METHOD FOR THE PREVENTION AND TREATMENT OF CORONAVIRUS DISEASE

FIELD OF THE INVENTION

The present disclosure relates to method for preventing or reducing the severity of COVID- 19.

BACKGROUND OF THE INVENTION

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

Coronaviruses (family Coronaviridae, order Nidovirales) are large, enveloped, positive- stranded RNA viruses with a typical crown-like appearance (website: en.wikipedia.org/wiki/Coronavirus). Their viral genomes (26 to 32 kb) are some of the largest known among all RNA viruses. Coronaviruses are classified into four subgroups

(Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus), initially based on antigenic relationships of the spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. The Betacoronavirus subgroup includes HCoV-OC43, HCoV-HKUl, SARS-CoV, MERS-CoV, and SARS-CoV-2. Genetic recombination readily occurs between members of the same and of different subgroups providing opportunity for increased genetic diversity.

There is an urgent need for the development of methods for preventing or treating COVID- 19.

SUMMARY OF THE INVENTION

The disclosure provides methods of preventing or reducing the severity of COVID-19 in a subject, the method comprising administering a first immunogenic composition against SARS- CoV-2 to the subject, followed by a second immunogenic composition against SARS-CoV-2, wherein second immunogenic composition comprises: a. an S-RBD-sFc protein comprising a receptor binding domain (RBD) of the S protein of a SARS-CoV-2 spike protein or a variant thereof; and b. a Th/CTL peptide; wherein the second immunogenic composition optionally comprises one or more of: c. an aluminum phosphate- or an aluminum hydroxide-based adjuvant; d. a CpG oligonucleotide; and e. one or more pharmaceutically-acceptable excipients; and wherein the first immunogenic composition is different from the second immunogenic composition.

In some embodiments, the second immunogenic composition comprises a. an S-RBD- sFc protein comprising a receptor binding domain (RBD) of the S protein of a SARS-CoV-2 spike protein or a variant thereof; b. a Th/CTL peptide; c. an aluminum phosphate- or an aluminum hydroxide-based adjuvant; d. a CpG oligonucleotide; and e. optionally, one or more pharmaceutically-acceptable excipients; and wherein the first immunogenic composition is different from the second immunogenic composition.

In some embodiments, the first immunogenic composition comprises one or more proteins or peptides, nucleic acid molecules (e.g., RNA or DNA), viral vectors, or whole viruses.

In some embodiments, the first immunogenic composition comprises a spike protein of SARS-CoV-2, or a variant and/or fragment thereof (e.g., an RBD-containing fragment thereof).

In some embodiments, the first immunogenic composition is selected from NVX- CoV2372 and MVC-COV1901.

In some embodiments, the first immunogenic composition comprises a nucleic acid molecule encoding a spike protein of SARS-CoV-2, or a variant and/or fragment thereof (e.g., an RBD-containing fragment thereof).

In some embodiments, the first immunogenic composition is selected from mRNA-1273 and BNT162b2.

In some embodiments, the first immunogenic composition comprises a viral vector which comprises a sequence encoding an immunogen of SARS-CoV-2, or a variant or fragment thereof, wherein the immunogen is optionally a spike protein or a fragment thereof (e.g., an RBD- containing fragment thereof).

In some embodiments, the viral vector is an adenoviral vector or a parainfluenza virus vector (e.g., hPIV2).

In some embodiments, the vector is an adenoviral vector.

In some embodiments, the first immunogenic composition is selected from the group consisting of AZD1222, Janssen COVID-19 vaccine (JNJ-78436735), and Sputnik V (Gam- CO VID- Vac).

In some embodiments, the first immunogenic composition comprises whole SARS-CoV- 2 virus.

In some embodiments, the first immunogenic composition is CoronaVac.

In some embodiments, the first immunogenic composition comprises a composition of (a)-(b) or (a)-(d) (see above), except that the S-RBD-sFc protein and/or the amount of one or more components of the composition is different from that of the second composition.

In some embodiments, the first immunogenic composition is administered one time before the second immunogenic composition is administered.

In some embodiments, the first immunogenic composition is administered two times before the second immunogenic composition is administered.

In some embodiments, the second immunogenic composition is administered within about 2.5 to 4.5 months after the first immunogenic composition; within about 3 to 4 months of the first immunogenic composition; about three months after the first immunogenic composition; or about six or more months (e.g., about 6, 7, 8, 9, 10, or 11 months, or about 1, 2, 3, 4, or 5 years) after the first immunogenic composition.

In some embodiments, the second immunogenic composition comprises: a. a S-RBD-sFc protein comprising a receptor binding domain (RBD) of the S protein of SARS-CoV-2 (SEQ ID NO: 20), an S-RBD-sFc protein comprising a RBD of the S protein of SARS-CoV-2 SA, beta variant, both, or a variant(s) thereof; and b. a Th/CTL peptide selected from the group consisting of SEQ ID NOs: 9-16, 19, 35-36, 39-100, 145-165, 345-348, 350, 351, 362-365, any combination thereof, variants thereof, or combinations including variants thereof; wherein the composition further optionally includes one or more of: c. an aluminum hydroxide-based adjuvant and a CpG oligonucleotide adjuvant; and d. one or more a pharmaceutically acceptable excipients.

In some embodiments, the second immunogenic composition comprises: a. a S-RBD-sFc protein comprising a receptor binding domain (RBD) of the S protein of SARS-CoV-2 (SEQ ID NO: 20), an S-RBD-sFc protein comprising a RBD of the S protein of SARS-CoV-2 SA, beta variant, both, or a variant(s) thereof; b. a Th/CTL peptide selected from the group consisting of SEQ ID NOs: 9-16, 19, 35-36, 39-100, 145-165, 345-348, 350, 351, 362-365, any combination thereof, variants thereof, or combinations including variants thereof; c. an aluminum hydroxide- based adjuvant and a CpG oligonucleotide adjuvant; and d. optionally one or more a pharmaceutically acceptable excipients.

In some embodiments, the S-RBD-sFc protein of the second immunogenic composition comprises a receptor binding domain (RBD) of the S protein of SARS-CoV-2 (SEQ ID NO: 20), or a variant thereof, and optionally wherein the S-RBD-sFc protein is of SEQ ID NO: 235, or a variant thereof.

In some embodiments, the S-RBD-sFc protein of the second immunogenic composition comprises a RBD of the S protein of SARS-CoV-2 SA, beta variant, or a variant thereof.

In some embodiments, the second immunogenic composition comprises an S-RBD-sFc protein comprising a receptor binding domain (RBD) of the S protein of SARS-CoV-2 (SEQ ID NO: 20), or a variant thereof, and an S-RBD-sFc protein comprising an RBD of the S protein of SARS-CoV-2 SA, beta variant, or both, or a variant thereof or of both.

In some embodiments, the second immunogenic composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the Th/CTL peptides.

In some embodiments, the second immunogenic composition comprises 6 of the Th/CTL peptides.

In some embodiments, the second immunogenic composition comprises Th/CTL peptides which comprise SEQ ID NOs: 345, 346, 347, 348, 361, and 66, or variants of one or more thereof.

In some embodiments, the second immunogenic composition comprises Th/CTL peptides which comprise SEQ ID NOs: 345, 346, 347, 348, 361, and 66.

In some embodiments, each of the Th/CTL peptides are present in the second immunogenic composition in equal-weight amounts.

In some embodiments, the ratio (w:w) of the S-RBD-sFc protein to the total weight of the mixture of Th/CTL peptides in the second immunogenic composition is 88:12.

In some embodiments, the composition comprises a pharmaceutically acceptable excipient which optionally is an adjuvant, buffer, surfactant, emulsifier, pH adjuster, saline solution, preservative, solvent, or any combination thereof.

In some embodiments, the second immunogenic composition comprises an aluminum phosphate-based adjuvant.

In some embodiments, the second immunogenic composition comprises an aluminum hydroxide-based adjuvant.

In some embodiments, the second immunogenic composition comprises a CpG oligonucleotide adjuvant.

In some embodiments, the second immunogenic composition comprises a pharmaceutically acceptable excipient which is optionally selected from the group consisting of a CpG oligonucleotide, an aluminum hydroxide-based adjuvant, histidine, histidine HC1*H2O, arginine HC1, TWEEN 80 (polyoxyethylene (20) sorbitan monooleate), hydrochloric acid, sodium chloride, 2-phenoxyethanol, water, and any combination thereof.

In some embodiments, the second immunogenic composition comprises about 0.5-20, 1- 10, or 2-5 pg of a CpG oligonucleotide.

In some embodiments, the second immunogenic composition comprises about 1-10 pg of a CpG oligonucleotide.

In some embodiments, the second immunogenic composition comprises about 2-5 pg of a CpG oligonucleotide.

In some embodiments, the second immunogenic composition comprises about 2 pg of a CpG oligonucleotide.

In some embodiments, the second immunogenic composition: (a) the Th/CTL peptide is a mixture of SEQ ID NOs: 345, 346, 347, 348, 361, and 66, and each peptide is present in the mixture in equal-weight amounts; and (b) the pharmaceutically acceptable excipient is a combination of a CpGl oligonucleotide, an aluminum hydroxide- or aluminum phosphate-based adjuvant, histidine, histidine HC1*H2O, arginine HC1, polyoxyethylene (20) sorbitan monooleate, hydrochloric acid, sodium chloride, and 2-phenoxyethanol in water.

In some embodiments, the second immunogenic composition, the total amount of the S- RBD-sFc protein is between about 2 pg to about 200 pg; and the total amount of the Th/CTL peptides is between about 1 pg to about 25 pg.

In some embodiments, in the second immunogenic composition, the total amount of the S- RBD-sFc protein is about 8.8 pg; and the total amount of the Th/CTL peptides is about 1.2 pg.

In some embodiments, in the second immunogenic composition, the total amount of the S- RBD-sFc protein is about 26.4 pg; and the total amount of the Th/CTL peptides is about 3.6 pg.

In some embodiments, in the second immunogenic composition, the total amount of the S- RBD-sFc protein is about 88 pg; and the total amount of the Th/CTL peptides is about 12 pg.

In some embodiments, the method reduces the severity of one or more symptoms of COVID-19, prevents hospitalization for COVID-19, reduces the length of hospitalization for COVID-19, and/or maintains vaccine-induced antibodies above protective threshold.

The disclosure further provides methods for preventing or reducing the severity of COVID-19 in a subject, the method comprising administering the second immunogenic composition is administered about 6.5-11 or 7-9 months after the first dose (or only) dose of the first immunogenic composition.

The disclosure further provides methods for producing antibodies in a subject, the method comprising administering first and second immunogenic compositions as set forth herein to the subject.

In some embodiments, the method protects against variants of SARS-CoV-2 and breakthrough cases thereof.

The disclosure further provides methods for preventing or reducing the severity of COVID-19 in a subject, the method comprising administering three doses of an immunogenic composition against SARS-CoV-2 to the subject, wherein the immunogenic composition is as described as a second immunogenic composition described herein, and the three doses are administered within about 5 months of one another.

In some embodiments, the second dose is administered within about 2 weeks to about 1.5 months after the first dose. In some embodiments, the second dose is administered within about 1 month after the first dose.

In some embodiments, the third dose is administered within about 2.5 months to about 4.5 months after the first dose.

In some embodiments, the third dose is administered about 3 to about 4 months after the first dose.

In some embodiments, the third dose is administered about 3 months after the first dose.

In some embodiments, the methods further comprise a booster dose at 6.5-11 or 7-9 months after the first dose.

In some embodiments of any of the methods listed or described herein, the vaccination regimen (e.g., heterologous boosting or accelerated dosing, optionally followed by a boost) is carried out on a seasonal basis (e.g., each fall or winter).

In some embodiments of any of the methods described herein, the heterologous boost is carried out as a seasonal boost after the primary boost.

BRIEF DESCRIPTION OF THE DRAWINGS

Figs. 1A and IB. Flow of the phase-1 trial of UB-612 trial with extended booster study. Sixty healthy young adults, male and female, aged 20 to 55 years were enrolled for the primary series of the open-label, 196-day phase-1 study of UB-612 [NCT04545749]. They were administered intramuscularly with two vaccine doses at 10, 30, or 100 pg. All, but one, participants completed the study. The extension study [NCT04967742] that involved 50 enrolees was conducted between Days 255 to 316, a time period over 6 months after the second vaccine shot. The 50 participants in 10-pg (n = 17), 30-pg (n = 15), and 100-pg (n = 18) dose groups received a booster UB-612 dose at 100 pg and were followed up for 14 days for interim evaluation. They are to be monitored until 84 days post-booster.

Figs. 2A and 2B. Selected solicited local and systemic reactions within 7 days of each vaccination. Both local and systemic reactions are shown as the percentage of participants who reported grade 1 (mild; yellow) or grade 2 (moderate, orange) local (Fig. 2A) and systemic (Fig. 2B) adverse reactions.

Fig. 3. T-cell response by IFN-y ELISpot after restimulation with RBD+Th/CTL or Th/CTL in the primary 2-dose vaccination series of the 196-day Phase- 1 trial. T-Cell responses were measured by IFN-y ELISpot with PBMC cells from young adults (20 to 55 years) in the phase- 1 trial of UB-612 with three dose groups of 10, 30, and 100 pg (n = 20 each). The Y-axis shows the number of cytokine secreting cells per 10 ⁶ PBMC cells in ELISpot assays and the X- axis depicts IFN-y (Thl) response to restimulation with Sl-RBD+Th/CTL and IFN-y response to pooled Th/CTL peptides. Brown dots represent Day 0, blue dots represent Day 7, dark green dots represent Day 28 (before second vaccination), gray dots represent Day 35 and green dots Day 196. Of note, in both stimulation cases, the T cell responses (SFU/l.OxlO ⁶ cells) at Day 196 retained half (50%) that at Day 35, i.e., 121-vs.-254 for RBD+Th/CTL and 86.8-vs.-173 for Th/CTL. This suggests the immune T-cell responses in vaccine recipients are substantially long-lasting, over 6 months. In addition, the bulk of the T cell responses (-70%) were contributed by the presence of Th/CTL as compared with RBD+Th/CTL.

Fig. 4. NHP studies showing over 5-fold increase of neutralizing antibodies in accelerated 3-dose regimen. Neutralizing antibodies were measured at days 42, 70, and 77 in NHPs vaccinated with the indicated amounts of vaccine.

Fig. 5. Three-dose vaccination raises neutralizing antibodies substantially. Levels of neutralizing antibodies are above a reported protective threshold, thus indicating a range for a third dose in a 3-dose accelerated vaccination regimen.

Fig. 6. Preclinical studies in non-human primates (NHPs) testing UB-612 against WA and Delta at the indicated amounts. VNT50 results are shown.

Fig. 7. Neutralizing antibody loss against variants of concern, Beta VOC and Delta VOC.

Figs. 8-23. Neutralization antibody data obtained in studies including a booster dose of UB-612.

Fig. 24. SARS-CoV-2 Omicron BA.l and BA.2 amino acid substitutions and neutralization antibody responses. Panel A shows the amino acid substitutions in Omicron’s BA.l and BA.2 sublineage spike protein. The upper part is the S protein diagram, and the lower part shows the substitutions. The and “+” represent sequence identical, deletion, and insertion in Omicron BA.l and BA.2 compared with the US-WA1/2020 virus, respectively. Panel B shows the GMT VNT50 neutralizing antibody titers against SARS-CoV-2 ancestral strain Victoria/ 1/2020 (VIC01/2020) and Omicron (B.1.1.529) variant sublineages BA.l and BA.2 in sera from Phase 1 trial (V123) participants (n=15). The sera were collected at 28 days after 2 doses and at 14 days after the booster dose with UB-612 (100 pg). Data expressed in the reciprocal dilutions for each serum sample and GMT (95% CI) are plotted. GMT, geometric mean titers; VNT, virus neutralization test.

Fig. 25. UB-612 stimulated durable immunity and boosted neutralizing antibodies 75- fold over pre-boost titers (V-123). Geometric mean titer (GMT) of neutralizing antibody in subjects vaccinated with 100 pg (N=18), 30 pg (N=15), or 10 pg (N=17) of UB-612 and boosted with 100 pg during Phi (V-122/V-123) study. Bars represent the GMT+ 95% CI. Neutralizing titers expressed in International Units by comparing to neutralizing titers of the WHO international standards against Wuhan live virus. Fig. 26. NAbs against SARS-CoV-2 or omicron variants after booster of UB-612* compared to booster dose of BNT vaccine.

Fig. 27. RBD-specific antibody responses. IgG binding titers against SARS-CoV-2 major VOCs in sera collected 28 days after 2 doses and 14 days after 3 doses with UB-612 (100 pg) from Phase 1 trial participants (n=15). The loss of antibody binding to the RBD of variants compared with the original RBD (ancestral strain) remains stable between 2 and 3 doses of UB- 612 vaccine, despite a high increase in levels of binding antibodies to RBD. The ratios of original RBD to variants are 0.9, 2.4, 1.3, 1.7, and 3.6 (after 2 doses) and 0.9, 1.8, 1.4, 1.5, and 3.7 (after booster, 3 doses) for Alpha, Beta, Delta, Gamma, and Omicron, respectively. IgG, immunoglobulin G; RBD, receptor-binding domain; VOC, Variant of Concern; WHO, world health organization.

Fig. 28. Spike protein- specific binding IgG against SARS-CoV-2 major VOCs in the sera of Phase 1 participants (n=15) collected 30 days after the second dose and at 14 days after the third dose of UB-612 vaccination. The results of CoV-2 N in binding assay indicate that participants were not naturally infected with SARS-CoV-2 (<10 BAU/mL). After 2 doses, the loss in binding titers to the spike protein of VOC compared with the original (ancestral) spike is 1.6, 3.3, 2.0, and 3.3 for Alpha, Beta, Gamma, and Delta, respectively. After a booster dose these numbers remain relatively stable at 1.3-, 1.7-, 2.0-, and 2.1-fold, for Alpha, Beta, Gamma, and Delta, respectively. Numbers above each bar represent GMT and 95% CI. GMT, geometric mean titer; IgG, immunoglobulin G; VOC, Variant of Concern.

Fig. 29. IgG binding titers against SARS-CoV-2 variants in individuals at 28 days post 2 doses (2x UB-612) and at 14 days post 3 doses (3x UB-612) of UB-612 vaccination. The loss of antibody bindings to RBD of variants compared with the original RBD (ancestral strain) remains stable between 2 doses and 3 doses of UB-612 vaccine despite a high increase in levels of binding antibodies to RBD. The ratios of original RBD to variants (from left to right, e.g., RBD E484K to RBD V367F) are 2.0, 3.0, 1.8, 1.3, 1.3, 2.1, 1.4, and 1.2 for 2 doses, respectively, and 2.0, 2.5, 1.9, 1.4, 1.4, 2.0, 1.6, and 1.4 for 3 doses, respectively. Numbers above each bar represent GMT and 95% CI. GMT, geometric mean titer; RBD, receptor-binding domain.

Fig. 30. ACE2 binding blocking antibody titers in vaccinated participants at 30 days post 2 doses (2x UB-612) and at 14 days post 3 doses (3x UB-612) of UB-612 vaccination (n=15). A: spike protein (S):ACE2 blocking Ab against VOCs; B: RBD:ACE2 blocking Ab against VOCs. After 2 doses, the loss in binding inhibition for the spike protein of VOC compared with the original (ancestral) spike is none, 2.0, and 2.0 for Alpha, Beta, and Gamma, respectively. After a booster dose these numbers remain relatively stable at none, 1.7, 2.1, and 2.3-fold for Alpha, Beta, and Gamma, respectively (Panel A). Binding inhibition for the RBD protein of VOC compared with the original (ancestral) spike is none, 1.3, and 1.3 for Alpha, Beta, and Gamma, respectively. After a booster dose, these numbers remain relatively stable at none, 2.6, 1.9-fold for Alpha, Beta, and Gamma, respectively (Panel B). Numbers above each bar represent GMT and 95% CI. GMT, geometric mean titer; RBD, receptor-binding domain; VOC, Variant of Concern.

Fig. 31. RBD binding IgG (Panel A) and spike binding IgG (Panel B) against SARS- CoV-2 original Wuhan isolate in vaccinated participants at 30 days post 2 doses (2x UB-612) and at 14 days post 3 doses (3x UB-612) of UB-612 vaccination from the Phase 1 study (V123 study) (n=15). Sera from a subset of UB-612 vaccinated participants from the Phase 2 trial (V205) (n=84) drawn at 14 days post 2 doses were also included. For EU-approved vaccines, sera were collected from vaccinated participants (given at 1 or 2 doses) after 7, 8, 8, and 34 days (median) for mRNA1273, BNT162b2, ChadOXl, and Ad26. COCV2.S, respectively. The median time between doses was 3-4 weeks (for ChadOXl vaccine median time was 66 days and Ad26. COCV2.S was given at a single dose) (7). Numbers above each bar represent GMT and 95% CI. Note: The GMT numbers in this figure are different than those published in Fig. 1 by Goldblatt et al (18). This is because the samples described in the Goldblatt et al paper were above the upper limit of the assay, and therefore were assigned arbitrary values. In this figure those samples were further diluted and retested to determine the exact titers with accurate concentrations at the upper limit. GMT, geometric mean titer; IgG, immunoglobulin G; RBD, receptor-binding domain.

Fig. 32. Estimated UB-612 efficacy after 2 and 3 doses. A model bridging vaccine- induced RBD IgG response to vaccine efficacy against symptomatic COVID- 19 caused by ancestral Wuhan (18). Estimated efficacy of UB-612 after 2 doses is -72% (CI, 70%-80%) based on RBD binding IgG antibodies from 15 participants (Phase 1) (GMT 235 BAU/mL, 95% CI, 158-350, -82% (CI, 80%-85%) based on RBD binding IgG antibodies (GMT 494 BAU/mL, 95% CI, 337-725, shown in this graph), and -95% (93%-97%) after a booster vaccination (GMT 6767 (95% CI, 4142-11,057). IgG, immunoglobulin G; RBD, receptor-binding domain.

Fig. 33. Graph showing live virus neutralization, V-123 (n=10), results as described in Example 6 herein.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is directed to methods of preventing or reducing the severity of COVID- 19 and involve the use of a SARS-CoV-2 multitope peptide/protein-based vaccine containing Sl-RBD-sFc as described herein. The invention utilizes amino acid sequences from SARS-CoV-2 proteins, peptide immunogen constructs, and formulations thereof vaccines for the prevention and treatment of COVID-19. In some embodiments, the disclosure provides heterologous boosting methods as described herein. In some embodiments, the heterologous boosting methods include the use of compositions comprising Sl-RBD-sFc fusion proteins and Th/CTL peptides, e.g., as described herein, as boosters to different, previously administered COVID- 19 vaccines, as described further below. In some embodiments, the disclosure provides accelerated dosing methods as described herein.

Aspects of the disclosed invention is discussed in further detail below.

General

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

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Hence, the phrase “comprising A or B” means including A, or B, or A and B. It is further to be understood that all amino acid sizes, and all molecular weight or molecular mass values, given for polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosed method, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

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

The term “COVID- 19”, as used herein, refers to the human infectious disease caused by the SARS-CoV-2 viral strain, and variants thereof (e.g., as described herein). COVID- 19 was initially known as SARS-CoV-2 acute respiratory disease. The disease may initially present with few or no symptoms, or may develop into fever, coughing, shortness of breath, pain in the muscles and tiredness. Complications may include pneumonia and acute respiratory distress syndrome. A. RECEPTOR-BASED ANTIVIRAL THERAPIES FOR THE TREATMENT OF COVID- 19 IN INFECTED PATIENTS

The present disclosure is directed to the use of fusion proteins comprising a bioactive molecule and portions of an immunoglobulin molecule in methods described herein. Various aspects of the present disclosure relate to fusion proteins, compositions thereof, and methods for making and using the disclosed fusion proteins. The disclosed fusion proteins are useful for extending the serum half-life of bioactive molecules in an organism. In some embodiments, the fusion proteins are components of a composition that is used in heterologous boosting methods described herein.

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

1. Fusion Protein

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

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

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

The disclosed fusion protein can have any of the following formulae: (B)-(Hinge)-(C _H 2-C _H 3) or

(C _H 2-C _H 3)-(Hinge)-(B) or

(B)-(L) _m -(Hinge)-(C _H 2-C _H 3) or

(C _H 2-C _H 3)-(Hinge)-(L) _m -(B) wherein

“B” is a bioactive molecule;

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

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

“L” is an optional linker; and

“m” may be an any integer or 0.

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

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

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

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

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

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

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

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

The Fc region and/or Fc fragment can include a hinge region found in some immunoglobulin isotypes (IgA, IgD, and IgG). In certain embodiments, the Fc fragment is modified by mutating the hinge region so that it does not contain any Cys and cannot form disulfide bonds. The hinge region is discussed further below. The Fc fragment of the disclosed fusion protein is preferably a single chain Fc. As used herein, “single chain Fc” (of “sFc”) means that the Fc fragment is modified in such a manner that prevents it from forming a dimer (e.g., by chemical modification or mutation addition, deletion, or substation of an amino acid).

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

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

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

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

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

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

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

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

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

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

In one embodiment, the biologically active molecule is the S protein of SARS-CoV-2 (SEQ ID NO: 20). In certain embodiments, the biologically active molecule is the receptor binding domain (RBD) of the S protein (S-RBD or Sl-RBD) of SARS-CoV-2 (SEQ ID NO: 226), which corresponds to amino acid residues 331-530 of the full-length S protein. In certain embodiments, the cysteine (C) residues at positions 61 and 195 of the S-RBD sequence of SEQ ID NO: 226 are mutated to alanine (A) residues, as shown in SEQ ID NO: 227 (residues 61 and 195 of S-RBD correspond to residues 391 and 525 of the full-length S protein of SEQ ID NO: 20). The mutated S-RBD sequence is also referred to as S-RBDa in this disclosure. The C61A and Cl 95 A mutations in the S-RBD sequence are introduced to avoid a mismatch of disulfide bond formation in the recombinant protein expression.

In another embodiment, the biologically active molecule is the S protein of SARS-CoV-2 SA, beta variant (see Figs. 33 and 34 of WO 2021/168305). In certain embodiments, the biologically active molecule is the receptor binding domain (RBD) of the S protein (S-RBD or S 1- RBD) of SARS-CoV-2 SA, beta variant (see Figs. 33 and 34 of WO 2021/168305), which corresponds to amino acid residues 331-530 of the full-length S protein. In certain embodiments, the cysteine (C) residues at positions 61 and 195 of the S-RBD sequence are mutated to alanine (A) residues, as shown in Figs 33 and 34 of WO 2021/168305 (residues 61 and 195 of S-RBD correspond to residues 391 and 525 of the full-length S protein of Figs. 33 and 34 of WO 2021/168305).

In another embodiment, the biologically active molecule includes both the S protein of SARS-CoV-2 (SEQ ID NO: 20) and the S protein of SARS-CoV-2 SA, beta variant (Figs. 33 and 34 of WO 2021/168305). In certain embodiments, the biologically active molecule is the receptor binding domain (RBD) of the S protein (S-RBD or Sl-RBD) of SARS-CoV-2 (SEQ ID NO: 20), and the RBD of the S protein of SARS-CoV-2 SA, beta variant (Figs. 33 and 34 of WO 2021/168305).

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

2. Compositions

In certain embodiments, the present invention relates to compositions, including pharmaceutical compositions, comprising the fusion protein and a pharmaceutically acceptable carrier, adjuvant, and/or other excipients such as diluents, additives, stabilizing agents, preservatives, solubilizing agents, buffers, and the like.

Pharmaceutical compositions can be prepared by mixing the fusion protein with optional pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers include solvents, dispersion media, isotonic agents and the like. Examples of carriers include water, saline solutions or other buffers (such as phosphate, citrate buffers), oil, alcohol, proteins (such as serum albumin, gelatin), carbohydrates (such as monosaccharides, disaccharides, and other carbohydrates including glucose, sucrose, trehalose, mannose, mannitol, sorbitol or dextrins), gel, lipids, liposomes, stabilizers, preservatives, antioxidants including ascorbic acid and methionine, chelating agents such as EDTA; salt forming counter-ions such as sodium; non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG), or combinations thereof.

Pharmaceutical compositions can contain one or more adjuvant that act(s) to accelerate, prolong, or enhance the immune response to the fusion protein without having any specific antigenic effect itself. Adjuvants used in the pharmaceutical composition can include oils, oil emulsions, aluminum salts, calcium salts, immune stimulating complexes, bacterial and viral derivatives, virosomes, carbohydrates, cytokines, polymeric microparticles. In certain embodiments, the adjuvant can be selected from alum (potassium aluminum phosphate), aluminum phosphate (e.g. ADJU-PHOS®), aluminum hydroxide (e.g. ALHYDROGEL®), calcium phosphate, incomplete Freund’s adjuvant (IFA), Freund’s complete adjuvant, MF59, adjuvant 65, Lipovant, ISCOM, liposyn, saponin, squalene, L121, EMULSIGEN®, EmulsIL-6n®, monophosphoryl lipid A (MPL), Quil A, QS21, MONTANIDE® ISA 35, ISA 50V, ISA 50V2, ISA 51, ISA 206, ISA 720, liposomes, phospholipids, peptidoglycan, lipopolysaccahrides (LPS), ASO1, ASO2, ASO3, ASO4, AF03, lipophilic phospholipid (lipid A), gamma inulin, algammulin, glucans, dextrans, glucomannans, galactomannans, levans, xylans, dimethyldioctadecylammonium bromide (DDA), as well as the other adjuvants and emulsifiers.

In some embodiments, the pharmaceutical composition contains MONTANIDE™ ISA 51 (an oil adjuvant composition comprised of vegetable oil and mannide oleate for production of water-in-oil emulsions), TWEEN® 80 (also known as: Polysorbate 80 or Polyoxyethylene (20) sorbitan monooleate), a CpG oligonucleotide, and/or any combination thereof. In other embodiments, the pharmaceutical composition is a water-in-oil-in-water (i.e., w/o/w) emulsion with EMULSIGEN or EMULSIGEN D as the adjuvant.

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

Pharmaceutical compositions can be formulated as immediate release or for sustained release formulations. Additionally, the pharmaceutical compositions can be formulated for induction of systemic, or localized mucosal, immunity through immunogen entrapment and coadministration with microparticles. Such delivery systems are readily determined by one of ordinary skill in the art. Pharmaceutical compositions can be prepared as injectables, either as liquid solutions or suspensions. Liquid vehicles containing the S-RBD peptide immunogen construct can also be prepared prior to injection. The pharmaceutical composition can be administered by any suitable mode of application, for example, i.d., i.v., i.p., i.m., intranasally, orally, subcutaneously, etc. and in any suitable delivery device. In certain embodiments, the pharmaceutical composition is formulated for subcutaneous, intradermal, or intramuscular administration. Pharmaceutical compositions suitable for other modes of administration can also be prepared, including oral and intranasal applications.

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

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

The pharmaceutical compositions can also contain more than one active compound. For example, the formulation can contain one or more fusion protein and/or one or more additional beneficial compound(s). The active ingredients can be combined with the carrier in any convenient and practical manner, e.g., by admixture, solution, suspension, emulsification, encapsulation, absorption and the like, and can be made in formulations such as powder (including lyophilized powder), suspensions that are suitable for injections, infusion, or the like. Sustained-release preparations can also be prepared. In certain embodiments, the pharmaceutical composition contains the fusion protein for human use. The pharmaceutical compositions can be prepared in an appropriate buffer including, but not limited to, citrate, phosphate, Tris, BIS-Tris, etc. at an appropriate pH and can also contain excipients such as sugars (50 mM to 500 mM of sucrose, trehalose, mannitol, or mixtures thereof), surfactants (e.g., 0.025% - 0.5% of TWEEN 20 or TWEEN 80), and/or other reagents. The formulation can be prepared to contain various amounts of fusion protein. In general, formulations for administration to a subject contain between about 0.1 pg/mL to about 400 pg/mL. In certain embodiments, the formulations can contain between about 0.5 pg/mL to about 50 pg/mL; between about 1.0 pg/mL to about 50 pg/mL; between about 1 pg/mL to about 25 pg/mL; or between about 10 pg/mL to about 25 pg/mL of fusion protein. In specific embodiments, the formulations contain about 1.0 pg/mL, about 5.0 pg/mL, about 10.0 pg/mL, or about 25.0 pg/mL of fusion protein. In other embodiments, the formulations can contain between about 50 pg/mL to about 300 pg/mL; between about 100 pg/mL to about 250 pg/mL; or between about 150 pg/mL to about 200 pg/mL of fusion protein. In other specific embodiments, the formulations include about 176 pg/mL of fusion protein and 0.5 mL is administered per dose.

3. Methods

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

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

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

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

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

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

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

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

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

In some embodiments, the fusion protein is administered in a composition that also includes Th/CTL epitopes, e.g., as described herein, as well as, optionally, one or more adjuvant and/or excipient.

Furthermore, as explained further below, in some embodiments, the fusion protein can be used in an immunogenic composition in heterologous boosting methods.

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

An aspect of the invention relates to the use of multitope protein/peptide vaccine compositions for the prevention of infection by SARS-CoV-2.

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

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

Due to the clear advantages of a strong Sl-RBD vaccine component, the multitope protein/peptide vaccine composition comprises the SI -receptor-binding region-based designer protein described in Part A above. As described above, Sl-RBD-sFc is a recombinant protein made through a fusion of Sl-RBD of SARS-CoV-2 to a single chain fragment crystallizable region (sFc) of a human IgGl. Genetic fusion of a vaccine antigen to a Fc fragment has been shown to promote antibody induction and neutralizing activity against HIV gpl20 in rhesus macaques or Epstein Barr virus gp350 in BALB/c mice (Shubin, Z., et al., 2017; and Zhao, B., et al., 2018). Moreover, engineered Fc has been used in many therapeutic antibodies as a solution to minimized non-specific binding, increase solubility, yield, thermostability, and in vivo half-life (Liu, H., et al., In some embodiments, the vaccine composition contains Sl-RBD-sFc fusion protein of SEQ ID NO: 235. The Sl-RBD-sFc protein (SEQ ID NO: 235) contains the Sl-RBD peptide (SEQ ID NO: 226), which corresponds to amino acid residues 331-530 of the full-length S protein of SARS-CoV-2, fused to the single chain Fc peptide (SEQ ID NO: 232) through a mutated hinge region from IgG (SEQ ID NO: 188).

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

In some embodiments, the amino acid sequence of an S-RBD-sFc fusion used in a composition of the disclosure is at least 80%, 85%, 90%, 95%, 96%, 97%, 97%, 98%, 99%, or more identical to a reference sequence described herein (e.g., SEQ ID NO: 235 or SEQ ID NO: 226), provided that immunogenicity is substantially maintained. In some embodiments, the amino acid sequence of an S-RBD-sFC fusion used in a composition of the disclosure has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid substitutions, deletions, or insertions compared to a reference sequence, provided that immunogenicity is substantially maintained. In regard to such variants, reference is made to the description of functional analogs, above.

The amount of the SI -receptor-binding region-based designer protein in the vaccine composition can vary depending on the need or application. The vaccine composition can contain between about 1 pg to about 1,000 pg of the SI -receptor-binding region-based designer protein. In some embodiments, the vaccine composition contains between about 10 pg to about 200 pg of the SI -receptor-binding region-based designer protein. In some embodiments, the vaccine composition contains between about 50 pg to about 150 pg of the SI -receptor-binding regionbased designer protein. In some embodiments, the vaccine composition contains between about 88 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 ORFlab, N, M, and ORF3a regions; only 3 are in S, with only 1 CD8+ epitope being located in the Sl-RBD (Ferretti, A.R, et al., 2020). The smaller M and N structural proteins are recognized by T cells of patients who successfully controlled their infection. In a study of nearly 3,000 people in the UK, it was found that individuals with higher numbers of T cells were more protected against SARS-CoV-2 compared to those with low T cell responses, suggesting that T cell immunity may play a critical role in preventing COVID- 19 (Wyllie, D., et al., 2020).

To provide immunogens to elicit T cell responses, Th/CTL epitopes from highly conserved sequences derived from S, N, and M proteins of SARS-CoV and SARS-CoV-2 (e.g., Ahmed, S.F., et al., 2020/0 were identified after extensive literature search. These Th/CTL peptides are shown in Tables 4 and 5. Several peptides within these regions were selected and subject to further designs. Each selected peptide contains Th or CTL epitopes with prior validation of MHC I or II binding and exhibits good manufacturability characteristics (optimal length and amenability for high quality synthesis). These rationally designed Th/CTL peptides were further modified by addition of a Lys-Lys-Lys tail to each respective peptide’s N-terminus to improve peptide solubility and enrich positive charge for use in vaccine formulation. The designs and sequences of the five final peptides and their respective HLA alleles are shown in Table 14.

To enhance the immune response, a proprietary peptide UBIThOla (SEQ ID NO: 66) can be added to the peptide mixture of the vaccine composition. UBIThOla 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 UBIThOla to a target “functional B epitope peptide” derived from a self-protein rendered the self-peptide immunogenic, thus breaking immune tolerance (Wang, C.Y., et al, 2017). The Th epitope of UBITh®l has shown this stimulatory activity whether covalently linked to a target peptide or as a free charged peptide, administered together with other designed target peptides, that are brought together through the “charge neutralization” effect with CpGl, to elicit site-directed B or CTL responses. Such immunostimulatory complexes have been shown to enhance otherwise weak or moderate response of the companion target immunogen (e.g., WO 2020/132275A1). CpGl is designed to bring the rationally designed immunogens together through “charge neutralization” to allow generation of balanced B cells (induction of neutralizing antibodies) and Th/CTL responses in a vaccinated host. In addition, Toll-like receptors (TLRs) play critical roles in the innate immune system by recognizing pathogen-associated molecular patterns derived from a variety of microbes. Activation of Toll-like receptor 9 (TLR-9) signaling by CpG is known to promote IgA production and favor Thl immune response. UBITh® 1 peptide is incorporated as one of the Th peptides for its “epitope cluster” nature to further enhance the SARS-CoV-2 derived Th and CTL epitope peptides for their antiviral activities. The amino acid sequence of UBITh® 1 is SEQ ID NO: 65 and the sequence of UBITh®la is SEQ ID NO: 66. The nucleic acid sequence of CpGl is SEQ ID NO: 104.

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

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

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

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

3. Excipients

The vaccine composition can also contain a pharmaceutically acceptable excipient.

As used herein, the term “excipient” or “excipients” refers to any component in the vaccine composition that is not (a) the 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 (potassium aluminum phosphate), aluminum phosphate (e.g. ADJU-PHOS®), aluminum hydroxide (e.g. ALHYDROGEL®), calcium phosphate, incomplete Freund’s adjuvant (IFA), Freund’s complete adjuvant, MF59, adjuvant 65, Lipovant, ISCOM, liposyn, saponin, squalene, L121, EMULSIGEN®, EmulsIL-6n®, monophosphoryl lipid A (MPL), Quil A, QS21, MONTANIDE® ISA 35, ISA 50V, ISA50V2, ISA 51, ISA 206, ISA 720, liposomes, phospholipids, peptidoglycan, lipopolysaccahrides (LPS), ASO1, ASO2, ASO3, ASO4, AF03, lipophilic phospholipid (lipid A), gamma inulin, algammulin, glucans, dextrans, glucomannans, galactomannans, levans, xylans, dimethyldioctadecylammonium bromide (DDA), as well as the other adjuvants and emulsifiers.

In some embodiments, the vaccine composition contains ALHYDROGEL® (aluminum hydroxide), MONTANIDE™ ISA 51 (an oil adjuvant composition comprised of vegetable oil and mannide oleate for production of water-in-oil emulsions), TWEEN® 80 (also known as: Polysorbate 80 or Polyoxyethylene (20) sorbitan monooleate), a CpG oligonucleotide, and/or any combination thereof. In other embodiments, the pharmaceutical composition is a water-in-oil-in- water (i.e., w/o/w) emulsion with EMULSIGEN or EMULSIGEN D as the adjuvant.

In certain embodiments, the multitope protein/peptide vaccine composition contains ALHYDROGEL® (aluminum hydroxide) and a CpG oligonucleotide as the adjuvant to improve the immune response. In particular embodiments, the CpG oligonucleotide is present in an amount of about 0.5-10 pg, of about 1-5 pg, of about 1.5-4 pg, or of about 2-3 pg. .

The vaccine composition can contain pH adjusters and/or buffering agents, such as hydrochloric acid, phosphoric acid, citric acid, acetic acid, histidine, histidine HC1*H2O, lactic acid, tromethamine, gluconic acid, aspartic acid, glutamic acid, tartaric acid, succinic acid, malic acid, fumaric acid, a-ketoglutaric acid, and arginine HC1.

The vaccine composition can contain surfactants and emulsifiers, such as olyoxyethylene sorbitan fatty acid esters (Polysorbate, TWEEN®), Polyoxyethylene 15 hydroxy stearate (Macrogol 15 hydroxy stearate, SOLUTOL HS 15®), Polyoxyethylene castor oil derivatives (CREMOPHOR® EL, ELP, RH 40), Polyoxyethylene stearates (MYRJ®), Sorbitan fatty acid esters (SPAN®), Polyoxyethylene alkyl ethers (BRU®), 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.v., i.p., i.m., intranasally, orally, subcutaneously, etc. and in any suitable delivery device. In certain embodiments, the vaccine composition is formulated for subcutaneous, intradermal, or intramuscular administration. The vaccine composition can also be prepared for other modes of administration, including oral and intranasal applications. The vaccine composition can also be formulated in a suitable dosage unit form. In some embodiments, the vaccine composition contains from about 1 pg to about 1,000 pg of the API (e.g., the SI -receptor-binding region-based designer protein and/or one or more of the Th/CTL peptides). Effective doses of the vaccine composition can vary depending upon many different factors, including means of administration, target site, physiological state of the subject, whether the subject is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the 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 a 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 a 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 a 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 a 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 peptide to Th/CTL peptide(s) can be 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, or 90:10. In specific embodiments, the ratio (w:w) of the designer peptide to Th/CTL peptide(s) is 95:5, 94:6, 93:7, 92:8, 91:9, 90:10, 89:11, 88:12, 87:13, 86:14, or 85:15. In specific embodiments, the ratio (w:w) of the designer peptide to Th/CTL peptide(s) is 88:12.

In some embodiments, the vaccine composition comprises the SI -receptor-binding regionbased designer protein of SEQ ID NO: 235. In other embodiments, the vaccine composition comprises one or more Th/CTL peptides. In some embodiments, the vaccine composition comprises the SI -receptor-binding region-based designer protein of SEQ ID NO: 235 in combination with Th/CTL peptides of SEQ ID NOs: 345, 346, 347, 348, 361, and 66. In certain embodiments, the vaccine composition comprises the SI -receptor-binding region-based designer protein of SEQ ID NO: 235, the Th/CTL peptides of SEQ ID NOs: 345, 346, 347, 348, 361, and 66, together with one or more adjuvant and/or excipient. In various embodiments, the vaccine composition comprises SEQ ID NO: 235 together with the Th/CTL peptides of SEQ ID NOs: 345, 346, 347, 348, 361, and 66, where the Th/CTL peptides are present in an equal- weight ratio to each other and the ratio (w:w) of SEQ ID NO: 235 to the combined weight of the Th/CTL peptides is 88:12. Specific embodiments of the vaccine composition containing 20 pg/mL, 60 pg/mL, and 200 pg/mL, based on the total weight of the S 1-RBD-sFC protein (SEQ ID NO: 235) together with the Th/CTL peptides of SEQ ID NOs: 345, 346, 347, 348, 361, and 66 are provided in Tables 15- 17, respectively. b. Pharmaceutical compositions

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

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

Pharmaceutical compositions can contain one or more adjuvant that act(s) to accelerate, prolong, or enhance the immune response to the vaccine composition without having any specific antigenic effect itself. Adjuvants used in the pharmaceutical composition can include oils, oil emulsions, aluminum salts, calcium salts, immune stimulating complexes, bacterial and viral derivatives, virosomes, carbohydrates, cytokines, polymeric microparticles. In certain embodiments, the adjuvant can be selected from alum (potassium aluminum phosphate), aluminum phosphate (e.g. ADJU-PHOS®), aluminum hydroxide (e.g. ALHYDROGEL®), calcium phosphate, incomplete Freund’s adjuvant (IFA), Freund’s complete adjuvant, MF59, adjuvant 65, Lipovant, ISCOM, liposyn, saponin, squalene, L121, EMULSIGEN®, EmulsIL- 6n®, monophosphoryl lipid A (MPL), Quil A, QS21, MONTANIDE® ISA 35, ISA 50V, ISA 50V2, ISA 51, ISA 206, ISA 720, liposomes, phospholipids, peptidoglycan, lipopolysaccahrides (LPS), ASO1, ASO2, ASO3, ASO4, AF03, lipophilic phospholipid (lipid A), gamma inulin, algammulin, glucans, dextrans, glucomannans, galactomannans, levans, xylans, dimethyldioctadecylammonium bromide (DDA), as well as the other adjuvants and emulsifiers.

In some embodiments, the pharmaceutical composition contains MONTANIDE™ ISA 51 (an oil adjuvant composition comprised of vegetable oil and mannide oleate for production of water-in-oil emulsions), TWEEN® 80 (also known as: Polysorbate 80 or Polyoxyethylene (20) sorbitan monooleate), a CpG oligonucleotide, and/or any combination thereof. In other embodiments, the pharmaceutical composition is a water-in-oil-in-water (i.e., w/o/w) emulsion with EMULSIGEN or EMULSIGEN D as the adjuvant.

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

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

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

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

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

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

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

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

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

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

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

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

In some embodiments, the pharmaceutical composition contains SEQ ID NOs: 345, 346, 347, 348, 361, and 66. In some embodiments, the pharmaceutical composition contains 1 or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of any Th epitope peptides described in one or more of the Tables herein, in any combinations.

5. Antibodies

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

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

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

6. Methods

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

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

In prophylactic applications, the disclosed multitope protein/peptide vaccine composition can be administered to a subject susceptible to, or at risk of, becoming infected with SARS-CoV- 2, the virus that causes COVID-19 to eliminate or reduce the risk, lessen the severity, or delay the onset of the disease. The amount of the vaccine composition that is adequate to accomplish prophylactic treatment is defined as a prophylactically-effective dose. The disclosed multitope protein/peptide vaccine composition can be administered to a subject in one or more doses to produce a sufficient immune response in order to prevent an infection by SARS-CoV-2. Typically, the immune response is monitored, and repeated dosages are given if the immune response starts to wane.

The 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.v., i.p., i.m., intranasally, orally, subcutaneously, etc. and in any suitable delivery device. In certain embodiments, the vaccine composition is formulated for subcutaneous, intradermal, or intramuscular administration. The vaccine composition can also be prepared for other modes of administration, including oral and intranasal applications.

The dose of the vaccine composition will vary depending upon the subject and the particular mode of administration. The dosage required will vary according to a number of factors known to those skilled in the art, including, but not limited to the species and size of the subject. The dosage may range from 1 pg to 1,000 pg of the combined weight of the designer protein and the Th/CTL peptides. The dosage can between about 1 pg to about 1 mg, between about 10 pg to about 500 pg, between about 20 pg to 200 pg, or between about 50 pg to 150 pg of the combined weight of the designer protein and the Th/CTL peptides. The dosage, as measured by the combined weight of the designer protein and the Th/CTL peptides is about 10 pg, about 20 pg, about 30 pg, about 40 pg, about 50 pg, about 60 pg, about 70 pg, about 80 pg, about 90 pg, about 100 pg, about 110 pg, about 120 pg, about 130 pg, about 140 pg, about 150 pg, about 160 pg, about 170 pg, about 180 pg, about 190 pg, about 200 pg, about 250 pg, about 300 pg, about 400 pg, about 500 pg, about 600 pg, about 700 pg, about 800 pg, about 900 pg, about 1,000 pg. The ratio (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 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 99:1, or with a fixed amount of the Th/CTL peptides per dose. In specific embodiments, the ratio (w:w) of the designer protein to Th/CTL peptide(s) is 95:5, 94:6, 93:7, 92:8, 91:9, 90:10, 89:11, 88:12, 87:13, 86:14, or 85:15. In specific embodiments, the ratio (w:w) of the designer peptide to Th/CTL peptide(s) is 88:12. In specific embodiments, the vaccine composition contains the components shown in Tables 15-17. The vaccine composition can be administered in a single dose, in multiple doses over a period of time. The effective doses may be extrapolated from dose-response curves obtained from animal models. In some embodiments, the vaccine composition is provided to a subject in a single administration. In other embodiments, the vaccine composition is provided to a subject in multiple administrations (two or more). When provided in multiple administrations, the duration between administrations can vary depending on the application or need. In some embodiments, a first dose of the vaccine composition is administered to a subject and a second dose is administered about 1 week to about 12 weeks after the first dose. In certain embodiments, the second dose is administered about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks after the first administration. In a specific embodiment, the second dose is administered about 4 weeks after the first administration.

A booster dose of the vaccine composition can be administered to a subject following an initial vaccination regimen to increase immunity against SARS-CoV-2. In some embodiments, a booster dose of the vaccine composition is administered to a subject about 6 months to about 10 years after the initial vaccination regimen. In certain embodiments, the booster dose of the vaccine composition is administered about 6 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years after the initial vaccination regimen or after the last booster dose. In other embodiments, the booster dose of the vaccine composition is administered about 7 to about 9 months after the initial vaccine dose or regimen. Advantageously, boosting can be carried out to protect against SARs- CoV-2 variants including, e.g., the delta variant. In some embodiments, the booster dose of the vaccine composition is different from the priming dose(s) administered to subjects, as described further below.

In other embodiments, three or more doses of one or more vaccine compositions described herein are administered to a subject in an accelerated 3-dose regimen. In these methods, three doses are typically administered within about 5 months of one another. In some embodiments, the second dose is administered within about 2 weeks to about 1.5 months (e.g., about 2-7 weeks, 3-6 weeks, 4-5 weeks, or 1 month) after the first dose. In some embodiments, the third dose is then administered within about 2.5 months to about 5 months (e.g., about 10-20 weeks, 12-18 weeks, 14-16 weeks, 3-4 months, 3 months, or 4 months) after the first dose. Accelerated regimens as described herein can advantageously be carried out to prevent symptomatic COVID-19, reduce the severity of one or more symptoms of COVID-19, prevent hospitalization for COVID-19, reduce the length of hospitalization for COVID-19, protect against death, and/or maintain vaccine- induced antibodies above a protective threshold. Furthermore, accelerated boosting can be carried out to protect against different SARS-CoV-2 variants, e.g., the delta variant.

Heterologous boosting

In other embodiments, one or more doses of one or more vaccine compositions described herein is administered as a booster to a different, heterologous vaccine composition. In some embodiments, the initially administered vaccine composition that is later boosted comprises one or more proteins or peptides. For example, the initially administered vaccine composition may comprise a spike protein of SARS-CoV-2 or a variant thereof (e.g., SA, beta variant) and/or a fragment of the spike protein (e.g., an RBD-containing fragment). In some examples, such vaccines include CpG oligonucleotides or other adjuvants and/or are in the form of nanoparticles. Exemplary vaccines of this type include NVX-CoV2372 (Novavax), NVX-CoV2373 (Novavax), and MVC-COV1901 (Medigen).

In other embodiments, the initially administered vaccine composition comprises one or more nucleic acid molecules (e.g., RNA or DNA). Accordingly, in such embodiments, the initially administered vaccine composition may comprise an mRNA encoding an immunogen of SARS- CoV-2, or a variant thereof (e.g., SA, beta variant), such as a spike protein or a fragment thereof (e.g., an RBD-containing fragment thereof). Exemplary vaccines of this type include mRNA-1273 (Moderna) or BNT162b2 (BioNTech, Pfizer).

In other embodiments, the initially administered vaccine composition comprises a viral vector which comprises a sequence encoding an immunogen of SARS-CoV-2, or a variant thereof (e.g., SA, beta variant), such as a spike protein or a fragment thereof (e.g., an RBD-containing fragment thereof). In some embodiments, the viral vector is an adenoviral vector. Exemplary vaccines of this type include AZD1222 (Vaxzevria, University of Oxford/Vaccitech/AstraZeneca), Janssen COVID- 19 vaccine (JNJ-78436735; Johnson & Johnson), and Sputnik V (Gam-COVID- Vac; Gamal eya Research Institute of Epidemiology and Microbiology).

In other embodiments, the viral vector is a recombinant human parainfluenza virus type 2 (hPIV2) (BC-PIV SARS-CoV-2 (MediciNova). In other embodiments, the first immunogenic composition comprises whole SARS-CoV-2 virus (e.g., a killed or attenuated SARS-CoV-2 virus, or a variant thereof, e.g., SA, beta variant). Exemplary vaccines of this type include CoronaVac (Sinovac) and BBIBP-CorV (Covilo; Sinopharm).

Additional examples of vaccines that can be boosted according to the heterologous boosting methods of the disclosure are described in WO 2021/154812; WO 2021/181100; U.S. Patent No. 10,703,789; U.S. Patent No. 10,702,600; U.S. Patent No. 10,577,403; U.S. Patent No. 10,442,756; U.S. Patent No. 10,266,485; U.S. Patent No. 10,064,959; and U.S. Patent No. 9,868,692; and US 2021/0246170.

The initially administered vaccine in a heterologous vaccination regimen can be administered one or more times prior to heterologous boosting. In some embodiments, the initially administered vaccine is administered in the same manner as it would be used on its own (without homologous boosting), whether in single or multiple (e.g., 2 or 3 doses). Thus, for example, before heterologous boosting as described herein, a protein or peptide-based vaccine may be administered in two doses about 3 or 4 weeks apart (e.g., 1-8, 2-6, 3-6, or 3-4 weeks apart); an mRNA vaccine may be administered in two doses about 3 or 4 weeks apart (e.g., 1-8, 2-6, 3-6, or 3-4 weeks apart); a viral-based vaccine (e.g., an adenoviral vectored vaccine, e.g., as described herein) may be administered only once; while an inactivated whole virus vaccine may be administered in two doses about 3 or 4 weeks apart (e.g., 1-8, 2-6, 3-6, or 3-4 weeks apart). Alternatively, the second dose of an initially administered vaccine that is typically administered in more than one dose can be replaced with a heterologous booster as described herein. In some embodiments, the heterologous booster is administered within about 2.5 to 4.5 months after the first immunogenic composition; within about 3 to 4 months of the first immunogenic composition; within about three months after the first immunogenic composition; or within about six or more months (e.g., about 6, 7, 8, 9, 10, or 11 months, or about 1, 2, 3, 4, or 5 years) after the first immunogenic composition. Heterologous boosting as described herein can advantageously be carried out to prevent symptomatic COVID-19, reduce the severity of one or more symptoms of COVID-19, prevent hospitalization for COVID-19, reduce the length of hospitalization for COVID-19, prevent against death, and/or maintain vaccine-induced antibodies above a protective threshold. Furthermore, heterologous boosting can be carried out to protect against different SARS-CoV-2 variants, e.g., the delta variant.

In some embodiments, an immunogenic composition of the invention used in a method described here in is UBV-612. c. Methods for the manufacturing of pharmaceutical compositions

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

In order for a pharmaceutical composition to be used by a large population, safety becomes another important factor for consideration. Despite there has been use of water-in-oil emulsions in many clinical trials, Alum remains the major adjuvant for use in formulations due to its safety. Alum or its mineral salts Aluminum phosphate (ADJUPHOS) are, therefore, frequently used as adjuvants in preparation for clinical applications.

In particular embodiments, the invention encompasses the use of aluminum phosphate (ADJUPHOS) and a CpG oligonucleotide to improve the immune response. In particular embodiments, the CpG oligonucleotide is present in an amount of about 0.5-10 pg, of about 1-5 pg, of about 1.5-4 pg, or of about 2-3 pg. In still other embodiments, the CpG oligonucleotide is present in an amount of about 2 pg. UB-612 is an exemplary embodiment using aluminum phosphate and about 2 pg of CpG oligonucleotide as adjuvant.

In particular embodiments, the invention encompasses the use of ALHYDROGEL® (aluminum hydroxide) and a CpG oligonucleotide as the adjuvant to improve the immune response. In particular embodiments, the CpG oligonucleotide is present in an amount of about 0.5-10 pg, of about 1-5 pg, of about 1.5-4 pg, or of about 2-3 pg.

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

The choice of an adjuvant depends on the stability of the immunogenic formulation containing the adjuvant, the route of administration, the dosing schedule, the efficacy of the adjuvant for the species being immunized, and, in humans, a pharmaceutically acceptable adjuvant is one that has been approved or is approvable for human administration by pertinent regulatory bodies. For example, alum, MPL or Incomplete Freund's adjuvant (Chang, J.C.C., et al., 1998), which is hereby incorporated by reference in its entirety) alone or optionally all combinations thereof are suitable for human administration. The compositions can include pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate- buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, non-immunogenic stabilizers, and the like.

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

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

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

The present disclosure also includes methods of using pharmaceutical compositions containing SI -receptor-binding region-based designer proteins, e.g., in the methods described herein (e.g., the heterologous boosting methods described above).

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

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

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

Details related to preparation of S-RBD peptides and related fusion proteins and formulations, as well as antigenic peptides, assay methods, and related materials and methods are set forth in Examples 1-18 of WO 2021/168305, the contents of which are incorporated herein by reference, and which may be consulted in reference to the preparation of compositions described and claimed herein. Certain sequences noted herein are set forth in Tables herein, which may be consulted for details of the sequences. Sequences from these tables can be used in the compositions described herein as determined to be appropriate based on the nature of sequence.

EXAMPLE 1

Summary

As vaccine immunity wanes, a booster has been recommended for maintenance of vaccine efficacy (VE) against pandemic Delta variant. We report unusually high neutralizing antibody titers against Delta variant by a booster dose of UB-612 vaccine, which contains Sl-RBD-sFc fusion protein and synthetic T-cell epitope peptides for activation of humoral and T-cell immunity.

A 100-pg booster dose of UB-612 was administered to 50 healthy adults, aged 22-55 years, over 6 months after a primary 2-dose series of 10-, 30-, or 100-pg dose given at 0 and 1 months. We evaluated safety and immunogenicity 14 days after the booster including live SARS-CoV-2 wild type (WT) and Delta variant neutralization, as well as antigen- specific T-cell responses.

Neutralizing antibody following the primary series of the 100-pg dose group, declined slowly with a long half-life of 187 days, along with a long-lasting T cell response. After boosting, the most common solicited adverse events (AEs) were injection- site pain (61%) and fatigue (11%) that were mild and transient, similar to those observed in the primary series and no serious AEs were recorded. In the cohort (N=17) who received three doses of 100-pg UB-612 the 50% virus neutralization test (VNT50) geometric mean titer (GMT) was 3992 against WT (37-fold increase over the peak after primary immunization), and 2358 against Delta variant (1.7-fold reduction relative to WT). Neutralization data are also presented in international units and predict a high level of efficacy of UB-612 based on published regression models.

UB-612 is safe and well tolerated and antibodies decay more slowly than reported for other vaccines. Priming with UB-612 led to robust memory, with rapid anamnestic responses after boosting against WT virus and Delta variant with neutralizing antibody levels predicting high VE.

Introduction

The COVID-19 pandemic continues to cost human lives and sap the world’s economy and healthcare system with a sizable pool of asymptomatic contagious coverts ^1,2 that sustain transmission. Amongst the globally dominant Variants of Concern (VoCs), Delta variant is as contagious as chickenpox, and those infected can carry up to 1,000 times more virus in their nasal passages than other variants ³ . Additional safe, effective and affordable vaccines are needed against emergent viral variants .

People fully-vaccinated with authorized COVID-19 vaccines can develop breakthrough cases, carry as much of the virus as unvaccinated people, and contribute to spread of the virus worldwide ^4,5 . To maintain the vaccine protection against the Delta variant, many regulatory agencies have approved or are considering a booster dose not only for the elderly and the high- risk populations, but also for healthy individuals ^{6 11} .

While neutralizing antibody levels correlate with vaccines’ protection efficacy ^12,13 , substantial activation of CD4 ⁺ and CD8 ⁺ T cells by viral antigens is also critical for better duration of robust immunity and immunological memory ^14,15 . Early induction of functional SARS-CoV-2- specific T cells is critical to rapid viral clearance and amelioration of disease ¹⁶ . Thus, T cell responses elicited by peptides representing viral structural and non- structural proteins are of increasing interest in the control of infection ¹⁷ , an immunological-base by which UB-612 is designed for clinical development.

UB-612 vaccine has a unique composition of matter, containing (a) a B cell immunogen as SARS-CoV-2 Sl-receptor binding domain (RBD) protein, constrained to preserve its ACE2 binding site, as a single chain Fc fusion protein; and (b) T cell immunogens as designer synthetic peptides, incorporating helper T-cell (Th) and cytotoxic T-cell (CTL) epitopes from the conserved regions of the M, N, and S2 proteins among available viral variants, to activate both B and T- cell immunities. This multitope composition represents different viral T epitopes known to bind to multiple Class I and Class II Major Histocompatability Complexes (MHC-I and MHC-II) ^19,20 , by which UB-612 is unique and distinct from other full length spike protein-based vaccines.

Here we report the interim 14-day results of UB-612 from the extension arm of a 196-day phase- 1 trial, where a booster (3 ^rd ) dose of 100 pg was administered to 50 healthy adults who had originally received 10-, 30-, and 100-pg in a 2-dose primary series. The booster dose induced the highest level of viral-neutralizing titers against the Delta variant reported for any vaccine. UB-612 is safe and well tolerated, and has induced long-lasting high neutralizing antibody titers and durable antigen-specific T-cell responses.

Methods

Trial Design and Oversight

Safety and immunogenicity of UB-612 vaccine was evaluated in an open-label dose ascension phase- 1 study, conducted at China Medical University Hospital, Taiwan, [ClinicalTrials.gov: NCT04545749] and an 85-day extension study to evaluate a 3rd booster dose [ClinicalTrials.gov: NCT04967742]. The primary- series 196-day Phase-1 study enrolled 60 healthy adults aged 20-55 years, who received two intramuscular (IM) injections (28 days apart) of 10-, 30-, or 100-pg (N=20/group). Seven to nine months following completion of the primary series, 50 participants were enrolled in the extension study to receive a booster dose of 100 pg UB- 612, with an interim analysis at 14 days and monitoring until Day 84 post-booster.

The Principal Investigators at the study sites agreed to conduct the study according to the specifics of the study protocol and the principles of Good Clinical Practice (GCP); and all the authors assured accuracy and completeness of the data and analyses presented. The protocols were approved by the ethics committee at the site and all participants provided written informed consent.

Trial Procedures and Safety

The phase- 1 trial was initiated with a sentinel group of 6 participants to receive the low 10- pg dose (n = 20), 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. Subjects 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. Complete details for solicited reactions are provided in the study protocols.

Immunogenicity

The primary immunogenicity endpoints were the geometric mean titers (GMT) of neutralizing antibodies against SARS-CoV-2 wild-type (Wuhan strain) and Delta variant. Viral- neutralizing antibody titers were measured by a cytopathic effect (CPE)-based assay using Vero- E6 cells challenged with SARS-CoV-2-TCDC#4 (Wuhan strain) and SARS-CoV-2-TCDC#1144 (B.1.617.2; Delta variant). The replicating virus neutralization test conducted at Academia Sinica was fully validated using internal reference controls and results expressed as VNT50. The WHO reference standard was also employed and results reported in international units (lU/mL). The secondary immunogenicity endpoints include binding IgG antibody responses to Sl-RBD, inhibitory titers against S1-RBD:ACE2 interaction, and T-cell responses assayed by ELISpot and Intracellular Cytokine Staining (ICS). The RBD IgG ELISA was fully validated using internal reference controls and results expressed in end-point titers. The WHO reference standard was also employed and results reported in Binding Antibody Units (BAU/mL). A panel of 20 human convalescent serum samples from COVID-19 hospitalized patients aged 20 to 55 years were also tested for comparison with those in the vaccinees. Human peripheral blood mononuclear cells (PBMCs) were used for analysis of T cell responses.

Statistics

As the studies were not powered for formal statistical comparisons of between-dose and between-phase vaccination, we report descriptive results of safety and immunogenicity. Immunogenicity results for GMT and geometric mean fold increase (GMFI) in titer are presented with the associated 95% confidence intervals. Seroconversion rate (SCR) is presented as counts and percentages along with the 95% exact (Clopper-Pearson) confidence intervals. SCR is defined as the percentage of study subjects with > 4-fold increase in antibody titers from baseline. 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. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.

Results

Trial Population

The booster vaccination was an 84-day extension from the 196-day open-label phase- 1 study involving 60 healthy adults, aged 20-55 years (Figs. 1A and IB). In the primary series, the participants in three dose groups (n = 20 each) received two doses (28 day-apart) of UB-612 at 10, 30, or 100 pg. Following the primary series, 50 participants were enrolled to receive additional one 100-pg booster over 6 months after the second shot, for the 10-pg (n = 17), 30-pg (n = 15), and 100-pg (n = 18) dose groups. The boosted participants were followed up for 14 days for interim evaluation on safety and immunogenicity, and to be monitored until 84 days post-booster. Reactogenicity and Safety

In the primary series and up to 14 days post-booster, AEs were solicited local and systemic AEs reported within 7 days in all vaccination groups (Fig. 2) were mild (grade 1) and transient, with lower frequencies for most systematic reactions than local reactions. The incidences of solicited local AE were comparable after the first and second vaccination and slightly increased after the booster dose (Fig. 2A), of which the most common post-booster solicited local AEs were pain at the injection site (60-71%). The incidence of solicited systemic AEs were similar after each vaccination (Fig. 2B), of which the most common post-booster solicited systemic AE was fatigue (11-33%). There were no serious AEs recorded.

Analysis of T-Cell Responses by ELISPOT and Intracellular Cytokine Staining

In primary vaccination series, peripheral blood mononuclear cells (PBMCs) were collected from vaccinees for evaluation by ELISpot of T cell responses. The highest antigen- specific spot forming unit (SFU)/10 ⁶ PBMC responses were observed with the 100-pg dose group: estimated to be 254 by stimulation with Sl-RBD+Th/CTL peptide pool and 173 by Th/CTL peptide pool (Fig. 6), demonstrating that the Th/CTL peptides in the vaccine were essential and principally responsible for T cell responses. At Day 196, the Interferon-y ⁺ -T cell responses for the 100-pg dose group remained significant at levels of -50% from the peak T-cell responses, which descend from 254 to 121 with RBD+Th/CTL peptide pool restimulation, or from 173 to 86.8 with Th/CTL peptide restimulation only (Fig. 6). This observation suggests that UB-612 vaccine elicited T cell responses after two shots are long-lasting as well that persist for at least 6 months.

Using a modified ELISpot method, preliminary analysis on vaccine-induced memory T cell response Day-14 post-booster vs. pre-boosting showed a modest elevation in IFN-ySFU/10 ⁶ PBMCs. The trend of increases from 19.9 to 68.1, 101 to 144 and 31.3 to 43.1 for the 10-, 30-, and 100-pg dose groups, respectively, in response to the restimulation of Sl-RBD+Th/CTL peptide pool, indicating substantial antigen- specific memory T cells were retained even at more than 6 months after the primary vaccination series and these T cells are further boosted by a subsequent vaccination. In contrast with the IFN-y, the IL-4 responses were far lower in all three dose groups, suggesting the induction of a Th 1 -predominant cellular response by UB-612 vaccination.

Substantial antigen- specific CD4 ⁺ T cells producing Thl cytokines (IFN-y and IL-2) were observed in PBMCs collected prior to the booster dose after restimulation with different pools of vaccine antigens in all three dose groups, while only minimal Th2 cytokine (IL-4)-producing cells were detected. Remarkably, robust antigen- specific CD8 ⁺ T cells expressing cytotoxic markers CD107a and Granzyme B were detected after two shots of UB-612 in participants prior to receiving booster dose. The results, consistent with that from ELISpot, showed a durable memory T cell response which is Thl prone in both helper CD4 ⁺ and cytotoxic CD8 ⁺ T cell responses assessed by ICS and flow cytometry.

As waning immunity infection after complete 2-dose vaccination has been associated with breakthrough infections with Delta variant, recommendations for a booster dose at 6 months after the primary series may be considered ^{6 11,21,22} . SARS-CoV-2 will continue to evolve and new variants may lead to immune escape resulting in reduced effectiveness of current vaccines ¹⁸ . New vaccines tailored to Delta variant have been evaluated ²³ as Delta is less sensitive than WT to the antibodies induced by Wuhan based vaccines, and by natural SARS-CoV-2 infection, and is both more transmissible and more virulent than the original virus. Thus, in the absence of a Deltaspecific vaccine, a booster dose with the currently available vaccines may be needed, in addition to continued public health measures, such as face masking and social distancing. The present invention provides a booster that can advantageously be used in this manner.

There have been a number of reports on booster vaccination ^25-30 revealing for some but not all vaccine platforms, substantial fold-increases in neutralizing antibody titers against live viruses, compared to peak responses after primary vaccination series. With such an unusually high neutralizing fold-increase, supported by the T helper immunity elicited by UB-612 during the primary vaccination, vaccines of the disclosure, such as UB-612, are potent boosters that can prompt recall of high levels of neutralizing antibodies. Since heterologous boosting may be more efficient at stimulating high antibody responses than homologous boosting, we consider that vaccines of the disclosure, such as UB-612, could be effective for boosting other vaccine platforms, particularly adenovirus vectored and inactivated vaccines that have shown modest homologous boosting. Additional data supporting the use of the s-RBD-sFc fusion protein and Th/CTL peptide- based vaccines described herein as heterologous boosters is found in the Examples that follow.

References for Example 1

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EXAMPLE 2

THREE-DOSE PRIMING REGIMEN

Vaccine given at a 3-dose regimen is able to stimulate high levels of neutralizing antibodies, and the antibodies are estimated to have long half-life. Furthermore, responses remained broad across various variants of concern or interest. Preclinical studies in non-human primates (NHPs) were carried out to assess an accelerated 3-dose regimen in consideration of possible dosing at 0, 1, and 3 months. NHPs were vaccinated at days 0, 28, and 70, and samples were taken at days 42, 70, and 77. As shown in Fig. 4, this regimen led to substantial increases in VNT50, with an increase of over 5-fold in animals given doses of 100 pg/dose. The fold increase was 8.6-fold for 10 pg/dose, 2.5-fold for 30 pg per dose, and 5.5-fold for 100 pg per dose. Vaccine utilized is UB- 612 as described herein.

Fig. 5 shows that a three-dose vaccination regimen raises neutralizing antibodies substantially. Levels of neutralizing antibodies are above a reported protective threshold, thus indicating a range for a third dose in a 3-dose accelerated vaccination regimen. As described herein, a third dose may be administered in the range of 3-4 months (see above). Vaccine utilized is UB- 612, as described herein.

EXAMPLE 3

ADDITIONAL SUPPORTING DATA

Preclinical studies in non-human primates (NHPs) shown that UB-612 is immunogenic and induces serum antibodies that efficiently neutralize both Wuhan and Delta strains (Fig. 6).

Fig. 7 shows that UB-612 reduces neutralizing antibody loss against variants of interest (beta and delta). The decrease for beta with UB-612 was -3.9-fold. The decrease for delta with UB-612 was -1.6-fold, while the difference for delta was +1.6-fold. These data show the advantages of the UB-612 vaccines in minimizing neutralizing antibody loss against variants of concern.

EXAMPLE 4

Data supporting the present invention is present in the accompanying figures (Figs. 8-23) and is based, in part, on studies of ex vivo sera from COVID19 patients. In brief summary, neutralization data show that a booster dose of UB-612 produced neutralizing antibodies which were 3.2-fold higher than those produced by a 3 ^rd dose of an mRNA vaccine (Pfizer). The results of binding and functional assays (2 doses) are shown in the figures. UB-612 generated high binding Abs against variants of concern (VOC) and variants of interest (VOI). UB-612 generated modest bAbs against Omicron after 2 doses but significant levels after 3 doses. Neutralizing antibody (Nab) data (V205 (n=84) (2 doses)) show that UB-612 generated NAbs against SARS-CoV-2 2019-nCoV/Italy-INMIl strain at a similar level to Delta variant. Furthermore, UB-612 generated modest NAbs against Omicron after 2 doses and significant NAbs against Omicron after 3 doses V123 (n=15). The titer of NAbs after 3 doses of UB-612 (7-9 m post second dose) was 3.2-fold higher than those generated after 3 doses (6-11 months post second dose) of Pfizer vaccine (Muik et al, Science 2022). In further data, the following neutralization results were found: for Wuhan: Pfizer 3-dose>UB-612 3-dose; for Omicron: UB- 612 3-dose >Pfizer 3-dose. Data from VNT550/HCS (Phi) showed VE of 82% for UB-612 (2 doses). Additional data predicted vaccine efficacy of -82% for 2 doses and 95% for 3 doses, including a booster given at 7-9 m against the prototype strain.

EXAMPLE 5

Omicron, a highly transmissible SARS-CoV-2, emerged in November 2021. The high mutation rates within its spike protein raised concerns about increased breakthrough infections among the vaccinated. We tested cross-reactivity of antibodies induced by UB-612 against Omicron and other variants. After 2 doses, UB-612 elicited modest levels of neutralization antibodies against ancestral virus, and very low levels against Omicron. A booster dose delivered 7-9 months after primary vaccination dramatically increased neutralizing antibody levels, with 131-, 61-, and 49-fold increases against the ancestral, Omicron BA.l, and BA.2, respectively. Using a model bridging vaccine efficacy with ancestral virus RBD binding antibody responses, predicted efficacy against symptomatic COVID- 19 caused by the ancestral strain after UB-612 booster is estimated at -95%. UB-612 is anticipated to be a potent booster against current and emerging SARS-CoV-2 variants.

In November 2021, the Omicron (B.1.1.529) Variant of Concern (VOC) was first reported in South Africa and Botswana and quickly spread globally, becoming the dominant SARS-CoV-2 variant worldwide. Omicron’s high transmissibility and potential for immune system evasion, as suggested by its ability to infect and be transmitted by previously infected and vaccinated individuals, predicted a transmission advantage over the Delta variant and the displacement of the latter as the dominant variant (7). The Omicron variant has three major sublineages (BA.l, BA.2, and BA.3). While BA.l caused most of the cases globally throughout November 2021 and February 2022, the BA.2 is now the main cause of COVID-19 globally (2).

The Omicron variant has over 50 new amino acid substitutions, >15 of which are in the receptor-binding domain (RBD) of the Spike (S) protein (3, 4). Although BA.2 shares many mutations with BA.l, these 2 sublineages differ by dozens of amino acid substitutions, especially at key portions of the virus S protein (Fig. 24A), some of which could be responsible for the rapid surge in BA.2 cases.

Given that over 90% of neutralizing antibodies are present in plasma of convalescent individuals and up to 99% of neutralizing antibodies elicited by vaccination with the mRNA- 1273 vaccine are directed to the RBD (5), these mutations could be largely responsible for Omicron’s ability to evade neutralizing antibodies induced by the approved COVID- 19 vaccines (6-9). Multiple studies have shown a 20- to 30-fold reduction in neutralization antibody activity against Omicron in the sera of primary vaccine recipients compared with the ancestral SARS- CoV-2 or D614G viruses (6-8,10-13'). The emergence of new variants, including Omicron, in addition to the rapidly waning immunity of vaccines over time, has raised concerns about breakthrough infections in vaccinated individuals, and highlights the need for booster doses worldwide. Homologous or heterologous booster vaccines, all based on the full-length S protein, restored protective neutralizing antibodies to levels achieved by the primary immunization; however, these titers were 7.1 -fold lower against Omicron BA.l than the ancestral strain, suggesting a continued risk of breakthrough infections in vaccinated individuals over time (7).

In contrast to most of the approved COVID-19 vaccines, which encode the full-length S protein, the UB-612 vaccine candidate is composed of Wuhan-Hu Sl-RBD-sFc fusion protein and is enriched with 5 rationally designed peptides representing Sarbecovirus-conserved Th and CTL epitopes on the S2 subunit, Membrane (M), and Nucleocapsid (N) proteins (14). A favorable safety and tolerability profile for UB-612 was demonstrated in -4000 participants in a Phase 1 trial and its extension, and a Phase 2 trial, both conducted in Taiwan (15). In both Phase 1 and Phase 2 trials, the UB-612 vaccine was found to have a favorable safety profile and low reactogenicity after every injected dose. Two immunizations with UB-612 were immunogenic and led to a seroconversion rate of neutralizing antibody in >90% of vaccine recipients. In these same studies, UB-612 was shown to elicit long-lasting neutralizing antibody titers similar to levels detected in convalescent patients (16) and B-cell and T-cell responses against Delta and Omicron variants (15).

The objectives of this study were to evaluate the neutralization potential of antibodies elicited by a third dose (booster) with the RBD-based vaccine UB-612 against Omicron and their reactivity to recombinant S and RBD protein antigens across various variants.

After receiving a 2-dose primary vaccine series or a booster given 7-9 months after the second dose, sera from 15 participants (who consented to be in this study) in the Phase 1 trial (UB-612, 100-pg dose), were tested in a live virus neutralization test (VNT) at Vismederi, Siena, Italy (a Coalition for Epidemic Preparedness Innovations central testing laboratory for COVID- 19 vaccines) . Previously, to establish an International Reference Standard for anti-SARS-CoV-2 antibody detection, the VNT used in our analysis and performed by Vismederi, was compared with other VNTs and found to be the most stringent assay, resulting in a lower geometric mean titer (GMT) than other plaque reduction-, foci reduction-, cytopathic effect (CPE)-, or pseudotyped virus-based neutralization assays (77).

Two doses of UB-612 showed modest neutralizing activity against the authentic wildtype SARS CoV-2 2019 ancestral strain (Victoria/ 1/2020) (GMT VNTso of 47.0), and very low activity detected against Omicron’s BA.l and BA.2 sublineages (GMT VNTsoof 10-11) (Fig. 24B) (n=15). Similarly, 2 dose immunization with mRNA vaccines resulted in low levels of Omicron neutralizing antibody responses: (i) mRNA-1273 on day 21 after immunization stimulated GMT pVNTso of 14, and (ii) BNT162b2 on day 28 after immunization lead to GMTs VNT of 7 (S, 72).

A booster dose of UB-612 delivered 7-9 months after the primary series increased neutralizing antibody titers against the ancestral strain, BA.l and BA.2 to GMT VNT50 of 6159, 670, and 485, respectively, which constitutes 131-, 61-, and 49-fold higher GMTs than those achieved after 2 doses (Fig. 24). The estimated decrease in neutralization titers against Omicron BA.l and BA.2 in our 15 UB-612 sera obtained 2 weeks after the booster, was 9.2- and 12.7- fold, respectively, compared with the ancestral Victoria strain in a live virus assay. Previously, only a 5.5-fold decrease against BA.l was reported when all 20 sera from UB-612 vaccinated participants were tested in a pseudovirus-based neutralization assay (GMT pVNTso of 12,778 against the Wuhan strain, vs 2325 against the BA.l strain) (75). These data support the breadth of UB-612-elicited neutralizing antibodies across multiple SARS-CoV-2 variants particularly after the booster dose, a differentiation property of UB-612 primarily attributed to its subunit protein RBD antigenic component (76).

Fig. 25 shows that UB612 stimulated durable immunity and boosted neutralizing antibodies 75-fold over pre-boost titers (V-123). Fig. 26 shows Nabs against SARS-CoV-2 or Omicron variants after booster of UB-612 as compared to booster dose of BNT vaccine. A booster dose of UB-612 induced comparable levels of Nabs, against BA.l and BA.2, to those obtained with the BNT162b2 vaccine.

We evaluated reactivity of UB-612-elicited antibodies to S and RBD protein antigens, using 15 sera from the Phase 1 trial (V123) participants and 84 randomly selected sera from Phase 2 trial participants (all consented to be in the trial) immunized with UB-612. These sera were tested in 2 ELISA-based assays for immunoglobulin G (IgG) direct binding to recombinant S and RBD protein antigens and inhibition of recombinant S and RBD protein binding to the human angiotensin-converting enzyme 2 (hACE2) receptor. A third dose of UB-612 booster immunization stimulated broadly reactive IgG antibodies, effectively binding to RBDs of 14 divergent SARS-CoV-2 variants, including Alpha, Beta, Gamma, Delta, and Omicron (Fig. 27 and Fig. 29).

Compared with the second UB-612 dose, IgG binding titers against Omicron’s RBD increased by over 40-fold, and the titers against RBDs of other SARS-CoV-2 variants were also increased in the range of 30- to 50-fold after the booster dose. When the IgG titer ratio (in binding antibody units (BAU)/mL) of several variants was compared to the ancestral Wuhan strain, the normalized RBD antibody-binding responses to the tested variants were found to be similar after 2 or 3 doses: Alpha (0.98-fold), Beta (2.44-fold), Delta (1.33-fold), Gamma (1.77- fold), and Omicron (3.3-fold) after 2 doses; and Alpha (0.91-fold), Beta (1.8-fold), Delta (1.4- fold), Gamma (1.55-fold), and Omicron (3.7-fold) after 3 doses.

Similar to RBD binding, the results of the S-protein binding antibody responses (S:ACE2- and RBD:ACE2-blocking antibody titers), confirmed the extent of stability in ratios of parental to variant IgG antibodies stimulated by 2 or 3 doses of UB-612, despite an up to 60-fold increase in titers against different variants after the booster dose (Fig. 29, Fig. 30).

We also compared the level of UB-612-elicited IgG antibodies with data previously reported for several authorized vaccines determined in equivalent S- and RBD-binding assays (18). After a 2-dose primary immunization series, the GMTs of UB-612-elicited IgG antibodies were 69 and 127 (BAU/mL) against the Wuhan S protein, and 235 and 494 (BAU/mL) against the RBD antigen in sera from Phase 1 and Phase 2 participants, respectively (Fig. 31). These IgG responses were comparable to those observed in individuals after the primary immunization with adenovirus vectored vaccines (1-dose Ad26.COV2.S or 2-dose ChAdOxl-S) but were lower than the response observed after 2 immunizations with mRNA vaccines. The additional booster dose with UB-612 increased levels of both S- and RBD-protein binding IgG antibodies in the Phase 1 participants by more than 16- and 13 -fold, and increased antibody GMTs to 2138 and 6767 (BAU/mL), respectively, matching those achieved by 2 immunizations with the mRNA vaccines.

We further utilized a vaccine efficacy prediction model based on the RBD activity of IgG antibodies to the ancestral strain, extending previous models based on neutralizing antibodies (79, 20) or S protein-binding activities (18). According to this model, the predicted vaccine efficacy of UB-612 against symptomatic disease caused by the prototype strain after 2 doses is -72% (235 BAU/mL with sera from 15 Phase 1 participants), -82% (494 BAU/mL with sera from 84 randomly selected Phase 2 participants) (Fig. 31), and -95% after the booster dose (6767 BAU/mL, with sera from 15 Phase 1 participants (Fig. 32).

It is difficult to compare neutralization activities of different vaccine platforms against variants because there are currently no international standard reagents available for variants, and each manufacturer uses a different assay, including live virus, pseudovirus-based, or chimeric recombinant virus, with different endpoint readouts, such as colorimetric or CPE-based evaluations. Moreover, each stock virus may contain different ratios of non-infectious to infectious particles and different virus concentrations for neutralizing sera from vaccinees, both of which could influence the resulting titers. Keeping these caveats in mind, we observed GMT titers after the UB-612 booster dose comparable to those elicited by a booster dose of mRNA vaccine BNT162b2 (72) and mRNA-1273-50 mg (8). After the booster dose (third vaccination), the live virus neutralizing antibody GMTs against the ancestral strain and Omicron BA.l were 763 and 106 for BNT162b2 (7.2-fold loss) (72), or 2423 and 850 for mRNA-1273-50 mg (2.8- fold loss) (S). The pseudovirus neutralization GMT titers were 6539, 1066, and 776 against the ancestral strain WA1/2020, Omicron BA.l, and BA.2, respectively, at 14 days after the third dose of BNT162b2 (a 6.1- and 8.4-fold loss against BA.l and BA.2, respectively, compared with the ancestral strain) (27). The homologous booster dose of mRNA-1273, BNT162b2, or Ad26.COV.S S-based vaccines dramatically increased neutralizing antibodies to Omicron (20- to 30-fold) compared with the modest increase reported for the ancestral strain (1- to 4-fold), which is likely due to a higher baseline titer for the ancestral strain compared with the variants (22).

The vaccination with UB-612 elicited highly cross -reactive IgG and neutralizing antibodies to Omicron variants (with 49- to 61 -fold increase in VNT50) and the ratio of binding antibodies to ancestral strain/Omicron and other variants remained stable after the second and booster immunizations. It was demonstrated that a booster with a full-length S protein vaccine would refocus/recall the memory B-cell pool to produce neutralizing antibodies to conserved RBD regions that have been affinity-matured after a long interval between the doses, enhancing the breadth of cross-variant neutralization (23). We believe that the UB-612 vaccine may be able to recall such memory B-cell responses targeting the RBD region carrying the major neutralizing epitopes.

In summary, a third booster dose of UB-612 elicited robust S- and RBD-specific binding and virus neutralizing antibodies against several SARS-CoV-2 variants, including Omicron BA.l and BA.2. The magnitude and extent of reactivity of the neutralizing antibody responses after the UB-612 booster match those reported for the authorized vaccines, including BNT162b2 and mRNA-1273. Additionally, UB-612 immunization has been shown to stimulate T-cell responses against conserved S2, N, and M peptides, included in the UB-612 vaccine formulation (14,15, 24), and may provide long-lasting antibody responses (16) that would further differentiate UB- 612 from many authorized vaccines. As SARS-CoV-2 continues to evolve, several strategies are being explored to effectively prevent COVID- 19 caused by newly emerging SARS-CoV-2 variants, including monovalent variant antigen matching, multivalent, or universal vaccine approaches. Our results indicate that UB-612 could offer an alternative strategy for the rapid development of a booster vaccine, eliciting high qualities of antibody responses with extensive activity across currently circulating and potentially future SARS-CoV-2 variants.

Reference list for Example 2

1. Vianaet al., Nature doi:10.1038/s41586-022-04411-y (2022).

2. World Health Organization, Global overview data as of 20 March 2022. COVID-19 Weekly Epidemiological Update 84th edition. https://www.who.int/publications/rn/item/weekly-epidemiologi cal-update-on-covid-19— -22-march-2022.

3. Rahimi et al., Arch. Med. Res. doi:10.1016/J.ARCMED.2022.01.001 (2022).

4. Dolgin, Nature 601, 311-311 (2022).

5. Kleanthous et al., NPJ Vaccines 6, 128. doi:10.1038/s41541-021-00393-6 (2021).

6. Planas et al., Nature doi:10.1038/s41586-021-04389-z (2021).

7. Perez-Then et al., Nat. Med. doi:10.1038/s41591-022-01705-6 (2022).

8. Pajon et al., N. Engl. J. Med. doi:10.1056/NEJMC2119912 (2022).

9. Collie et al., NEJM 386, 494-496 doi:10.1056/NEJMc2119270 (2021).

10. Edara et al., N. Engl. J. Med. 385, 664-666 (2021).

11. Hoffmann et al., Cell 185, 447-456.ell (2022).

12. Muik et al., Science 375, 678-680 doi: 10.1126/science.abn7591 (2022).

13. Schmidt et al., Nature 600, 512-516 (2021).

14. Guirakhoo et al., bioRxiv doi:10.1101/2020.11.30.399154 (2022).

15. Wang et al., J. Clin. Invest. https://doi.org/10.1172/JCI157707 (2022). Wang et al., doi:10.21203/RS.3.RS-944205/Vl (2021).

16. Mattiuzzo et al., Establishment of the WHO International Standard and Reference Panel for anti-SARS-CoV-2 antibody on behalf of the ISARIC4C Investigators. https://cdn.who.int/media/docs/default-source/biologicals/ec bs/bs-2020-2403-sars-cov- 2-ab-ik- 17 -nov-2020_4ef4fdae-e 1 ce-4ba7 -b21 a- d725c68bl52b.pdf?sfvrsn=662b46ae_8&download=true (2020).

17. Goldblatt et al., Vaccine 40, 306-315 (2022).

18. Khoury et al., Nat. Med. 27, 1205-1211 (2021).

19. Cromer et al., Lancet Microbe 3, e52-e61 (2022).

20. Yu et al., N. Engl J Med. 2022 Mar 16. doi: 10.1056/NEJMc2201849.

21. Dejnirattisai et al., Cell. 185, 467-484 (2022). 22. Wesemann, Cell 185, 457-466.e4 (2022).

23. Wang et al., Provisional US patent application 62,978,596. February 19, 2020.

24. Manenti et al., J. Med. Virol. 92, 2096-2104 (2020).

25. Reed et al., Am. J. Epidemiol. 27, (1938).

26. Johnson et al., J. Clin. Virol. 130, 104572 (2020).

EXAMPLE 6

Booster immunization with UB-612 stimulates cross-reactive neutralizing antibodies against Omicron BA.5 virus.

Selected serum samples from Phase 1 study V-123 (prime-boost immunization with UB- 612, n=10) were tested against BA.5 Omicron. The results of the live BA.5 virus neutralization test were compared to the data generated against BA.l and Wuhan variants in a similar test using the same set of sera samples. In comparison to BA.l, the BA.5 titers were approximately 2-3- fold lower and compared to Wuhan they were ~ 12- fold lower. The results were in line with the expectations based on reports for other vaccines, i.e. after a 3 ^rd dose booster immunization with Pfizer BNT162b2 (Durability of Booster mRNA Vaccine against SARS-CoV-2 BA.2.12.1, BAA, and BA.5 Subvariants (nejm.org).). The results are shown in Fig. 33.

Table 1

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

Table 2

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

Table 3

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

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

Table 4

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

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

Table 5

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

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

61

SUBSTITUTE SHEET ( RULE 26) Table 6

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

Table 7

Examples of Optional Heterologous Spacers, CpG Oligonucleotides, and RT-PCR Primers/Probes 51615-003W0

Table 8

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

51615-003W0

51615-003W0

51615-003W0

Table 9

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

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

Table 10

Examples of Amino Acid Sequences of Mutated Hinge Regions Derived from IgGl Table 11

Amino Acid Sequences of sFC and Fc Fusion Proteins

Table 12

Nucleic Acid Sequences of sFc and Fc Fusion Proteins 51615-003W0

Table 13

SARS-CoV-2 antigenic peptides

51615-003W0

51615-003W0 The cysteine residues were replaced by serine that are underlined.

51615-003W0

Table 14

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 15

Composition of UB-61220 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-612 60 pg/mL

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

Table 17

Composition of UB-612200 pg/mL

¹ Materials to be used for the Phase 2 and 2/3 clinical trials will be manufactured to cGMP The following Table provides exemplary sequences of molecules that can be used in vaccine compositions described herein (including as heterologous boosters or as components of an accelerated dosing method as described herein.

Exemplary Sequences

References:

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

1. AHMED, S.F., et al., “Preliminary identification of potential vaccine targets for 2019-nCoV based on SARS-CoV immunological studies.” DOI: 10.1101/2020.02.03.933226 (2020)

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

3. BLUMBERG, R.S., et al., “Receptor specific transepithelialus transport of therapeutics.” US Patent Nos. 6,030,613 (2000), 6,086,875 (2000), and 6,485,726 (2002)

4. BLUMBERG, R.S., et al., “Central airway administration for systemic delivery of therapeutics.” WO 03/077834 (2002) and US Patent Publication US2003-0235536A1 (2003)

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

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

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

8. CHAN, J.F.W., et al., “Broad-spectrum antivirals for the emerging Middle East respiratory syndrome coronavirus.” J. Infect., 67(6):606- 616 (2013) CHANG, J.C.C., et al., “Adjuvant activity of incomplete Freund’s adjuvant.” Advanced Drug Delivery Reviews, 32(3): 173-186 (1998) CHEN, Y, et al., “Emerging coronaviruses: Genome structure, replication, and pathogenesis.” J Med Virol. DOI: 10.1002/jmv.25681 (2020) CHENG, K.W., et al., “Thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of Middle East respiratory syndrome coronavirus.” Antiviral Res., 115: 9-16 (2015) DE WIT, E., et al., “SARS and MERS: recent insights into emerging coronaviruses.” Nat. Rev. Microbiol., 14(8):523-534 (2016) FERRETTI, A.P., et al., “Unbiased Screens Show CD8(+) T Cells of COVID-19 Patients Recognize Shared Epitopes in SARS-CoV-2 that Largely Reside outside the Spike Protein.” Immunity, (2020) doi:10.1016/j.immuni.2020.10.006. FIELDS, G.B., et al., Chapter 3 in Synthetic Peptides: A User’s Guide, ed. Grant, W.H. Freeman & Co., New York, NY, p.77 (1992) GOEBL, N.A., et al., “Neonatal Fc Receptor Mediates Internalization of Fc in Transfected Human Endothelial Cells.” Mol. Biol. Cell, 19(12):5490-5505 (2008) GRAHAM, R.L., et al., “A decade after SARS: strategies for controlling emerging coronaviruses.” Nat. Rev. Microbiol., 11(12):836- 848 (2013) JUNGHANS, R.P., et al., “The protection receptor for IgG catabolism is the beta2- microglobulin-containing neonatal intestinal transport receptor.” Proc. Natl. Acad. Sci. USA, 93(11):5512-5516 (1996) LE BERT, N., et al., “SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls.” Nature, 584, 457-462 (2020). LEI, C., et al., “Potent neutralization of 2019 novel coronavirus by recombinant ACE2-Ig.” DOI: 10.1101/2020.02.01.929976 (2020) LIU, H., et al., “Fc Engineering for Developing Therapeutic Bispecific Antibodies and Novel Scaffolds.” Frontiers in Immunology ., 8, 38 (2017). LONG, Q.-X., et al., “Clinical and immunological assessment of asymptomatic SARS-CoV- 2 infections.” Nat. Med. 26, 1200-1204 (2020). MAIR-JENKINS, J., et al., “The effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis.” J. Infect. Dis., 211 (l):80-90 (2015) NG, O.-W., et al., “Memory T cell responses targeting the SARS coronavirus persist up to 11 years post-infection.” Vaccine, 34, 2008-2014 (2016). OSBORN, B.L., et al., “Pharmacokinetic and pharmacodynamic studies of a human serum albumin-interferon- alpha fusion protein in cynomolgus monkeys.” J. Pharmacol. Exp. Then, 303(2):540-8 (2002) PERLMAN, S., “Another decade, another coronavirus.” N. Engl. J. Med., DOI: 10.1056/NEJMe2001126 (2020) SHUBIN, Z., et al., “An HIV Envelope gpl20-Fc Fusion Protein Elicits Effector Antibody Responses in Rhesus Macaques.” Clin. Vaccine Immunol., 24, (2017). SUI, J., et al. “Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to S 1 protein that blocks receptor association.” Proc. Natl. Acad. Sci. USA, 101, 2536-2541 (2004). WANG, C.Y., et al., “UB-311, a novel UBITh(®) amyloid P peptide vaccine for mild Alzheimer’s disease.” Alzheimer ’s Dement., 3, 262-272 (2017). WANG, C.Y., “Artificial promiscuous T helper cell epitopes as immune stimulators for synthetic peptide immunogens.” PCT Publication No. WO 2020/132275A1 (2020). WANG, Y, et al., “Coronavirus nspl0/nspl6 methyltransferase can be targeted by nsplO- derived peptide in vitro and in vivo to reduce replication and pathogenesis.” J. Virol., 89(16): 8416-8427 (2015) WIKIPEDIA, The free encyclopedia, “Coronavirus” available at website: en.wikipedia.org/wiki/Coronavirus (accessed February 17, 2020). WYLLIE, D., et al., “SARS-CoV-2 responsive T cell numbers are associated with protection from COVID- 19: A prospective cohort study in key workers.” medRxiv 2020.11.02.20222778 (2020) doi: 10.1101/2020.11.02.20222778. ZHAO, B., et al., “Immunization With Fc-Based Recombinant Epstein-Barr Virus gp350 Elicits Potent Neutralizing Humoral Immune Response in a BALB/c Mice Model.” Front. Immunol., 9, 932 (2018). ZHOU, P, et al., “Discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin.” DOI: 10.1101/2020.01.22.914952 (2020) ZHU, N., et al., “A novel coronavirus from patients with pneumonia in China, 2019.” N. Engl. J. Med., DOI: 10.1056/NEJMoa2001017 (2020) GIANDHARI, J., et al. IRANZADEH, A., et al. PCT International Patent Application No: PCT/US2021/018855, filed February 19, 2021. Some embodiments are within the following numbered paragraphs. 1. A method of preventing or reducing the severity of COVID-19 in a subject, the method comprising administering a first immunogenic composition against SARS-CoV-2 to the subject, followed by a second immunogenic composition against SARS-CoV-2, wherein second immunogenic composition comprises: a. an S-RBD-sFc protein comprising a receptor binding domain (RBD) of the S protein of a SARS-CoV-2 spike protein or a variant thereof; and b. a Th/CTL peptide; wherein the second immunogenic composition optionally comprises one or more of: c. an aluminum phosphate- or an aluminum hydroxide-based adjuvant; d. a CpG oligonucleotide; and e. one or more pharmaceutically-acceptable excipients; and wherein the first immunogenic composition is different from the second immunogenic composition.

2. The method of paragraph 1, wherein the second immunogenic composition comprises a. an S-RBD-sFc protein comprising a receptor binding domain (RBD) of the S protein of a SARS-CoV-2 spike protein or a variant thereof; b. a Th/CTL peptide; c. an aluminum phosphate- or an aluminum hydroxide-based adjuvant; d. a CpG oligonucleotide; and e. optionally, one or more pharmaceutically-acceptable excipients; and wherein the first immunogenic composition is different from the second immunogenic composition.

3. The method of paragraph 1 or 2, wherein the first immunogenic composition comprises one or more proteins or peptides, nucleic acid molecules (e.g., RNA or DNA), viral vectors, or whole viruses.

4. The method of any one of paragraphs 1 to 3, wherein the first immunogenic composition comprises a spike protein of SARS-CoV-2, or a variant and/or fragment thereof (e.g., an RBD-containing fragment thereof).

5. The method of paragraph 4, wherein the first immunogenic composition is selected from NVX-CoV2372 and MVC-COV1901.

6. The method of any one of paragraphs 1 to 3, wherein the first immunogenic composition comprises a nucleic acid molecule encoding a spike protein of SARS-CoV-2, or a variant and/or fragment thereof (e.g., an RBD-containing fragment thereof).

7. The method of paragraph 6, wherein the first immunogenic composition is selected from mRNA-1273 and BNT162b2.

8. The method of any one of paragraphs 1 to 3, wherein the first immunogenic composition comprises a viral vector which comprises a sequence encoding an immunogen of SARS-CoV-2, or a variant or fragment thereof, wherein the immunogen is optionally a spike protein or a fragment thereof (e.g., an RBD-containing fragment thereof).

9. The method of paragraph 8, wherein the viral vector is an adenoviral vector or a parainfluenza virus vector (e.g., hPIV2).

10. The method of paragraph 9, wherein the vector is an adenoviral vector.

11. The method of paragraph 9 or 10, wherein the first immunogenic composition is selected from the group consisting of AZD1222, Janssen COVID-19 vaccine (JNJ-78436735), and Sputnik V (Gam-COVID-Vac).

12. The method of any one of paragraphs 1 to 3, wherein the first immunogenic composition comprises whole SARS-CoV-2 virus.

13. The method of paragraph 12, wherein the first immunogenic composition is CoronaVac.

14. The method of any one of paragraphs 1 to 13, wherein the first immunogenic composition comprises a composition of (a)-(b) or (a)-(d) of paragraph 1, except that the S-RBD- sFc protein and/or the amount of one or more components of the composition is different from that of the second composition.

15. The method of any one of paragraph 1 to 14, wherein the first immunogenic composition is administered one time before the second immunogenic composition is administered.

16. The method of any one of paragraphs 1 to 15, wherein the first immunogenic composition is administered two times before the second immunogenic composition is administered.

17. The method of any one of paragraphs 1 to 16, wherein the second immunogenic composition is administered within about 2.5 to 4.5 months after the first immunogenic composition; within about 3 to 4 months of the first immunogenic composition; about three months after the first immunogenic composition; or about six or more months (e.g., about 6, 7, 8, 9, 10, or 11 months, or about 1, 2, 3, 4, or 5 years) after the first immunogenic composition.

18. The method of any one of paragraphs 1 to 17, wherein the second immunogenic composition comprises: a. a S-RBD-sFc protein comprising a receptor binding domain (RBD) of the S protein of SARS-CoV-2 (SEQ ID NO: 20), an S-RBD-sFc protein comprising a RBD of the S protein of SARS-CoV-2 SA, beta variant, both, or a variant(s) thereof; and b. a Th/CTL peptide selected from the group consisting of SEQ ID NOs: 9-16, 19, 35-36, 39-100, 145-165, 345-348, 350, 351, 362-365, any combination thereof, variants thereof, or combinations including variants thereof; wherein the composition further optionally includes one or more of: c. an aluminum hydroxide-based adjuvant and a CpG oligonucleotide adjuvant; and d. one or more a pharmaceutically acceptable excipients.

19. The method of any one of paragraphs 1 to 18, wherein the second immunogenic composition comprises: a. a S-RBD-sFc protein comprising a receptor binding domain (RBD) of the S protein of SARS-CoV-2 (SEQ ID NO: 20), an S-RBD-sFc protein comprising a RBD of the S protein of SARS-CoV-2 SA, beta variant, both, or a variant(s) thereof; b. a Th/CTL peptide selected from the group consisting of SEQ ID NOs: 9-16, 19, 35-36, 39-100, 145-165, 345-348, 350, 351, 362-365, any combination thereof, variants thereof, or combinations including variants thereof; c. an aluminum hydroxide-based adjuvant and a CpG oligonucleotide adjuvant; and d. optionally one or more a pharmaceutically acceptable excipients.

20. The method of any one of paragraphs 1 to 19, wherein the S-RBD-sFc protein of the second immunogenic composition comprises a receptor binding domain (RBD) of the S protein of SARS-CoV-2 (SEQ ID NO: 20), or a variant thereof, and optionally wherein the S-RBD-sFc protein is of SEQ ID NO: 235, or a variant thereof.

21. The method of any one of paragraphs 1 to 20, wherein the S-RBD-sFc protein of the second immunogenic composition comprises a RBD of the S protein of SARS-CoV-2 SA, beta variant, or a variant thereof.

22. The method of any one of paragraphs 1 to 21, wherein the second immunogenic composition comprises an S-RBD-sFc protein comprising a receptor binding domain (RBD) of the S protein of SARS-CoV-2 (SEQ ID NO: 20), or a variant thereof, and an S-RBD-sFc protein comprising an RBD of the S protein of SARS-CoV-2 SA, beta variant, or both, or a variant thereof or of both.

23. The method of any one of paragraphs 1 to 22, wherein the second immunogenic composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the Th/CTL peptides.

24. The method of any one of paragraphs 1 to 23, wherein the second immunogenic composition comprises 6 of the Th/CTL peptides.

25. The method of any one of paragraphs 1 to 24, wherein the second immunogenic composition comprises Th/CTL peptides which comprise SEQ ID NOs: 345, 346, 347, 348, 361, and 66, or variants of one or more thereof.

26. The method of any one of paragraphs 1 to 25, wherein the second immunogenic composition comprises Th/CTL peptides which comprise SEQ ID NOs: 345, 346, 347, 348, 361, and 66.

27. The method of any one of paragraphs 1 to 26, wherein each of the Th/CTL peptides are present in the second immunogenic composition in equal-weight amounts.

28. The method of any one of paragraphs 1 to 27, wherein the ratio (w:w) of the S-RBD- sFc protein to the total weight of the mixture of Th/CTL peptides in the second immunogenic composition is 88:12.

29. The method of any one of paragraphs 1 to 28, wherein the composition comprises a pharmaceutically acceptable excipient which optionally is an adjuvant, buffer, surfactant, emulsifier, pH adjuster, saline solution, preservative, solvent, or any combination thereof.

30. The method of any one of paragraphs 1 to 29, wherein the second immunogenic composition comprises an aluminum phosphate-based adjuvant.

31. The method of any one of paragraphs 1 to 30, wherein the second immunogenic composition comprises an aluminum hydroxide-based adjuvant.

32. The method of any one of paragraphs 1 to 31, wherein the second immunogenic composition comprises a CpG oligonucleotide adjuvant.

33. The method of any one of paragraphs 1 to 32, wherein the second immunogenic composition comprises a pharmaceutically acceptable excipient which is optionally selected from the group consisting of a CpG oligonucleotide, an aluminum hydroxide-based adjuvant, histidine, histidine HC1*H2O, arginine HC1, TWEEN 80 (polyoxyethylene (20) sorbitan monooleate), hydrochloric acid, sodium chloride, 2-phenoxyethanol, water, and any combination thereof.

34. The method of any one of paragraphs 1 to 33, wherein the second immunogenic composition comprises about 0.5-20, 1-10, or 2-5 pg of a CpG oligonucleotide.

35. The method of any one of paragraphs 1 to 34, wherein the second immunogenic composition comprises about 1-10 pg of a CpG oligonucleotide.

36. The method of any one of paragraphs 1 to 35, wherein the second immunogenic composition comprises about 2-5 pg of a CpG oligonucleotide.

37. The method of any one of paragraphs 1 to 36, wherein the second immunogenic composition comprises about 2 pg of a CpG oligonucleotide.

38. The method of any one of paragraphs 1 to 37, wherein, in the second immunogenic composition: (a) the Th/CTL peptide is a mixture of SEQ ID NOs: 345, 346, 347, 348, 361, and 66, and each peptide is present in the mixture in equal- weight amounts; and

(b) the pharmaceutically acceptable excipient is a combination of a CpGl oligonucleotide, an aluminum hydroxide- or aluminum phosphate -based adjuvant, histidine, histidine HC1*H2O, arginine HC1, polyoxyethylene (20) sorbitan monooleate, hydrochloric acid, sodium chloride, and 2-phenoxyethanol in water.

(a) the Th/CTL peptide is a mixture of SEQ ID NOs: 345, 346, 347, 348, 361, and 66, and each peptide is present in the mixture in equal- weight amounts; and

(b) the pharmaceutically acceptable excipient is a combination of a CpGl oligonucleotide, an aluminum hydroxide- or aluminum phosphate-based adjuvant, histidine, histidine HC1*H2O, arginine HC1, polyoxyethylene (20) sorbitan monooleate, hydrochloric acid, sodium chloride, and 2-phenoxyethanol in water.

39. The method of any one of paragraphs 1 to 38, wherein in the second immunogenic composition, the total amount of the S-RBD-sFc protein is between about 2 pg to about 200 pg; and the total amount of the Th/CTL peptides is between about 1 pg to about 25 pg.

40. The method of any one of paragraphs 1 to 39, wherein in the second immunogenic composition, the total amount of the S-RBD-sFc protein is about 8.8 pg; and the total amount of the Th/CTL peptides is about 1.2 pg.

41. The method of any one of paragraphs 1 to 38, wherein in the second immunogenic composition, the total amount of the S-RBD-sFc protein is about 26.4 pg; and the total amount of the Th/CTL peptides is about 3.6 pg.

42. The method of any one of paragraphs 1 to 38, wherein in the second immunogenic composition, the total amount of the S-RBD-sFc protein is about 88 pg; and the total amount of the Th/CTL peptides is about 12 pg.

43. The method of any one of paragraphs 1 to 43, wherein the method reduces the severity of one or more symptoms of COVID-19, prevents hospitalization for COVID-19, reduces the length of hospitalization for COVID-19, and/or maintains vaccine-induced antibodies above protective threshold.

44. A method for preventing or reducing the severity of COVID-19 in a subject, the method comprising administering the second immunogenic composition is administered about 6.5-11 or 7-9 months after the first dose (or only) dose of the first immunogenic composition.

45. A method for producing antibodies in a subject, the method comprising administering first and second immunogenic compositions as set forth in any one of paragraphs 1 to 45 to the subject. 46. The method of any one of paragraphs 1 to 46, wherein the method protects against variants of SARS-CoV-2 and breakthrough cases thereof.

47. A method for preventing or reducing the severity of COVID-19 in a subject, the method comprising administering three doses of an immunogenic composition against SARS- CoV-2 to the subject, wherein the immunogenic composition is as described as a second immunogenic composition in any one of paragraphs 1 to 47, and the three doses are administered within about 5 months of one another.

48. The method of paragraph 47, wherein the second dose is administered within about 2 weeks to about 1.5 months after the first dose.

49. The method of paragraph 47 or 48, wherein the second dose is administered within about 1 month after the first dose.

50. The method of any one of paragraphs 47 to 49, wherein the third dose is administered within about 2.5 months to about 4.5 months after the first dose.

51. The method of any one of paragraphs 47 to 50, wherein the third dose is administered about 3 to about 4 months after the first dose.

52. The method of any one of paragraphs 47 to 51, wherein the third dose is administered about 3 months after the first dose.

53. The method of any one of paragraphs 47 to 52, further comprising a booster dose at 6.5-11 or 7-9 months after the first dose.

54. The method of any one of paragraphs 1 to 53, wherein the vaccination (e.g., the heterologous boosting or the accelerated dosing, optionally followed by a booster) is carried out on a seasonal basis.

55. The method of any one of paragraphs 1 to 54, wherein the heterologous boost is carried out as a seasonal boost.