: 2, 9, 22, 27, 34, 35, 36, are provided in Table 15. Specific embodiments of the vaccine composition containing 20 pg/mL, 40 ug/mL, 60 pg/mL, and 200 pg/mL, based on the total weight of one of the Sl-RBD Omicron BA.4/BA.5-SFC proteins (SEQ ID NO: 53) together with the Th/CTL peptides of SEQ ID NOs: 2, 9, 27, 34, 35, 36 are provided in Table 19. Specific embodiments of the vaccine composition containing 20 pg/mL, 40 ug/mL, 60 pg/mL, and 200 pg/mL, based on the total weight of one of the Sl-RBD-sFc(Wuhan) proteins (SEQ ID NO: 49) and Sl-RBD Omicron BA.4/BA.5-SFC proteins (SEQ ID NO: 53) together with the Th/CTL peptides of SEQ ID NOs: 2, 9, 27, 34, 35, 36, are provided in Table 20.

5. Methods

The present disclosure is also directed to methods for making and using the vaccine compositions and formulations thereof. a. Methods for Manufacturing the Si-Receptor-Binding Region-Based Designer Protein and Th/CTL Peptides.

The disclosed SI -receptor-binding region-based designer protein can be manufactured according to the methods described according to Examples 2 and 3. In addition, the disclosed Th/CTL peptides can be manufactured according to the methods described in Example 1. b. Methods for making the vaccine compositions.

The disclosed vaccine compositions can be manufactured according to the Methods described in EXAMPLES 5 and 6 for their compounding processes. c. Methods for Using the Vaccine Composition

In prophylactic applications, the disclosed protein/peptide vaccine compositions can be administered to a subject susceptible to, or at risk of, becoming infected with SARS-CoV-2 and its variants of concern, the virus that causes COVID 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 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 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 co- administration 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.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 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 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 protein to Th/CTL peptide(s) can be 70:30, 80:20, or 90: 10. In specific embodiments, the ratio (w:w) of the designer protein to Th/CTL peptide(s) is 95:5, 90:10, 88: 12 or 85: 15 etc. In specific embodiments, the ratio (w:w) of the designer protein to Th/CTL peptide(s) is 88: 12. In specific embodiments, the vaccine composition contains the components shown in Tables 15-20.

The vaccine composition can be administered in a single dose, in multiple doses over a period of time. The vaccine composition can be administered according to a specific dosage schedule. 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 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 3 months, 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.

SPECIFIC EMBODIMENTS OF THE PRESENT INVENTION INCLUDE, BUT NOT LIMITED TO, THE FOLLOWING EXAMPLES

1. The fusion protein comprising the Fc fragment of the IgG molecule and the bioactive molecule, wherein the Fc fragment is the single chain Fc (sFc), wherein the Fc fragment comprises the hinge region, wherein the hinge region is mutated and does not form disulfide bonds, wherein the hinge region comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 38 and 39, wherein the bioactive molecule is the receptor binding domain (RBD) of the S protein (S-RBD) from SARS- CoV-2 of SEQ ID NO: 40 or the variant form of S-RBD of SEQ ID NOs:41-44.

2. The fusion protein according to (1), wherein the hinge region comprises the amino acid sequence of SEQ ID NO: 39.

3. The fusion protein according to (1), wherein the fusion protein is selected from the group consisting of SEQ ID NOs: 49-53.

4. The pharmaceutical composition comprising the fusion protein according to (1) and the pharmaceutically acceptable carrier or excipient.

5. The method for producing a fusion protein according to (1) comprising: a) providing the bioactive molecule, wherein the bioactive molecule is the receptor binding domain (RBD) of the S protein (S-RBD) from SARS-CoV-2 Wuhan or one of its Variants of Concern, wherein the receptor binding domain (RBD) of the S protein (S- RBD) is selected from the group consisting of SEQ ID NOs: 40-44, b) providing the Fc fragment of an IgG molecule, wherein the Fc fragment comprises the hinge region, wherein the hinge region is mutated by substitution and/or deletion of the cysteine residue to form the mutated Fc, and the mutated Fc does not form disulfide bonds, wherein the hinge region comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 38 and 39, and c) combining the bioactive molecule and the mutated Fc through the hinge region.

6. The fusion protein selected from the group consisting of Sl-RBDVoC-sFc of SEQ ID

NOs: 49-53.

7. The composition comprising the fusion protein according to (6).

8. The composition according to (7), further comprising a Th/CTL peptide, wherein the

Th/CTL peptide is derived from the SARS-CoV-2 M, N, or S protein, the pathogen protein, or any combination thereof, wherein the Th/CTL peptide is selected from the group consisting of SEQ ID NOs: 2-5, 7-12, 14-35,36 and any combination thereof.

9. The composition according to (8), wherein the Th/CTL peptides is selected from the group consisting of SEQ ID NOs: 2, 9, 22, 23, 27, 34, 35, 36 and any combination thereof.

10. The COVID vaccine composition comprising: a), the S-RBDVoC-sFc protein selected from the group of SEQ ID NOs:49-53; b).the Th/CTL peptide selected from the group consisting of SEQ ID NOs:2-5, 7-12, 14-36 and any combination thereof; c). the pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient is the adjuvant, buffer, surfactant, emulsifier, pH adjuster, saline solution, preservative, solvent, or any combination thereof.

11. The COVID vaccine composition according to (10), wherein the Th/CTL peptides in (b) is selected from the group consisting of SEQ ID NOs: 2, 9, 22, 23, 27, ,34, 35,36 and any combination thereof.

12. The COVID vaccine composition according to (11), wherein the pharmaceutically acceptable excipient is the combination of the CpGl oligonucleotide, ALUM(aluminum phosphate or aluminum hydroxide), histidine, histidine HCEH2O, arginine HC1, TWEEN 80 (polyoxyethylene (20) sorbitan monooleate), hydrochloric acid, sodium chloride, and 2-phenoxyethanol in water.

13. The COVID vaccine composition according to (12), wherein the pharmaceutically acceptable excipient is CpGl (SEQ ID NO: 67).

14. The method for preventing COVID in a subject comprising administering the pharmaceutically effective amount of the vaccine composition according to (10) to the subject.

15. The method for preventing COVID in the subject comprising administering the pharmaceutically effective amount of the vaccine composition according to (11) to the subject.

16. The method for generating antibodies against SARS-CoV-2 comprising administering the pharmaceutically effective amount of the vaccine composition according to (10) to the subject.

17. The method for generating antibodies against SARS-CoV-2 comprising administering the pharmaceutically effective amount of the vaccine composition according to (11) to the subject.

18. The COVID vaccine composition compositing the components in the amounts shown in any one of Tables 15-20.

19. The cell line transfected with a cDNA sequence encoding the fusion protein according to

(6).

20. The cell line according to (19) that is Chinese Hamster Ovary (CHO) cell line.

21. The cell line according to (19), wherein the cDNA sequence is selected from the group consisting of SEQ ID NOs: 60-64.

22. The Global CO VID T vaccine composition comprising: a), the Th/CTL peptide, wherein the Th/CTL peptide is derived from the SARS-CoV-2 M,

N, or S protein, a pathogen protein, or any combination thereof, wherein the Th/CTL peptide is selected from the group consisting of SEQ ID NOs:2-5, 7-12, 14-36 and any combination thereof; b). the pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient is the adjuvant, buffer, pH adjuster, saline solution, preservative, solvent, or any combination thereof.

23. The Global CO VID T vaccine composition according to (22), wherein the Th/CTL peptide is selected from the group consisting of SEQ ID NOs: 2, 9, 22, 23, 27, 34, 35, 36, 3, 4, 7, 8, 25, 26 and any combination thereof.

24. The Global COVID T vaccine composition according to (23), wherein the pharmaceutically acceptable excipient is the combination of a CpGl oligonucleotide, ALUM (aluminum phosphate or aluminum hydroxide), hydrochloric acid, sodium chloride, and 2-phenoxyethanol in water.

25. The Global CO VID T vaccine composition compositing the components in the amounts shown in Tables 21-24.

26. The fusion protein comprising the Fc fragment of an IgG molecule and the bioactive molecule, wherein the Fc fragment is the single chain Fc (sFc), wherein the Fc fragment comprises the hinge region, wherein the hinge region is mutated and does not form disulfide bonds, wherein the hinge region comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 38 and 39, wherein the bioactive molecule is the receptor binding domain (RBD)(SEQ ID NOs: 40 or 44) of the S protein (Sl-RBD) from SARS- CoV-2 wherein the Wuhan strain is of SEQ ID NO: 40, wherein the Omicron BA.4/BA.5 variant form is of SEQ ID NO: 44.

27. The fusion protein according to (1), wherein the fusion protein is selected from the group consisting of SEQ ID NOs: 49 and 53.

28. The fusion protein according to (1), wherein the hinge region comprises the amino acid sequence of SEQ ID NO: 39.

29. The pharmaceutical composition comprising the fusion protein according to (1) and the pharmaceutically acceptable carrier or excipient.

30. The method for producing a fusion protein according to (1) comprising: a) providing the bioactive molecule, wherein the bioactive molecule is the receptor binding domain (RBD) of the S protein (S-RBD) from SARS-CoV-2 Wuhan (SEQ ID NO: 40) or one of its Omicron BA.4/BA.5 variant, wherein the receptor binding domain (RBD) of the S protein (S-RBD) is of SEQ ID NO: 44, b) providing the Fc fragment of an IgG molecule, wherein the Fc fragment comprises the hinge region, wherein the hinge region is mutated by substitution and/or deletion of the cysteine residue to form the mutated Fc, and the mutated Fc does not form disulfide bonds, wherein the hinge region comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 38 and 39, and c) combining the bioactive molecule and the mutated Fc through the hinge region.

31. The fusion protein selected from the group consisting of Sl-RBD Omicron BA.4/BA.5 variant -sFc of SEQ ID NO: 53. 32. The composition comprising the fusion protein according to (31).

33. The composition according to (32), further comprising the Th/CTL peptide, wherein the Th/CTL peptide is derived from the SARS-CoV-2 M, N, or S protein, the pathogen protein, or any combination thereof, wherein the Th/CTL peptide is selected from the group consisting of SEQ ID NOs: 2, 9, 22, 23, 27, ,34, 35,36 and any combination thereof.

34. The composition according to (33), wherein the Th/CTL peptides is selected from the group consisting of SEQ ID NOs: 2, 9, 22, 23, 27, ,34, 35,36 and any combination thereof.

35. The COVID vaccine composition comprising: a), the S-RBD Omicron BA.4/BA.5 variant protein selected from the group of SEQ ID NO:

53; b). the Th/CTL peptide selected from the group consisting of SEQ ID NOs: 2, 9, 22, 23,

27, ,34, 35,36 and any combination thereof; c). the pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient is an adjuvant, buffer, surfactant, emulsifier, pH adjuster, saline solution, preservative, solvent, or any combination thereof.

36. The COVID vaccine composition according to (35), wherein the Th/CTL peptides in (b) is selected from the group consisting of SEQ ID NOs: 2, 9, 22, 23, 27, 34, 35,36 and any combination thereof.

37. The COVID vaccine composition according to (36), wherein the pharmaceutically acceptable excipient is the combination of a CpGl oligonucleotide, ALUM(aluminum phosphate or aluminum hydroxide), histidine, histidine HC1»H2O, arginine HC1, TWEEN 80 (polyoxyethylene (20) sorbitan monooleate), hydrochloric acid, sodium chloride, and 2- phenoxyethanol in water.

38. The CO VID vaccine composition according to (37), wherein the pharmaceutically acceptable excipient is CpGl (SEQ ID NO: 67).

39. The method for preventing CO VID in the subject comprising administering the pharmaceutically effective amount of the vaccine composition according to (35) to the subject.

40. The method for preventing COVID in the subject comprising administering the pharmaceutically effective amount of the vaccine composition according to (36) to the subject.

41. The method for generating antibodies against SARS-CoV-2 Omicron BA.4/BA.5 variant comprising administering the pharmaceutically effective amount of the vaccine composition according to (35) to the subject.

42. The method for generating antibodies against SARS-CoV-2 Omicron BA.4/BA.5 variant comprising administering the pharmaceutically effective amount of the vaccine composition according to (36) to the subject.

43. The COVID vaccine composition comprising the components in the amounts shown in any one of Tables 15, 19 and 20.

44. The cell line transfected with a cDNA sequence encoding the fusion protein according to (31).

45. The cell line according to (44) that is Chinese Hamster Ovary (CHO) cell line.

46. The cell line according to (44), wherein the cDNA sequence is selected from the group consisting of SEQ ID NO: 64.

EXAMPLE 1

Synthesis of SARS-CoV-2 related peptides

SARS-CoV-2 related Th and CTL peptides as immunogens for vaccine development can be synthesized in small-scale amounts that are useful for serological assays, laboratory pilot and field studies, as well as in large-scale (kilogram) amounts for use in commercial production of pharmaceutical compositions. A large repertoire of SARS-CoV-2 related Th/CTL epitope peptides having sequences with lengths from approximately 9 to 40 amino acids were designed and selected as peptide immunogen constructs for use in vaccine formulations.

Tables 1-3, 8 and 10 provide the sequences of Th/CTL peptides derived from SARS- CoV-2 M, N, and S proteins with known MHC binding activities as designer peptides (e.g. with KKK as a linker at the N-terminus to increase its positive charges for better formulation) for inclusion in the final SARS-CoV-2 vaccine formulations. An idealized artificial Th epitope peptide (SEQ ID NO: 36) is also used as a catalyst for T cell activation in vaccine compositions.

All peptides that can be used for immunogenicity studies or related serological tests were synthesized on a small-scale using F-moc chemistry by peptide synthesizers of Applied BioSystems Models 430A, 431 and/or 433. Each peptide was 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 were cleaved from the solid support with side chain protecting groups removed with 90% Trifluoroacetic acid (TFA). Synthetic peptide preparations were evaluated by Matrix-Assisted Laser Desorption/Ionization-Time-Of-Flight (MALDI-TOF) Mass Spectrometry to ensure correct molecular weights and amino acid content. Each synthetic peptide was 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 of the coupling efficiency, peptide analogues were also produced due to unintended events during elongation cycles, including amino acid insertion, deletion, substitution, and premature termination. Thus, synthesized preparations typically included multiple peptide analogues, though in minute amounts, along with the targeted peptide.

Despite the inclusion of such unintended peptide analogues, the resulting synthesized peptide preparations were nonetheless suitable for use in immunological applications and as peptide immunogens. Typically, such peptide analogues were frequently as effective as the purified peptide, as long as a discerning QC procedure is developed to monitor both the manufacturing and the evaluation processes to assure the reproducibility and efficacy of the final product employing these peptides. Large scale peptide syntheses in the multi-hundred to kilo gram quantities were conducted on a customized automated peptide synthesizer UB 12003 at 15 mmole to 150 mmole scale.

For active ingredients used in the final pharmaceutical composition for clinical trials or commercial use, peptide immunogen constructs were purified by preparative RP-HPLC under a shallow elution gradient and characterized by MALDI-TOF mass spectrometry, amino acid analysis and RP-HPLC for purity and identity.

EXAMPLE 2

Design, plasmid construction, and expression of S-RBD fusion proteins in CHO cells

1. Design of the cDNA sequence

The cDNA sequences encodes the SARS-CoV-2-RBD wuhan SEQ ID NO: 54), SARS- CoV-2-RBD VoC Beta (SEQ ID NO: 55), SARS-RBD VoC Omicron (SEQ ID NO: 56), SARS-RBD- VoC Delta (SEQ ID NO: 57), and SARS-CoV-2-RBD Omicron BA.4/BA.5 (SEQ ID NO: 58), are optimized for CHO cell expression. To produce the S-RBD Wuhan-sFc (DNA SEQ ID NO: 60), SARS-CoV-2-RBD VoC Beta-sFc (DNA SEQ ID NO: 61), SARS-RBD VoC Omicron-sFc (DNA SEQ ID NO: 62), SARS-RBD-VoC Delta-sFc (DNA SEQ ID NO: 63), and SARS-CoV-2-RBD Omicron variant BA.4/BA.5-sFc (DNA SEQ ID NO: 64) fusion proteins, the nucleic acid sequences encoding S-RBDWuhan of SARS-CoV-2 (DNA SEQ ID NO: 54), four VoCs (aa331-530) of SARS-CoV-2 (DNA SEQ ID NO: 55-58) including Omicron BA.4/BA.5 (aa331-530) of SARS- CoV-2 (DNA SEQ ID NO: 58) are respectively fused to the N-terminus of the single chain of the immunoglobulin Fc (DNA SEQ ID NO: 59), with the plasmid map shown in Figure 2. The corresponding plasmids carrying the gene of respective S-RBD VoC sFc proteins including S- RBD Omicron BA.4/BA.5- sFc protein would transfect into CHO cell system and produce the respective S-RBD-sFc fusion protein.

Since no disulfide bonds form in the hinge region, the large protein fused with sFc would not constrain the expression of corresponding S-RBD proteins. The structure of single chain Fc also has the advantage of being purified through “protein A binding and elution” purification process.

2. Plasmid construction and protein expression a. Plasmid construction

To express the S-RBD-sFc fusion proteins, the respective cDNA sequences encoding these target proteins were each produced in an appropriate cell line. The N-terminus of the cDNA fragment was added a leader signal sequence for protein secretion, and the C-terminus can be linked to single-chain Fc (sFc). The cDNA fragments were inserted into the pND expression vector, which contained a neomycin-resistance gene for selection and a dhfr gene for gene amplification. The vector and the cDNA fragments were digested with PacI/EcoRV restriction enzymes, and then ligated to yield the expression vectors each for its corresponding pS-RBD VoC-sFc including pS-RBD Omicron BA.4/BA.5-SFC. b. Host cell line

CHO-S™ cell line (Gibco, Al 134601) is a stable aneuploid cell line established from the ovary of an adult Chinese hamster. The host cell line CHO-S™ is 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 anti-clumping 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 EXPIFECT AMINE™ CHO Kit (Gibco, Cat. A29129). On day 1 posttransfection, EXPIFEC FAMINE™ CHO Enhancer and first feed is added, and the cells are transferred from a 37°C incubator with a humidified atmosphere of 8% CO2 to a 32°C incubator with a humidified atmosphere of 5% CO2. Then, the second feed is added on day 5 post-transfection, 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 incubated 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 (Yeastern Biotech Co., Ltd Cat.YC203). Finally, the cDNA sequence is confirmed by DNA sequencing. g. Stability of the expressing cellsl 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 3

Purification and biochemical characterization of sFc fusion proteins

1. Purification of sFc Fusion proteins

All sFc fusion proteins were 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 was adjusted to 3.5. The protein solution was then neutralized to pH 6.0 by the addition of 1 M Tris base buffer, pH 10.8. The purity of the fusion protein was determined by polyacrylamide gel electrophoresis. The protein concentration was measured according to the UV absorbance at a wavelength of 280 nm.

2. Biochemical characterization of Sl-RBDVoCs-sFc fusion proteins including Sl-RBD Omicron variants BA.4/BA.5-SFC fusion protein as immunogens for the prevention and treatment of COVID

Sl-RBDVoCs-sFc proteins including S-RBD Omicron BA.4/BA.5- sFc protein (SEQ ID NOs: 49-53) were prepared and purified according to the methods described in Example 2 above for use as representative immunogen fusion proteins in a high precision designer vaccine formulation for immunogenicity assessment.

After purification of the sFc fusion proteins, the purity of the proteins was determined by SDS- PAGE using Coomassie blue staining under non-reducing and reducing conditions). Figure 8 is an image showing a highly purified preparation of the Sl-RBD-sFc protein under non-reducing conditions (lane 2) and reducing conditions (lane 3).

The purified proteins were further characterized by mass spectrometry analysis and glycosylation analysis. a. S-RBD-sFc - LC Mass Analysis and Glycosylation Analysis i. Glycosylation

Glycoproteins can have two types of glycosylation linkages: N-linked glycosylation and O-linked glycosylation. N-linked glycosylation usually occurs on an asparagine (Asn) residue within a sequence: Asn-Xaa-Ser/Thr, where Xaa is any amino acid residue except Pro, and the carbohydrate moiety attaches on the protein through the NH2 on the side chain of asparagine. O-linked glycosylation makes use of side chain OH group of a serine or threonine residue.

Glycosylation sites of Sl-RBD-sFc were investigated by trypsin digestion followed by LC-MS and MS/MS which shows that Sl-RBD-sFc has one N-linked glycosylation site on the arginine residue at amino acid position 13 (N13) and O-glycosylation sites on the serine residues at amino acid positions 211 (S211) and 224 (S224). ii. N-glycosylation

The N-linked glycan structure of Sl-RBD-sFc was analyzed by mass spectrometry (MS) spectra technology. In brief, PNGase F was used to release N-oligosaccharides from the purified protein. Then the portions of N-linked glycans were further labeled with 2- aminobenzamide (2-AB) to enhance the glycan signals in the mass spectrometry. iii. O-glycosylation

The O-linked glycans of Sl-RBD-sFc were investigated by trypsin digestion followed by mass spectrometry spectra technology. After trypsin digestion, the peaks containing O- linked glycans were collected and their molecular weights were determined by mass spectrometry. iv. LC Mass Spectrometry Analysis The purified Sl-RBD-sFc protein was characterized by LC mass spectrometry analysis. The theoretical molecular weight of the Sl-RBD-sFc protein based on its amino acid sequence is 48,347.04 Da. The mass spectrometry profile of the Sl-RBD-sFc protein, with a major peak at 49,984.51 Da. The difference between the theoretical molecular weight and the weight observed by LC mass spectrometry is 1,637.47 Da, which suggests that the purified Sl-RBD-sFc protein contains N- and/or O- glycans.

Figure 7. illustrates the general manufacturing process of drug substance (DS) Sl- RBDVoC-sFc. The process starts with the Working Cell Bank (WCB) to inoculate the cell seed and expand the culture in 2000 L fed-batch bio-reactor. After the cell-culture process, the unprocessed bulk is collected and clarified by sterile filtration to produce the clarified bulk. To purify the Drug Substance (DS), the bulk goes through the processes of Protein A affinity chromatography, Depth Filtration and Ion-exchange (IEX) chromatography, followed by Tangential Flow Filtration (TFF) for the buffer exchange to arrive at the formulated DS. To avoid the adventitious virus contamination, the clarified bulk also goes through the process with solvent detergent treatment, acid-inactivation in Protein A chromatography and nano-filtration. Finally, the formulated Sl-RBDVoCs-sFc DS concentrate including S-RBD Omicron BA.4/BA.5- sFc DS concentrates is produced after the sterile filtration.

EXAMPLE 4

Compounding process for the manufacturing of designer protein/peptide vaccines against SARS-CoV-2

Figure 10A and 10B. illustrate the compounding process for the manufacturing of Designer Multitope COVID Vaccine against VoCs of SARS-CoV-2 including Omicron BA.4/BA.5. To produce the vaccine product, the process is sequential addition of the peptides, CpGl, Alum adjuvant and the protein components in the solution. At first, the designer Th/CTL peptides are added to WFI and then followed by addition of CpGl in the peptide solution to form the peptides/CpGl complexes. Thereafter, the protein buffer, Alum and NaCl are added to the solution containing complexes of peptides/CpGl/Alum/NaCl. Finally, the Sl-RBDVoCs-sFc protein solution or Sl-RBD Omicron BA.4/BA.5-SFC protein solution is added, mixed well and adjusted for protein concentration, pH and other buffer conditions to arrive at the final Vaccine Product.

EXAMPLE 5

Designer Protein/Peptide COVID Vaccine product and Placebo

Designer COVID vaccine used in the preclinical, phase 1, 2 and 3 clincial trials or extension booster vaccination are designed to activate both humoral and cellular responses. For SARS-CoV-2 immunogens, COVID vaccine product combines a CHO-expressed Sl-RBD-sFc fusion protein (Wuhan strain or Omicron BA.4/BA.5 variant) and a mixture of synthetic T helper (Th) and cytotoxic T lymphocyte (CTL) epitope peptides, which are selected from immunodominant M, S2 and N regions known to bind to human major histocompatibility complexes (MHC) I and II. The preparation of the vaccine product consists of compounding, filtration, mixing, and filling operations. Before addition of the subunit protein Sl-RBD-sFc, the individual components of the vaccine are filtered through a 0.22 micron membrane filter, including the peptide solution (2 pg/mL), CpGl, a proprietary oligonucleotide (ODN), solution (2 pg/mL), 10X protein buffer containing 40 mM Histidine, 500 mM Arginine and 0.6% Tween 80, 20% sodium chloride stock solution. After sequentially addition of each component, the Sl- RBD-sFc fusion protein and peptides are formulated with components described as above to form a protein-peptide complex and then is adsorbed to aluminum phosphate (Adju-Phos®) adjuvant. The last step would be addition of water for injection containing the 2- phenoxyethanol preservative solution to make final drug product at 200 pg/mL. The finished vaccine product is stored at 2 to 8 °C.

Placebo used in all trials was sterile 0.9% normal saline.

EXAMPLE 6

Supplementary Methods employed for assessment of COVID vaccine’s B-cell or T-cell Immunogenicity

1. Anti-Sl-RBDwT binding IgG antibody by ELISA.

The 96-well ELISA plates were coated with 2 pg/mL recombinant Sl-RBDwr-His protein antigen (100 pL/well in coating buffer, 0.1 M sodium carbonate, pH 9.6) and incubated overnight (16 to 18 hr) at room temperature. One hundred pL/well of serially diluted serum samples (diluted from 1 :20, 1 : 1,000, 1 : 10,000 and 1 : 100,000, total 4 dilutions) in 2 replicates were added and plates are incubated at 37 °C for 1 hr. The plates were washed six times with 250 pL Wash Buffer (PBS-0.05% Tween 20, pH 7.4). Bound antibodies were detected with HRP- rProtein A/G at 37 °C for 30 min, followed by six washes. Finally, 100 pL/well of TMB (3,3’,5,5’-tetramethylbenzidine) prepared in Substrate Working Solution (citrate buffer containing hydrogen peroxide) was added and incubated at 37 °C for 15 min in the dark, and the reaction stopped by adding 100 pL/well of H2SO4, 1.0 M. Sample color developed was measured on ELISA plate reader (Molecular Device, VersaMax). UBI® EIA Titer Calculation Program was used to calculate the relative titer. The anti-Sl-RBD antibody level is expressed as Logio of an end point dilution for a test sample (SoftMax Pro 6.5, Quadratic fitting curve, Cut-off value 0.248).

2. Inhibition of RBDWT binding to ACE2 by ELISA.

The 96-well ELISA plates were coated with 2 pg/mL ACE2-ECD-Fc antigen (100 pL/well in coating buffer, 0.1M sodium carbonate, pH 9.6) and incubated overnight (16 to 18 hr) at 4 °C. Plates were washed 6 times with Wash Buffer (25-fold solution of phosphate buffered saline, pH 7.0-7.4 with 0.05% Tween 20, 250 pL/well/wash) using an Automatic Microplate Washer. Extra binding sites were blocked by 200 pL/well of blocking solution (5 N HC1, Sucrose, Triton X-100, Casein, and Trizma Base). Five-fold dilutions of immune serum or a positive control (diluted in a buffered salt solution containing carrier proteins and preservatives) were mixed with a 1 : 100 dilution of RBDWT-HRP conjugate (horseradish peroxidase- conjugated recombinant protein Sl-RBD-His), incubated for 30+2 min at 25+2 °C, washed and TMB substrate (3,3’,5,5’-tetramethylbenzidine diluted in citrate buffer containing hydrogen peroxide) is added. Reaction is stopped by stop solution (diluted sulfuric acid, H2SO4, solution, 1.0 M) and the absorbance of each well is read at 450nm within 10 min using the Microplate reader (VersaMax). Calibration standards for quantitation ranged from 0.16 to 2.5 pg/mL. Samples with titer value below 0.16 pg/mL were defined as being half of the detection limit. Samples with titer exceed 2.5 pg/mL were further diluted for reanalysis.

3. Viral-neutralizing antibody titers against SARS-CoV-2 wild-type and variants by CPE based live virus neutralization assay.

Neutralizing antibody titers were measured by CPE-based live virus neutralization assay using Vero-E6 cells challenged with wild type (SARS-CoV-2-Taiwan-CDC#4, Wuhan) and Delta variant (SARS-CoV-2-Taiwan-CDC#1144, B.1.617.2), which was conducted in a BSL-3 lab at Academia Sinica, Taiwan. Vero-E6 (ATCC® CRL-1586) cells were cultured in DMEM (Hyclone) supplemented with 10% fetal bovine serum (FBS, Gibco) and lx Penicillin- Streptomycin solution (Thermo) in a humidified atmosphere with 5% CO2 at 37°C. The 96-well microtiter plates are seeded with 1.2* 10⁴ cells/100 pL/well. Plates are incubated at 37° C in a CO2 incubator overnight. The next day tested sera were heated at 56 °C for 30 min to inactivate complement, and then diluted in DMEM (supplemented with 2% FBS and lx Penicillin/Streptomycin). Serial 2-fold dilutions of sera were carried out for the dilutions. Fifty pL of diluted sera were mixed with an equal volume of virus (100 TCID50) and incubated at 37°C for 1 hr. After removing the overnight culture medium, 100 pL of the sera-virus mixtures were inoculated onto a confluent monolayer of Vero-E6 cells in 96-well plates in triplicate. After incubation for 4 days at 37 °C with 5% CO2, the cells were fixed with 10% formaldehyde and stained with 0.5% crystal violet staining solution at room temperature for 20 min. Individual wells were scored for CPE as having a binary outcome of ‘infection” or ‘no infection’. Determination of SARS-CoV-2 virus specific neutralization titer was to measure the neutralizing antibody titer against SARS-CoV-2 virus based on the principle of VNT50 titer (>50% reduction of virus-induced cytopathic effects). Virus neutralization titer of a serum was defined as the reciprocal of the highest serum dilution at which 50% reduction in cytopathic effects are observed and results are calculated by the method of Reed and Muench.

4. Neutralizing titers against Omicron BA.1/BA.2/BA.5 by pesudovirus assay.

Neutralizing antibody titers were measured by neutralization assay using HEK-293T- ACE2 cells challenged with SARS-CoV-2 pseudovirus variants. The study was conducted in a BSL2 lab at RNAi core, Biomedical Translation Research Center (BioTReC), Academia Sinica. Human embryonic kidney (HEK-293T/17; ATCC® CRL-11268™) cells were obtained from the American Type Culture Collection (ATCC). Cells were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (Hyclone) and 100 U/mL of Penicillin-Streptomycin solution (Gibco), and then incubated in a humidified atmosphere with 5% CO2 at 37 °C. HEK- 293T-ACE2 cells were generated by transduction of VSV-G pseudotyped lentivirus carrying human ACE2 gene. To produce SARS-CoV-2 pseudoviruses, a plasmid expressing C-terminal truncated wild-type Wuhan-Hu-1 strain SARS-CoV-2 spike protein (pcDNA3.1-nCoV-SA18) was co-transfected into HEK-293T/17 cells with packaging and reporter plasmids (pCMVA8.91, and pLAS2w.FLuc.Ppuro, respectively) (BioTReC, Academia Sinica), using TransIT-LTl transfection reagent (Minis Bio). Site-directed mutagenesis was used to generate the Omicron BA.1, BA.2, and BA.4/BA.5 variants by changing nucleotides from Wuhan-Hu-1 reference strain. For BA.1 variant, the mutations of spike protein are A67V, A69-70, T95I, G142D/A143- 145, A211/L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F. For BA.2 variant, the mutations of spike protein are T19I, L24S, A25-27, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K. For BA.4/5 variant, the mutations of spike protein are T19I, L24S, A25-27, A69-70, G142D, V213G, G339D, S371F, S373P, S375F, T,376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, L486V, Q493, Q498R, N501Y, Y505H, D614G, H655Y, N679K, N764K, D796Y, N856K, and Q954H, & L969K.

Indicated plasmids were delivered into HEK-293T/17 cells by using TransITR-LTl transfection reagent (Minis Bio) to produce different SARS-CoV-2 pseudoviruses. At 72 hours post-transfection, cell debris were removed by centrifugation at 4,000 xg for 10 minutes, and supernatants were collected, filtered (0.45 pm, Pall Corporation) and frozen at -80 °C until use. HEK-293-hACE2 cells (IxlO⁴ cells/well) were seeded in 96-well white isoplates and incubated for overnight. Tested sera were heated at 56°C for 30 min to inactivate complement, and diluted in medium (DMEM supplemented with 1% FBS and 100 U/ml Penicillin/Streptomycin), and then 2-fold serial dilutions were carried out for a total of 8 dilutions. The 25 pL diluted sera were mixed with an equal volume of pseudovirus (1,000 TU) and incubated at 37 °C for 1 hr before adding to the plates with cells. After 1-hr incubation, the 50 pL mixture added to the plate with cells containing with 50 pL of DMEM culture medium per well at the indicated dilution factors. On the following 16 hours incubation, the culture medium was replaced with 50 pL of fresh medium (DMEM supplemented with 10% FBS and 100 U/ml Penicillin/Streptomycin). Cells were lysed at 72 hours post-infection and relative light units (RLU) was measured by using Bright-GloTM Luciferase Assay System (Promega). The luciferase activity was detected by Tecan i-control (Infinite 500). The percentage of inhibition was calculated as the ratio of RLU reduction in the presence of diluted serum to the RLU value of virus only control and the calculation formula was shown below: (RLU ^Control - RLU ^Serum) / RLU ^Contro1. The 50% protective titer (NT50 titer) was determined by Reed and Muench method.

5. T cell responses by ELISPOT.

Human peripheral blood mononuclear cells (PBMCs) were used in the detection of the T cell response. For the booster-series third-dose series extension study, ELISpot assays were performed using the human IFN-y/IL-4 FluoroSpot^PLUS kit (MABTECH). Aliquots of 250,000 PBMCs were plated into each well and stimulated, respectively, with 10 pg/mL (each stimulator) of RBD-WT+T1I/CTL, Th/CTL, or Th/CTL pool without UBIThla (CoV2 peptides), and cultured in culture medium alone as negative controls for each plate for 24 hours at 37 °C with 5% CO2. The analysis was conducted according to the manufacturer’s instructions. Spot-forming units (SFU) per million cells was calculated by subtracting the negative control wells.

6. Intracellular cytokine taining (ICS).

Intracellular cytokine staining and flow cytometry was used to evaluate CD4⁺ and CD8⁺ T cell responses. PBMCs were stimulated, respectively, with Sl-RBD-His recombinant protein plus with Th/CTL peptide pool, Th/CTL peptide pool only, CoV2 peptides, PMA + Inonmycin (as positive controls), or cultured in culture medium alone as negative controls for 6 hours at 37°C with 5% CO2. Following stimulation, cells were washed and stained with viability dye for 20 minutes at room temperature, followed by surface stain for 20 minutes at room temperature, cell fixation and permeabilization with the BD cytofix/cytoperm kit (Catalog # 554714) for 20 minutes at room temperature, and then intracellular stain for 20 minutes at room temperature. Intracellular cytokine staining of IFN-y, IL-2 and IL-4 was used to evaluate CD4⁺ T cell response. Intracellular cytokine staining of IFN-y, IL-2, CD 107a and Granzyme B was used to evaluate CD8⁺ T cell responses. Upon completion of staining, cells were analyzed in a FACSCanto II flow cytometry (BD Biosciences) using BD FACSDiva software.

7. Statistics.

For the phase 2 extension booster vaccination study, the immunogenicity results for Geometric Mean Titer (GMT) are presented with the 95% confidence intervals. Statistical analyses were performed using SAS® Version 9.4 (SAS Institute, Cary, NC, USA) or Wilcoxon sign rank test. Spearman correlation was used to evaluate the monotonic relationship between non-normally distributed data sets. For the phase II primary 2-dose series, the sample size of the trial design meets the minimum safety requirement of 3000 study participants in the vaccine group with healthy adults, as recommended by the US FDA and WHO. EXAMPLE 7

High precision designer vaccine against SARS-CoV-2 infection containing respective Sl-RBD fusion proteins (Sl-RBDvoCs-sFc and Sl-RBD Omicron BA.4/BA.5 variant)

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 ALHYDROGEL, ADJUPHOS, MONTANIDE ISA, CpG, etc. and other excipients to enhance the immunogenicity of the high-precision designer immunogens.

2. A representative designer COVID-19 vaccine UB-612 employing CHO cell expressed S- RBD-sFc protein(amino acid sequence of SEQ ID NO: 49 and nucleic acid sequence of SEQ ID NO: 60) as the B cell immunogen.

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 epitope peptides 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 ALUM (ALHYDROGEL/CpG, ADJU-PHOS®/CpG and MONTANIDE™ ISA/CpG) for induction of optimal anti-SARS-CoV-2 immune responses. ALUM (ADJUPHOS and ALHYDROGEL) 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 TLR9 agonists that improve antigen presentation and the induction of vaccine-specific cellular and humoral responses. In general, the negatively 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 (e.g. UB-612) prepared according to the vaccine composition as shown in Table 11 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 in EXAMPLE 8 and EXAMPLE 9 set forth the approach in designing the disclosed high precision SARS-CoV-2 vaccine that are SAFE, 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.

EXAMPLE 8

Potent Booster and Long-lasting Immunity demonstrated in UB-612 (a SARS- CoV-2 (Wuhan) Protein-Peptide Vaccine) Phase-1/2 Trials

1. Trial procedures and safety a. Phase-1 trial of primary and booster third-dose series. The phase- 1 trial was initiated with a sentinel group of 6 participants to receive the low 10- pg dose, followed with the remaining 14 participants if without vaccine-related > grade 3 adverse reaction. The same procedure was extended for the escalating 30- and 100-pg dose groups. Additional follow-up visits were scheduled for all participants on Days 14, 28, 35, 42, 56, 112, and 196. Study participants were scheduled for visits 14 and 84 days after the booster. Electronic diaries were provided to the participants to be completed for the 7-day period after each injection to record solicited local reactions at the injection site (pain, induration/swelling, rash/redness, itch, and cellulitis) and solicited systemic reactions (17 varied constitutional symptoms). Severity was graded using a 5-level (0 to 4) scale from none to life-threatening. In addition, participants recorded their axillary temperature every evening starting on the day of the vaccination and for the 6 subsequent days. Safety endpoints include unsolicited AEs reported for up to 14 days post-booster in this interim phase-1 extension report. b. Phase-2 trial of primary series.

The primary safety endpoints the phase-2 trial were to evaluate the safety and tolerability of all participants receiving study intervention from Days 1 to 57 (28 days after the second dose). Vital signs were assessed before and after each injection. Participants were observed for 30 minutes after each injection for changes in vital signs or any acute anaphylactic reactions. After each injection, participants had to record solicited local and systemic AEs in their self-evaluation e-diary for up to seven days while skin allergic reactions were recorded in their e-diary for up to fourteen days. Safety endpoints include unsolicited AEs reported for Days 1 to Day 57 in this interim phase-2 report. ESULTS a. Trial populations i. Phase-1 primary and booster third-dose series.

The characteristics of the open-label phase- 1 trial participants included the 196-day primary series study involving 60 healthy adults (aged 20-55 years old.) in three dose groups (n = 20 each) who received two doses (28 day-apart) of UB-612 at 10, 30, or 100 pg; and the 84-day extension booster vaccination following the primary series, where 50 participants were enrolled to receive an additional one 100-pg booster between 7.6 and 9.6 months after the second shot for the 10-pg (n = 17), 30-pg (n = 15), and 100-pg (n = 18) groups. The boosted participants were followed for 14 days for assessment of safety and immunogenicity in this interim report, and subsequently monitored until 84 days after booster. ii. Phase-2 primary 2-dose series.

The phase-2 trial was of a randomized and double-blind design. A total of 3,875 participants who received at least one vaccine dose at 100 pg (3,321 received UB-612 and 554 received placebo at a 6: 1 ratio) were enrolled and included in the Safety Population, of which 1012 participants (vaccine 871 and placebo 141) were included in the Evaluable Immunogenicity Population. The mean age of the participants receiving UB-612 was 44.9 years (18 to 83 yr) and that of placebo was 44.4 years (19 to 84 yr). The ratio of younger adults (18 to 65 yr.) vs. elderly adults (>65 yr.) was approximately 80:20 for both UB-612 and placebo groups. All participants but 5 were Taiwanese. b. Reactogenicity and safety i. Phase-1 primary 2-dose and booster third-dose series.

In the 196-day primary series and up to 14 days after booster, neither vaccine-related severe adverse events (SAEs, including grade 3/4 AEs) nor dose-limited increase in incidence or severity were recorded. The solicited local and systemic AEs reported within 7 days in all vaccination groups were mild to moderate (grade 1/2) and transient, with lower frequencies for most systematic reactions than local reactions. The incidence of solicited local AEs was comparable after the first and second vaccination and slightly increased after the booster dose, the most common post-booster solicited local AE being pain at the injection site (60-71%). The incidence of solicited systemic AEs was similar after each vaccination, with the most common post-booster solicited systemic AE being fatigue (11-33%). The safety profile observed in the primary 2-dose vaccination series and the booster phase was similar. ii. Phase-2 primary 2-dose series.

There were no vaccine-related SAEs. Both local and systemic AEs were mild and transient, and were self-limited in a few days. Overall, 2546 of participants reported solicited local AEs, of which 2386 (72.0%) were from UB-612 and 160 (28.9%) from the placebo group after 1 and 2 doses. These local AEs were mild (grade 1) to moderate (grade 2) in severity, and the most common event was injection-site pain in 2246 (67.8%) participants of vaccine group, and occasional skin allergic reaction. There was no significant difference in the incidence of solicited systemic AEs between UB-612 vaccine and placebo groups across age strata (P > 0.05). Solicited systemic AEs were reported by 38.6% of the elderly participants (65-85 years old) among the vaccine groups, compared with 63.3% of the overall safety population. The most common solicited systemic AE was fatigue/tiredness reported in 1,488 (44.9%) of UB-612-treated participants and was generally mild. c. Neutralizing antibodies against live SARS-CoV-2 wild-type v.s Delta variant, and against pseudo-SARS-CoV-2 wild-type r.v VoCs including Alpha, Beta, Gamma and Omicron i. Phase-1 primary 2-dose and booster third-dose series.

A booster dose of 100 pg given 7.6-9.6 months after the 2^nd dose induced robust neutralizing antibodies against live SARS-CoV-2 wild type (WT, Wuhan strain) and Delta variant in 100% of the participants (Figure 11). In the 10-, 30- and 100-pg UB-612 dose groups, the booster elicited geometric mean 50% virus-neutralizing titers (VNTso) against WT of 4643, 3698 and 3992, respectively (Figures 11A-11D) representing (a) 104-, 118- and 37-fold respective increase (geometric mean fold increases, GMFIs) over the peak responses in the primary series (14 days after dose 2, i.e., Day 42), and (b) GMFIs of 465, 216 and 65, respectively, over the pre-boost levels. Compared with a panel of human convalescent sera (HCS) collected ~1 month after onset in hospitalized COVID-19 cases, the post-booster neutralizing antibody levels were 45.5-, 36.2- and 39.1-fold (GMFIs) higher. Neutralizing antibody titers in the same live virus test standardized with the WHO reference antiserum and expressed in international units (lU/mL) were similar.

The booster dose induced remarkably high VNTso titers against live Delta variant as well, reaching at 2854, 1646 and 2358 (Figure 12A), which represent modest GMFRs of 1.6-, 2.4-, and 1.7 (i.e., a preservation of -63%, -42% and -60% neutralizing strength, respectively) for the 10-, 30- and 100-pg groups, respectively, relative to the WT strain. Comparison of post-booster viral neutralizing antibody titers against SARS-CoV-2 wild type (WT) and Delta variant from sera collected from vaccinees receiving a third (booster) shot from different vaccine platforms is shown in Table 11 where UB-612 demonstrated the highest GMT against Delta when compared to vaccines from other platforms (NVX- CoV2373, mRNA-1273, BNT 162B2, MVC-COV1901, CoronaVac and AZD1222) along with the demonstration of a high preservation in Delta neutralizing capability by these UB-612 booster sera as expressed in WTZDelta(GMFR) (Table 11) . The pVNTso observed 14 days after booster of the 100-pg group (n = 18) were assessed for their cross-reactive neutralizing antibody titers against pseudo-SARS-CoV-2 wild type (WT) and other VoCs including Omicron as shown in Figure 12B. The pVNTso against WT, Omicron, Alpha, Gamma and Beta were 12,778, 2,325, 9,300, 13,408 and 4,974, respectively when compared with the wild type of 14,171 with a modest respective GMRFs of 5.5, 1.4, 1.0, and 2.6 (i.e., a preservation of 18.2%, 72.7%, 105%, and 38.9% neutralizing strength, respectively) relative to the WT strain.

The neutralizing antibodies in the primary series were long-lasting for the 100-pg group, associated with the highest increase in VNTso against WT observed at 14 to 28 days after dose 2, as compared with the lower-dose 10- and 30-pg groups (Figures 11A-11C). The peak neutralizing antibody GMT (108 at Day 42; 103 at Day 56) (Figure 11C) in the 100- pg group was close to the GMT of 102 for the panel of control human convalescent sera (HCS). Seroconversion rate based on the SARS-CoV-2 neutralizing antibody titers at Day 57 in Phase 1 was 100% for the 100 pg dose and remained 100% thereafter throughout the period monitored.

Prior to boosting (Days 255 to 316), none of the 18 participants (0%) in the 100-pg group with VNTso titers fell below the assay limit (LLOQ), suggesting that the induced neutralizing effect could persist for a long period of time. Antibody persistence after 2 doses for the 100-pg group from the phase 1 trial was calculated using first-order exponential model fitting (SigmaPlot) for the anti-WT neutralizing VNT50 over Days 42 to 196 (r² = 0.9877, the decay rate constant K_ei = -0.0037; ti/2 = 0.693/K_ei). The neutralizing antibody VNT50 GMT slowly declined, with a ti/2 of 187 days (Figure 12C).

We also investigated the neutralizing effects against Delta and other VoCs during the Phase- 1 primary vaccination phase with all serum samples (n = 20) from the primary series of Phase 1 trial of 100-pg UB-612 dose group (Figure 13). The results showed preserved viral-neutralizing activities, in particular against the Delta B.1.617.2 variant, to which a 63% neutralizing activity (GMFR of 1.6) was retained relative to the wild type Wuhan strain. Significant neutralizing antibodies were preserved as well against the Alpha (B. l.1.7) variant, with 91% retained (GMFR of 1.1), and Gamma (P. l) variant with 56% retained (GMFR of 1.8), while that against Beta B.1.351 was weaker, with 20% retained (GMFR of 5.1). ii. Phase-2 primary 2-dose. At Day 57 (4 weeks after the second dose), across participants of all ages (18 to 85 years), the anti-Sl-RBD titer with a GMT of 518.8 (Figure 14A) and the viral-neutralization titer against the original wild-type (WT Wuhan) strain was age-dependent with an overall VNTso of 87.2 (Figure 14B). The younger adults (18 to 65 years) had a higher VNT50 of 96.4, which is reproducibly close to that observed in Phase-1 study participants aged 20-55 years old (VNT50 of 103) (Figure 11C), while the elderly adults (=65 year old) exhibited a lower VNT50 of 51.6. An extension study of the Phase 2 trial with a booster third dose is being investigated. Seroconversion rate based on the Wild Type SARS-CoV-2 Neutralizing Antibody Titers at Day 57 (or Day 56 after dose 1) across participants of all ages (18 to 85 years old) in Phase 2 were from 88.6% for the elderly to 96.4% for the young adults.

At Day 57, a substantial level of anti-Delta neutralizing antibodies was observed. A pool of 48 serum samples randomly selected from vaccinees across age groups (n = 39 for young adults aged 18-65 years old; n = 9 for elderly adults >65 years old) were subjected to an ad hoc live virus assay analysis in two independent laboratories (Academia Sinica and the California Department of Viral and Rickettsial Diseases). The results were concordant and revealed that immune sera could neutralize two key SARS-CoV-2 prototypes with a similar VNT50: 329 against Wuhan WT obtained in Taiwan and 308 against USA WA 1/2020 strains in the United States (Figure 15). The VNT50 against the Alpha B.l.1.7 and Delta B.1617.2 were estimated to be 122 and 222, respectively, representing, a 2.7-fold and 1.4-fold reduction, relative to USA WA 1/2020 variant. d. Neutralizing antibodies against SI -RUD binding to ACE2 receptor i. Phase-1 primary 2-dose and booster third-dose series.

ELISA results of the functional inhibition (neutralization) against the S1-RBD:ACE2 interaction (Figure 16) were largely consistent with the VNT50 data (Figure 11). The 100- pg dose group exhibited the highest neutralizing titers (Figure 16C), with an anti-Sl- RBD:ACE2 quantitative neutralizing antibody (qNeuAb) level of 6.4 pg/mL at Day 112, a 4.6-fold increase as compared with 1.4 pg/mL from the 20 human convalescent sera (HCS). Upon booster vaccination, the anti-Sl-RBD:ACE2 qNeuAb levels reached 303 to 521 pg/mL, representing 77- to 168-fold increase over the peaks after the primary vaccination series; similarly, profound 82- to 579-fold increases were observed as compared with the pre-boost levels (Figures 16A-16C). Thus, the UB-612 booster can elicit significant immune responses in vaccinated subjects regardless of how low their preboost levels are.

The neutralization of S1-RBD:ACE2 binding on ELISA correlates well with VNT50 findings (Spearman’s r = 0.9012) (Figure 16D), thus corroborating the validity of the anti- WT VNTso results by the cytopathic effect (CPE) assay (Figures 16A-16C). Furthermore, the post-booster anti-Sl-RBD:ACE2 qNeuAb levels of 303 to 521 pg/mL (Figures 16A- 16C) were 216- to 372-fold higher than for human convalescent sera (HCS). This suggests that the majority of antibodies in HCS appears to bind more to the allosteric sites (N- or C- terminal domain of SI) than to the orthosteric (RBD) sites where viral Sl-RBD interacts with the ACE2 receptor. e. Sl-RBD IgG antibody ELISA responses i. In the Phase 1 trial,

Sl-RBD binding antibodies measured by ELISA (Figure 17) showed again that the 100- pg vaccinated group elicited the highest immune responses over the 196-day primary series, with GMT of 2,240 at Day 42, which far exceeded the GMT of 141 from the 20 human convalescent sera (HCS). Upon booster vaccination, the anti-Sl-RBD GMT in the three dose groups peaked at 7,154 to 9,863 (3- to 28-fold increase (GMFIs) over the peaks during the primary series); similarly, profound 37- to 378-fold increases were observed as compared with the pre-boos levels. f T cell responses by ELISpot i. Phase 1 trial.

In the primary vaccination series of the phase- 1 trial, peripheral blood mononuclear cells (PBMCs) were collected from vaccinees for evaluation by Interferon- y + (IFN-y+)- ELISpot (Figures 18A-18C). The highest antigen-specific responses were observed in the 100-pg dose group: on Day 35, 254 spot-forming unit (SFU)/106 PBMC after stimulation with Sl-RBD+Th/CTL peptide pool and 173 by Th/CTL peptide pool alone (Figure 18C), demonstrating that the Th/CTL peptides in the UB-612 vaccine were principally responsible for the T cell responses.

On Day 196, the IFN-y+ ELISpot responses for the 100-pg dose group remained at levels of -50% of the peak responses, which decreased from 254 to 121 SFU/106 cells with RBD+Th/CTL peptide pool re-stimulation, or from 173 to 86.8 with Th/CTL peptide pool re-stimulation only. This observation suggests that UB-612 vaccine elicited T cell responses after two vaccine doses persisted for at least 6 months. This is in concert with the persistence of neutralizing antibodies noted earlier (Figure 11C). ii. Phase 2 trial.

In the Phase 2 trial, the Day-57 strong IFN-y⁺-ELISpot responses were also observed: geometric mean of 370 (SFU/10⁶ cells) with Sl-RBD+Th/CTL re-stimulation, 322 with Th/CTL re-stimulation, and 181 with Th/CTL peptide pool without UBIThla (Figure 18D), which were all far higher than the counterparts in the placebo group (p<0.0001). In contrast with IFN-y, the IL-4 responses were far lower: 13.6, 7.5, and 5.4, respectively (Figure 18E). The overall ELISpot results indicate that the inclusion of the Th/CTL peptides is essential and principally responsible for the T-cell responses, while the recombinant protein, Sl-RBD plays only a minor role. Importantly, the orientation of the T cell response is predominantly Thl oriented. UBIThla plays a catalyst role as usual to trigger the Thl responses by the viral-specific Th/CTL peptide pool. g. CD4⁺ and CD8⁺ T cell responses by Intracellular Cytokine Staining (ICS). i. Phase 2 trial.

T cell responses by Intracellular Cytokine Staining (ICS) were evaluated (Figure 19). Substantial increase in IFN-y- and IL-2-producing CD4⁺ and CD8⁺ cells were observed across the three peptide-re-stimulation groups; and, consistent with ELISpot findings (Figures 18D-18E), lower IL-4-producing CD4⁺ T cells were detected, confirming the Thl -predominance of the T-cell response.

CD8⁺ T cells expressing cytotoxic markers, CD 107a and Granzyme B, were observed, accounted for 3.5%, 2.1%, and 1.8% of circulating CD8⁺ T cells after re-stimulation with Sl-RBD+Th/CTL, Th/CTL, and Th/CTL pools without UBIThla, respectively. Overall, UB-612 elicited Thl -oriented immunity with a robust CD8⁺ cytotoxic T cell response, which would be favorable for clearance of the viral infection, and the re-stimulation results indicated that Th/CTL peptides, which include non-spike nucleocapsid (N) and membrane (M) structure proteins, are the principal factor responsible for the T cell immunity.

3, CONCLUSIONS

No vaccine-related serious adverse events (SAE) were recorded. The most common solicited AEs were injection site pain and fatigue, mostly mild and transient. In both trials, UB- 612 elicited respective neutralizing antibody titers similar to a panel of human convalescent sera. The most striking findings were: long-lasting viral-neutralizing antibodies and broad T-cell immunity against SARS-CoV-2 VoCs including Delta and Omicron, and a strong booster- recalled memory immunity with high cross-reactive neutralizing titers against the Delta and Omicron variants.

UB-612 has presented a favorable safety profile, potent booster effect against VoCs, and long-lasting B and broad T cell immunity that warrants further development for both primary immunization and heterologous boosting of other COVID-19 vaccines.

Of special note, the five precision-designed T cell epitope peptides represent the Th and CTL epitopes from Sarbecovirus regions of the N, M and S2 proteins. These epitope peptides are highly conserved across all Variants of Concern including Delta and Omicron and are promiscuous epitopes that allow for induction in a broad population of memory recall, T cell activation and effector functions. Thus, the long-lasting and robust T cell immunity could be efficacious against all VoCs including Omicron, in addition to a potent anti-Delta and anti- Omicron effect upon a booster 3^rd-dose of UB-612. As non-spike structure M and N proteins fall beyond recognition by the currently authorized CO VID vaccines, UB-612 vaccine has a good stance to fend off new Variants of Concern such as Delta and Omicron, which warrants large scale field trial for assessment.

EXAMPLE 9

UB-612 Phase 2 booster vaccination can protect against Omicron infection better than those “Spike” only COVID vaccine

As with the outcome of the Phase-1 booster vaccination against Delta and Omicron BA. l variants shown in EXAMPLE 8, the Phase-2 booster results affirm that UB-612 may serve as a universal (pan-Sarbecovirus) vaccine protecting against Omicron variants and other ever- emergent new mutants.

In addition to Spike Sl-RBD (the restricted ACE2 receptor binding domain) as the immunogen that activates B cells (durable neutralizing antibodies), UB-612 is enriched with five sequence-conserved, promiscuous Th/CTL epitopes on Spike S2 and non-Spike (Nucleocapsid N and Membrane M) structure proteins for promotion of a fuller T cell (helper and cytotoxic) memory immunity.

Due to the uniqueness in vaccine design, UB-612 was included on the July 26^th agenda of the White House Next Generation COVID-19 Vaccine Summit, joining with Pfizer BNT and Modema vaccines, to showcase the vanguard vaccine platform. UB-612 booster vaccination can elicit potent, broadly-recognizing and durable B cell (neutralizing antibodies) and T cell (helper and cytotoxic) memory inmmunity, which could mimick infection with any SARS-CoV-2 variant.

1. Comparison of UB-612 vaccines with COVID vaccines employing other platform technologies and using “Spike” protein only as the immunogen

The recent approval for use of mRNA bivalent vaccines (Moderna and Pfizer) as the fourth dose (the 2^nd booster) in UK, US and here in Taiwan has prompted attention and skepticism. Thus, amid the concerns on use of the upcoming imported bivalent mRNA vaccines, it is time to contrast the performance of the current vaccines in their combat against Omicron variants on the levels of viral-neutralizing antibody and T cell immunity strength. a. Pseudovirus-neutralizing antibody activity.

After the booster shot with each vaccine (third-dose, homologous boosting) to non-infected persons, the immune sera of UB-612 against BA.1/BA.2/BA.5 variants measured by “pseudovirus-neutralizing assay” (50% geometric mean GMT, i.e., pVNTso/IDso titer) was shown to be greater than by Moderna (mRNA-1273), Pfizer (BNT162b2), and NVX- CoV2373; and far greater than by MVC-COV1901, AZD1222, CoronaVac, and BBIBIP vaccines (Table 12). Taking the most contagious BA.5 pVNTso as an example, the neutralizing titer was reported to be 854 for UB-612, 582 for NVX-CoV2373, 378 for Modema mRNA-1273, 360 for Pfizer BNT162b2, 75 for CoronaVac, and 43 for AZD1222. These data suggest that UB-612 vaccine materializes the design concept for synergizing B- cell and T-cell immunity, and that the third dose (the first booster) has been able to substantially neutralize the Omicron BA.5 variant strain, which is currently a formidable, dominant SARS-CoV-2 variant Taiwan is facing. UB-612 booster performed superior to that of the AZ vaccine.

It is important to note that, pseudovirus assay is set up with an artificial (fake) virus crowned with Spike proteins only, while the clinical isolate of live virus is an actual one containing Spike proteins plus non-spike proteins on the virus main body. All current licensed vaccines that are designed with a Spike-only immunogen would fail to identify the ontological structure of the virus's non-Spike proteins.

Non-Spike proteins also mutate along the trail of viral evolution (Table 13), the antibodies produced by the current Spike-only vaccines would be neither able to home in on, nor to induce B and T cell memory immunity to recognize non-Spike proteins. Thus, data inconsistency would arise between pseudovirus and live virus assays for those Spike protein only vaccines, unless the vaccine antigen is designed to take into account both Spike and non-Spike proteins. The inconsistency is verified below. b. Live virus-neutralizing activity.

After the booster shot with each vaccine (third-dose, homologous boosting) administered to non-infected persons, the immune sera against BA. l variant measured by “live virusneutralizing assay” (50% geometric mean GMT, i.e., VNT50/FRNT50 titer) showed a much greater GMT potency for UB-612 (value at 670) than those by other vaccines (values at 46 to 106) (mRNA-1273, BNT162b2, and AZDI 222 vaccines), representing a 6- to 12-fold higher titer strength (Table 13) .

Upon comparison of the live and the artificial pseudovirus neutralization assay methods (Tables 12 and 13), only UB-612 vaccine presents between-method consistency, and the UB-612 viral-neutralizing strength by either viral assay method excels over all the other EUL listed vaccines.

A data discrepancy exists between the pseudovirus and the live virus assay for these brand- named vaccines. Note, with anti-BA.l pVNTso as example (Table 13), the gap in pVNTso (pseudovirus neutralization assay) between UB-612 (value at 1,196-2,325) and mRNA- 1273/BNT162b2 ( value at 945-1,116) is small; while that gap with live virus assay for anti- BA. l VNT50 is much wider, about 6- to 12-fold different (Table 12).

2. Behind the superior viral-neutralizing activity by UB-612.

The superiority of UB-612 over other vaccines in regard of pseudovirus- and live virusneutralizing strength could be attributable to its recognition of targets on both Spike and nonSpike proteins (conserved and promiscuous Th/CTL epitopes on S2, M and N proteins), producing striking, broadly-recognizing full-scale T-cell immune memory that intensifies the B- cell immune response with cross-neutralizing antibodies against BAI, BA.2 to BA.5. B cell and T cell immunity react in a synergistic manner.

The brand-named vaccines’ booster vaccination (third dose) has exposed their weakness in fighting against even the BA. l live virus, let alone against BA.2 and BA.5. As seen with the difficulty for the current mRNA vaccines to produce a post-booster peak VNT50 titer of >100 in combating live BA. l virus, it can be predicted that the vaccines will be further weakened in neutralizing the live BA.2 and BA.5 viruses.

3. Mutation sites on both Spike and non-Spike proteins.

In addition to more than 30 mutations on Spike protein, there are also mutation sites on non-Spike proteins (E, M, and N) of BA. l, BA.2, and BA.5 variants as shown in Table 14, which are beyond recognition by the Spike-only vaccines, namely, they bear an intrinsic shortfall for promoting a fuller T cell immunity. And, in contrast, by targeting smaller protein fragment of Sl-RBD within the Spike protein and the designed T immunogens with conserved and promiscuous T epitopes, the UB-612-induced immunity would meet less viral mutant resistance than those other vaccines, and thus making Omicron evasion less likely as the UB-612 vaccine induced immunity could behave closer to the breath of infection-induced immunity.

The observations above also implicate that 1) only the live virus test assay is actually reliable when comparing the viral-neutralization potency of distinct vaccine platforms; and 2) the artificial pseudovirus assay is good for comparison amongst those Spike-focused vaccines.

4. A potential pan-Sarbecovirus vaccine.

Of special note, the non-spike structure proteins of envelope (E), membrane (M) and nucleocapsid (N), are critically involved in the host cell interferon response and induction of T- cell memory. The profound T cell memory immunity recalled by UB-612 vaccine could thus play a critical role in long-term control of SARS-CoV-2 infection. UB-612 as a booster could thus potentially benefit most for infected persons for protection against reinfection.

Unlike other vaccines that use the Spike (S) protein as the only B and T immunogen, the composition of UB-612 includes immunogen Sl-RBD to trigger B cells for production of neutralizing antibodies, and five conserved, nonmutable promiscuous epitopes (S2x3, N and M proteins) as T immunogens as well (Table 15). The unique, rational vaccine design confers UB- 612 a potential of heading toward a pan-Sarbecovirus vaccine.

5. Strong T cell immunity to prevent immune escape.

As vaccines capable of producing a strong T-cell immune response against conserved, nonmutable epitope can prevent immune escape. It is of high interest to look into the T cell immune responses exerted by different vaccine plateforms. Given as a booster (third dose, homologus boosting), UB-612appears to elicit a degree of T cell immunity (SFU/10⁶ PBMC cells) far greater than by mRNA vaccine (BNT162b2) or adeno-vectored DNA vaccine (ChAdOxl or AZDI 222) as decribed below. The SFU units at pre-booter/post-booster for 3 doses of ChAd/ChAd/ChAd are 38/45, and 3 doses of BNT/BNT/BNT are 28/82, respectively, which are lower than the 261/374 SFU by UB-612 booster observed in the Phase-2 extension study. These results are consistent with the fact that UB-612 has a strong anti -BA.1 live virus-neutralization titer, while those for BNT162b2 and ADZ 1222 are weak (Table 13). It is generally known that a strong T cell immunity is also critical for protection against severe diseases and for long-term vaccine success.

6. UB-612 vaccine effectiveness against infection.

While the degree of real-world vaccine effectiveness against infection is not available, the positive functional correlation between UB-612’s strong blockade of ACE2:RBDWT interaction and viral-neutralizing VNTso (live virus WT and Delta) and pVNTso (pseudovirus BA. l) infers a substantial clinical efficacy against COVID-19. Indeed, using models of S protein binding activities and neutralizing antibodies, the clinical efficacy of 2-dose primary immunization of UB-612 is predicted to be 70-80% against Wuhan/Delta and a booster vaccination may lead to 95% efficacy against symptomatic COVID-19 caused by the ancestral Wuhan/Delta or Omicron strain.

7. Ongoing investigation of UB-612 effectiveness in Taiwan and USA.

It should be noted that, of the 1,480 subjects who received two or three doses of the UB- 612 vaccine (Phase-2 trial in Taiwan), no occurrence of infection cases are reported, as of this March at the conclusion of the designed Phase 2 clinical study.

In addition, at the time of the rapid escalation of the domestic Omicron infection in Taiwan in May of this year, telephone interviews of the subjects of the Phase-2 clinical trial are being conducted and the initial vaccine protection efficacy of the subjects receiving two or three doses of UB-612 vaccine against Omicrons during the unprecendeted outbreaks in Taiwan was estimated to be greater than 95% (this trial is ongoing).

Further, the phase 3 clinical efficacy protecting against infection of circulating subvariants including the dominant Omicron B5 would await outcome of an ongoing Phase-3 trial that compares UB-612 with authorized vaccines under homologous and heterologous boosting [ClinicalTrials.gov ID: NCT05293665],

Use of mRNA bivalent vaccines as the fourth dose. At present, the Moderna bivalent vaccine mRNA- 1273.214 (original Spike plus Omicron BA.l Spike) as the fourth dose (second booster needle) has lately received Emergency Use Authorization. The pseudovirus-neutralizing titer (pVNTso) against BA.5 was reported to be 727, which is 50-60% higher than the original mRNA-1273 vaccine (fourth dose) and 90% higher than the third dose of mRNA-1273 vaccine (Table 12); none is greater than 2 times. This small increase in pVNTso has been shown not to lead to an elevation of vaccine effectiveness. Another bivalent vaccine containing BA.5 has received Emergency Use Authorization as well on September 6; the approval was made based on an 8 -mice study only.

Unfortunately, Moderna did not present the crucial "live virus-neutralization potency" data at the US FDA Review meeting on June 28. At present, only the anti-BA.1 live virus VNTso data is available, reported to be 81.0 for the original mRNA-1273 after the third dose (Table 13); and that of anti-BA.5 live virus after the fourth dose of the bivalent vaccine mRNA-1273.214 could be lower.

Based on the overall estimate of Table 12 data for anti-pseudovirus pVNTso, the neutralizing potency against BA. l is 1.3 times higher than that against BA.2; and that against BA.5 is within 2- to 3-fold higher, relative to anti-BA. l/andi-BA.2. Without mask wearing, frequent hand washing, and proper social distancing, there is a good likelihood of re-infection or breakthrough infection by Omicron BA.5 variant. While full vaccination plus booster shot is needed for protection against infection, the question remains as to whether one can get both the vaccine and the dose regimen right.

8. Potential benefit to prevention against long-haul COVID.

Finally, regardless of vaccination status or hybrid immunity, each reinfection would add risks of mortality, hospitalization and other health hazards including burden of long-haul COVID, and immunization with the currently authorized vaccines could present only a limited benefit to alleviate long CO VID.

As long-haul COVID is found to be associated with a decline in IFNy-producing CD8⁺ T cell, a vaccine platform capable of eliciting strong and durable T cell immunity for clearance of residual systemic infection (sustained viral reservoirs) would be desirable for prevention of long- haul CO VID, to which UB-612 may play a positive role. EXAMPLE 10

Development of a Pan-Sarbeco Vaccine to prevent and treat SARS-CoV-2 infection, to clear viral infection and treat long-haul COVID

The SARS-CoV-2 Omicron lineage has swept the globe from the original Wuhan strain with a rapid succession of dominating subvariants from BA.1, BA.2 and to the current BA.4/BA.5 that makes up more than 90% of SARS infection cases with overriding edges in transmissibility and neutralizing antibody escape.

Omicron BA.l is heavily mutated from the original SARS-CoV-2 Wuhan strain, including more than 35 amino acid changes in S protein. Compared to 2 mutations associated with Delta at S-l receptor binding domain (Sl-RBD, residues 319-541), BA.l and BA.2 share 12 mutations, with BA. l and BA.2 each having additional 3 and 4 unique ones, respectively, that confers BA.2 a higher immune evasion. BAA and BA.5 have identical spike protein. They differ from BA.2 by having additional mutations at 69-70del, L452R, F486V and wild type amino acid at position Q493 within the spike protein (Table 14), contributing to their higher degree of immune escape than BA.2. BA.2 exhibits a 1.3- to 1.5-fold higher transmissibility and a 1.3-fold immune evasion than BA.1, consistent with the finding that BA.1 -immune sera neutralizes BA.2 with lower titers by a factor of 1.3 to 1.4 and that BA.2 reinfection can occur after BA.1. BA.4/BA.5 are more transmissible and resistant to BA. l/BA.2-immunity and monoclonal antibodies.

Furthermore, immune sera after a booster shot from UB-612 vaccinees would still have a lower ability to neutralize the live Wuhan virus with a titer reduction (50% geometric mean GMT, i.e., VNT50/FRNT50 titer) in the range from 10 to 50 folds (GMFR= ~10 to 50) as shown in Table 13, which fall short of the goal to develop a Pan-Sarbeco Vaccine.

Development of composition-updated (variant-specific) vaccines has been strongly advocated so as to fulfill this urgent need to prevent individuals from contracting SARS-CoV-2 Omicron BA.4/BA.5 to control the outbreaks and to reduce the resulting sufferings, including long- haul COVID and death.

The disclosed high precision designer vaccines (e.g. UB-612, UniCoVac-2, UniCoVac-3) prepared according to the vaccine composition as shown in Tables 15, 19 and 20 have 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 antibodydependent 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 leading to both increased infectivity and virulence and has been observed with mosquito-borne flaviviruses, HIV, and coronaviruses. The disclosed high precision vaccine compositions, employing only the Sl-RBD-sFc proteins as the B cell immunogens, are designed to avoid vaccine-induced disease enhancement by monitoring the quality and quantity of the antibody responses as they would dictate functional outcomes.

In additional to the shown advantages of UB-612 over other COVID vaccine using different platforms employing “Spike” only protein as the vaccine target as discussed extensively in EXAMPLE 9), our monovalent UniCoVac 2 and bivalent UniCoVac 3 would have the added benefits of variant-specific (Omicron BA.4/BA.5) composition-updated vaccines meeting the urgently called for COVID vaccine need. UniCoVac 2 would complement the existing UB-612 monovalent vaccine employing the original strain WuHan sequence derived Sl-RBD Wuhan-sFc protein as the B cell immunogen by providing a highly complementary Sl-RBD-Omicron BA.4/BA.5 variant specific sFc as the B cell immunogen so as to induce neutralizing antibodies that can effectively neutralize the currently prevalent BA.4/BA.5 variants.

A combo vaccine prepared according to Table 20 employing both Sl-RBD Wuhan-sFc (SEQ ID NO: 49) and Sl-RBD Omicron-BA.4/BA.5-sFc protein (SEQ ID NO:53) as the B cell immunogen would allow generation of complementary neutralizing antibodies directed against a broad spectrum of RBD of SARS-CoV-2. Such a breadth of neutralizing antibodies, coupled with the long duration immunological memories afforded by the conserved T cell immunity, through the incorporation of the SARS-CoV Th/CTL peptides (SEQ ID NOs: 27, 9, 34, 2, 35) and an idealized artificial Th peptide (SEQ ID NO: 36) as the catalyst for T cell activation, would allow the development of the most ideal Pan-Sabeco Virus vaccine compositions that are (1) SAFE due to the high precision and subunit nature of the vaccine platform; (2) can facilitate the elicitation of antibodies that bind to the CHO-expressed Sl-RBD-sFc protein covering from the original Wuhan strain to the latest Omicron BA.4/BA.5 strains and neutralize viral mediated cytopathic effect in a cell mediated neutralization assay; (3) can generate Thl prone T cell immunity to immediately fend off invading SARS-CoV-2 variants by activating IFN-y producing Th cells upon mucosal contact; (4) can generate viral antigen (M. M and S2) specific CD8+ cytolytic cells to clear the virally infected cells; and (5) provide long lasting immunity due to far enhanced immunological memory recall, to arrive at the ultimate goal of an ideal Pan-Sabeco Virus vaccine to prevent individuals from contracting SARS-CoV-2 Omicron BA.4/BA.5, to control the SARS-COV-2 outbreaks, and to reduce the resulting sufferings, including long-haul COVID and death.

EXAMPLE 11

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: 49) 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 COVID-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. 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. Since a decent level of pre-existing memory T cell immunity through prior infection or primary vaccination would be critical for control of SARS-CoV-2 reinfection and breakthrough infections, the incorporation in UB-612 multitope vaccine of the selected Th/CTL epitope peptides derived from highly conserved regions of Sarbevirus membrane (M), nucleocapsid (N) and spike S2 proteins (Table 10), that are structurally constrained from mutation and recognized by individuals who have recovered from COVID disease would be critical for such a global T vaccine to fend off SARS-CoV-2 infected cells regardless of Variants of Concern(VoCs). Specific multitope protein/peptide vaccine compositions for the prevention of infection by SARS-CoV-2 containing 20 pg/mL, 40ug/mL, 60 pg/mL, and 200 pg/mL (combined weight of the Sl-RBD-sFc fusion protein (e.g. Wuhan and/or Omicron strains) and Th/CTL peptides) are shown in Tables 15, 19 and 20. Since T cell vaccines based on this current invention can also be formulated independently for combinational injections with other B immunogen oriented vaccines, their representative formulations are also shown as Global T1-T4 vaccines (with only the Th/CTL peptides) at formulations with only Th/CTL epitope peptides at lOug/mL, 25ug/mL, or 50ug/mL as examples shown in Tables 21- 24

1. Immunogenicity Study in Rats

In a set of experiments conducted in rats, a proprietary mixture of Th/CTL peptides were added to the Sl-RBD-sFc fusion protein(s) for further assessment of optimal formulations and adjuvants and establishment of the cellular immunity components of the vaccine. These vaccine compositions were utilized in the following studies. a. Humoral Immunogenicity Testing in Rats

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 with adjuvant systems. 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 the S1-RBD:ACE2 binding inhibition ELISA test, the most potent inhibitory activity was seen as the best candidate formulations for further iterative experiments for next level of immunogenicity improvement. In the replicating virus neutralization assay against the Wuhan SARS-CoV-2 isolate, the 4 WPI immune sera induced by the vaccine composition could neutralize viral infection at VNT50 of >10,240 dilution fold. 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

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 UBI Asia. 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 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 Th 1 -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.

2. Representative Toxicity Studies in Preparation for Clinical Trials of vaccine products as shown with their respective formulations in Tables 15-20 and 21-24.

To enable clinical trials, the vaccine compositions were/are tested in a GLP-compliant repeatdose toxicology study in Sprague-Dawley rats for each of the products with the standard design and procedures 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 IFN-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 repeat-dose 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 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 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). A series of products through the multitope designs are to be tested based on the above procedures for safety before entering clinical trials for later generation product approvals.

EXAMPLE 12

Representative UB-SARS-CoV-2 Global T vaccines with long lasting immunity in Phase-1/2 Trials to eliminate SARS-CoV-2 variant-infected cells

Since a decent level of pre-existing memory T cell immunity through prior infection or primary vaccination would be critical for control of SARS-CoV-2 re-infection and breakthrough infections, UB-612 multitope vaccine, with the incorporation of the selected Th/CTL multitope peptides (Table 10) derived from highly conserved regions of Sarbevirus membrane (M), nucleocapsid (N) and spike S2 proteins, that are structurally constrained from mutation and recognized by individuals who have recovered from COVID-19 disease, would set forth the preexisting memory T cell immunity in the primary vaccination series that would dictate a significant potential of booster immunity for cross-protection against infection by VoCs including Delta and especially Omicron (B.1.1.529) which has a heavily-mutated S protein difficult to be cross-neutralized by antibodies.

Furthermore, virus-specific B-humoral and T-cellular responses act synergistically to protect the host from viral infection. Using humoral antibody response as a sole metric of protective immunity lacks full understanding of post-vaccination immune responses, as antibody response is shorter-lived than virus-reactive T cells.

As mentioned in Example 6, T-cell responses was assayed by ELISpot and Intracellular Cytokine Staining. The results indicated that, in phase 1 and 2 trials, UB-612 induced a long-lasting, robust Th 1 -predominant IFN-y⁺-T cell response measured by ELISpot, which confirmed that the precision-designed Th/CTL peptide pool is essential and principally responsible for the T cell responses while a more focused “S-l RBD” functional domain, lacking major Th/CTL epitopes, is used mainly as its B immunogen component. Overall, in the combined three clinical trials of a phasel primary series, an extended booster third-dose vaccination, and a phase-2 primary series, we have demonstrated that UB-612 vaccination (100 pg dose group) can induce substantial viral neutralizing antibodies with a long half-life that go in parallel with a long-lasting cellular immunity. As memory B and T cells are critical in secondary responses to infection, a successful vaccine must generate and maintain immunological memory), and to mount a rapid recall of effective humoral and cellular responses upon natural exposure or vaccine boosting. UB-612 has indeed demonstrated such important vaccine design features through these clinical studies.

While neutralizing antibody level correlates well with vaccine’s protection efficacy, substantial activation of CD4⁺ and CD8⁺ T cells by viral antigens are also critical for better duration of immunity and immunological memory. Early induction of functional SARS-CoV-2-specific T cells have also been found to be critical for rapid viral clearance and amelioration of disease. Thus, T cell responses elicited by Th/CTL peptides representing viral structural and non- structural proteins are of increasing interest for assessment in the control of infection as the virus derived peptides defines heterologous and COVID-19 induced T cell recognition.

The development of immunogens that can induce CD4+/CD8+ T cell responses to highly conserved epitopes across the SARS-CoV- 2 proteome, which are structurally constrained from mutation, conserved across VoCs and Sarbecoviruses, and recognized by individuals who have recovered from COVID-19 disease, could greatly augment current vaccines for SARS-CoV-2 given the emergence of variants that escape convalescent plasma and vaccine induced antibodies responses. UB-612, the first rationally designed multitope protein/peptide subunit COVID vaccine to activate both B- and T-cell immunities, contains an SI -receptor binding domain (Sl-RBD) - single chain Fc fusion protein produced in CHO cells, formulated with five designer Th and CTL epitope peptides known to bind to multiple Class I and Class II Major Histocompatibility Complexes (MHC-I and MHC-II) representing helper T-cell (Th) and cytotoxic T-cell (CTL) epitopes from Sarbecovirus conserved regions of the viral spike (S2), nucleocapsid (N) and membrane (M) proteins, and an extrinsic MHC class II epitope (UBIThOla) modified from measles virus fusion (MVF) protein which would serve as a catalyst for T cell activation (Figure 9A). The Th and CTL peptides are promiscuous epitopes that would allow for induction of memory recall, T cell activation and effector functions.

Based on the results of ELISpot and ICS mentioned in Example 6, the conclusion for UB-SARS- CoV-2 Global T vaccine could be provided as below. As seen in Table 10, amino acid sequences of the designed Th/CTL epitope peptides incorporated in our UB- SARS-CoV-2 Global T vaccine series (e.g. Wuhan, Alpha, Beta, Gamma, Delta, Omicron) are highly conserved throughout all SARS-CoV-2 VoCs. Subjects receiving UB-SARS-CoV-2 Global T vaccines would elicit Thl-oriented immunity with a robust CD8⁺ cytotoxic T cell response as observed in our phase 1 and 2 trials of UB-612, which is favorable for clearance of all Variants of Concern (VoCs) of SARS-CoV-2, an unprecedented powerful feature of our multitope SARS-COV-2 vaccine series over other currently employed SARS-CoV-2 vaccines (e.g. mRNA, DNA, inactivated viral lysates, or Vector based SARS-CoV-2).

Specific formulations for UB-Global T 1 to T 4 vaccines are shown in Tables 21-24 which can be co-administered in 0.1 to 0.5mL per dose with any other SARS-COV-2 vaccines derived from different platforms where humoral immune responses are more the focus for detection of neutralizing antibodies while optimal T cellular responses are being overlooked. Timely supply of optimal viral T immunogens would allow substantial activation of CD4⁺ and CD8⁺ T cells, critical for better duration of immunity and immunological memory. Early induction of functional SARS-CoV-2-specific T cells have also been found to be critical for rapid viral clearance and amelioration of disease. The employment of SARS-CoV-2 Th/CTL peptides as shown in Table 10 and formulations in Tables 21-24 that can induce CD4+/CD8+ T cell responses to highly conserved epitopes across the SARS-CoV-2 proteome, which are structurally constrained from mutation, conserved across VoCs and Sarbecoviruses, and recognized by individuals who have recovered from COVID-19 disease, could greatly augment current vaccines for SARS-CoV-2 given the emergence of variants that escape convalescent plasma and vaccine induced antibodies responses.

Table 1

Amino Acid Sequences of Membrane Glycoprotein M from SARS-CoV-2 and highly conserved CTL epitopes (previously validated by PBMC binding and stimulation assays) for use in vaccine design with KKK linker at the N-terminus

Table 2

Amino Acid Sequences of Nucleocapsid Phosphoprotein N from SARS-CoV-2 and highly conserved CTL and Th epitopes (previously validated by PBMC binding and stimulation assays) for use in vaccine design with KKK linker at the N-terminus

Table 3

Amino Acid Sequences of Surface Glycoprotein S from SARS-CoV-2 and highly conserved CTL and Th epitopes (previously validated by PBMC binding and stimulation assays) for use in vaccine design with KKK linker at the N-terminus

Table 4

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

Table 5

Wild-Type and Mutated Hinge Regions from human IgGl

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

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

Table 6

Amino Acid Sequences of Sl-RBD-sFc Fusion Proteins for SARS-CoV-2 VoCs (Beta, Delta and Omicron)

Table 7

Nucleic Acid Sequences of Sl-RBD-sFc Fusion Proteins for SARS-CoV-2 VoCs (Beta, Delta and Omicron)

Table 8

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 9

Examples of Optional Heterologous Spacers and CpG Oligonucleotides

Table 10

Conserved Th/CTL epitopes on membrane (M), nucleocapsid (N), and Spike-2 (S2) proteins across SARS-CoV-2 Variants of Concern (VoCs)used in T cell vaccine against COVID

a The presence of T cell epitopes is critical for the induction of B and T cell memory responses against viral antigens. SARS-CoV-2 CTL and Th epitopes, validated by HLA binding and T cell functional assays, are highly conserved between SARS-CoV-2 and SARS-CoV-1 viruses, with minor between- variant differences seen only at S957-984. The Wuhan wild-type peptides (M, N and S2x3) are employed for precision-design of UB-612 vaccine against COVID-19. Identification of T cell epitopes on SARS- CoV-1 (2003), determined using HLA -binding assays, were used to determine corresponding T cell epitopes in SARS-CoV-2 (2019) by sequence alignment. b Except for N969K (on BA. 1 through BA.5) and E98 IF (on BA. 1) within S957-984 peptide on the S2 spike protein, none of the other four designer epitope peptides for UB-612 vaccine has an aa-residue that overlaps with the reported mutation sites on Spike, M, and N proteins protein (Table 14). c At S957-984, there are minor sequence differences between Omicron BA.l and BA.2/BA.4/BA.5, marked in bold. Table 11

Comparison of post-booster viral-neutralizing antibody titers against SARS-CoV-2 wild-type (WT) and Delta variant by vaccines from different platforms

Abbreviation: MNA=Microneutralization assay; PNA=pseudotyped virus neutralization assay; PRNT=plaque reduction neutralization test; FRNT=focus reduction neutralization test; NA=not available; GMT=geometric mean titer; GMFI=geometric mean fold increase; GMFR=geometric mean fold reduction; WT=wild type virus; and Delta=the Delta variant of the SAR-CoV-2 WT. ^a Vaccine reported of post-booster GMT for NVX-CoV2373, mRNA-1273, BNT16b2, MVC-Covl901, Corona Vac, ADZ1222 (ChAdOxl nCov-19), and UB-612 in the present report. ^b GMTs against WT measured at 14 or 28 days post-booster third dose. ^c Post-2^nd dose GMTs at peak/pre-booster against WT. ^d Post-2^nd dose GMFIs at peak/pre-booster against WT. ^e Sources of Delta strain for assay: MNA & FRNT/live clinical isolate: PNA/pseudovirus: PRNT/WT recombinantly engineered with Delta spike. ^f GMFR, a fold value that the post-booster anti-Delta titer is reduced relative to the anti-WT titer.

Table 12. Pseudovirus-neutralization assay (pVNTso/IDso)

Abbreviation: PNA = pseudotyped virus neutralization assay; GMT = geometric mean titer; GMFR = geometric mean fold reduction relative to WT; WT = wild type strain of SARS-CoV-2; Omicrons = Omicron subvariants BA.1/BA.2/BA.5; ND = not determined. pVNT₅o & ID50 = 50% neutralization GMT by pseudoviruss assay ^a Vaccines reported of homologous booster (third dose) vaccination. ^b GMTs against WT measured at 14 or 28 days post-booster third dose. ^c UB-612 - Pseudovirus assays conducted with sera of subset participants (Phase-2 booster extension study) when Omicron infection were dominated sequentially by BA.2 and BA.5 subvariant.

Table 13. Live virus-neutralization assay (VNT50/FRNT50/ID50)

Abbreviation: MNA = Microneutralization assay; PRNT = plaque reduction neutralization test; FRNT = focus reduction neutralization test; GMT = geometric mean titer; GMFR = geometric mean fold reduction relative to WT; WT = wild type strain of SARS-CoV-2; Omicrons = Omicron subvariants BA.1/BA.2; ND = not determined. pVNTso & ID50 = 50% neutralization GMT by live virus assay; VNT50, ID50 & FRNT₅o = 50% neutralization GMT by live virus assay. ^a Vaccines reported of homologous booster (third dose) vaccination. ^b GMTs against WT measured at 14 or 28 days post-booster third dose.

Table 14. Mutation sites on SARS-CoV-2 Spike (S), and non-Spike envelope

(E), membrane (M) and nucleocapsid (N) proteins

a Reported mutation sites on Spike, E, M, and N proteins (Bold). b Omicron BA.4 and BA.5 have identical mutation site profile on Spike protein, which are more related to BA.2 than B A.1. Among BA.2, BAA and BA.5, the between-variant differences in mutation sites on S, E, M, and N proteins are marked in red. c Except for N969K (on BA.1 through BA.5) and L98 IF (on BA.1) within S_957-984_peptide on the S2 spike protein, none of the other four designer epitope peptides for UB-612 vaccine has an aa-residue that overlaps with the reported mutation sites on Spike, M, and N proteins protein.

Table 15

Composition of UB-612 (Wuhan) 200 pg/mL

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

Table 16

Composition of UB-613 (Wuhan) 40 pg/mL

Materials to be used for the Phase 2 and 2/3 clinical trials will be manufactured to cGMP Table 17

Composition of UB-614 (Omicron B.1.1.529) 40 pg/mL

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

Table 18

Composition of UB-615 (Wuhan+Omicron B.l.1.529) 40 pg/mL

Materials to be used for Phase 2 and 2/3 clinical trials are manufactured to cGMP Table 19

Composition of UniCoVac 2 Monovalent (Omicron BA.4/BA.5) 40 pg/mL

Materials to be used for the Phase 2 and 2/3 clinical trials will be manufactured to cGMP Table 20

Composition of UniCoVac 3 Bivalent (Wuhan+Omicron BA.4/BA.5) 40 pg/mL

Materials to be used for the Phase 2 and 2/3 clinical trials will be manufactured to cGMP Table 21

Composition of UB-Global T1 10 pg/mL

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

Table 22

Composition of UB-Global T2 25 pg/mL

Materials to be used for Phase 2 and 2/3 clinical trials are manufactured to cGMP Table 23

Composition of Global T3 vaccine 20 pg/mL

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

Table 24

Composition of Global T4 vaccine 50 pg/mL

¹ Materials to be used for Phase 2 and 2/3 clinical trials

Claims

1. The fusion protein comprising the Fc fragment of the IgG molecule and the bioactive molecule, wherein the Fc fragment is the single chain Fc (sFc), wherein the Fc fragment comprises the hinge region, wherein the hinge region is mutated and does not form disulfide bonds, wherein the hinge region comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 38 and 39, wherein the bioactive molecule is the receptor binding domain (RBD) of the S protein (S-RBD) from SARS-CoV-2 of SEQ ID NO: 40 or the variant form of S-RBD of SEQ ID NOs:41-44.

2. The fusion protein according to claim 1, wherein the hinge region comprises the amino acid sequence of SEQ ID NO: 39.

3. The fusion protein according to claim 1, wherein the fusion protein is selected from the group consisting of SEQ ID NOs: 49-53.

4. The pharmaceutical composition comprising the fusion protein according to claim 1 and the pharmaceutically acceptable carrier or excipient.

5. The method for producing a fusion protein according to claim 1 comprising: a) providing the bioactive molecule, wherein the bioactive molecule is the receptor binding domain (RBD) of the S protein (S-RBD) from SARS-CoV-2 Wuhan or one of its Variants of Concern, wherein the receptor binding domain (RBD) of the S protein (S- RBD) is selected from the group consisting of SEQ ID NOs: 40-44, b) providing the Fc fragment of an IgG molecule, wherein the Fc fragment comprises the hinge region, wherein the hinge region is mutated by substitution and/or deletion of the cysteine residue to form the mutated Fc, and the mutated Fc does not form disulfide bonds, wherein the hinge region comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 38 and 39, and c) combining the bioactive molecule and the mutated Fc through the hinge region.

6. The fusion protein selected from the group consisting of Sl-RBDVoC-sFc of SEQ ID NOs: 49-53.

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7. The composition comprising the fusion protein according to claim 6.

8. The composition according to claim 7, further comprising a Th/CTL peptide, wherein the Th/CTL peptide is derived from the SARS-CoV-2 M, N, or S protein, the pathogen protein, or any combination thereof, wherein the Th/CTL peptide is selected from the group consisting of SEQ ID NOs: 2-5, 7-12, 14-35,36 and any combination thereof.

9. The composition according to claim 8, wherein the Th/CTL peptides is selected from the group consisting of SEQ ID NOs: 2, 9, 22, 23, 27, 34, 35, 36 and any combination thereof.

10. The COVID vaccine composition comprising: a), the S-RBDVoC-sFc protein selected from the group of SEQ ID NOs:49-53; b). the Th/CTL peptide selected from the group consisting of SEQ ID NOs:2-5, 7-12, 14-36 and any combination thereof; c). the pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient is the adjuvant, buffer, surfactant, emulsifier, pH adjuster, saline solution, preservative, solvent, or any combination thereof.

11. The COVID vaccine composition according to claim 10, wherein the Th/CTL peptides in (b) is selected from the group consisting of SEQ ID NOs: 2, 9, 22, 23, 27, ,34, 35,36 and any combination thereof.

12. The COVID vaccine composition according to claim 11, wherein the pharmaceutically acceptable excipient is the combination of the CpGl oligonucleotide, ALUM(aluminum phosphate or aluminum hydroxide), histidine, histidine HC1»H2O, arginine HC1, TWEEN 80 (polyoxyethylene (20) sorbitan monooleate), hydrochloric acid, sodium chloride, and 2-phenoxyethanol in water.

13. The CO VID vaccine composition according to claim 12, wherein the pharmaceutically acceptable excipient is CpGl (SEQ ID NO: 67).

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14. The method for preventing COVID in a subject comprising administering the pharmaceutically effective amount of the vaccine composition according to claim 10 to the subject.

15. The method for preventing COVID in the subject comprising administering the pharmaceutically effective amount of the vaccine composition according to claim 11 to the subject.

16. The method for generating antibodies against SARS-CoV-2 comprising administering the pharmaceutically effective amount of the vaccine composition according to claim 10 to the subject.

17. The method for generating antibodies against SARS-CoV-2 comprising administering the pharmaceutically effective amount of the vaccine composition according to claim 11 to the subject.

18. The COVID vaccine composition compositing the components in the amounts shown in any one of Tables 15-20.

19. The cell line transfected with a cDNA sequence encoding the fusion protein according to claim 6.

20. The cell line according to claim 19 that is Chinese Hamster Ovary (CHO) cell line.

21. The cell line according to claim 19, wherein the cDNA sequence is selected from the group consisting of SEQ ID NOs: 60-64.

22. The Global COVID T vaccine composition comprising: a), the Th/CTL peptide, wherein the Th/CTL peptide is derived from the SARS-CoV-2 M, N, or S protein, a pathogen protein, or any combination thereof, wherein the Th/CTL

91 peptide is selected from the group consisting of SEQ ID NOs:2-5, 7-12, 14-36 and any combination thereof; b). the pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient is the adjuvant, buffer, pH adjuster, saline solution, preservative, solvent, or any combination thereof.

23. The Global COVID T vaccine composition according to claim 22, wherein the Th/CTL peptide is selected from the group consisting of SEQ ID NOs: 2, 9, 22, 23, 27, 34, 35, 36, 3, 4, 7, 8, 25, 26 and any combination thereof.

24. The Global COVID T vaccine composition according to claim 23, wherein the pharmaceutically acceptable excipient is the combination of a CpGl oligonucleotide, ALUM (aluminum phosphate or aluminum hydroxide), hydrochloric acid, sodium chloride, and 2-phenoxyethanol in water.

25. The Global CO VID T vaccine composition compositing the components in the amounts shown in Tables 21-24.

26. The fusion protein comprising the Fc fragment of an IgG molecule and the bioactive molecule, wherein the Fc fragment is the single chain Fc (sFc), wherein the Fc fragment comprises the hinge region, wherein the hinge region is mutated and does not form disulfide bonds, wherein the hinge region comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 38 and 39, wherein the bioactive molecule is the receptor binding domain (RBD)(SEQ ID NOs: 40 or 44) of the S protein (Sl-RBD) from SARS-CoV-2 wherein the Wuhan strain is of SEQ ID NO: 40, wherein the Omicron BA.4/BA.5 variant form is of SEQ ID NO: 44.

27. The fusion protein according to claim 1, wherein the fusion protein is selected from the group consisting of SEQ ID NOs: 49 and 53.

28. The fusion protein according to claim 1, wherein the hinge region comprises the amino acid sequence of SEQ ID NO: 39.

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29. The pharmaceutical composition comprising the fusion protein according to claim 1 and the pharmaceutically acceptable carrier or excipient.

30. The method for producing a fusion protein according to claim 1 comprising: a) providing the bioactive molecule, wherein the bioactive molecule is the receptor binding domain (RBD) of the S protein (S-RBD) from SARS-CoV-2 Wuhan (SEQ ID NO: 40) or one of its Omicron BA.4/BA.5 variant, wherein the receptor binding domain (RBD) of the S protein (S-RBD) is of SEQ ID NO: 44, b) providing the Fc fragment of an IgG molecule, wherein the Fc fragment comprises the hinge region, wherein the hinge region is mutated by substitution and/or deletion of the cysteine residue to form the mutated Fc, and the mutated Fc does not form disulfide bonds, wherein the hinge region comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 38 and 39, and c) combining the bioactive molecule and the mutated Fc through the hinge region.

31. The fusion protein selected from the group consisting of SI -RBD Omicron BA.4/BA.5 variant -sFc of SEQ ID NO: 53.

32. The composition comprising the fusion protein according to claim 31.

33. The composition according to claim 32, further comprising the Th/CTL peptide, wherein the Th/CTL peptide is derived from the SARS-CoV-2 M, N, or S protein, the pathogen protein, or any combination thereof, wherein the Th/CTL peptide is selected from the group consisting of SEQ ID NOs: 2, 9, 22, 23, 27, ,34, 35,36 and any combination thereof.

34. The composition according to claim 33, wherein the Th/CTL peptides is selected from the group consisting of SEQ ID NOs: 2, 9, 22, 23, 27, ,34, 35,36 and any combination thereof.

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35. The COVID vaccine composition comprising: a), the S-RBD Omicron BA.4/BA.5 variant-sFc protein selected from the group of SEQ ID

NO: 53; b). the Th/CTL peptide selected from the group consisting of SEQ ID NOs: 2, 9, 22, 23, 27,

34, 35,36 and any combination thereof; c). the pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient is an adjuvant, buffer, surfactant, emulsifier, pH adjuster, saline solution, preservative, solvent, or any combination thereof.

36. The COVID vaccine composition according to claim 35, wherein the Th/CTL peptides in (b) is selected from the group consisting of SEQ ID NOs: 2, 9, 22, 23, 27, 34, 35, 36 and any combination thereof.

37. The COVID vaccine composition according to claim 36, wherein the pharmaceutically acceptable excipient is the combination of a CpGl oligonucleotide, ALUM(aluminum phosphate or aluminum hydroxide), histidine, histidine HC1»H2O, arginine HC1, TWEEN 80 (polyoxyethylene (20) sorbitan monooleate), hydrochloric acid, sodium chloride, and 2-phenoxyethanol in water.

38. The CO VID vaccine composition according to claim 37, wherein the pharmaceutically acceptable excipient is CpGl (SEQ ID NO: 67).

39. The method for preventing COVID in the subject comprising administering the pharmaceutically effective amount of the vaccine composition according to claim 35 to the subject.

40. The method for preventing COVID in the subject comprising administering the pharmaceutically effective amount of the vaccine composition according to claim 36 to the subject.

41. The method for generating antibodies against SARS-CoV-2 Omicron BA.4/BA.5 variant comprising administering the pharmaceutically effective amount of the vaccine

94 composition according to claim 35 to the subject. The method for generating antibodies against SARS-CoV-2 Omicron BA.4/BA.5 variant comprising administering the pharmaceutically effective amount of the vaccine composition according to claim 36 to the subject. The COVID vaccine composition comprising the components in the amounts shown in any one of Tables 15, 19 and 20. The cell line transfected with a cDNA sequence encoding the fusion protein according to claim 31. The cell line according to claim 44 that is Chinese Hamster Ovary (CHO) cell line. The cell line according to claim 44, wherein the cDNA sequence is selected from the group consisting of SEQ ID NO: 64.

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