WO2022013609A1

WO2022013609A1 - Sars-cov-2 vaccine compositions and methods of preparation and use

Info

Publication number: WO2022013609A1
Application number: PCT/IB2021/000464
Authority: WO
Inventors: Marianne Stanford; Olga HRYTSENKO; Kelcey Grace PATTERSON; Rajkannan RAJAGOPALAN; Moamen BYDOUN
Original assignee: Immunovaccine Technologies, Inc.
Priority date: 2020-07-13
Filing date: 2021-07-13
Publication date: 2022-01-20

Abstract

The present application relates generally to compositions and methods for preventing COVID- 19, and in particular to vaccine compositions comprising at least one epitope on the spike protein of SARS-CoV-2 that can elicit production of antibodies, including neutralizing antibodies and antibodies that inhibit steps of viral infection (e.g., phagocytosis).

Description

SARS-COV-2 VACCINE COMPOSITIONS AND METHODS OF PREPARATION AND

USE

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application Nos. 63/051,223, filed on July 13, 2020, and 63/062,702, filed on August 7, 2020, the disclosures of each of which are herein incorporated by reference in their entireties.

[0003] SEQUENCE LISTING

[0004] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on July 12, 2021, is named 249979_000094_SL.txt and is 21,575 bytes in size.

[0005] FIELD OF THE INVENTION

[0006] The present application relates generally to SARS-CoV-2 vaccine compositions, and in particular to vaccine compositions comprising B cell epitopes and/or T cell epitopes unique to SARS-CoV-2 that are derived from certain proteins, such as the highly immunogenic extra-virionic spike (S) protein of SARS-CoV-2, the nucleocapsid protein, and ORF1AB, as well as methods of making and administering the vaccine compositions.

BACKGROUND

[0007] In December 2019, a novel strain of coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), emerged in Wuhan, Hubei Province, China. While initially the outbreak was largely concentrated in China and caused significant disruptions to the economy, it has now spread to over 220 countries and territories across the world which resulted in the World Health Organization (WHO) declaring a pandemic on March 11, 2020. COVID-19, the disease caused by the SARS-CoV-2 virus, is a serious and life-threatening disease. As of July 7th, 2021, there are around 185 million confirmed cases worldwide with around 4 million deaths as a result of SARS-CoV-2 infections. [0008] Research in coronaviruses has identified the benefit of humoral and cellular (B and T cell) immune responses for treatment and protection from infection. For example, the spike (S) protein of SARS-CoV-2 has been identified as likely being highly immunogenic based on in silico and predictive studies based on known viral proteins including SARS-CoV proteins. The S protein forms a homotrimer to mediate viral entry into cells by binding to the cell surface protein ACE2. The receptor-binding domain (RBD) of the S protein undergoes hinge-like conformational movements that transiently hide (close) or expose (open) residues involved in ACE2 interactions (receptor binding motif) on RBD. Each of the monomers in the homotrimer can be open or closed. The RBD should be open in order to expose the receptor binding motif of the RBD and to enable the S protein monomer(s) to engage in ACE2 binding.

[0009] An approach to vaccine development to treat or prevent COVID-19 is to develop vaccines to improve the B cell and/or T cell immune responses to SARS-CoV-2.

[0010] An exemplary method of improving the B cell immune response is to develop antibodies (Abs), including antibodies that can moderate neutralizing activity (i.e., neutralizing antibodies) and antibodies that can inhibit steps in viral infection, e.g., phagocytic activity, to SARS-CoV-2. Neutralizing antibodies (NAbs) are antibodies that defend a cell from a pathogen or infectious particle by neutralizing any effect that the pathogen or particle has biologically. NAbs are part of the humoral response of the adaptive immune system against viruses, intracellular bacteria and bacterial toxins. Vaccines that result in the production of Abs can, thus, be an effective therapeutic against COVID-19.

[0011] T cell-mediated immunity can also play a role in controlling persistent viral infections through processes known as cellular immunity. As T cells recognize and respond to viral antigens, they may produce many protective responses and effector molecules. One such molecule is the cytokine interferon γ, secreted by CD4+ and CD8+ T cells and their memory cells.

[0012] There is, therefore, a need in the art for new and effective vaccines against SARS-

CoV-2. Such a vaccine can comprise viral epitopes that are predicted to elicit an improved B cell and/or T cell immune response against the vims, such as the generation of Abs including, but not limited to, NAbs and antibodies that can inhibit steps of viral infection (e.g., phagocytosis). SUMMARY OF THE INVENTION

[0013] Applicants have now surprisingly discovered that an effective SARS-CoV-2 vaccine can be generated by selecting a combination of epitopes on the spike (S) protein of SARS- CoV-2, optionally in combination with epitopes on the nucleocapsid (N) protein and epitopes in ORF1AB, that elidt Ab production, including NAb production, (also referred to herein as “B cell epitopes” and “T cell epitopes”) in combination with liposomes. In some examples, the combination of epitopes are present in a lipid-in-oil water-free formulation that optionally includes an adjuvant. For example, the selected B cell epitopes and/or T cell epitopes can be found in different portions of the S protein, including in the receptor binding domain (RBD) region, the Sl- CTD and Sl-NTD, and the S2 region of the S protein. The selected T cell epitopes can also be found in different portions of the N protein and/or ORF1AB. Selected epitopes are outside of the S protein 614 mutation which, according to recent research has been demonstrated to increase the virus’ ability to infect cells in vitro and has been suggested to reduce vaccine-induced immunity. A vaccine candidate could keep its potential efficacy independently from current/future mutations of the virus at this site. Selected epitopes of the invention are also outside of areas identified as potentially responsible for vaccine-enhanced disease.

[0014] In one aspect, the invention relates to compositions comprising one or more B cell epitopes of SARS-CoV-2 and/or T cell epitopes of SARS-CoV-2, a carrier, liposomes, and optionally an adjuvant.

[0015] In one aspect, the invention relates to methods of preparing the invented compositions.

[0016] In one aspect, the invention relates to methods of administering the invented compositions to a subject in order to generate antibodies, including neutralizing antibodies, against SARS-CoV-2 and/or to prevent COVID-19.

[0017] In certain embodiments of the compositions and methods disclosed herein, the B cell epitope is a peptide present in the SARS-CoV-2 spike protein. In certain embodiments, the B cell epitope comprises an amino add sequence from the SARS-CoV-2 spike protein (SEQ ID NO: 1) that is capable of eliciting antibodies, such as neutralizing antibodies (NAbs) and/or antibodies that inhibit other steps in viral infection (e.g., phagocytosis), in the subject. In certain embodiments, the B cell epitope is present in the receptor binding site (RBD) and/or the S1 region and/or S2 region of the SARS-CoV-2 spike protein. In certain embodiments, a combination of one or more B cell epitopes are included in the invented compositions. In certain embodiments, the B cell epitope can be a peptide antigen and/or can comprise at least one of amino acid sequences

or a nucleic acid molecule encoding said peptide antigen/epitope. In

certain embodiments, the at least one B cell epitope comprises one or more of the amino acid sequences of SEQ ID NOs: 5, 7, 11, 14, 17, 19, 23, 24, and/or 26. In certain embodiments, the at least one B cell epitope comprises one or more of the amino add sequences of SEQ ID NOs: 5, 7, 14, and/or 19. In certain embodiments, the at least one B cell epitope comprises a mixture of four B cell epitopes comprising the amino acid sequences of SEQ ID NOs: 5, 7, 14, and 19. In certain embodiments, the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 5. In certain embodiments, the at least one B cell epitope comprises the amino add sequence of SEQ ID NO: 7. In certain embodiments, the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the at least one B cell epitope is present as a dimer.

[0018] In certain embodiments of the compositions and methods disclosed herein, the at least one B cell epitope is administered at a concentration of about 0.25 mg/ml to about 1 mg/ml, for example about 0.25 mg/ml to about 0.5 mg/ml, or about 0.5 mg/ml to about 1 mg/ml for each B cell peptide. In certain embodiments, the at least one B cell epitope is administered at a dose of about 0.01 ml to about 1 ml. In certain embodiments, the at least one B cell epitope is administered in at least two doses. In certain embodiments, the at least one B cell epitope is administered at a priming dose of about 0.01 ml to about 1 ml. In certain embodiments, the at least one B cell epitope is administered at a booster dose of about 0.01 ml to about 1 ml. In certain embodiments, the mixture of the four B cell epitopes comprising the amino acid sequences of SEQ ID Nos: 5, 7, 14 and 19 is administered at a dose of 10 μg to 50 μg, for example 10 μg to 25 μg or 25 μg to 50 μβ·

[0019] In certain embodiments of the compositions and methods disclosed herein, the B cell epitope is present in combination with a T cell epitope. The T cell epitope is a peptide present in the SARS-CoV-2 spike protein and/or the nucleocapsid protein and/or in ORF1AB. In certain embodiments, the T cell epitope comprises an amino acid sequence from the SARS-CoV-2 spike protein (SEQ ID NO: 1) that is capable of eliciting antibodies, such as neutralizing antibodies (NAbs) and/or antibodies that inhibit other steps in viral infection (e.g., phagocytosis), in the subject. In certain embodiments, a combination of one or more T cell epitopes are included in the invented compositions. In certain embodiments, the at least one T cell epitope can be a peptide antigen and/or can comprise at least one of amino acid sequences of SEQ ID NOs: 27-40 and/or 42-43 or a nucleic acid molecule encoding said peptide antigen/epitope. In certain embodiments, the at least one T cell epitope comprises one or more of the amino acid sequences of SEQ ID NOs: 27, 28, 32, 34, 38, 42, 43, and combinations thereof. In certain embodiments, the at least one T cell epitope comprises a mixture of T cell epitopes comprising the amino acid sequences of SEQ ID NOs: 27, 28, 32, 34, 38, 42, 43, and combinations thereof. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 27. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 28. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 32. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 34. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 38. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 42. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 43. In certain embodiments, the at least one T cell epitope is present as a dimer.

[0020] In certain embodiments of the compositions and methods disclosed herein, the at least one T cell epitope is administered at a concentration of about 0.25 mg/ml to about 1 mg/ml, for example about 0.25 mg/ml to about 0.5 mg/ml, or about 0.5 mg/ml to about 1 mg/ml for each T cell peptide. In certain embodiments, the at least one T cell epitope is administered at a dose of about 0.01 ml to about 1 ml. In certain embodiments, the at least one T cell epitope is administered in at least two doses. In certain embodiments, the at least one T cell epitope is administered at a priming dose of about 0.01 ml to about 1 ml. In certain embodiments, the at least one T cell epitope is administered at a booster dose of about 0.01 ml to about 1 ml. In certain embodiments, the mixture of the four T cell epitopes comprising the amino acid sequences of SEQ ID Nos: 27-40 and/or 42-43 is administered at a dose of 10 μg to 50 μg, for example 10 μg to 25 μg or 25 μg to 50 μg.

[0021] In certain embodiments of the methods disclosed herein, the method comprises administering the invented composition by injection to the subject. In certain embodiments of the methods disclosed herein, the injection is a subcutaneous or intramuscular injection. In certain embodiments, the injection is a subcutaneous injection.

[0022] In certain embodiments of the compositions and methods disclosed herein, the at least one B cell epitope and/or T cell epitope is present in a composition comprising the at least one B cell epitope and/or T cell epitope, liposomes, and a carrier. In certain embodiments, the carrier comprises a continuous phase of a hydrophobic substance. In certain embodiments, the carrier is a hydrophobic substance such as a vegetable oil, nut oil, or mineral oil. In certain embodiments, the carrier is mineral oil or is a mannide oleate in a mineral oil solution. In certain embodiments, the carrier is Montanide® ISA 51. In certain embodiments, the composition further comprises an adjuvant. In certain embodiments, the composition is water-free or substantially water-free. In certain embodiments of the compositions and methods disclosed herein, the at least one B cell epitope and/or T cell epitope is present in a lipid-in-oil water-free formulation and optionally further includes an adjuvant.

[0023] In certain embodiments of the compositions and methods disclosed herein, the at least one B cell epitope and/or T cell epitope enhances the immune response against the B cell epitope and/or T cell epitope, such as by stimulating the production of antibodies, including NAbs and antibodies that inhibit other steps in viral infection (e.g., phagocytosis).

[0024] In certain embodiments of the compositions and methods disclosed herein, the subject is a human.

[0025] These and other aspects described herein will be apparent to those of ordinary skill in the art in the following description, claims, and drawings.

BRIEF DESCRIPTION OF THE FIGURES

[0026] The accompanying Figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0027] Figure 1A-1B show the effects of the different formulations on mouse body weight changes. Mice (CD-1) received one of eight water-free oil-based formulations containing SARS COV-2 peptides (vaccine candidate), Formulation A (irrelevant control, a water-free oil-based formulation containing SheA peptide (Seq ID NO: 41)), or Formulation Z (a water-free oil-based formulation containing no peptides, vehicle only) or were untreated. Body weights were monitored once every week for the duration of the study. (1A) CD-1 mice (n=10 mice per each treatment) were treated with one of eight Formulations (1 through 8, inclusive). Each formulation contained 2-4 SARS-CoV-2 peptides (50 μg peptide/dose). (1B) Mice (n=10 mice per each treatment) were treated with Formulation 1 or 4 formulated with or without Pam3CSK4 (also referred to as Pam). Formulation 1 contained two SARS-CoV-2 peptides (50 μg peptide/dose) and Formulation 4 contained four SARS-CoV-2 peptides (50 μg peptide/dose). Results are shown as mean ± SD per group. (1 A) Changes in body weights in each group were compared to the changes observed in naive group using a linear regression analysis of the slopes of the regression lines plotted over six or seven weekly timepoints; * p<0.05 (1B) there were no significant differences in body weights between groups treated with Pam vs. those which were not treated (p-values 0.258 and 0.314 in formulations 1 and 4). Note: mice treated with Formulation 8 (1 A) were older (9-11 weeks vs 6-9 weeks) and their weights were higher than the average weights in other groups.

[0028] Figure 2A-2C shows induration grades observed at SOIs after vaccinations of CD- 1 mice with one of eight water-free oil-based formulations containing SARS COV-2 peptides (vaccine candidate), Formulation A (irrelevant control, a water-free oil-based formulation containing SheA peptide Seq ID NO: 41), or with Formulation Z (a water-free oil-based formulation containing no peptides, vehicle only), or in untreated (naive) mice (n=10 per each treatment). Each of eight Formulations (1-8) contained 2-4 SARS-CoV-2 peptides (50 μg peptide/dose). Indurations were monitored every day for 3 days after prime vaccination (2A), every day for 3 days after boost vaccination (2B) and once every week for the duration of the study (2C). Percent of grade 1, 2, 3, and 4 indurations and present of SOIs without indurations (0) are shown for groups of mice vaccinated with Formulation 1-8, Formulation A, Formulation Z and in naive mice.

[0029] Figures 3A-3C show erythema grades observed at SOIs after vaccinations of CD- 1 mice with one of eight water-free oil-based formulations containing SARS COV-2 peptides (vaccine candidate), Formulation A (irrelevant control, a water-free oil-based formulation containing SheA peptide Seq ID NO: 41), or with Formulation Z (a water-free oil-based formulation containing no peptides, vehicle only), or in untreated (naive) mice (n=10 per each treatment). Each of eight Formulations (1-8) contained 2-4 SARS-CoV-2 peptides (50 μg peptide/dose). Erythema was monitored every day for 3 days after prime vaccination (3 A), every day for 3 days after boost vaccination (3B) and once every week for the duration of the study (3C). Percent of grade 1, 2, and 3 erythema and present of SOIs without erythema (0) are shown for groups of mice vaccinated with Formulations 1-8, Formulation A, Formulation Z and in naive mice. [0030] Figure 4A-4D show the effect of Pam3CSK4 adjuvant on induration and erythema grades observed at SOIs after vaccinations with Formulation 1 and 4. CD-1 mice (n=10 per each treatment) were treated with Formulation 1 or 4 formulated with (+) or without (-) Pam3CSK4. Formulation 1 contained two SARS-CoV-2 peptides (50 μg peptide/dose) and Formulation 4 contained four SARS-CoV-2 peptides (50 μg peptide/dose). Indurations (4A and 4C) and erythema (4B and 4D) were monitored every day for 3 days after prime vaccination, every day for 3 days after boost vaccination and once every week for the duration of the study. Percent of grade 1, 2, and 3 erythema, percent of grade 1, 2, 3, and 4 indurations and present of SOI without erythema or indurations (0) are shown for groups of mice vaccinated with Formulation 1 (4 A and 4B) and 4 (4C and 4D).

[0031] Figure 5A-5B show the peptide-antibody titers in response to individual peptides in Formulation 1 without (1 A) and with (1B) the adjuvant Pam3CSK4. CD-1 mice (n=10) were vaccinated with Formulation 1 with (+) or without (-) Pam3CSK4 on SDO and were boosted on SD14. Formulation 1 contained two SARS-CoV-2 peptides (50 μg peptide/dose) including COV2B-494 and -S807. Antibody titers were determined using indirect ELISA on SDH (pre- boost), SD21, SD28, and SD42. Data are presented as Log₁₀ mean end point titer; titers below LLOQ are assigned values of 10^1·85 which is Log 10 (LLOQ/√2) (Croghan et al. 2003). Dots represent individual animal antibody titers.

[0032] Figure 6 shows the peptide-antibody titers in response to individual peptides in Formulation 2. CD-1 mice (n=10) were vaccinated with Formulation 2 on SDO and were boosted on SDH. Formulation 2 contained three SARS-CoV-2 peptides (50 μg peptide/dose) including COV2B-404, -S327 and -S555. Antibody titers were determined using indirect ELISA on SDH (pre-boost), SD21, SD28, and SD42. Data are presented as Logic mean end point titer. Titers below LLOQ are assigned values of 10^1·85 which is Log 10 (LLOQ/√2) (Croghan et al. 2003).Dots represent individual animal antibody titers.

[0033] Figure 7 shows the peptide-antibody titers in response to individual peptides in Formulation 3. CD-1 mice (n=10) were vaccinated with Formulation 3 on SDO and were boosted on SDH. Formulation 3 contained three SARS-CoV-2 peptides (50 μg peptide/dose) including COV2B-461, -S496 and -S516. Antibody titers were determined using indirect ELISA on SDH (pre-boost), SD21, SD28, and SD42. Data are presented as Logic mean end point titer. Titers below LLOQ are assigned values of 10^1·85 which is Log 10 (LLOQ/√2) (Croghan et al. 2003). Dots represent individual animal antibody titers.

[0034] Figure 8A-8B show the peptide-antibody titers in response to individual peptides in Formulation 4 without (8A) and with (8B) the adjuvant Pam3CSK4. CD-1 mice (n=10) were vaccinated with Formulation 4 with (+) or without (-) Pam3CSK4 on SD0 and were boosted on SDH. Formulation 4 contained four SARS-CoV-2 peptides (50 μg peptide/dose) including COV2B-1157, -S821, -S616 and -S369. Antibody titers were determined using indirect ELISA on SDH (pre-boost), SD21, SD28, and SD42. Data are presented as Logic mean end point titer. Titers below LLOQ are assigned values of 10^1·85 which is Log 10 (LLOQ/√2) (Croghan et al. 2003).Dots represent individual animal antibody titers.

[0035] Figure 9 shows the peptide-antibody titers in response to individual peptides in Formulation 5. CD-1 mice (n=10) were vaccinated with Formulation 5 on SD0 and were boosted on SDH. Formulation 5 contained three SARS-CoV-2 peptides (50 μg peptide/dose) including COV2B-S524, -S1165 and -S250. Antibody titers were determined using indirect ELISA on SDH (pre-boost), SD21, SD28, and SD42. Data are presented as Logic mean end point titer. Titers below LLOQ are assigned values of 10^1·85 which is Log 10 (LLOQ/√2) (Croghan et al. 2003). Dots represent individual animal antibody titers.

[0036] Figure 10 shows the peptide-antibody titers in response to individual peptides in

Formulation 6. CD-1 mice (n=10) were vaccinated with Formulation 6 on SD0 and were boosted on SDH. Formulation 6 contained four SARS-CoV-2 peptides (50 μg peptide/dose) including COV2B-S1252, -S672, -S431 and -S373. Antibody titers were determined using indirect ELISA on SDH (pre-boost), SD21, SD28, and SD42. Data are presented as Logic mean end point titer. Titers below LLOQ are assigned values of 10^1·85 which is Log 10 (LLOQ/√2) (Croghan et al. 2003). Dots represent individual animal antibody titers.

[0037] Figure 11 shows the peptide-antibody titers in response to individual peptides in Formulation 7. CD-1 mice (n=10) were vaccinated with Formulation 7 on SD0 and were boosted on SDH. Formulation 7 contained two SARS-CoV-2 peptides (50 μg peptide/dose) including COV2B-S329 and -S684. Antibody titers were determined using indirect ELISA on SD14 (pre- boost), SD21, SD28, and SD42. Data are presented as Log₁₀ mean end point titer. Titers below LLOQ are assigned values of 10^1·85 which is Log 10 (LLOQ/√2) (Croghan et al. 2003). Dots represent individual animal antibody titers.

[0038] Figure 12 shows the peptide-antibody titers in response to individual peptides in Formulation 8. CD-1 mice (n=10) were vaccinated with Formulation 8 on SD0 and were boosted on SD14. Formulation 8 contained two SARS-CoV-2 peptides (50 μg peptide/dose) including COV2B-S486 and -S1182. Antibody titers were determined using indirect ELISA on SD14 (pre- boost), SD21, SD28, and SD42. Data are presented as Log₁₀ mean end point titer. Titers below LLOQ are assigned values of 10^1·85 which is Log 10 (LLOQ/√2) (Croghan et al. 2003). Dots represent individual animal antibody titers.

[0039] Figure 13 shows the peptide-antibody titers in response to individual peptides in

Formulation 9. CD-1 mice (n=8) were vaccinated with Formulation 9 on SD0 and were boosted on SD14. Formulation 9 contained two SARS-CoV-2 peptides (50 μg peptide/dose) including COV2B-S553 and -S809. Antibody titers were were determined using indirect ELISA on SD14 (pre-boost), SD21, SD28, and SD42. Data are presented as Log₁₀ mean end point titer. Titers below LLOQ are assigned values of 10^1·85 which is Log 10 (LLOQ/√2) (Croghan et al. 2003). Dots represent individual animal antibody titers.

[0040] Figure 14 shows serum peptide-antibody titers for the nine SARS-CoV-2 epitopes demonstrating moderate to high immunogenicity. Outbred CD-1 mice (n=8-10) were vaccinated with positive control B cell epitope Formulation A (She A) or Formulations containing SARS- CoV-2 peptides COV2B-S329 (Formulation 7), COV2B-S369, COV2B-S821, COV2B-S616 (Formulation 4), COV2B-S373 (Formulation 6), COV2B-S1165 (Formulation 5), COV2B-S486 (Formulation 8), COV2B-S461 (Formulation 3), or COV2B-S809 (Formulation 9) on SD0 and were boosted on SD14. Serum antibody titers were determined using indirect ELISA. Data are presented as Log₁₀ mean end point titer and are considered significant (**** indicates p-value <

0.0001, *** indicates p-value < 0.001, ** indicates p-value < 0.01, * indicates p-value <0.05 in comparison to time matched irrelevant control, Formulation A (SheA) titers) by two-tailed unpaired t-test. Titers below LLOQ are assigned values of 10^1·85 which is Log 10 (LLOQ/√2) (Croghan et al. 2003). Dots represent individual antibody titers.

[0041] Figure 15 shows neutralization activity in serum samples collected from mice vaccinated with screening Formulation 4 (containing peptides COV2B-S369, -S616, -S821, - S1157 at 50 μg each), Formulation 3 (containing peptides COV2B-S461, -S496, -S516 at 50 μg each), and Formulation 6 (containing peptides COV2B-S373, -S431, -S672, -S1252 at 50 μg each) and with irrelevant control Formulation A (containing SheA peptide). Outbred CD-1 mice were vaccinated on SD0 and were boosted on SD14. On SD42-SD51, the presence of neutralizing antibodies was evaluated in serum samples by pseudo-particle neutralizing assay using a pseudoviruses expressing the SARS-CoV-2 spike protein and HEK293T cells expressing the human SARS-CoV-2 receptor ACE2. Data presented as geometric mean of Log2 IC50 titers; dots represent individual animal titers. Samples were serially diluted two-fold starting at a 1 :4 dilution. IC50 titer values are defined as the inverse of the greatest dilution of sera providing 50% viral load reduction relative to virus-only infection. Lines indicate lower and upper limit of quantification of the assay (LLOQ=1:42²; ULOQ=1:32; 2⁵); titers below LLOQ are assigned values of 2^1.5 which is Log₂ (LLOQ/√2) (Croghan et al. 2003).

[0042] Figure 16A-16B. Antibody responses to Formulation X prepared using in house dimers. Outbred CD-1 mice (n=10-12 per group) were vaccinated with Formulation X (16A) or with Formulation Z (vehicle only) (16B) on SD0 and were boosted on SD14. Formulation X contained COV2B-S373D, -S461D, -S616D, -S821D peptide dimers (left to right across the x- axis) prepared in house and were evaluated at 25 μg of each peptide per dose. Antibody titers in response to individual peptides in Formulation X were determined using indirect ELISA on SD14 (pre-boost), SD21, SD28, SD42, SD84, SD112 and SD140. Figure presents geometric mean of Log₁₀ end point titers; dots represent individual animal antibody titers. Samples were serially diluted two-fold starting at a 100-fold dilution. Endpoint titer values are defined as the inverse of the greatest dilution above the assay cutoff determined using a calculation method described by Frey et al. (Frey et al. 1998). Line indicates lower limit of quantification of the assay (LLOQ=1: 100 or 10²); titers below LLOQ are assigned values of 1.85 which is Log10 (LLOQ/√2) (Croghan et al. 2003). Note: 3 mice per group were terminated on SD21 for an additional analysis of serum samples. [0043] Figure 17A-17B. Antibody responses to Formulation X prepared using commercial dimers. Outbred CD-1 mice (n=10-12 per group) were vaccinated with Formulation X (17A) or with vehicle only, Formulation Z (17B) on SDO and were boosted on SD14. Formulation X contained COV2B-S373D, -S461D, -S616D, -S821D peptide dimers (left to right across the x- axis) was evaluated at two strengths: 25 μg of each peptide per dose and 10 μg of each peptide per dose. Antibody titers in response to individual peptides in Formulation X were determined using indirect ELISA on SD14 (pre-boost), SD21, SD28, SD42, SD56, SD84, SD112 and SD140. Figure presents geometric mean of Log₁₀ end point titers; dots represent individual animal antibody titers. Samples were serially diluted two-fold starting at a 100-fold dilution. Endpoint titer values are defined as the inverse of the greatest dilution above the assay cutoff determined using a calculation method described by Frey et al. (Frey et al. 1998). Line indicates lower limit of quantification of the assay (LLOQ=1:100=10²); titers below LLOQ are assigned values of 10^1·85 which is Log10 (LLOQ/√2) (Croghan et al.2003). In some cases, the endpoint titers exceeded the highest prepared dilution on the ELISA plate. The highest prepared dilution was then used to determine the corresponding titer and used for further downstream analysis. Repeats were performed for samples where a endpoint titer was not achieved during the initial assay run. Note: 3 mice per group were terminated on SD43 for an additional analysis of serum samples.

[0044] Figure 18. Analysis of cytokine levels in sera from mice vaccinated with Formulation X prepared with in house dimers. Outbred CD-1 mice (n=3-5 per group) were vaccinated with Formulation X, with Formulation A (irrelevant control), or with Formulation Z (vehicle only) on SDO and were boosted on SD14. Formulation X contained COV2B-S373D, COV2B-S461D, COV2B-S616D, COV2B-S821D peptide dimers and was evaluated at 25 μg of each peptide per dose. Multiplex analysis evaluated levels of the following cytokines: IL-1α, IL- 1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12(p70), IL-13, IL-17A, IFN-γ, TNFα, GM-CSF, KC/CXCL1, LIX/CXCL-5, MCP-1/CCL-2, and MIP-2 in sera samples collected on SD21. Data shown as mean of group and individual animal values; means were not statistically different by one-way ANOVA with Tukey’s post hoc (p < 0.05).

[0045] Figure 19. Analysis of cytokine levels in sera from mice vaccinated with Formulation X. Outbred CD-1 mice (n=3-5 per group) were vaccinated with Formulation X, with Formulation A (irrelevant control) or with Formulation Z (vehicle only) on SDO and were boosted on SDH. Formulation X contained COV2B-S373D, COV2B-S461D, COV2B-S616D, COV2B- S821D peptide dimers prepared commercially and evaluated at two strengths: 25 μg of each peptide per dose and 10 μg of each peptide per dose. Multiplex analysis evaluated levels of the following cytokines: IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12 (p70), IL-13, IL-17A, IFN-γ, TNFα , GM-CSF, KC/CXCL1, LIX/CXCL-5, MCP-1/CCL-2, and MIP-2 in sera samples collected on SD43. Data shown as mean of group and individual animal values; statistics by one- way ANOVA with Tukey’s post hoc,** indicates p-value < 0.01, * indicates p-value <0.05.

[0046] Figure 20. Structural organization of the SARS-CoV-2 S-protein. The positioning of the linear epitopes evaluated in this study are marked on the sequence map. Gycosylation sites are marked in pink and amino acid residues involved in contact with the ACE2 receptor are marked in orange italics. The four peptide epitopes included in the final candidate formulation are coloured dark purple.

[0047] Figure 21A-21B. Immunogenicity assessment of the antigen targeted immune responses in human sera samples to peptides in Formulation X. Formulation X contains COV2B- S373D, -S461D, -S616D, -S821D peptide dimers. The line indicates background of the assay. Human sera samples from normal healthy subjects (Figure 21 A) or from SARS-CoV-2 convalescent samples (Figure 21B) were assessed for antibody detection by indirect ELISA to peptide dimers contained in Formulation X. Figure 21A-21B presents OD values obtained at the lowest sera dilution in indirect ELISA. The background of the assay is calculated as average OD of blank wells (collected on plates for all four peptides) + 2 standard deviations and is 0.057, represented by the dotted line. Statistics by one-way Mann-Whitney U test to compare the response between healthy (21A) and convalescent (21B) samples for each of the peptides. ** indicates p- value < 0.01, * indicates p-value <0.05.

[0048] Figure 22. Peptide-specific antibody responses to Formulation X. Outbred CD-1 mice (n=10 per group) were vaccinated with Formulation X or with Formulation Z (vehicle only) on SD0 and were boosted on SDH. Formulation X contained COV2B-S373D, -S461D, -S616D, -S821D peptide dimers. Antibody titers were determined one week after boost vaccination (SD21) and presented as geometric mean of Log₁₀ end point titers; dots represent individual animal antibody titers. Samples were serially diluted two-fold starting at a 100-fold dilution. Endpoint titer values are defined as the inverse of the greatest dilution above the assay cutoff determined using a calculation method described by Frey et al. (Frey et al. 1998). Line indicates lower limit of quantification of the assay (LLOQ=1: 100 10²;); titers below LLOQ are assigned values of 10^1·85 which is Log 10 (LLOQ/V2) (Croghan et al. 2003).

[0049] Figure 23. Spike-specific antibody responses to Formulation X. Full-length S protein (S1+S2 ECD) (Sino Biologicals, aa Vall6-Prol213) coated and pre-coated plates with S1 (AcrobioSystems aa Vall6-Arg685) and RBD (AcrobioSystems amino adds Arg319-Phe541) were utilized to measure S-protein-specific IgG antibody levels in sera. Outbred CD-1 mice (n=10 per group) were vaccinated with Formulation X or with Formulation Z (vehicle only) on SD0 and were boosted on SD14. Formulation X contained COV2B-S373D, -S461D, -S616D, -S821D peptide dimers. Antibody titers were determined one week after boost immunization (SD21) and presented as geometric mean of Log₁₀ end point titers; dots represent individual animal antibody titers. Samples were serially diluted two-fold starting at a 100-fold dilution. Endpoint titer values are defined as the inverse of the greatest dilution above the assay cutoff determined using a calculation method described by Frey et al. (Frey et al. 1998). Line indicates lower limit of quantification of the assay (LLOQ=1:100 10²;); titers below LLOQ are assigned values of 10^1·85 which is Log 10 (LLOQ/√2) (Croghan et al. 2003).

[0050] Figure 24A-24D. Isotypes of peptide-specific immunoglobulins (Ig). Outbred CD- 1 mice were vaccinated with Formulation X or with Formulation Z (vehicle only) on SD0 and were boosted on SD14 or remained untreated (naive). Formulation X contained COV2B-S373D, -S461D, -S616D, -S821D peptide dimers. Isotypes of peptide-specific Igs were determined in mouse sera samples one week after boost immunization (SD21) with Formulation X (n=10). Serum samples were incubated with beads that were coated with individual Formulation X peptides to capture peptide-specific antibodies. Fluorescently labelled antibodies were then used to detect IgA, IgG, IgM and IgE and samples were acquired on a Flow Cytometer (FACSCelesta™, BD Biosciences). Data was analysed using FlowJo software. Levels of peptide-specific immunoglobulins are quantified as the median fluorescent intensity (MFI) of each sample, normalized to the MFI levels of the no serum control . Results are presented as group means and dots represent individual animal immunoglobulin levels. [0051] Figure 25. IFN-γ ELISPOT responses in spleens of CD-1 mice one week after boost vaccination (SD21) with Formulation X. Outbred CD-1 mice (n=10 per group) were vaccinated with Formulation X or with Formulation Z (vehicle only) on SDO and were boosted on SD14 or remained untreated (naive). One week after boost immunization, spleens were collected and used in an IFN-γ ELISPOT assay. Data are presented as the number of spot forming units (SFU) of duplicate wells in response to peptide stimulation for each individual mouse. The bar represents the average SFU per group. Media represents responses in non-stimulated splenocytes and “Irr Pep” represents responses in splenocytes stimulated with an irrelevant peptide.

[0052] Figure 26A-26C. Changes in NK cells, mature B cells and plasmablasts in spleen and in vaccine draining lymph nodes of Formulation X-vaccinated mice. Outbred CD-1 mice (n=10 per group) were vaccinated with Formulation X or with Formulation Z (vehicle only) on SDO and were boosted on SDH or remained untreated (naive). Formulation X contained COV2B- S373D, -S461D, -S616D, and -S821D peptide dimers and was evaluated at 25 μg of each peptide per dose. One week after boost immunization (SD21), spleens and vaccine draining lymph nodes were collected and used for IMF staining. (26A) Total NK (NK1.1+) cells are presented as percent of CD45+ cells. (26B) Plasmablasts were identified as CD19⁺ CD38⁺ CD138⁺ cells, and (26C) mature B cells were identified as CD19⁺ IgD⁺ IgM ^+/- Statistics by ANOVA with Tukey’s post hoc; ***indicates p-value < 0.001, ** indicates p-value < 0.01, * indicates p-value <0.05.

[0053] Figure 27A-27B. MHC class II expression on B cell subsets in immune organs after Formulation X vaccination. Outbred CD-1 mice (n=10 per group) were vaccinated with Formulation X or with Formulation Z (vehicle only) on SDO and were boosted on SDH or remained untreated (naive). Formulation X contained COV2B-S373D, -S461D, -S616D, and - S821D peptide dimers and was evaluated at 25 μg of each peptide per dose. Seven days after boost immunization (SD21), spleens and vaccine draining lymph nodes were collected and used for IMF staining. Mean fluorescence intensity (MFI) of B cells subsets is reported for spleen-resident B cells (27 A), and lymph node resident B cells (27B). Note: two mice from naive group were removed from analysis, as they did not have any detectable MHC- II expression. Statistics by two- way ANOVA with Tukey’s post hoc, ** indicates p-value < 0.01, * indicates p-value <0.05. [0054] Figure 28A-28R. Cytokine secretion by splenocytes from mice vaccinated with Formulation X. Outbred CD-1 mice (n=10 per group) were vaccinated with Formulation X, or with Formulation Z (vehicle only) on SDO and were boosted on SDH or remained untreated (naive). Formulation X contained COV2B-S373D, -S461D, -S616D, -S821D peptide dimers and was evaluated at 25 μg of each peptide per dose. Splenocytes were extracted on SD21 and stimulated with individual peptides; with pool of four Formulation X peptides, with irrelevant peptide (COV2B-S1157) or remained unstimulated. Supernatants were collected 18 hours post- stimulation and profiled by multiplex analysis to evaluate levels of the following cytokines: IL- 1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12 (p70), IL-13, IL-17A, IFN-γ, TNF-α, GM- CSF, KC/CXCL1, MIP-2/CXCL-2, LIX/CXCL-5, and MCP-1/CCL-2 (Figure 28A-28R, respectively). Data shown as mean of group and individual mouse values of fold-change from respective media alone control; **** indicates p-value <0.0001, *** indicates p-value < 0.001, ** indicates p-value < 0.01, * indicates p-value <0.05. All statistics were conducted using two-way ANOVA with post-hoc Tukey HSD.

[0055] Figure 29. Peptide-specific antibody responses to Formulation X GMP clinical lot. Outbred CD-1 mice (n=10 per group) were vaccinated with Formulation X, with Formulation Z (vehicle only), or with phosphate saline buffer (PBS; background control) on SDO and were boosted on SDH. Formulation X contained COV2B-S373D, -S461D, -S616D, -S821D peptide dimers and was evaluated at 25 μg of each peptide per dose. Peptide-specific antibody titers were determined on SD56 and presented as geometric mean of Log₁₀ end point titers; dots represent individual animal antibody titers. Samples were serially diluted two-fold starting at a 100-fold dilution. Endpoint titer values are defined as the inverse of the greatest dilution above the assay cutoff determined using a calculation method described by Frey et al. (Frey et al. 1998). Line indicates lower limit of quantification of the assay (LLOQ=1:100 10²;); titers below LLOQ are assigned values of 10^1·85 which is Log 10 (LLOQ/√2) (Croghan et al. 2003).

[0056] Figure 30. Spike-specific antibody response determined by indirect ELISA in serum samples collected from mice vaccinated with Formulation X GMP clinical lot. Outbred CD- 1 mice (n=10 per group) were vaccinated with Formulation X, with Formulation Z (vehicle only), or with phosphate saline buffer (PBS; background control) on SDO and were boosted on SDH. Formulation X contained COV2B-S373D, -S461D, -S616D, -S821D peptide dimers and was evaluated at 25 μg of each peptide per dose. Spike-specific antibody titers were determined on SD56 and presented as geometric mean of Log₁₀ end point titers; dots represent individual animal antibody titers. Samples were serially diluted two-fold starting at a 100-fold dilution. Endpoint titer values are defined as the inverse of the greatest dilution above the assay cutoff determined using a calculation method described by Frey et al. (Frey et al. 1998). Line indicates lower limit of quantification of the assay (LLOQ=1: 100 10²;); titers below LLOQ are assigned values of 10^1·85 which is Log 10 (LLOQ/√2) (Croghan et al. 2003).

[0057] Figure 31. Neutralization activity in serum samples collected from mice vaccinated with Formulation X GMP clinical lot. Outbred CD-1 mice (n=10 per group) were vaccinated with Formulation X, with Formulation Z (vehicle only), or with phosphate saline buffer (PBS; background control) on SD0 and were boosted on SDH. Formulation X contained COV2B- S373D, -S461D, -S616D, -S821D peptide dimers and was evaluated at 25 μg of each peptide per dose. Neutralization activity was determined using PNA on SD56 and presented as geometric mean of Logz IC50 titers; dots represent individual animal titers. Samples were serially diluted two-fold starting at a 1:4 dilution. IC50 titer values are defined as the inverse of the greatest dilution of sera providing 50% viral load reduction relative to virus-only infection. Lines indicate lower and upper limit of quantification of the assay (LLOQ=1:42²; ULOQ=1:32; 2⁵); titers below LLOQ are assigned values of 2^1.5 which is Log2 (LLOQ/√2) (Croghan et al. 2003).

[0058] Figure 32. Phagocytic activity of serum samples collected from mice vaccinated with Formulation X GMP clinical lot. Outbred CD-1 mice (n=10 per group) were vaccinated with Formulation X, with Formulation Z (vehicle only), or with phosphate saline buffer (PBS; background control) on SD0 and were boosted on SD14. Formulation X contained COV2B- S373D, -S461D, -S616D, -S821D peptide dimers and was evaluated at 25 μg of each peptide per dose. Bone marrow-derived cells isolated from independent untreated CD-1 mice were stimulated with M-CSF for 6 days to promote monocytic differentiation. On Day 7, Spike protein coated fluorescent beads were treated with sera from mice vaccinated with Formulation X or Formulation Z, or with sera from naive mice. Treated beads were then added to macrophages to allow bead uptake and/or antibody-dependent phagocytosis to take place. The assay measured the ability of antibody-containing sera to bind spike protein-coated fluorescent beads and mediate Fc-recep tor- dependent phagocytosis by M-CSF-differentiated macrophages, using flow cytometry. Phagocytic score was determined as % bead positive × MFI bead positive, after subtraction of assay background. Bars represent group means and dots represent individual animals, **** indicates p- value <0.0001,*** indicates p-value < 0.001, ** indicates p-value < 0.01, * indicates p-value <0.05. All statistics were conducted using two-way ANOVA with post-hoc Tukey HSD.

[0059] Figure 33. Cytotoxic T cell response by IFN-γ ELISPOT in mice vaccinated with Formulation X GMP clinical lot. Outbred CD-1 mice (n=10 per group) were vaccinated with Formulation X, with Formulation Z (vehicle only), or with phosphate saline buffer (PBS; background control) on SD0 and were boosted on SDH. Formulation X contained COV2B- S373D, -S461D, -S616D, -S821D peptide dimers and was evaluated at 25 μg of each peptide per dose. Six weeks after boost immunization (SD56) spleens were collected and used in an IFN-γ ELISPOT assay. Data are presented as mean number of spot forming units (SFU) of duplicate wells in response to peptide stimulation for each individual mouse. The bar represents the average SFU per group. Media represents responses in non-stimulated splenocytes and S1157 represents responses in splenocytes stimulated with an irrelevant peptide.

[0060] Figure 34A-34B. Ability of antibodies induced by Formulation X to recognize and bind presently available SARS-CoV-2 variant subunits and mutations found in COV2B peptides. CD-1 mice (n=10 per group) were vaccinated with Formulation X on SD0 and were boosted on SDH. Serum samples were collected on SD56 and assessed for ability to bind to (34A) mutated COV2B-S461 peptide or (34B) spike subunits from SARS-CoV-2 variants Alpha (B.1.17) and Beta (B 1.351) compared to wild-type counterparts using indirect ELISA. Based on the alignment of four Formulation X epitopes with the locations of known spike mutations, currently, only binding of one of the four Formulation X antibodies can be directly impacted by known spike mutations: binding of the COV2B-S461D antibodies to its epitope that contains point spike mutation E484K in Beta (B.1.351) and Gamma (P.1) variants (and occasionally Alpha (B.1.1.7)). Serum samples were serially diluted two-fold starting at a 100-fold dilution. Endpoint titer values are defined as the inverse of the greatest dilution above the assay cutoff determined using a calculation method described by Frey et al. (Frey et al. 1998). Line indicates lower limit of quantification of the assay (LLOQ=1:100 10²); titers below LLOQ are assigned values of 10^1·85 which is Log 10 (LLOQ/V2) (Croghan et al. 2003). [0061] Figure 35A-35B. Peptide-specific antibody titers in rats vaccinated with Formulation X. Sprague-Dawley rats (n=8-20 per group) were vaccinated with Formulation X (35A) or with Formulation Z (vehicle only) (35B) on SDO and were boosted on SD14 and SD28. Formulation X contained COV2B-S373D, -S461D, -S616D, -S821D peptide dimers prepared commercially and was evaluated at two doses obtained with a single formulation, dosed by volume: 25 μg of each peptide per dose and 50 μg of each peptide per dose. Serum samples were collected on SD30 and on SD56. Antibody titers in response to individual peptides in Formulation X were determined with indirect ELISA. Figure presents geometric mean of Log₁₀ end point titers; dots represent individual animal antibody titers. Samples were serially diluted two-fold starting at a 400-fold dilution. Endpoint titer values are defined as the inverse of the greatest dilution above the assay cutoff (mean naive OD + 2SD). Line indicates lower limit of quantification of the assay (LLOQ=1:400 or 10²·⁶); titers below LLOQ are assigned values of 10²·⁴⁵ which is Log₁₀ (LLOQ/√2) (Croghan et al. 2003).

[0062] Figure 36. Spike binding antibody titers in rats vaccinated with Formulation X.

Sprague-Dawley rats (n=8-20 per group) were vaccinated with Formulation X or with Formulation Z (vehicle only) on SDO and were boosted on SD14 and SD28. Formulation X contained COV2B- S373D, -S461D, -S616D, -S821D peptide dimers prepared commercially and was evaluated at two doses obtained with a single formulation, dosed by volume: 25 μg of each peptide per dose and 50 μg of each peptide per dose. Serum samples were collected on SD30 and on SD56. Antibody titers in response to SARS-CoV-2 spike protein were determined with indirect ELISA. Figure presents geometric mean of Log₁₀ end point titers; dots represent individual animal antibody titers. Samples were serially diluted two-fold starting at a 100-fold dilution. Endpoint titer values are defined as the inverse of the greatest dilution above the assay cutoff determined using a calculation method described by Frey et al. (Frey et al. 1998). Lines indicates lower and upper limit of quantification of the assay (LLOQ=1: 100 10²; ULOQ=12800; 10⁴¹¹); titers below LLOQ are assigned values of 1.85 which is Log₁₀ (LLOQ/√2) (Croghan et al. 2003).

[0063] Figure 37A-37C. IFN-γ ELISPOT responses in spleens of HLA-A1 (37A), HLA- A2 (37B) and HLA-A24 (37C) transgenic mice 8 days following injection of screening Formulation A1, Formulation A2, and Formulation A24. T cell epitope screening Formulation A1 contained COV2T-ORF1AB-4163 and COV2T-ORF1AB-5299. T cell epitope screening Formulation A2 contained COV2T-N222, COV2T-S996, COV2T-S1185, and COV2T-S1220. T cell epitope screening Formulation A24 contained COV2T-S444 and COV2T-S508. Transgenic mice (n=8 per group) were vaccinated with Formulation A1 (37A), Formulation A2 (37B), or Formulation A24 (37C), or with Formulation Z (vehicle only) (37 A-C) on SDO. Eight days after immunization (SD8), spleens were collected and used in an IFN-γ ELISPOT assay. Data are presented as the number of spot forming units (SFU) of duplicate wells in response to the indicated peptide stimulation or peptide pool for each individual mouse. The bar represents the average SFU per group. Media represents responses in non-stimulated splenocytes and P5 represents responses in splenocytes stimulated with an irrelevant peptide.

[0064] Figure 38A-38C. IFN-γ ELISPOT responses in spleens of HLA-A1 (38 A), HLA- A2 (38B) and HLA-A24 (38C) transgenic mice four weeks after boost vaccination (SD42) with Formulation Y. Transgenic mice (n=10 per group) were vaccinated with Formulation Y or Formulation Z (vehicle only) on SDO and were boosted on SD14. Formulation Y contained COV2B-S373D, -S461D, -S616D, -S821D peptide dimers at 25 μg each peptide per dose and COV2T-S1220, -S444, -ORF 1 AB-3906 (25 μg dose), and -ORF1AB-5299 each at 50 μg each peptide per dose. Twenty-eight days after boost immunization (SD42), spleens were collected and used in an IFN-γ ELISPOT assay. Data are presented as the number of spot forming units (SFU) of duplicate wells in response to the indicated peptide stimulation for each individual mouse. The bar represents the average SFU per group. Media represents responses in non-stimulated cell conditions and P5 represents responses stimulated with an irrelevant peptide.

[0065] Figure 39. Peptide-specific antibody responses to Formulation Y. Transgenic mice with either HLA-A1, HLA-A2 or HLA-A24 (n=10 per group) were vaccinated with Formulation Y on SDO and were boosted on SD14. Formulation Y contained COV2B-S373D, -S461D, -S616D, -S821D peptide dimers at 25 μg each peptide per dose and COV2T-S 1220, -S444, -ORF 1 AB-3906 (25 μg dose), and -ORF1AB-5299 each at 50 μg each peptide per dose. Peptide-specific antibody titers were determined on SD42 and presented as geometric mean of Logic end point titers; dots represent individual animal antibody titers. Samples were serially diluted two-fold starting at a 100-fold dilution. Endpoint tier values are defined as the inverse of the greatest dilution above the assay cutoff determined using a calculation method described by Frey et al. (Frey et al. 1998). Line indicates lower limit of quantification of the assay (LLOQ=1:100 10²;); titers below LLOQ are assigned values of 10^1·85 which is Log 10 (LLOQ/√2) (Croghan et al. 2003).

DETAILED DESCRIPTION

[0066] Before the present invention is described, it is to be understood that this invention is not limited to particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0067] Vaccines are used to elicit immune responses against desired pathogens. These immune responses include generation of protective antibodies to confer immunity against the pathogen. The vaccines generally comprise antigens unique to the pathogen, and the antigens can be selected to activate certain immune responses in the subject. For example, it is possible to select antigens that interfere with the pathogen’s ability to bind to and interact with the subject’s cells, and to administer these antigens to the subject in order to stimulate production of neutralizing antibodies.

[0068] The compositions and methods of the present invention relate to the development of vaccine compositions against SARS-CoV-2. These compositions comprise combinations of antigens as well as liposomes, a carrier, and optionally an adjuvant. The antigens are portions of immunogenic proteins unique to SARS-CoV-2, and specifically the spike protein, N protein, and/or ORFIAB of SARS-CoV-2. These epitopes were selected by in silico analysis and various prediction systems.

[0069] In one aspect, the invention relates to compositions comprising one or more B cell epitopes and/or T cell epitopes of SARS-CoV-2, a carrier, liposomes, and optionally an adjuvant.

[0070] In one aspect, the invention relates to methods of preparing the invented compositions.

[0071] In one aspect, the invention relates to methods of administering the invented compositions to a subject in order to generate antibodies, including neutralizing antibodies, against SARS-CoV-2 and/or to prevent COVID-19. [0072] Definitions

[0073] It must be noted that as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Unless defined otherwise all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.

[0074] The phrase “and/or”, as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0075] As used throughout herein, the term “about" means reasonably close. For example, “about” can mean within an acceptable standard deviation and/or an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend on how the particular value is measured. Further, when whole numbers are represented, about can refer to decimal values on either side of the whole number. When used in the context of a range, the term “about” encompasses all of the exemplary values between the one particular value at one end of the range and the other particular value at the other end of the range, as well as reasonably close values beyond each end.

[0076] Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0077] As used herein, whether in the specification or the appended claims, the transitional terms “comprising”, “including”, ‘carrying”, “having”, “containing”, “involving”, and the like are to be understood as being inclusive or open-ended (i.e., to mean including but not limited to), and they do not exclude unrecited elements, materials or method steps. Only the transitional phrases “consisting of’ and “consisting essentially of’, respectively, are closed or semi-closed transitional phrases with respect to claims and exemplary embodiment paragraphs herein.

[0078] As used herein, “B cell epitope” or the like refers to an epitope on the S protein of

SARS-CoV-2 that can be used to generate antibodies against the SARS-CoV-2 virus, including neutralizing antibodies and antibodies that inhibit steps in viral infection (e.g., phagocytosis). The term also includes nucleic acids encoding the epitope.

[0079] As used herein, “T cell epitope” or the like refers to an epitope on the S protein, N protein, or in ORF1 AB of SARS-CoV-2 that can be used to generate antibodies against the SARS- CoV-2 virus, including neutralizing antibodies and antibodies that inhibit steps in viral infection (e.g., phagocytosis). The term also includes nucleic acids encoding the epitope.

[0080] As used herein, the term “DPX” or the like refers to a lipid-in-oil water-free formulation or a water-free oil-based composition.

[0081] “Treating” or “treatment of’, or “preventing” or “prevention of’, as used herein, refers to an approach for obtaining beneficial or desired results. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilisation of the state of disease, prevention of development of disease, prevention of spread of disease, delay or slowing of disease progression (e.g., suppression), delay or slowing of disease onset, conferring protective immunity against a disease- causing agent and amelioration or palliation of the disease state. “Treating” or “preventing” can also mean prolonging survival of a patient beyond that expected in the absence of treatment and can also mean inhibiting the progression of disease temporarily or preventing the occurrence of disease, such as by preventing infection in a subject.

[0082] “Treating” may be distinguished from “preventing” in that “treating” typically occurs in a subject who already has a disease or disorder, or is known to have already been exposed to an infectious agent, whereas “preventing” typically occurs in a subject who does not have a disease or disorder, or is not known to have been exposed to an infectious agent. As will be appreciated, there may be overlap in treatment and prevention. For example, it is possible to be “treating” a disease in a subject, while at same time “preventing” symptoms or progression of the disease. Moreover, “treating” and “preventing” may overlap in that the treatment of a subject to induce an immune response (e.g., vaccination) may have the subsequent effect of preventing infection by a pathogen or preventing the underlying disease or symptoms caused by infection with the pathogen.

[0083] As used herein, a “therapeutically effective amount” means an amount of the at least one B cell epitope, the liposome, optional adjuvant, and/or any additional therapeutic (e.g., a T cell epitope) effective to provide a therapeutic, prophylactic, or diagnostic benefit to a subject, and/or an amount sufficient to modulate an immune response and/or humoral response in a subject. As used herein, to “modulate” an immune and/or humoral response is distinct and different from activating an immune and/or humoral response. By “modulate”, it is meant that the at least one B cell epitope, the liposome, optional adjuvant and/or additional therapeutic agent herein enhance an immune and/or humoral response that is activated by other mechanisms or compounds (e.g., by an antigen or immunogen). In an embodiment, the immune and/or humoral response was activated before the at least one B cell epitope, the liposome, optional adjuvant, and/or any additional therapeutic herein are administered. In another embodiment, the immune and/or humoral response may be activated commensurately to administration of at least one B cell epitope, the liposome, optional adjuvant, and/or any additional therapeutic described herein. In another embodiment, the immune and/or humoral response may be activated subsequently to administration of at least one B cell epitope, the liposome, optional adjuvant, and/or any additional therapeutic described herein. [0084] The terms “subject”, “patient”, “individual”, and “animal” are used interchangeably herein and refer to mammals, including, without limitation, human and veterinary animals (e.g., primates, cats, dogs, cows, horses, sheep, pigs, rabbits, mice, rats, etc.) and experimental animal models. In a preferred embodiment, the subject is a human.

[0085] Broadly, an “antibody” refers to a polypeptide or protein that consists of or comprises antibody domains, which are understood as constant and/or variable domains of the heavy and/or light chains of immunoglobulins, with or without a linker sequence. In an embodiment, polypeptides are understood as antibody domains if they comprise a beta-barrel sequence consisting of at least two beta-strands of an antibody domain structure connected by a loop sequence. Antibody domains may be of native structure or modified by mutagenesis or derivatization, e.g., to modify binding specificity or any other property.

[0086] The term “antibody” refers to an intact antibody. In an embodiment, an “antibody” may comprise a complete (i.e., full-length) immunoglobulin molecule, including e.g., polyclonal, monoclonal, chimeric, humanized and/or human versions having full length heavy and/or light chains. The term “antibody” encompasses any and all isotypes and subclasses, including without limitation the major classes of IgA, IgD, IgE, IgG and IgM, and the subclasses IgGl, IgG2, IgG3, IgG4, IgA1 and IgA2. In an embodiment, the antibody is an IgG. The antibody may be one that is naturally occurring or one that is prepared by any means available to the skilled person, such as for example by using animals or hybridomas, and/or by immunoglobulin gene fragment recombinatorial processes. Antibodies are generally described in, for example, Greenfield, 2014).

[0087] An antibody can be in an isolated form, meaning that the antibody is substantially free of other antibodies against a different target antigen and/or comprising a different structural arrangement of antibody domains. In an embodiment, the antibody can be an antibody isolated from the serum sample of mammal. In an embodiment, the antibody is in a purified form, such as provided in a preparation comprising only the isolated and purified antibody as the active agent. This preparation may be used in the preparation of a composition of the invention. In an embodiment, the antibody is an affinity purified antibody.

[0088] The antibody may be of any origin, including natural, recombinant and/or synthetic sources. In an embodiment, the antibody may be of animal origin. In an embodiment, the antibody may be of mammalian origin, including without limitation human, murine, rabbit and goat. In an embodiment, the antibody may be a recombinant antibody.

[0089] The antibody may be a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a human antibody or a fully human antibody. The meaning applied to these terms and the types of antibodies encompassed therein will be well understood by the skilled person.

[0090] Briefly, and without limitation, the term “chimeric antibody” as used herein refers to a recombinant protein that contains the variable domains (including the complementarity determining regions (CDRs)) of an antibody derived from one species, such for example a rodent, while the constant domains of the antibody are derived from a different species, such as a human. For veterinary applications, the constant domains of the chimeric antibody may be derived from that of an animal, such as for example a cat or dog.

[0091] Without limitation, a “humanized antibody” as used herein refers to a recombinant protein in which the CDRs from an antibody from one species; e.g., a rodent, are transferred from the heavy and light variable chains of the rodent antibody into human heavy and light variable domains, including human framework region (FR) sequences. The constant domains of the humanized antibody are likewise derived from a human antibody.

[0092] Without limitation, a “human antibody” as used herein refers to an antibody obtained from transgenic animals {e.g, mice) that have been genetically engineered to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci. The transgenic animal can synthesize human antibodies specific for human antigens, and the animal can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described e.g., by Green, 1994; Lonberg, 1994; and Taylor, 1994. A fully human antibody also can be constructed by genetic or chromosomal transfection methods, as well as phage display technology, all of which are known in the art. (See, e.g., McCafferty, 1990, for the production of human antibodies and fragments thereof in vitro, from immunoglobulin variable domain gene repertoires from unimmunized donors). In this technique, antibody variable domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. In this way, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats, for their review, see, e.g., Johnson and Chiswell, 1993. Human antibodies may also be generated by in vitro activated B cells (see, e.g., U.S. Patent Nos. 5,567,610 and 5,229,275).

[0093] As used herein, the term “functional fragment”, with respect to an antibody, refers to an antigen-binding portion of an antibody. In this context, by “functional” it is meant that the fragment maintains its ability to bind to the target antigen. In an embodiment, the binding affinity may be equivalent to, or greater than, that of parent antibody. In an embodiment, the binding affinity may be less than the parent antibody, but nevertheless the functional fragment maintains a specificity and/or selectivity for the target antigen.

[0094] In addition to the functional fragment maintaining its ability to bind to the target antigen of the parent antibody, the functional fragment also maintains the effector function of the antibody, if applicable (e.g., activation of the classical complement pathway; antibody-dependent cellular phagocytosis (ADCP), antibody dependent cellular cytotoxicity (ADCC); other downstream signalling processes).

[0095] Functional fragments of antibodies include, without limitation, a portion of an antibody such as a F(ab')2, a F(ab)2, a Fab', a Fab, a Fabz, a Fabs, a single domain antibody (e.g., a Dab or VHHs) and the like, including half-molecules of IgG4 (van der Neut Kolfschoten, 2007).

Regardless of structure, a functional fragment of an antibody binds with the same antigen that is recognized by the intact antibody. The term “functional fragment”, in relation to antibodies, also includes isolated fragments consisting of the variable regions, such as the “Fv” fragments consisting of the variable regions of the heavy and light chains and recombinant single chain polypeptide molecules in which light and heavy chain variable regions are connected by a peptide linker (“scFv proteins”). As used herein, the term “functional fragment” does not include fragments such as Fc fragments that do not contain antigen-binding sites. [0096] Antibody fragments, such as those described herein, can be incorporated into single domain antibodies (e.g., nanobodies), single-chain antibodies, maxibodies, evibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, vNAR, bis-scFv and other like structures (see e.g., Hollinger and Hudson, 2005). Antibody polypeptides including fibronectin polypeptide monobodies, also are disclosed in U.S. Patent No. 6,703,199. Other antibody polypeptides are disclosed in U.S. Patent Publication No.20050238646. Each reference cited herein is incorporated by reference in their entirety for all purposes.

[0097] Another form of a functional fragment is a peptide comprising one or more CDRs of an antibody or one or more portions of the CDRs, provided the resultant peptide retains the ability to bind the target antigen.

[0098] A functional fragment may be a synthetic or genetically engineered protein. For example, functional fragments include isolated fragments consisting of the light chain variable region, “Fv” fragments consisting of the variable regions of the heavy and light chains, and recombinant single chain polypeptide molecules which light and heavy regions are connected by a peptide linker (scFv proteins).

[0099] As used herein, the terms “antibody” and “functional fragments” of antibodies encompass any derivatives thereof. By “derivatives” it is meant any modification to the antibody or functional fragment, including both modifications that occur naturally (e.g., in vivo ) or that are artificially introduced (e.g, by experimental design). Non-limiting examples of such modifications include, for example, sequence modifications (e.g., amino acid substitutions, insertions or deletions), post-translational modifications (e.g., phosphorylation, N-linked glycosylation, O-linked glycosylation, acetylation, hydroxylation, methylation, ubiquitylation, amidation, etc.), or any other covalent attachment or incorporation otherwise of a heterologous molecule (e.g., a polypeptide, a localization signal, a label, a targeting molecule, etc.). In an embodiment, modification of the antibody or functional fragment thereof may be made to generate a bispecific antibody or fragment (i.e., having more than one antigen-binding specificity) or a bifunctional antibody or fragment (i.e., having more than one effector function).

[00100] As used herein, a “functional equivalent” in the context of an antibody refers to a polypeptide or other compound or molecule having similar binding characteristics as an antibody to a particular target, but not necessarily being a recognizable “fragment” of an antibody. In an embodiment, a functional equivalent is a polypeptide having an equilibrium dissociation constant (KD) for a particular target in the range of 1 O^-7 to 10^-12. In an embodiment, the functional equivalent has a KD for a particular target of 10^-8 or lower. In an embodiment, the functional equivalent has a KD for a particular target of 10^-10 or lower. In an embodiment, the functional equivalent has a KD for a particular target of 10^-11 or lower. In an embodiment, the functional equivalent has a KD for a particular target of 10^-12 or lower. The equilibrium constant (KD) as defined herein is the ratio of the dissociation rate (K-ofi) and the association rate (K-on) of a compound to its target

[00101] The antibody, functional fragment thereof or functional equivalent thereof, may be one that has an immunomodulatory activity or function. By “immunomodulatory activity or function”, it is meant that the antibody, functional fragment thereof or functional equivalent thereof can enhance (upregulate), suppress (downregulate), direct, redirect or reprogram the immune response. The antibody, functional fragment thereof or functional equivalent thereof, may be one that binds to a stimulatory checkpoint molecule and/or an inhibitory checkpoint molecule, such has for example, and without limitation, those described herein. In an embodiment, the antibody, functional fragment thereof or functional equivalent thereof, is an agonist or an antagonist of a stimulatory checkpoint molecule and/or an inhibitory checkpoint molecule. In an embodiment, the antibody, functional fragment thereof or functional equivalent thereof, is an antagonist of an inhibitory checkpoint molecule. In an embodiment, the antibody, functional fragment thereof or functional equivalent thereof, is an agonist or super agonist of a stimulatory checkpoint molecule.

[00102] As used herein, the term “antibody mimetic” refers to compounds which, like antibodies, can specifically and/or selectively bind antigens or other targets, but which are not structurally related to antibodies. Antibody mimetics are usually artificial peptides or proteins, but they are not limited to such embodiments. Typically, antibody mimetics are smaller than antibodies, with a molar mass of about 3-20 kDa (whereas antibodies are generally about 150 kDa). Non-limiting examples of antibody mimetics include peptide aptamers, affimers, affilins, affibodies, affitins, alphabodies, anticalins, avimers, DARPins™, fynomers, Kunits domain peptides, nanoCLAMPs™, affinity reagents and scaffold proteins. Nucleic acids and small molecules may also be antibody mimetics. [00103] Other embodiments of antibody mimetics include, without limitation, Z domain of Protein A, Gamma B crystalline, ubiquitin, cystatin, Sac7D from Sulfolobus acidocaldarius, lipocalin, A domain of a membrane receptor, ankyrin repeat motive, SH3 domain of Fyn, Kunits domain of protease inhibitors, the 10^th type ΙII domain of fibronectin, 3- or 4- helix bundle proteins, an armadillo repeat domain, a leucine-rich repeat domain, a PDZ domain, a SUMO or SUMO-like domain, an immunoglobulin-like domain, phosphotyrosine-binding domain, pleckstrin homology domain, or src homology 2 domain.

[00104] As used herein, the term “functional fragment”, with respect to an antibody mimetic, refers any portion or fragment of an antibody mimetic that maintains the ability to bind to its target molecule. The functional fragment of an antibody mimetic may be, for example, a portion of any of the antibody mimetics as described herein. In an embodiment, the binding affinity may be equivalent to, or greater than, that of parent antibody mimetic. In an embodiment, the binding affinity may be less than the parent antibody mimetic, but nevertheless the functional fragment maintains a specificity and/or selectivity for the target antigen.

[00105] In an embodiment, in addition to the functional fragment of an antibody mimetic maintaining its ability to bind to the target molecule of the parent antibody mimetic, the functional fragment also maintains the effector function of the antibody mimetic, if applicable (e.g, downstream signalling).

[00106] As used herein, a “functional equivalent” in the context of an antibody mimetic refers to a polypeptide or other compound or molecule having similar binding characteristics to an antibody mimetic, but not necessarily being a recognizable “fragment” of an antibody mimetic. In an embodiment, a functional equivalent is a polypeptide having an equilibrium dissociation constant (KD) for a particular target in the range of 10^-7 to 10^-12. In an embodiment, the functional equivalent has a KD for a particular target of 10^-8 or lower. In an embodiment, the functional equivalent has a KD for a particular target of 10^-10 or lower. In an embodiment, the functional equivalent has a KD for a particular target of 10^-11 or lower. In an embodiment, the functional equivalent has a KD for a particular target of 10^-12 or lower. The equilibrium constant (KD) as defined herein is the ratio of the dissociation rate (K-ofi) and the association rate (K-on) of a compound to its target. [00107] In certain embodiments, one or more of the B cell epitope, liposomes, carrier, any additional therapeutic (e.g., a T cell epitope) and optional adjuvant is an immunomodulatory agent. As used herein, an “immunomodulatory agent” is a compound or molecule that modulates the activity and/or effectiveness of an immune response. “Modulate”, as used herein, means to enhance (upregulate), direct, redirect or reprogram an immune response. The term “modulate” is not intended to mean activate or induce. By this, it is meant that the immunomodulatory agent modulates (enhances or directs) an immune response that is activated, initiated or induced by a particular substance (e.g., an antigen), but the immunomodulatory agent is not itself the substance against which the immune response is directed, nor is the immunomodulatory agent derived from that substance.

[00108] The term “polypeptide” encompasses any chain of amino acids, regardless of length (e.g., at least 6, 8, 10, 12, 14, 16, 18, or 20 amino acids) or post-translational modification (e.g., glycosylation or phosphorylation), and includes, for example, natural proteins, synthetic or recombinant polypeptides and peptides, epitopes, hybrid molecules, variants, homologs, analogs, peptoids, peptidomimetics, etc. A variant or derivative therefore includes deletions, including truncations and fragments; insertions and additions, for example conservative substitutions, site- directed mutants and allelic variants; and modifications, including peptoids having one or more non-amino acyl groups (for example, sugar, lipid, etc.) covalently linked to the peptide and post- translational modifications. As used herein, the term "conserved amino add substitutions" or "conservative substitutions" refers to the substitution of one amino acid for another at a given location in the peptide, where the substitution can be made without substantial loss of the relevant function. In making such changes, substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobidty, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide by routine testing. Specific, non-limiting examples of a conservative substitution include the following examples:

Table 1: Conservative Amino Acid Substitutions

[00109] Polypeptides or peptides that have substantial identity to a preferred antigen sequence may be used. Two sequences are considered to have substantial identity if, when optimally aligned (with gaps permitted), they share at least approximately 50% sequence identity, or if the sequences share defined functional motifs. In alternative embodiments, optimally aligned sequences may be considered to be substantially identical (i.e., to have substantial identity) if they share at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity over a specified region. The term "identity" refers to sequence similarity between two polypeptides molecules. Identity can be determined by canparing each position in the aligned sequences. A degree of identity between amino acid sequences is a function of the number of identical or matching amino acids at positions shared by the sequences, for example, over a specified region. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, as are known in the art, including the ClustalW program, available at http ://clustalw.qenome. ad.j p, the local homology algorithm of Smith and Waterman, 1981 , Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, and the computerised implementations of these algorithms (such as GAP, BESTFIT, PASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI, U.S.A.). Sequence identity may also be determined using the BLAST algorithm, described in Altschul etal., 1990, J. Mol. Biol. 215:403-10 (using the published default settings). For example, the "BLAST 2 Sequences" tool, available through the National Center for Biotechnology Information (through the internet at http://www.ncbi.nlm.nih.gov/ BLAST/bl2seq/wblast2.cqi) may be used, selecting the "blastp" program at the following default settings: expect threshold 10; word size 3; matrix BLOSUM 62; gap costs existence 11, extension 1. In another embodiment, the person skilled in the art can readily and properly align any given sequence and deduce sequence identity and/or homology by mere visual inspection.

[00110] Vaccine Compositions

[00111] The vaccine compositions disclosed herein comprise at least one B cell epitope, the liposome, optional adjuvant, and/or any additional therapeutic (e.g., a T cell epitope) effective to provide a therapeutic, prophylactic, or diagnostic benefit to a subject, in an amount sufficient to modulate an immune response and/or humoral response in a subject. For example, and not limitation, the at least one B cell epitope (or T cell epitope) can be present in the receptor binding site (RBD) portion of the spike protdn of SARS-CoV-2 and/or the S1 region and/or the S2 region of the spike protein of SARS-CoV-2 and/or the N protein of SARS-CoV-2 and/or ORF1AB of SARS-CoV-2.

[00112] The B cell epitopes can be a combination of any of the amino add sequences of SEQ ID NOs: 2-26. For example and not limitation, the combination of B cell epitopes and/or T cell epitopes can include more than one epitope and up to five epitopes. In certain embodiments, the at least one B cell epitope comprises one or more of the amino acid sequences of SEQ ID NOs: 5, 7, 11, 14, 17, 19, 23, 24, and/or 26 or a nucldc acid molecule encoding said epitope. In certain embodiments, the at least one B cell epitope comprises one or more of the amino acids of SEQ ID NOs: 5, 7, 14, and/or 19. In certain embodiments, the at least one B cell epitope comprises a mixture of four B cell epitopes comprising the amino acid sequences of SEQ ID NOs: 5, 7, 14, and 19. In certain embodiments, the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 5. In certain embodiments, the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 7. In certain embodiments, the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the at least one B cell epitope is present as a dimer.

[00113] The T cell epitopes can be a combination of any of the amino add sequences of SEQ ID NOs: 27-40 and/or 42-43 or a nucleic acid molecule encoding said epitope. For example, and not limitation, the combination of B cell epitopes and/or T cell epitopes can include more than one epitope and up to five epitopes. In certain embodiments, the at least one T cell epitope comprises at least one of amino add sequences of SEQ ID NOs: 27-40 and/or 42-43 or a nucldc acid molecule encoding said epitope. In certain embodiments, the at least one T cell epitope comprises one or more of the amino acid sequences of SEQ ID NOs: 27, 28, 32, 34, 38, 42, 43, and combinations thereof. In certain embodiments, the at least one T cell epitope comprises a mixture of T cdl epitopes comprising the amino add sequences of SEQ ID NOs: 27, 28, 32, 34, 38, 42, 43, and combinations thereof. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 27. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 28. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 32. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 34. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 38. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 42. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 43. In certain embodiments, the at least one T cell epitope is present as a dimer.

[00114] Without wishing to be bound by theory, it is suggested that an optimal combination of B cell epitopes and/or T cell epitopes can induce several antibodies, such as NAbs, that can bind to different domains of the S protein and interfere with the infection at multiple steps, e.g., interfering with the S protein homotrimer binding to the ACE2 protein (steric hindrance; stabilizing the RBD of the S protein in the closed position to prevent opening and exposure of the receptor binding motif; allosteric effect on ACE2 binding); and/or interfering with the fusion between the cell membrane and the viral particle.

[00115] A vaccine composition in accordance with the invention also encompasses compositions containing one or more of the B cell epitopes and/or T cell epitopes, where the B cell epitope and/or T cell epitope can be present individually or as a construct containing multiple copies of the same or different B cell epitopes and/or T cell epitopes. For example, the B cell epitope and/or T cell epitope can be present as a homopolymer (e.g., a dimer) comprising multiple copies of the same B cell epitope and/or T cell epitope, or a heteropolymer of various different B cell epitopes and/or T cell epitopes, may be used. Such polymers may have the advantage of providing an increased immunological reaction as they comprise multiple copies of B cell epitopes and/or T cell epitopes, such that the resultant effect may be an enhanced ability to induce an immune response with the one or more antigenic determinants of the S protein and/or N protein and/or ORF1AB of SARS-CoV-2. The composition can comprise a naturally occurring region of one or more B cell epitopes and/or T cell epitopes or can comprise prepared antigens, e.g., recombinantly or by chemical synthesis.

[00116] To obtain vaccine compositions of the invention, it may be suitable to combine the B cell epitope with various materials such as adjuvants, excipients, surfactants, immunostimulatory components and/or carriers. Adjuvants may be included in the composition to enhance the specific immune response. Different carriers may be used depending on the desired route of administration or the desired distribution in the subject, e.g., systemic or localized.

[00117] In a particular embodiment, the vaccine composition for use in the methods of the invention is a composition comprising at least one B cell epitope and/or at least one T cell epitope, liposomes, and a carrier comprising a continuous phase of a hydrophobic substance. In a further embodiment, the vaccine composition may additionally comprise an adjuvant. In a further embodiment, the composition may additionally comprise an additional therapeutic.

[00118] Thus, in an embodiment, the vaccine composition comprises one or more B cell epitopes and/or T cell epitopes; liposomes; a carrier comprising a continuous phase of a hydrophobic substance; and optionally an adjuvant. The B cell epitope may, for example, be a peptide antigen and can be selected from a group of peptides comprising the amino add sequence

ID NO:26; COV2B-S809), and nucldc adds encoding these peptides. The adjuvant may be, by way of example and not limitation, a lipid-based adjuvant such as Pam3CSK4. In certain embodiments, the B cell epitopes comprise a combination of peptides comprising one or more of SEQ ID NOs: 5, 7, 11, 14, 17, 19, 23, 24 and/or 26. In certain embodiments, the at least one B cell epitope comprises one or more of SEQ ID NOs: 5, 7, 14, and/or 19. In certain embodiments, the at least one B cell epitope comprises a mixture of four B cell epitopes comprising the amino acid sequences of SEQ ID NOs: 5, 7, 14, and 19. In certain embodiments, the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 5. In certain embodiments, the at least one B cell epitope comprises the amino add sequence of SEQ ID NO: 7. In certain embodiments, the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the at least one B cell epitope is present as a dimer. [00119] In certain embodiments, the T cell epitope included in any of the vaccine compositions described herein can be a combination of any of the amino acid sequences of SEQ ID NOs: 27-40 and/or 42-43 and/or a nucleic acid molecule encoding said epitope. For example, and not limitation, the combination of B cell epitopes and/or T cell epitopes can include more than one epitope and up to five epitopes. In certain embodiments, the at least one T cell epitope comprises at least one of amino acid sequences of SEQ ID NOs: 27-40 and/or 42-43 and/or a nucleic acid molecule encoding said epitope. In certain embodiments, the at least one T cell epitope comprises one or more of the amino acid sequences of SEQ ID NOs: 27, 28, 32, 34, 38, 42, 43, and combinations thereof. In certain embodiments, the at least one T cell epitope comprises a mixture of T cell epitopes comprising the amino add sequences of SEQ ID NOs: 27, 28, 32, 34, 38, 42, 43, and combinations thereof. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 27. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 28. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 32. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 34. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 38. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 42. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 43. In certain embodiments, the at least one T cell epitope is present as a dimer.

[00120] In a further embodiment, the B cell epitope and/or T cell epitope for use in the vaccine compositions and methods of the invention comprises at least one B cell epitope and/or T cell epitopes, together with IMV, Inc's liposome-based and/or amphipathic compound-based vaccine adjuvanting platform, including, but not limited to, the DPX™ platform technologies (see e.g., US Patent Nos. 6,793,923 and 7,824,686; US Patent Publication No. 20160067335, WO 2002/038175; WO 2007/041832; WO 2009/039628; WO 2009/043165 WO 2009/146523, WO 2013049941, WO 2014/153636, WO 2016/176761, WO 2016/109880, WO 2017/190242, WO 2017/083963, WO 2018/058230, each of which is incorporated herein by reference in their entirety for all intended purposes.). The DPX platform is a therapeutic delivery formulation that provides controlled and prolonged exposure of antigens plus adjuvant to the immune system. The platform is capable of providing a strong, specific and sustained immune response and is capable of single- dose effectiveness.

[00121] In certain embodiments, the vaccine composition of the invention comprises at least one B cell epitope and/or at least one T cell epitope, wherein each B cell epitope and/or T cell epitope is at a concentration of about 50 μg/ml to about 10 mg/ml.

[00122] In certain embodiments, the vaccine composition of the invention comprises at least one B cell epitope and/or at least one T cell epitope; liposomes; a carrier comprising a continuous phase of a hydrophobic substance; and optionally an adjuvant. The at least one B cell epitope may, for example, be a peptide/peptide antigen comprising one or more of the amino acid sequences SYGFQPTNGVGYQPY (SEQ ID NO: 2); GDEVRQIAPGQTGKIADYNYKLP (SEQ ID NO: 3); VRFPNITNLCPFGE (SEQ ID NO: 4); LLFNKVTLADAGFIKQYGDCLGDIAA (SEQ ID NO: 5); GCVIAWNSNNLDSKVGG (SEQ ID NO: 6); LKPFERDISTEIYQAGSTPCNGVEGFN (SEQ ID NO: 7); GFQPTNGV GYQPY (SEQ ID NO: 8); ELLHAPATVCGPKKSTNLVKN (SEQ ID NO: 9); RVYSTGSNVFQ (SEQ ID N: 10); DLGDISGINASWNIQK (SEQ ID NO: 11); VCGPKKSTNLVKN (SEQ ID NO: 12); KNHTSPDVDLGDISGIN (SEQ ID NO: 13); NCTEVPVAIHADQLTPT (SEQ ID NO: 14); SCCKFDEDDSEPVLKG (SEQ ID NO: 15); ASYQTQTNSPRRARSVASQ (SEQ ID NO: 16); YNSASFSTFKCYGVSPTKLNDLCFT (SEQ ID NO: 17); TPGDSSSGWTA (SEQ ID NO: 18); SFSTFKCYGVSPTKLNDL (SEQ ID NO: 19); SNKKFLPF (SEQ ID NO: 20); PDPSKPSK (SEQ ID NO: 21); EIDRLNEVAKNLNESLIDLQELGKYEQY (SEQ ID NO: 22);

FNCYFPLQS YGFQPTNGV GY QPYRVWLSFE (SEQ ID NO: 23);

FPNITNLCPFGEVFNATRFASVYAWNRKRISNCVA (SEQ ID NO: 24);

TESNKKFLPFQQFGRDIA (SEQ ID NO:25; COV2B-S553); PSKPSKRSFIEDLLFNKV (SEQ ID NO:26; COV2B-S809), and nucleic acids encoding these peptides. In certain embodiments, the B cell epitopes comprise a combination of peptides comprising one or more of SEQ ID NOs: 5, 7, 11, 14, 17, 19, 23, 24 and/or 26. In certain embodiments, the at least one B cell epitope comprises one or more of SEQ ID NOs: 5, 7, 14, and/or 19. In certain embodiments, the at least one B cell epitope comprises a mixture of four B cell epitopes comprising the amino add sequences of SEQ ID NOs: 5, 7, 14, and 19. In certain embodiments, the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 5. In certain embodiments, the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 7. In certain embodiments, the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the at least one B cell epitope is present as a dimer.

[00123] In certain embodiments, the T cell epitope included in any of the vaccine compositions described herein can be a combination of any of the amino acid sequences of SEQ ID NOs: 27-40 and/or 42-43 or a nucleic acid molecule encoding said epitope. For example, and not limitation, the combination of B cell epitopes and/or T cell epitopes can include more than one epitope and up to five epitopes. In certain embodiments, the at least one T cell epitope comprises at least one of amino acid sequences of SEQ ID NOs: 27-40 and/or 42-43 and/or a nucleic acid molecule encoding said epitope. In certain embodiments, the at least one T cell epitope comprises one or more of the amino acid sequences of SEQ ID NOs: 27, 28, 32, 34, 38, 42, 43, and combinations thereof. In certain embodiments, the at least one T cell epitope comprises a mixture of T cell epitopes comprising the amino acid sequences of SEQ ID NOs: 27, 28, 32, 34, 38, 42, 43, and combinations thereof. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 27. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 28. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 32. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 34. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 38. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 42. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 43. In certain embodiments, the at least one T cell epitope is present as a dimer.

[00124] The adjuvant may, for example, be a lipid-based adjuvant (e.g., Pam3CSK4, etc.). The liposomes may, for example, be comprised of l,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC; synthetic phospholipid) and cholesterol. The hydrophobic carrier may, for example, be Montanide® ISA51 VG. In certain embodiments, the vaccine composition can be a lipid-in-oil water-free formulation. [00125] In a particular embodiment, the vaccine composition of the invention may comprise four B cell epitopes having the amino acid sequences of SEQ ID NOs: 5, 7, 14, and 19; liposomes consisting of DOPC and cholesterol; the hydrophobic carrier Montanide® ISA 51 VG; and optionally the adjuvant Pam3CSK4. Exemplary amounts of each component (per ml of the vaccine composition) include, without limitation, 0.5 mg of each B cell epitope; 120.0 mg of synthetic DOPC phospholipid; 12.0 mg of cholesterol; 0.87 ml of hydrophobic carrier (e.g., Montanide® ISA51 VG); and optionally 0.04 mg of Pam3CSK4.

[00126] The vaccine composition may optionally further comprise additional components such as, for example, emulsifiers. A more detailed disclosure of exemplary embodiments of the vaccine composition, and the components thereof, are described as follows.

[00127] B Cell Enitopes

[00128] The vaccine compositions of the invention comprise at least one B cell epitope, optionally with at least one T cell epitope as discussed herein. The expression “at least one” is used herein interchangeably with the expression “one or more”. These expressions, unless explicitly stated otherwise herein, refer to the number of different B cell epitopes in the vaccine composition, and not to the quantity of any particular B cell epitope. In accordance with the ordinary meaning of “at least one” or “one or more”, the vaccine composition of the invention contains a minimum of one B cell epitope.

[00129] B cell epitopes of the invention also encompass variants and functional equivalents of the listed B cell epitopes of the spike protein of SARS-CoV-2. Variants or functional equivalents of a B cell epitope encompass peptides that exhibit amino acid sequences with differences as compared to the specific sequence of the B cell epitope, such as one or more amino acid substitutions, deletions or additions, or any combination thereof. The difference may be measured as a reduction in identity as between the B cell epitope sequence and the B cell epitope variant or B cell epitope functional equivalent.

[00130] The identity between amino acid sequences may be calculated using algorithms well known in the art. B cell epitope variants or functional equivalents are to be considered as falling within the meaning of a “B cell epitope” of the invention when they are, preferably, over their entire length, at least 70% identical to a peptide sequence of a B cell epitope, such as at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical, including 96%, 97%, 98% or 99% identical with a peptide sequence of a B cell epitope. In a particular embodiment, the B cell epitope variant has a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a consecutive amino acid sequence of SEQ ID NO: 1.

[00131] The SARS-CoV-2 spike protein from which the B cell epitope can be derived is given in SEQ ID NO: 1. Based on the sequence of the selected SARS-CoV-2 spike protein, the B cell epitope may be derived by any appropriate chemical or enzymatic treatment of the SARS- CoV-2 spike protein or coding nucleic acid. Alternatively, the B cell epitope may be synthesized by any conventional peptide or nucleic acid synthesis procedure with which the person of ordinary skill in the art is familiar.

[00132] The B cell epitope of the invention (peptide or nucleic add) may have a sequence which is a native sequence of SARS-CoV-2 spike protein. Alternatively, the B cell epitope may be a peptide or nucldc add sequence modified by one or more substitutions, deletions or additions, such as e.g., the B cell epitope variants or functional equivalents described herein.

[00133] In some embodiments, the B cell epitopes can comprise one or more peptide sequences from the spike protdn of SARS-CoV-2. For example, the peptide sequences include portions of the spike protein at one or more of amino acid positions 494-508; 404-426; 327-340; 821-846; 431-448; 461-487; 496-508; 516-536; 634-644; 1165-1181; 524-536; 1157-1173; 616- 632; 1252-1268; 672-690; 369-393; 250-260; 373-390; 555-562; 807-814; 1182-1209; 486-516; and 329-363. The B cell epitopes may comprise a peptide in the RBD and/or S1 region and/or S2 region of the SARS-CoV-2 spike protein.

[00134] In some embodiments, the B cell epitope variants can comprise modifications to the peptide sequences listed in SEQ ID Nos 2-26. For example, SYGFQPTNGVGYQPY (SEQ ID NO: 2) can have a substitution of Y508H; GDEVRQIAPGQTGKIADYNYKLP (SEQ ID NO: 3) can have substitutions of Q409E and/or R408I; GCVIAWNSNNLDSKVGG (SEQ ID NO: 6) can have a substitution of A435S; LKPFERDISTEIYQAGSTPCNGVEGFN (SEQ ID NO: 7) can have substitutions of V483A and/or G476S; GFQPTNGVGYQPY (SEQ ID NO: 8) can have a substitution of Y508H; ELLHAPATVCGPKKSTNLVKN (SEQ ID NO: 9) can have a substitution of H519P; DLGDISGINASWNIQK (SEQ ID NO: 11) can have a substitution of V1176F; KNHTSPDVDLGDISGIN (SEQ ID NO: 13) can have a substitution of P1162L or P1162A; NCTEVPVAIHADQLTPT (SEQ ID NO: 14) can have a substitution of V622I and/or D614G (which is just upstream of the sequence); SCCKFDEDDSEPVLKG (SEQ ID NO: 15) can have substitutions of C1254F, D1259H, and/or P1263L; ASYQTQTNSPRRARSVASQ (SEQ ID NO: 16) can have substitutions of Q675H and/or R682Q; YNSASFSTFKCYGVSPTKLNDLCFT (SEQ ID NO: 17) can have a substitution of K378R; TPGDSSSGWTA (SEQ ID NO: 18) can have substitutions of S254F and/or P251H; SFSTFKCYGVSPTKLNDL (SEQ ID NO: 19) can have a substitution of K378R; SNKKFLPF (SEQ ID NO: 20) can have a substitution of P561L; PDPSKPSK (SEQ ID NO: 21) can have substitutions of P809S and/or P812S;

can have substitutions of K1205N and/or

can have a substitution of Y508H;

24) can have substitutions of V341S and/or S359N.

[00135] In a particular embodiment, the vaccine composition of the invention may comprise one or more of the B cell epitopes selected from:

(SEQ ID NO:26; COV2B-S809), and nucleic acids encoding these peptides. In certain embodiments, the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 5. In certain embodiments, the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 7. In certain embodiments, the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the at least one B cell epitope is present as a dimer.

[00136] In a further embodiment, the vaccine composition of the invention comprises any one or more of the B cell epitopes listed below, in any suitable combination: SEQ ID NOs: 5, 7, 11, 14, 17, 19, 23, 24, and/or 26. In a further embodiment, the vaccine composition of the invention consists of one or more of the B cell epitopes, in any suitable combination: SEQ ID NOs: 5, 7, 11, 14, 17, 19, 23, , and/or 26. In some embodiments, the vaccine composition consists of only one B cell epitope selected from the group consisting of SEQ ID NOs: 5, 7, 11, 14, 17, 19, 23, 24, and/or 26. In certain embodiments, the at least one B cell epitope comprises one or more of SEQ ID NOs: 5, 7, 14, and/or 19. In certain embodiments, the at least one B cell epitope comprises a mixture of four B cell epitopes comprising the amino acid sequences of SEQ ID NOs: 5, 7, 14, and 19. In certain embodiments, the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 5. In certain embodiments, the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 7. In certain embodiments, the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the at least one B cell epitope is present as a dimer.

[00137] In a particular embodiment, the vaccine composition of the invention comprises all four of the B cell peptides of SEQ ID NOs: 5, 7, 14, and/or 19, as found in IMV Inc’s DPX platform technologies, or any combination of one or more of the B cell epitopes, optionally in combination with at least one T cell epitope of SEQ ID NOs: 27, 28, 32, 34, 38, 42 and/or 43. In a preferred embodiment, the composition comprises all four of the B cell epitopes of SEQ ID NOs: 5, 7, 14, and/or 19 in combination with the DPX platform technology, optionally in combination with one or more T cell epitope of SEQ ID NOs: 27, 28, 32, 34, 38, 42 and/or 43. [00138] T Cell Epitopes

[00139] In certain embodiments, the vaccine compositions of the invention comprise at least one T cell epitope, in addition to at least one B cell epitope. The expression “at least one” is used herein interchangeably with the expression “one or more”. These expressions, unless explicitly stated otherwise herein, refer to the number of different T cell epitopes in the vaccine composition, and not to the quantity of any particular T cell epitope. In accordance with the ordinary meaning of “at least one” or “one or more”, the vaccine composition of the invention contains a minimum of one B cell epitope.

[00140] T cell epitopes of the invention also encompass variants and functional equivalents of the listed T cell epitopes of the spike protein, nucleocapsid protein, and/or ORF1AB of SARS- CoV-2. Variants or functional equivalents of a T cell epitope encompass peptides that exhibit amino acid sequences with differences as compared to the specific sequence of the T cell epitope, such as one or more amino acid substitutions, deletions or additions, or any combination thereof. The difference may be measured as a reduction in identity as between the T cell epitope sequence and the T cell epitope variant or T cell epitope functional equivalent.

[00141] The identity between amino acid sequences may be calculated using algorithms well known in the art. T cell epitope variants or functional equivalents are to be considered as falling within the meaning of a “T cell epitope” of the invention when they are, preferably, over their entire length, at least 70% identical to a peptide sequence of a T cell epitope, such as at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical, including 96%, 97%, 98% or 99% identical with a peptide sequence of a T cell epitope. In a particular embodiment, the T cell epitope variant has a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a consecutive amino add sequence of SEQ ID NO: 1.

[00142] The SARS-CoV-2 spike protein from which the T cell epitope can be derived is given in SEQ ID NO: 1. Based on the sequence of the selected SARS-CoV-2 spike protein, the T cell epitope may be derived by any appropriate chemical or enzymatic treatment of the SARS- CoV-2 spike protein or coding nucleic acid. Alternatively, the T cell epitope may be synthesized by any conventional peptide or nucleic acid synthesis procedure with which the person of ordinary skill in the art is familiar. Alternatively, the T cell epitope can be derived from the SARS-CoV-2 nucleocapsid protein and/or ORF1AB as discussed herein.

[00143] The T cell epitope of the invention (peptide or nucleic add) may have a sequence which is a native sequence of SARS-CoV-2 spike protein, nucleocapsid protein, and/or ORF1AB. Alternatively, the T cell epitope may be a peptide or nucleic acid sequence modified by one or more substitutions, deletions or additions, such as e.g., the T cell epitope variants or functional equivalents described herein.

[00144] In some embodiments, the T cell epitopes can comprise one or more peptide sequences from the spike protein, nucleocapsid protein, and/or ORF 1 AB of SARS-CoV-2. In some embodiments, the T cell epitopes can be a combination of any of the amino acid sequences of SEQ ID NOs: 27-40 and/or 42-43. For example, and not limitation, the combination of B cell epitopes and/or T cell epitopes can include more than one epitope and up to five epitopes. In certain embodiments, the at least one T cell epitope comprises at least one of amino add sequences of SEQ ID NOs: 27-40 and/or 42-43. In certain embodiments, the at least one T cell epitope comprises one or more of the amino acid sequences of SEQ ID NOs: 27, 28, 32, 34, 38, 42, 43, and combinations thereof. In certain embodiments, the at least one T cell epitope comprises a mixture of T cell epitopes comprising the amino acid sequences of SEQ ID NOs: 27, 28, 32, 34, 38, 42, 43, and combinations thereof. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 27. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 28. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 32. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 34. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 38. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 42. In certain embodiments, the at least one T cell epitope comprises the amino acid sequence of SEQ ID NO: 43. In certain embodiments, the at least one T cell epitope is present as a dimer.

[00145] Liposomes [00146] In some embodiments, the vaccine composition of the invention comprises liposomes. In a particular embodiment, liposomes are included when the vaccine compositions comprise a carrier comprising a continuous phase of a hydrophobic substance as described herein.

[00147] Liposomes represent a particular embodiment of an adjuvanting system encompassed by the present invention. In certain embodiments, however, the vaccine compositions of the invention may not include liposomes. For example, in some embodiments of the vaccine compositions, the one or more B cell epitopes may be combined with any suitable, active agent, additional therapeutic agent and/or an adjuvant for delivery of the B cell epitopes to a subject.

[00148] A general discussion of liposomes can be found in Gregoriadis G. Immunol. Today,

11 :89-97, 1990; and Frezard, F., Braz. J. Med. Bio. Res., 32:181-189, 1999, each of which are incorporated by reference herein in their entirety for all purposes. As used herein and in the claims, the term "liposomes" is intended to encompass all such vesicular structures as described above, including, without limitation, those described in the art as “niosomes”, “transfersomes” and “virosomes”.

[00149] Although any liposomes may be used in this invention, including liposomes made from archaebacterial lipids, particularly useful liposomes use phospholipids and unesterified cholesterol in the liposome formulation. When cholesterol is used, the cholesterol may be used in any amount sufficient to stabilize the lipids in the lipid membrane. In an embodiment, the cholesterol may be used in an amount equivalent to about 10% of the weight of phospholipid (e.g., in a DOPC:cholesterol ratio of 10:1 w/w). The cholesterol may stabilize the formation of phospholipid vesicle particles. If a compound other than cholesterol is used, one skilled in the art can readily determine the amount needed. Other liposome stabilizing compounds are known to those skilled in the art. For example, saturated phospholipids produce liposomes with higher transition temperatures indicating increased stability.

[00150] Phospholipids that are preferably used in the preparation of liposomes are those with at least one head group selected from the group consisting of phosphoglycerol, phosphoethanolamine, phosphoserine, phosphocholine (e.g., DOPC; 1 ,2-Dioleoyl-sn-glycero- 3- phosphocholine) and phosphoinositol. More preferred are liposomes that comprise lipids which are 94-100% phosphatidylcholine. Such lipids are available commercially in the lecithin Phospholipon® 90 G. When unesterified cholesterol is also used in liposome formulation, the cholesterol is used in an amount equivalent to about 10% of the weight of phospholipid. If a compound other than cholesterol is used to stabilize the liposomes, one skilled in the art can readily determine the amount needed in the composition. In an embodiment, the phospholipid may be phosphatidylcholine or a mixture of lipids comprising phosphatidylcholine. In an embodiment, the lipid may be DOPC (Lipoid GmbH, Germany) or Lipoid S100 lecithin. In some embodiments, a mixture of DOPC and unesterified cholesterol may be used. In other embodiments, a mixture of Lipoid S100 lecithin and unesterified cholesterol may be used.

[00151] Liposome compositions may be obtained, for example, by using natural lipids, synthetic lipids, sphingolipids, ether lipids, sterols, cardiolipin, cationic lipids and lipids modified with poly (ethylene glycol) and other polymers. Synthetic lipids may include the following fatty acid constituents; lauroyl, myristoyl, palmitoyl, stearoyl, arachidoyl, oleoyl, linoleoyl, erucoyl, or combinations of these fatty acids.

[00152] In an embodiment, the compositions disclosed herein comprise about 120 mg/ml of DOPC and about 12 mg/ml of cholesterol.

[00153] Another common phospholipid is sphingomyelin. Sphingomyelin contains sphingosine, an amino alcohol with a long unsaturated hydrocarbon chain. A fatty acyl side chain is linked to the amino group of sphingosine by an amide bond, to form ceramide. The hydroxyl group of sphingosine is esterified to phosphocholine. Like phosphoglycerides, sphingomyelin is amphipathic.

[00154] Lecithin, which also may be used, is a natural mixture of phospholipids typically derived from chicken eggs, sheep's wool, soybean and other vegetable sources.

[00155] All of these and other phospholipids may be used in the practice of the invention. Phospholipids can be purchased, for example, from Avanti lipids (Alabastar, AL, USA), Lipoid LLC (Newark, NJ, USA) and Lipoid GmbH (Germany), among various other suppliers. [00156] There are various lipid-based structures which may form, and the compositions disclosed herein may comprise a single type of lipid-based structure or comprise a mixture of different types of lipid-based structures.

[00157] In an embodiment, the lipid-based structures may be closed vesicular structures. They are typically spherical or substantially spherical in shape, but other shapes and conformations may be formed and are not excluded. By “substantially spherical” it is meant that the lipid-based structures are close to spherical, but may not be a perfect sphere. Other shapes of the closed vesicular structures include, without limitation, oval, oblong, square, rectangular, triangular, cuboid, crescent, diamond, cylinder, or hemisphere shapes. Any regular or irregular shape may be formed. Exemplary embodiments of closed vesicular structures include, without limitation, single layer vesicular structures {e.g, micelles or reverse micelles) and bilayer vesicular structures {e.g., unilamellar or multilamellar vesicles), or various combinations thereof.

[00158] By “single layer" it is meant that the lipids do not form a bilayer, but rather remain in a layer with the hydrophobic part oriented on one side and the hydrophilic part oriented on the opposite side. By “bilayer” it is meant that the lipids form a two-layered sheet, such as with the hydrophobic part of each layer internally oriented toward the center of the bilayer with the hydrophilic part externally oriented. Alternatively, the opposite configuration is also possible, i.e., with the hydrophilic part of each layer internally oriented toward the center of the bilayer with the hydrophobic part externally oriented. The term “multilayer” is meant to encompass any combination of single and bilayer structures. The form adopted may depend upon the specific lipid that is used, and whether the composition is or is not water-free.

[00159] The closed vesicular structures may be formed from single layer lipid membranes, bilayer lipid membranes and/or multilayer lipid membranes. The lipid membranes are predominantly comprised of and formed by lipids but may also comprise additional components. For example, and without limitation, the lipid membrane may include stabilizing molecules to aid in maintaining the integrity of the structure. Any available stabilizing molecule may be used.

[00160] In an embodiment, the lipid-based structure is a bilayer vesicular structure, such as for example, a liposome. Liposomes are completely closed lipid bilayer membranes. Liposomes may be unilamellar vesicles (possessing a single bilayer membrane), multilamellar vesicles (characterized by multimembrane bilayers whereby each bilayer may or may not be separated iron the next by an aqueous layer) or multivesicular vesicles (possessing one or more vesicles within a vesicle). In an embodiment, the lipid-based structures are liposomes when the compositions herein are not water-free. In an embodiment, the composition is a lipid-in-oil water-free formulation.

[00161] In an embodiment, the one or more lipid-based structures are comprised of a single layer lipid assembly. There are various types of these lipid-based structures which may form, and the compositions disclosed herein may comprise a single type of lipid-based structure having a single layer lipid assembly or comprise a mixture of different such lipid-based structures.

[00162] In an embodiment, the lipid-based structures herein have a single layer lipid assembly when the compositions herein are water-free.

[00163] In an embodiment, the lipid-based structure having a single layer lipid assembly partially or completely surrounds the at least one B cell epitope and/or at least one T cell epitope and optionally the adjuvant. As an example, the lipid-based structure may be a closed vesicular structure surrounding the at least one B cell epitope and/or T cell epitope. In an embodiment, the hydrophobic part of the lipids in the vesicular structure is oriented outwards toward the hydrophobic carrier.

[00164] As another example, the one or more lipid-based structures having a single layer lipid assembly may comprise aggregates of lipids with the hydrophobic part of the lipids oriented outwards toward the hydrophobic carrier and the hydrophilic part of the lipids aggregating as a core. These structures do not necessarily form a continuous lipid layer membrane. In an embodiment, they are an aggregate of monomeric lipids.

[00165] In an embodiment, the one or more lipid-based structures having a single layer lipid assembly comprise reverse micelles. Atypical micelle in aqueous solution forms an aggregate with the hydrophilic parts in contact with the surrounding aqueous solution, sequestering the hydrophobic parts in the micelle center. In contrast, in a hydrophobic carrier, an inverse/reverse micelle forms with the hydrophobic parts in contact with the surrounding hydrophobic solution, sequestering the hydrophilic parts in the micelle center. A spherical reverse micelle can package a B cell epitope with hydrophilic affinity within its core (i.e., internal environment). [00166] Without limitation, the size of the lipid-based structures having a single layer lipid assembly is in the range of from 2 nm (20 A) to 20 nm (200 A) in diameter. In an embodiment, the size of the lipid-based structures having a single layer lipid assembly is between about 2 nm to about 10 nm in diameter. In an embodiment, the size of the lipid-based structures having a single layer lipid assembly is about 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, about 7 nm, about 8 nm, about 9 nm, or about 10 nm in diameter. In an embodiment, the maximum diameter of the lipid-based structures is about 4 nm or about 6 nm. In an embodiment, the lipid-based structures of these sizes are reverse micelles.

[00167] In an embodiment, one or more of B cell epitopes and/or T cell epitopes are inside the lipid-based structures after solubilization in the hydrophobic carrier. By “inside the lipid-based structure” it is meant that the at least one B cell epitope and/or T cell epitope is substantially surrounded by the lipids such that the hydrophilic components of the B cell epitope and/or T cell epitope are not exposed to the hydrophobic carrier. In an embodiment, the at least one B cell epitope and/or at least one T cell epitope inside the lipid-based structure is predominantly hydrophilic.

[00168] In an embodiment, one or more of the B cell epitopes and/or T cell epitopes are outside the lipid-based structures after solubilization in the hydrophobic carrier. By “outside the lipid-based structure”, it is meant that the B cell epitope and/or T cell epitope is not sequestered within the environment internal to the lipid membrane or assembly. In an embodiment, the B cell epitope and/or T cell epitope projects from the surface of the lipid-based structure. In an embodiment, the B cell epitope and/or T cell epitope outside the lipid-based structure is predominantly hydrophobic.

[00169] Carriers

[00170] In some embodiments, the vaccine composition of the invention comprises a pharmaceutically acceptable carrier, excipient or diluent. As used herein, a pharmaceutically acceptable carrier refers to any substance suitable for delivering a B cell epitope and/or T cell epitope of the invention, and which is useful in the method of the present invention. [00171] Carriers that can be used with the vaccine compositions of the invention are well known in the art, and include, but are by no means limited to, e.g., water, phosphate buffered saline, Ringer's solution, dextrose solution, serum-containing solutions, Hank's solution, other aqueous physiologically balanced solutions, oil-in-water emulsions, oils, water-in-oil emulsions, esters, poly(ethylene-vinyl acetate), copolymers of lactic acid and glycolic acid, poly(lactic acid), gelatin, collagen matrices, polysaccharides, poly(D,L lactide), poly(malic acid), poly(caprolactone), celluloses, albumin, starch, casein, dextran, polyesters, ethanol, methacrylate, polyurethane, polyethylene, vinyl polymers, glycols, thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly L-lysine, poly L-glutamic acid, influenza, hepatitis B virus core protein, mixtures thereof and the like. See, for example, Remington: The Science and Practice of Pharmacy, 2000, Gennaro, A R ed., Eaton, Pa.: Mack Publishing Co.

[00172] In a particular embodiment, the carrier of the vaccine composition is a carrier that comprises a continuous phase of a hydrophobic substance, preferably a liquid hydrophobic substance. The continuous phase may be an essentially pure hydrophobic substance or a mixture of hydrophobic substances. In addition, the carrier may be an emulsion of water in a hydrophobic substance or an emulsion of water in a mixture of hydrophobic substances, provided the hydrophobic substance constitutes the continuous phase. Further, in another embodiment, the carrier may function as an adjuvant.

[00173] Hydrophobic substances that are useful in the compositions as described herein are those that are pharmaceutically and/or immunologically acceptable. The carrier is preferably a liquid but certain hydrophobic substances that are not liquids at atmospheric temperature may be liquefied, for example by warming, and are also useful in this invention. In one embodiment, the hydrophobic carrier may be a Phosphate Buffered Saline/Freund's Incomplete Adjuvant (PBS/FIA) emulsion.

[00174] Oil or water-in-oil emulsions are particularly suitable carriers for use in the vaccine composition of the invention. Oils should be pharmaceutically and/or immunologically acceptable. Suitable oils include, for example, mineral oils (especially light or low viscosity mineral oil such as Drakeol® 6VR), vegetable oils (e.g., soybean oil), nut oils (e.g., peanut oil), or mixtures thereof. Thus, in a particular embodiment the carrier is a hydrophobic substance such as vegetable oil, nut oil or mineral oil. Animal fats and artificial hydrophobic polymeric materials, particularly those that are liquid at atmospheric temperature or that can be liquefied relatively easily, may also be used.

[00175] To enhance immunogenicity of therapeutic compositions, IMV Inc. has developed an adjuvanting therapeutic platform designed to facilitate a strong and robust immune response to peptide antigens. DepoVax™ (DPX) is a liposome-in-oil formulation that can be formulated with any epitope, or mixture of epitopes, to induce an adaptive cellular immune response (Karkada et al., J Immimother 33(3):2050-261, 2010; Berinstein NL., et al., J Transl Med. 2012;10:156; Berinstein NL et al., Oncoimmunology. 2015;4(8):el026529) and/or a humoral immune response (Langley JM et al., J Infect Dis. 2018;218(3):378-87). DPX forms a strong depot at the site of immunization which prolongs antigen exposure to the immune system.

[00176] It has been shown that a single vaccination with peptides in DPX results in equivalent or better immune responses than multiple vaccinations with peptides in other conventional formulations, such as Montanide ISA51 VG emulsions, similar to VacciMax® which was a first-generation emulsion-based T cell activation therapeutic platform (Daftarian et al., J Transl Med 5:26, 2007; Mansour et al., J Transl Med 5:20, 2007). A DPX based peptide- T cell activation therapeutic called DPX-0907 has completed a phase I clinical trial in breast, ovarian and prostate cancer patients demonstrating safety and immunogenicity in these advanced patients (Berinstein et al., J Transl Med 10(1): 156, 2012). A DPX platform for use with respiratory syncytial virus (RSV) has also been developed (Langley J.M. et al. A Respiratory Syncytial Virus Vaccine Based on the Small Hydrophobic Protein Ectodomain Presented With a Novel Lipid- Based Formulation Is Highly Immunogenic and Safe in Adults: A First-in-Humans Study. J Infect Dis. 2018 Aug 1; 218(3): 378-387).

[00177] Thus, in a particular embodiment, the carrier of the vaccine composition of the invention may be IMV, Inc's liposomal-based adjuvanting system. Unlike water-in-oil emulsion- based immune cell activation therapeutics, which rely on oil entrapping water droplets containing antigen and adjuvant, DPX based formulations rely on liposomes to facilitate the incorporation of antigens and adjuvants directly into the oil, without the need for emulsification. Advantages of this approach include: (1) enhancing the solubility of hydrophilic antigens/adjuvant in oil diluents which otherwise would normally have maximum solubility in aqueous based diluents, and (2) the elimination of cumbersome emulsification procedures prior to B cell activation therapeutic administration.

[00178] In a preferred embodiment, the carrier is mineral oil or is a mannide oleate in mineral oil solution, such as that commercially available as Montanide® ISA 51 (SEPPIC, France).

[00179] In certain embodiments, the compositions may be substantially free of water (e.g., "water-free"). It is possible that the hydrophobic carrier of these "water-free" compositions may still contain small quantities of water, provided that the water is present in the non-continuous phase of the carrier. For example, individual components of the composition may have bound water that may not be completely removed by processes such as lyophilization or evaporation and certain hydrophobic carriers may contain small amounts of water dissolved therein. Generally, compositions of the invention that are "water-free" contain, for example, less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.5%, 0.1 %, 0.05% or 0.01 % water on a weight/weight basis of the total weight of the carrier component of the composition.

[00180] Optional Adjuvants

[00181] In some embodiments, the vaccine composition of the invention comprises one or more pharmaceutically acceptable adjuvants. A large number of adjuvants have been described and are known to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985) and The United States Pharmacopoeia: The National Formulary (USP 24 NF19) published in 1999.

[00182] Exemplary adjuvants include, without limitation, alum, other compounds of aluminum, Bacillus of Calmette and Guerin (BCG), TiterMax™, Ribi™, Freund's Complete Adjuvant (FCA), CpG-containing oligodeoxynucleotides (CpG ODN), lipopeptides and polynucleotides (e.g., polyI:C, poly dldC, etc.). An exemplary CpG ODN is 5 '- TCCATGACGTTCCTGACGTT-3 ' (SEQ ID NO: 28). The skilled person can readily select other appropriate CpG ODNs on the basis of the target species and efficacy. An exemplary lipopeptide includes, without limitation, Pam3Cys-SKKK (Pam3CSK4) (EMC Microcollections, Germany) or variants, homologs and analogs thereof. The Pam2 family of lipopeptides has been shown to be an effective alternative to the Pam3 family of lipopeptides.

[00183] The lipid-based adjuvant may comprise palmitic acid (PAM) as at least one of the lipid moieties or components of the adjuvant. Such lipid-based adjuvants are referred to herein as a "palmitic acid adjuvant". Palmitic acid is a low molecular weight lipid found in the immunologically reactive Braun's lipoprotein of Escherichia coli. Other common chemical names for palmitic acid include, for example, hexadecanoic acid in IUPAC nomenclature and 1- Pentadecanecarboxylic acid. The molecular formula of palmitic acid is CH₃(CH₂)₁₄CO₂H. As will be understood to those skilled in the art, it is possible that the lipid chain of palmitic acid may be altered. Exemplary compounds which may be used herein as palmitic acid adjuvants, and methods for their synthesis, are described for example in United States Patent Publications US 2008/0233143; US 2010/0129385; and US 2011/0200632, each of which are incorporated herein in their entirety for all intended purposes.

[00184] As described above for lipid moieties generally, a palmitic acid adjuvant contains at a minimum at least one palmitic acid moiety, which can be coupled onto an amino add, an oligopeptide or other molecules. A palmitic acid moiety or a structure containing palmitic acid can be coupled covalently or non-covalently to an antigen to create antigenic compounds with built-in adjuvanting properties. The palmitic acid moiety or a chemical structure containing palmitic acid can be conjugated to a cysteine peptide (Cys) to allow for various structural configurations of the adjuvant, including linear and branched structures. The cystdne residue has been commonly extended by polar residues such as Serine (Ser) and/ or lysine (Lys) at the C terminus to create adjuvant compounds with improved solubility. Palmitic acid containing adjuvant compounds could be admixed with an antigen, associated with antigen through non-covalent interactions, or alternatively covalently linked to an antigen, either directly or with the use of a linker/spacer, to generate enhanced immune responses. Most commonly, two palmitic acid moieties are attached to a glyceryl backbone and a cysteine residue to create dipalmitoyl-S-glyceryl-cysteine (PAM2Cys) or tripalmitoyl-S-glyceryl-cysteine (PAM3Cys), which can also be used in multiple configurations as described above. [00185] Therefore, in an embodiment, the adjuvant of the composition may comprise a palmitic acid moiety or component. The palmitic acid moiety may be modified or manipulated to improve its stability in vitro or in vivo, enhance its binding to receptors (such as for example toll- like receptors as described below) or enhance its biological activity.

[00186] In a particular embodiment, the palmitic acid adjuvant may comprise PAM2Cys or PAM3Cys. In another particular embodiment, the palmitic acid adjuvant may be Pam-2- Cys-Ser- (Lys)4 or Pam-3 -Cys-Ser-(Lys)4 (Pam3CSK4). Such palmitic add adjuvants are available, for example, as research reagents from EMC Microcollections GmbH (Germany) and InvivoGen (San Diego, California, USA). Also available from EMC Microcollections are various analogs of Pam- 2-Cys-Ser-(Lys)4 and Pam-3 -Cys-Ser-(Lys)4, including labelled analogs.

[00187] The composition of the invention may comprise an adjuvant as described above in combination with at least one other suitable adjuvant. Exemplary embodiments of the at least one other adjuvant encompasses, but is by no means limited to, organic and inorganic compounds, polymers, protdns, peptides, sugars from synthetic, non-biological or biological sources (including but not limited to virosomes, virus-like particles, viruses and bacteria of their components). [0189] Further examples of compatible adjuvants may include, without limitation, chemokines, Toll like receptor agonists, colony stimulating factors, cytokines, 1018 ISS, aluminum salts, Amplivax, AS04, AS 15, ABM2, Adjumer, Algammulin, AS01 B, AS02 (SBASA), AS02A, BCG, Calcitriol, Chitosan, Cholera toxin, CP-870,893, CpG, polylC, CyaA, Dimethyldioctadecylammonium bromide (DDA), Dibutyl phthalate (DBP), dSLIM, Gamma inulin, GM-CSF, GMDP, Glycerol, IC30, IC31 , Imiquimod, ImuFactIMP321 , IS Patch, ISCOM, ISCOMATRIX, Juvlmmune, LipoVac, LPS, lipid core protein, MF59, monophosphoryl lipid A, Montanide® IMS1312, Montanide® based adjuvants, OK-432, OM- 174, OM-197-MP-EC, ONTAK, PepTel vector system, other palmitoyl based molecules, PLG microparticles, resiquimod, squalene, SLR172, YF-17 DBCG, QS21 , QuilA, P1005, Poloxamer, Saponin, synthetic polynucleotides, Zymosan, pertussis toxin.

[00188] Accordingly, the composition may comprise one or more pharmaceutically acceptable adjuvants. In some embodiments, at least one of the one or more B cell epitopes and/or T cell epitopes may be coupled to at least one of the adjuvants. [00189] The amount of adjuvant used depends on the amount of antigen and on the type of adjuvant. One skilled in the art can readily determine the amount of adjuvant needed in a particular application by empirical testing.

[00190] Additional Therapeutic

[00191] In some embodiments, the composition may comprise an additional therapeutic. In some embodiments, the additional therapeutic comprises a T cell epitope that comprises one or more of the amino acid sequences of SEQ ID NOs: 27-40 in the Table 2 below. The additional therapeutic can be present in a therapeutically effective amount.

[00192] Table 2. T cell epitopes

[00193] Methods of Making the Compositions

[00194] Disclosed herein are methods of making the compositions comprising the at least one B cell epitope. [00195] The vaccine compositions may be prepared by known methods in the art having regard to the present disclosure. Exemplary embodiments for preparing the compositions disclosed herein are described below without limitation.

[00196] In certain embodiments, the vaccine composition of the invention is one that comprises at least one B cell epitope from the spike protein of SARS-CoV-2, liposomes and a carrier comprising a continuous phase of a hydrophobic substance.

[00197] Methods for making liposomes are well known in the art. See e.g., Gregoriadis

(1990) and Frezard (1999) both cited previously. Any suitable method for making liposomes may be used in the practice of the invention, or liposomes may be obtained from a commercial source. Liposomes are typically prepared by hydrating the liposome components that will form the lipid bilayer (e.g., phospholipids and cholesterol) with an aqueous solution, which may be pure water or a solution of one or more components dissolved in water, e.g., phosphate-buffered saline (PBS), phosphate-free saline, or any other physiologically compatible aqueous solution.

[00198] In an embodiment, a liposome component or mixture of liposome components, such as a phospholipid (e.g., Phospholipon® 90G) or DOPC and cholesterol, may be solubilized in an organic solvent, such as a mixture of chloroform and methanol, tert-butanol or mixture of tert-butanol and water followed by filtering (e.g., a PTFE 0.2 pm filter) and drying, e.g., by rotary evaporation, freeze-drying to remove the solvents. Hydration of the resulting lipid mixture may be affected by e.g., injecting the lipid mixture into an aqueous solution or sonicating the lipid mixture and an aqueous solution. During formation of liposomes, the liposome components form single bilayers (unilamellar) or multiple bilayers (multilamellar) surrounding a volume of the aqueous solution with which the liposome components are hydrated.

[00199] In some embodiments, the liposomes are then dehydrated, such as by freeze- drying or lyophilization.

[00200] In some embodiments, the liposomes are combined with an appropriate carrier, such as a carrier comprising a continuous hydrophobic phase. This can be done in a variety of ways. [00201] If the carrier is composed solely of a hydrophobic substance or a mixture of hydrophobic substances (e.g., use of a 100% mineral oil carrier), the liposomes may simply be mixed with the hydrophobic substance, or if there are multiple hydrophobic substances, mixed with any one or a combination of them.

[00202] If instead the carrier comprising a continuous phase of a hydrophobic substance contains a discontinuous aqueous phase, the carrier will typically take the form of an emulsion of the aqueous phase in the hydrophobic phase, such as a water-in-oil emulsion. Such compositions may contain an emulsifier to stabilize the emulsion and to promote an even distribution of the liposomes. In this regard, emulsifiers may be useful even if a water-free carrier is used, for the purpose of promoting an even distribution of the liposomes in the carrier. Typical emulsifiers include mannide oleate (Arlacel™ A), lecithin (e.g., S100 lecithin), a phospholipid, Tween™ 80, and Spans™ 20, 80, 83 and 85. Typically, the volume ratio (v/v) of hydrophobic substance to emulsifier is in the range of about 5:1 to about 15:1 with a ratio of about 10: 1 being preferred.

[00203] In some embodiments, the liposomes may be added to the finished emulsion, or they may be present in either the aqueous phase or the hydrophobic phase prior to emulsification.

[00204] The B cell epitopes as described herein may be introduced at various different stages of the formulation process. More than one type of antigen may be incorporated into the composition. As used in this section, the term “antigen” is used generally and can refer to a B cell epitope, a T cell epitope, or a combination of B cell epitopes and/or T cell epitopes as described herein. The term is used generally to describe how any antigen may be formulated in the vaccine compositions of the invention. The term “antigen” encompasses both the singular form “antigen” and the plural “antigens”. It is not necessary that all antigens be introduced into the vaccine composition in the same way.

[00205] In some embodiments, the antigen is present in the aqueous solution used to hydrate the components that are used to form the lipid bilayers of the liposomes (e.g., phospholipid(s) and cholesterol). In this case, the antigen will be encapsulated in the liposome, present in its aqueous interior. If the resulting liposomes are not washed or dried, such that there is residual aqueous solution present that is ultimately mixed with the carrier comprising a continuous phase of a hydrophobic substance, it is possible that additional antigen may be present outside the liposomes in the final product. In a related technique, the antigen may be mixed with the components used to form the lipid bilayers of the liposomes, prior to hydration with the aqueous solution. The antigen may also be added to pre-formed liposomes, in which case the antigen may be actively loaded into the liposomes or bound to the surface of the liposomes or the antigen may remain external to the liposomes. In such embodiments, prior to the addition of antigen, the pre-formed liposomes may be empty liposomes (e.g., not containing encapsulated antigen or lipid-based adjuvant) or the pre- formed liposomes may contain lipid-based adjuvant incorporated into or associated with the liposomes. These steps may preferably occur prior to mixing with the carrier comprising a continuous phase of a hydrophobic substance.

[00206] In an alternative approach, the antigen may instead be mixed with the carrier comprising a continuous phase of a hydrophobic substance, before, during, or after the carrier is combined with the liposomes. If the carrier is an emulsion, the antigen may be mixed with either or both of the aqueous phase or hydrophobic phase prior to emulsification. Alternatively, the antigen may be mixed with the carrier after emulsification.

[00207] The technique of combining the antigen with the carrier may be used together with encapsulation of the antigen in the liposomes as described above, such that antigen is present both within the liposomes and in the carrier comprising a continuous phase of a hydrophobic substance.

[00208] Exemplary methods of making the vaccine compositions are found in U.S. Patent

No. 6,793,923, PCT Publication No. WO2019/090411, U.S. Patent Publication No. 2019/0224312

A1 and PCT Patent Publication No. W02019090411. Alternative methods are discussed below.

[00209] The above-described procedures for introducing the antigen into the composition apply also to the B cell epitope and/or the adjuvant and/or additional therapeutic (e.g., T cell epitope) of the compositions as described herein, in embodiments where they are included. That is, the B cell epitope and/or adjuvant and/or additional therapeutic may be introduced into e.g., one or more of: (1) the aqueous solution used to hydrate the components that are used to form the lipid bilayers of the liposomes; (2) the aqueous solution after formation of the lipid bilayers of the liposomes; (3) the components used to form the lipid bilayers of the liposomes; or (4) the carrier comprising a continuous phase of a hydrophobic substance, before, during, or after the carrier is combined with the liposomes. If the carrier is an emulsion, the B cell epitope and/or adjuvant and/or additional therapeutic may be mixed with either or both of the aqueous phase or hydrophobic phase before, during or after emulsification. In some embodiments, the B cell epitope and/or adjuvant and/or additional therapeutic is introduced into the aqueous solution after formation of the lipid bilayers of the liposomes.

[00210] The technique of combining the B cell epitope and/or adjuvant and/or additional therapeutic with the carrier may be used together with encapsulation of these components in the liposomes, or with addition of these components to the liposomes, such that B cell epitope and/or adjuvant and/or additional therapeutic is present inside and/or outside the liposomes and in the carrier comprising a continuous phase of a hydrophobic substance.

[00211] The B cell epitope and/or adjuvant and/or additional therapeutic can be incorporated in the composition together in the same processing step, or separately, at a different processing step. For instance, the B cell epitope and adjuvant and/or additional therapeutic may all be present in the aqueous solution used to hydrate the lipid bilayer-forming liposome components, such that the components become encapsulated in the liposomes. Alternatively, the B cell epitope may be encapsulated in the liposomes, and the adjuvant and/or additional therapeutic mixed with the carrier comprising a continuous phase of a hydrophobic substance. The B cell epitope and/or adjuvant may also be incorporated into the composition after the liposomes have been formed, such that the B cell epitope and adjuvant and/or additional therapeutic may be associated or remain external to the liposomes. In such embodiments, the resulting preparation may be lyophilized and then reconstituted in the carrier comprising a continuous phase of a hydrophobic substance. It will be appreciated that many such combinations are possible.

[00212] In an embodiment, the B cell epitope and/or adjuvant and/or additional therapeutic can be incorporated in the composition separately at a different processing step. For example, the B cell epitope and/or adjuvant and/or additional therapeutic can be incorporated into the composition after the liposomes have been formed, such that the B cell epitope and adjuvant and/or additional therapeutic may be associated with or remain external to the liposomes. In this embodiment, the resulting preparation can be lyophilized and then reconstituted in the carrier comprising a continuous phase of a hydrophobic substance. [00213] If the composition contains one or more further adjuvants, such additional adjuvants can be incorporated in the composition in similar fashion as described above for the adjuvant or by combining several of such methods as may be suitable for the additional adjuvant(s).

[00214] Stabilizers such as sugars, anti-oxidants, or preservatives that maintain the biological activity or improve chemical stability to prolong the shelf life of antigen, adjuvant, the liposomes or the continuous hydrophobic carrier, may be added to such compositions.

[00215] In some embodiments, an antigen/adjuvant mixture may be used, in which case the antigen and adjuvant are incorporated into the composition at the same time. An “antigen/adjuvant mixture” refers to an embodiment in which the antigen and adjuvant are in the same diluent at least prior to incorporation into the composition. The antigen and adjuvant in an antigen/adjuvant mixture may, but need not necessarily be chemically linked, such as by covalent bonding.

[00216] In an embodiment for preparing the composition, a lipid preparation is prepared by dissolving lipids, or a lipid-mixture, in a suitable solvent with gently shaking. The B cell epitope may then be added to the lipid preparation, by first preparing a stock of the B cell epitope dissolved in a suitable solvent. In certain embodiments, the B cell epitope is added to, or combined with, the lipid preparation with gently shaking. The B cell epitope preparation is then dried to form a dry cake, and the dry cake is resuspended in a hydrophobic carrier. The step of drying may be performed by various means known in the art, such as by freeze-drying, lyophilization, rotary evaporation, evaporation under pressure, etc. Low heat drying that does not compromise the integrity of the components can also be used.

[00217] The “suitable solvent” is one that is capable of dissolving the respective component (e.g., lipids, agents, or both), and can be determined by the skilled person.

[00218] In respect of the lipids, in an embodiment the suitable solvent is a polar protic solvent such as an alcohol (e.g., tert-butanol, n-butanol, isopropanol, n-propanol, ethanol or methanol), water, acetate buffer, formic add or chloroform. In an embodiment, the suitable solvent is 40% tertiary-butanol. The skilled person can determine other suitable solvents depending on the lipids to be used. [00219] In a particular embodiment to prepare the compositions, a lipid-mixture containing DOPC and cholesterol in a 10:1 ratio (w:w) (Lipoid GmBH, Germany) can be dissolved in 40% tertiary-butanol by shaking at 300 RPM at room temperature imtil dissolved. An active agent/immunomodulatory agent stock can be prepared in DMSO and diluted with 40% tertiary- butanol prior to mixing with the dissolved lipid-mixture. The B cell epitope stock can then be added to the dissolved lipid-mixture with shaking at 300 RPM for about 5 minutes. The preparation can then be freeze-dried. The freeze-dried cake can then be reconstituted in Montanide® ISA 51 VG (SEPPIC, France) to obtain a clear solution. Typically, the freeze-dried cake is stored (e.g, at -20°C) imtil the time of administration, when the freeze-dried cake is reconstituted in the hydrophobic carrier.

[00220] In another embodiment, for example using procedures as discussed in U.S. Patent

Publication No. 2019/0224312 A1 and modified for the specific peptides, peptide dimers of the B cell epitopes were formed and formulated in DPX. For example, the B-cell epitopes COV2B-S373, COV2B-S461, COV2B-S616, COV2B-S821 (SEQ ID NOs: 5, 7, 14 and 19) may be produced in dimer form under appropriate solvents and conditions. The solvents and conditions used to prepare the COV2B-S373, COV2B-S461, COV2B-S616 and COV2B-S821 peptide dimers were tabulated below in Table A.

[00221] Table A. Peptide solubilities.

[00222] The prepared peptide dimers were then formulated in a water-free oil-based composition at 0.5 mg and 1.0 mg/mL concentration without any adjuvant using lipid nanoparticles prepared in sodium acetate buffer, pH 7.5. These components are then lyophilized to form a dry cake. Just prior to injection, the dry cake is resuspended in ISA51 VG oil (SEPPIC, France) to prepare a water-free oil-based composition.

[00223] For GMP Formulation X manufacturing, COV2B-S373, COV2B-S461, COV2B- S616 and COV2B-S821 peptides were purchased in dimer form directly from the commercial peptide supplier and were formulated in a water-free oil-based composition using the formulation method described in US 2019/0224312 A1. In brief, the synthesized peptide dimers were added sequentially to lipid nanoparticles previously prepared in sodium acetate buffer, pH 7.5, sterile filtered using 0.22 pm Polyethersulfone (PES) filter and then lyophilized to form a dry cake. Just prior to injection, the dry cake is resuspended in ISA51 VG oil (SEPPIC, France) to prepare a water-free oil-based composition.

[00224] In another embodiment, to prepare the compositions the B cell epitope is dissolved in sodium phosphate or sodium acetate buffer with S100 lipids and cholesterol (Lipoid, Germany). These components are then lyophilized to form a dry cake. Just prior to injection, the dry cake is resuspended in ISA51 VG oil (SEPPIC, France) to prepare a water-free oil-based composition.

[00225] In another embodiment, to prepare the compositions the B cell epitope and/or immunomodulatory agent is dissolved in sodium phosphate or sodium acetate buffer with DOPC and cholesterol (Lipoid, Germany). These components are then lyophilized to form a dry cake. Just prior to injection, the dry cake is resuspended in ISA51 VG oil (SEPPIC, France) to prepare a water-free oil-based composition.

[00226] In another embodiment, to prepare the compositions the dry cake is mixed with lipid/cholesterol nanoparticles (size <110 nm) in sodium phosphate or sodium acetate buffer (100 mM, pH 6.0). The lipid may be DOPC. The components are then lyophilized to form a dry cake. Just prior to injection, the dry cake is resuspended in ISA51 VG oil (SEPPIC, France) to prepare a water-free oil-based composition.

[00227] In some embodiments, it may be appropriate to include an emulsifier in the hydrophobic carrier to assist in stabilizing the components of the dry cake when they are resuspended in the hydrophobic carrier. The emulsifier is provided in an amount sufficient to resuspend the dry mixture of active agent and/or immunomodulatory agent and lipids in the hydrophobic carrier and maintain the active agent and/or immunomodulatory agent and lipids in a dissolved state in the hydrophobic carrier. For example, the emulsifier may be present at about 5% to about 15% weight/weight or weight/volume of the hydrophobic carrier. [00228] Stabilizers such as sugars, anti-oxidants, or preservatives that maintain the biological activity or improve chemical stability to prolong the shelf life of any of the components, may be added to the compositions.

[00229] In an embodiment, methods for preparing the compositions herein may include those disclosed in WO 2009/043165, as appropriate in the context of the present disclosure. In such instances, the active agents and/or immunomodulatory agents as described herein would be incorporated into the compositions in similar fashion as described for antigens in WO 2009/043165.

[00230] In an embodiment, methods for preparing the compositions herein may include those disclosed in the publications of PCT/CA2017/051335 and PCT/CA2017/051336 involving the use of sized lipid vesicle particles. In such instances, the active agents and/or immunomodulatory agents as described herein would be incorporated into the compositions in similar fashion as described for therapeutic agents in the publications of PCT/CA2017/051335 and PCT/CA2017/051336, both of which are incorporated herein by reference in their entirety for all intended purposes.

[00231] An exemplary method to prepare a vaccine composition of the invention follows. However, it will be appreciated that alternate embodiments are also encompassed herein, such as those described above where the B cell epitope and optional adjuvant may be introduced at any stage in the formulation of the vaccine composition, in any order and may ultimately be found inside, outside or both inside and outside the liposomes.

[00232] In certain embodiments, the vaccine composition is formed with a combination of fourB cell epitopes (SEQ ID Nos: 5, 7, 14, and/or 19); an optional adjuvant (e.g., Pam3CSK4), an additional therapeutic, and liposomes (DOPC and cholesterol) in an aqueous buffer by a process of mixing and hydrating lipid components in the presence of the B cell epitopes, additional therapeutic, and adjuvant, extruded to achieve a particle size that can be sterile filtered, then filled into vials and lyophilized to a dry cake. The dry cake is then re-suspended in the hydrophobic carrier Montanide ISAS 1 VG before inj action. This exemplary method of preparation may be used with any combination of B cell epitopes and any suitable adjuvant. [00233] In certain embodiments, to prepare vaccine compositions of the invention, the four

B cell epitopes (SEQ ID NOs: 5, 7, 14, and/or 19) and optional adjuvant (e.g., Pam3CSK4) are added to previously sized liposomes (<100 nm, pdi <0.1) prepared in sodium acetate buffer, 50 mM, pH 7.5, sterile filtered and freeze-dried. The dry cake is then re-suspended in the hydrophobic carrier Montanide ISAS 1 VG before inj ection. This exemplary method of preparation may be used with any combination of B cell epitopes and any suitable adjuvant.

[00234] In some embodiments, the carrier comprising a continuous phase of a hydrophobic substance may itself have adjuvanting-activity. Incomplete Freund's adjuvant and Montanide® ISA 51 VG, are examples of a hydrophobic carrier with adjuvanting effect. As used herein and in the claims, when the term "adjuvant" is used, this is intended to indicate the presence of an adjuvant in addition to any adjuvanting activity provided by the carrier comprising a continuous phase of a hydrophobic substance.

[00235] Methods of Administering the Compositions

[00236] The methods disclosed herein comprise administering the at least one B cell epitope, the liposome, and/or optional adjuvant to elicit an immune response against SARS-CoV- 2 in the subject. In certain embodiments, the invention further comprises administering an additional therapeutic agent. In certain embodiments, the at least one B cell epitope, the liposome, optional adjuvant and additional therapeutic agent are administered simultaneously. In certain embodiments, the at least one B cell epitope, the liposome, optional adjuvant and additional therapeutic agent are administered at different times.

[00237] The at least one B cell epitope, the liposome, optional adjuvant and/or additional therapeutic agent as disclosed herein may be administered to a subject in a therapeutically effective amount. In certain embodiments, the effective amount of the at least one B cell epitope, the liposome, optional adjuvant and/or additional therapeutic agent is an amount sufficient to provide an immune-modulating effect.

[00238] The term “agent” includes any substance, molecule, element, compound, entity, or a combination thereof. It can be a natural product, a synthetic compound, or a combination of two or more substances. Unless otherwise specified, the terms “agent”, “substance”, and “compound” are used interchangeably herein.

[00239] As used herein, an “additional therapeutic” or “additional therapeutic agent” refers to a pharmaceutical or therapeutic agent. The additional therapeutic agent can comprise one or more T cell epitopes, such as the T cell epitope amino add sequences listed in SEQ ID Nos: 29- 42. The additional therapeutic agent can be an antiviral drug, a small molecule drug, an antibody, an antibody mimetic, or a functional equivalent or functional fragment of any one thereof.

[00240] In the methods disclosed herein, the amount of any of the at least one B cell epitope, the liposome, optional adjuvant and/or additional therapeutic agent may depend on the type of ingredient, the disease or disorder to be treated, and/or particular characteristics of the subj ect (e.g., age, weight, sex, immune status, etc.). One skilled in the art can readily determine the amount of the ingredient needed in a particular application by empirical testing.

[00241] A vaccine composition according to the invention may be administered by any suitable means, such as e.g., injection (e.g., intramuscular, intradermal, subcutaneous, or intraperitoneal), aerosol, oral, nasal, topical, intravaginal, transdermal, transmucosal, or any other suitable routes. The vaccine composition may be formulated for systemic or localized distribution in the body of the subject Systemic formulations include those designed for administration by injection, as well as those designed for transdermal, transmucosal or oral administration.

[00242] For injection, the vaccine compositions may be formulated in a carrier comprising a continuous phase of a hydrophobic substance as described herein, such as a water-in-oil emulsion or an oil-based carrier. In some embodiments, liposomes may be used together with the carrier. The vaccine composition may also be formulated as aqueous solutions such as in Hank's solution, Ringer's solution or physiological saline buffer.

[00243] The methods disclosed herein comprise administering a vaccine composition comprising at least one B cell epitope from the spike protein of SARS-CoV-2 to a subject in order to elicit an immune response against the SARS-CoV-2 virus. In certain embodiments, the invention further comprises administering an additional therapeutic agent, In certain embodiments, the active agent and additional therapeutic agent are administered with the same regimen. In certain embodiments, the active agent and additional therapeutic agent are administered with different regimens.

[00244] As used herein, the terms “combination”, “co-administration”, or “combined administration” or the like are meant to encompass administration of the vaccine composition and optional additional therapeutic to a single patient, and are intended to include instances where the vaccine composition and optional additional therapeutic are not necessarily administered by the same route of administration or at the same time. For example, the vaccine composition and optional additional therapeutic may be administered separately, sequentially, or using alternating administration.

[00245] In certain embodiments, the vaccine composition is administered before, at the same time, and/or after the administration of the optional additional therapeutic.

[00246] The vaccine composition is typically administered in an amount sufficient to provide an immune-modulating effect.

[00247] In certain embodiments, the vaccine composition is administered at a dose of about 10 μg to 50 μg, for example 10 μg to 25 μg or 50 μg.

[00248] In certain embodiments, the “amount sufficient to provide an immune-modulating effect” may be a “low dose” amount. Thus, in certain embodiments, the methods of the invention involve the use of a low dose of at least one B cell epitope, optionally in combination with an adjuvant

[00249] As it relates to certain embodiments of the invention “low dose” may refer to a dose of at least one B cell epitope that is less than about 25 mg/m². In terms of daily administration, a “low dose” of active agent is between about 25-300 mg/day or about 50-150 mg/day. In certain embodiments, a daily dosage amount is about 100 mg of active agent. In certain embodiments, a daily dosage amount is about 50 mg of active agent per dose.

[00250] The “low dose” amounts of other active agents, as encompassed herein, would be known to those skilled in the art, or could be determined by routine skill. [00251] In certain embodiments, the methods of the invention comprise the administration of at least one priming dose of the vaccine composition and then subsequently administering a boosting dose of the vaccine composition. The at least two doses may be separated by any suitable amount of time.

[00252] By “subsequently administering”, it is meant that the administration of the priming dose starts before the administration of the boosting dose. In an embodiment, the minimum amount of time separating the priming dose and the boosting does may be any amount of time sufficient to provide an immune-modulating effect. The skilled artisan will appreciate and take into consideration the amount of time sufficient to provide an immune-modulating effect based on the vaccine composition.

[00253] In some embodiments, the priming dose is administered at least 12 hours before the boosting dose, and preferably at least two, four or six days before the boosting dose. In a further embodiment, the priming dose may be provided about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days, or more, before the boosting dose. In a particular embodiment the administration of the priming dose occurs 1-4 days prior to the administration of the boosting dose. In certain embodiments, the administration of the priming dose occurs about one week before the administration of the boosting dose.

[00254] In an embodiment, the boosting dose may be followed by one or more maintenance doses. As used herein, the term “maintenance dose” is meant to encompass a dose of the vaccine composition that is given at such an interval and/or amount so as to maintain a sufficient amount of the antibodies, e.g., neutralizing antibodies, in the body of the subject (e.g., avoid total systemic clearance of the antibodies or NAbs). By providing a maintenance dose, it may be possible to prolong and/or maintain the immune-modulating effect of the antibodies or NAbs for an extended period of time before, during, and/or after the course of administration of the vaccine composition.

[00255] As the skilled person will appreciate, the frequency and duration of the administration of the vaccine composition may be adjusted as desired for any given subject. Factors that may be taken into account include, e.g.: the nature of the one or more B cell epitopes in the vaccine composition, the longevity of the antibodies or NAbs elicited, the age, physical condition, body weight, sex and diet of the subject; and other clinical factors. [00256] In certain embodiments, an additional therapeutic agent is administered. In certain embodiments, the additional therapeutic agent comprises one or more T cell epitopes, for example and not limitation, the T cell epitopes of SEQ ID Nos: 29-42.

[00257] In certain embodiments, administration of the additional therapeutic agent and the vaccine composition to a single patient and are intended to include instances wherein the agent and vaccine composition are not necessarily administered by the same route of administration or at the same time. For example, the additional therapeutic agent and the vaccine composition may be administered separately, sequentially, or using alternating administration.

[00258] In certain embodiments, the active agent is administered before, at the same time, or after the administration of the vaccine composition.

[00259] The additional therapeutic agent is typically administered in an amount sufficient to provide an immune-modulating effect.

[00260] In certain embodiments, the additional therapeutic agent is administered at a dose of about 5 μg to about 5 g.

[00261] Treatment Indications

[00262] As described herein, the methods of the present invention relate to the prevention and/or treatment of COVID-19, which is caused by the novel coronavirus SARS-CoV-2.

[00263] In some embodiments, the methods of the invention may be used to prevent a COVID-19 infection by inducing a humoral and/or cell-mediated immune response using the invented vaccine composition.

[00264] A humoral immune response, as opposed to cell-mediated immunity, is mediated by secreted antibodies which are produced in the cells of the B lymphocyte lineage (B cells). Such secreted antibodies bind to antigens, such as for example those on the surfaces of foreign substances and/or pathogens (e.g., viruses, bacteria, etc.) and flag them for destruction.

[00265] Antibodies are the antigen-specific glycoprotein products of a subset of white blood cells called B lymphocytes (B cells). Engagement of antigen with antibody expressed on the surface of B cells can induce an antibody response comprising stimulation of B cells to become activated, to undergo mitosis and to terminally differentiate into plasma cells, which are specialized cells for synthesis and secretion of antigen-specific antibodies.

[00266] B cells are the sole producers of antibodies during an immune response and are thus a key element to effective humoral immunity. In addition to producing large amounts of antibodies, B cells also act as antigen-presenting cells and can present antigen to T cells, such as T-helper CD4 or cytotoxic CDS, thus propagating the immune response. B cells, as well as T cells, are part of the adaptive immune response which may assist in B cell activation therapeutic efficacy. During an active immune response, induced either by vaccination or natural infection, antigen-specific B cells are activated and clonally expand. During expansion, B cells evolve to have higher affinity for the epitope. Proliferation of B cells can be induced indirectly by activated T-helper cells, and also directly through stimulation of receptors, such as the toll-like receptors (TLRs).

[00267] Antigen presenting cells, such as dendritic cells and B cells, are drawn to vaccination sites and can interact with antigens and adjuvants contained in the B cell activation therapeutic. The adjuvant stimulates the cells to become activated and the antigen provides the blueprint for the target. Different types of adjuvants provide different stimulation signals to cells. For example, polyI:C polynucleotide (a TLR3 agonist) can activate dendritic cells, but notB cells. Adjuvants such as Pam3CSK4, Pam2Cys and FSL-1 are especially adept at activating and initiating proliferation of B cells, which is expected to facilitate the production of an antibody response (Moyle et al., Curr Med Chem, 2008; So., J Immunol, 2012).

[00268] Kits and Reagents

[00269] For practicing the methods of the present invention, the compositions as described herein may optionally be provided to a user as a kit. For example, a kit of the invention contains one or more components of the compositions of the invention. The kit can further comprise one or more additional reagents, packaging material, containers for holding the components of the kit, and an instruction set or user manual detailing preferred methods of using the kit components.

[00270] In a particular embodiment, the vaccine composition of the invention (is supplied as a kit containing at least one container. Container 1, for example, may comprise the lyophilized adjuvant system (e.g., liposomes), at least one B cell epitope and/or at least one T cell epitope and optional adjuvant. Container 2, for example, may contain the oil component (Montanide® ISA51 VG) alone. An appropriate volume (e.g., 0.05 to 0.5 ml) of the reconstituted vaccine composition may be injected IΜ or subcutaneously.

[00271] In certain embodiments, the kit may additionally contain an additional therapeutic. The additional therapeutic may be included in the kit with a third container, or the additional therapeutic may be included in container 1 or container 2, as described above.

[00272] Illustrative Embodiments

[00273] In certain embodiments, the at least one B cell epitope comprises one or more of the amino add sequences of SEQ ID NOs: 5, 7, 11, 14, 17, 19, 23, 24, and/or 26, optionally in the form of a dimer. In certain embodiments, the at least one B cell epitope comprises one or more of the amino acid sequences of SEQ ID NOs: 5, 7, 14, and/or 19, optionally in the form of a dimer. In certain embodiments, the at least one B cell epitope comprises a mixture of four B cell epitopes comprising the amino add sequences of SEQ ID NOs: 5, 7, 14, and/or 19, optionally in the form of a dimer. In certain embodiments, the composition further comprises at least one T cell epitope comprising one or more of the amino acid sequences of SEQ ID NOs: 27, 28, 32, 34, 38, 42 and/or 43, optionally in the form of a dimer. In certain embodiments the at least one T cell epitope comprises a mixture of two T cell epitopes comprising the amino add sequences of SEQ ID NOs: 27, 28, 32, 34, 38, 42 and/or 43, optionally in the form of a dimer.

[00274] The present invention is also described and demonstrated by way of the following illustrative embodiments and in no way limits the scope and meaning of the invention.

EXAMPLES

[00275] The present invention is also described and demonstrated by way of the following examples. However, the use of these and other examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to any particular preferred embodiments described here. Indeed, many modifications and variations of the invention may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the invention in spirit or in scope. The invention is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which those claims are entitled.

[00276] Example 1 - Animal Testing of Group I B Cell Epitopes

[00277] This Example focuses on nine (9) formulations of the 25 B cell epitopes identified in the SARS-CoV-2 spike protein and their ability to elicit a humoral immune response in mice.

[00278] The B-cell epitopes were formulated in groups of 2-4 epitopes per water-free oil- based formulation and administered to CD-1 mice to assess the presence of epitope-specific antibodies in mice sera using indirect enzyme-linked immunosorbent assays (indirect ELISA).

[00279] Materials

[00280] Eighty-eight CD-1 female mice (Charles River Laboratories; 10 per group vaccinated with Formulations 1-8, 8 per group vaccinated with Formulations 9) between 6-11 weeks old and weighing 20-40 grams were obtained for this study. The study was conducted as four separate experiments. The first experiment evaluated Formulations 1-3; Formulations 4-7 were evaluated in the second experiment, the third experiment evaluated Formulation 8 and the fourth experiment evaluated Formulation 9. Experiments 1 and 2 also included positive and negative control groups (Formulation A and Formulation Z, N=5 per experiment). Experiments 3 and 4 shared controls with experiments 1 and 2. The data from the positive and the negative control groups from experiments 1 and 2 were combined to generate analyses of 10 mice per control group. Experiment 1 and 2 also included non-vaccinated naive group (N=10 per experiment). Experiments 3 and 4 shared naive mice with experiments 1 and 2. Formulations 1 and 4 were evaluated in two compositions: with and without Pam3CSK4 adjuvant. The mice were divided into treatment groups as follows.

Table 3. Treatment Groups

[00281] The formulations administered to the treatment groups were based on the water- free oil-based composition platform technologies discussed herein. Specific formulations are as follows.

Table 4. Vaccine Formulations

[00282] The following B cell epitopes were tested.

Table 5. Epitope Sequences and Formulations

[00283] The following combinations of epitopes were selected based on predicted coverage of the spike protein and in silico prediction of likely NAb binding sites. The combinations of epitopes were predicted to bind to (i) the receptor binding motif of the RBD of SARS-CoV-2 spike protein; (ii) other portions of the spike protein that were predicted to indirectly affect binding by allosteric hindrance, steric hindrance, and/or preventing the opening and exposure of the receptor binding motif of the RBD; (iii) other portions of the spike protein that were predicted to prevent fusion of SARS-COV-2 and cell membrane. In Formulation 1, COV2B-S494, COV2B-S634, and COV2B-S807 were initially selected, but COV2B-S634 had compatibility issues with the formulation buffer and was reformulated for use in Formulation 7. Thus, Formulation 1 included only COV2B-S494 and COV2B-S807. Formulation 1 was prepared in two compositions with and without Pam3Cys adjuvant. Formulation 2 included COV2B-S404, COV2B-S327 and COV2B- S555. Formulation 3 included COV2B-S461, COV2B-S496, and COV2B-S516. Formulation 4 included COV-2B-S369, COV-2B-S616, COV-2B-S821, and COV-2B-S1157. Formulation 4 was prepared in two compositions with and without Pam3Cys adjuvant. Formulation 5 included COV- 2B-S250, COV-2B-S524, and COV-2B-S1165. Formulation 6 included COV-2B-S373, COV-2B- S431, COV-2B-S672, and COV-2B-S1252. Formulation 7 included COV-2B-S329 and COV-2B- S634. Formulation 8 included COV-2B-S486 and COV-2B-S1182. Formulation 9 included COV- 2B-S553 and COV-2B-S809. Solubility of the different formulations was determined. All groups showed no signs of precipitation for the monitored 4-5 h at room temperature (RT).

Table 6. Solubility of Epitopes

[00284] Formulation preparation· [00285] Based on the peptide theoretical solubility profile and isoelectric point (pi), the solubilities of the peptides were tested in solvents such as sterile water, sodium hydroxide, sodium acetate, acetic acid, and DMSO, and observed for clarity and precipitation at T=0-4 hrs.

[00286] Peptides were added to the formulation buffer (sodium acetate, 50 mM, pH 9.5). The addition order of the peptides was determined based on the theoretical pi and pH of the peptide stock solution. The peptides were added to the formulation buffer one after another and pH was measured after each peptide addition and looked for signs of precipitation before continuing with the addition of the next peptide. The compatibility of the added peptides in the formulation buffer was observed for precipitation at T=0-4 hrs.

[00287] Each of the different formulations of peptides were formulated in DPX following the sequential addition order identified from the pre-formulation screening step.

[00288] Lipid nanoparticles (LNPs) of the selected size (<100 nm, pdi <0.1) prepared in sodium acetate, 50 mM, pH 7.5 were added to the peptide combination solution and mixed well. The mixture was then aseptically filled into 3 mL vials within 2 hrs of preparation and lyophilized. Samples were taken for Quality Control (QC) testing before filling and after lyophilization.

[00289] Freeze-dried water-free oil-based formulations were prepared with or without toll- like receptor agonist Pam3CSK4 adjuvant (PolyPeptide Group, San Diego) and a mixture of DOPC (1,2-dioleonyl-sn-glycero-3-phosphocholine): cholesterol (Lipoid GmBH, Germany) lipid nanoparticles with peptides (each at 50 μg/dose). The samples were then reconstituted in Montanide ISA 51 VG (Seppic, France) oil diluent for animal administration.

[00290] Methods

[00291] Injection sites on the mice were swabbed with alcohol prior to injection. Immunization start time and end time was noted. Animals were anesthetized by isoflurane (2-4%, lL/min O₂) for treatment on Study Day (SD) 0 and 14. Each mouse in groups 1-13 received a vaccine injection (I.M. : intramuscular); 25 μL dose in both caudal thigh muscles (50 μL total dose). Mouse body weight was monitored over the study (Figure 1A-1B). [00292] To address the safety of Formulation X, induration severity at the site of injection (SOI) was monitored for three days after each prime vaccination (study day 0, SD0) and after boost vaccination (SD14) and on weekly basis thereafter (Figures 2A-2C; 3A-3C, and 4A-4D). Induration is defined as a hardening of an area of the body as a reaction to inflammation, hyperemia, or neoplastic infiltration. SOI reactions are given a quantitative induration grade (0, 1, 2, 3) based on an adapted CTCAE table. “0” indicates no induration, “1” indicates an increased density on palpation, “2” indicates a marked increase in density and firmness on palpation and “3” indicates a very marked density or fixation (immoveable). Erythema scores are given a grade (0, 1, 2, 3 or 4) based on the Draize Dermal Irritation Scoring System. A “0” indicated no erythema or redness of the skin, “1” is a very slight erythema that is barely perceptible, “2” is a well-defined erythema, “3” is a moderate to severe erythema, and “4” is a severe erythema. In addition, detailed clinical exams including body weights and SOI evaluations were performed weekly for the duration of the study.

[00293] Formulations were administered on SD 0 and SDH, with blood collections occurring by facial venipuncture or cardiac venipuncture (on termination) on SDH, SD21, SD28, and SD42. On SDH, blood collection occurred prior to immunization.

[00294] The modified CTCAE table and Draize Dermal Irritation Scoring showed mild observations of erythema and indurations at SOIs in all of groups vaccinated with water-free oil- based composition formulations. The erythema and indurations in the groups exposed to Formulation X or to formulations 1-9 were similar in frequency, timing, and severity to those observed in the Formulation Z group suggesting that the SARS-COV-2 B-cell peptides formulated in water-free oil-based compositions did not elicit severe adverse inflammatory reactions.

[00295] The majority of mice developed transient mild erythema at SOI following prime and/or boost vaccinations (score 1). The development began as barely perceptible erythema detected in 38% of SOI observations on the 1st day after vaccination, frequency of mild erythema events was increased to 63% on the second day and was reduced to 34% on the 3rd day. No cases of Grade 3 erythema were reported. Almost all erythema developments were resolved by study week five (Figure 2A-2C). [00296] In addition, transient, mild-to-moderate indurations of SOI area were detected after prime vaccination in 80% of SOI observations with increase to 99% of SOI observations after the boost vaccination. Induration development was mild with scores primarily of one (57%) or two (43%) following initial vaccination (SD1-SD3), with progression towards increase density and firmness (Score 2 (75%) or 3 (3.6%) across all groups including Formulation Z after the boost vaccination (SD15-SD17). Only one SOI in one mouse (1/20, 5%) in group treated with formulation 7 developed a grade 3 induration on SD3. Figure 3A-3C). The majority of indurations were resolved by the end of each study.

[00297] The addition of the adjuvant Pam3CSK4 to Formulation 1 did not appear to cause a substantial change in SOI erythema; however an increase in frequency of erythema from 20% to 62% was noted after the boost vaccination (SD15-SD24) with Formulation 4 containing the adjuvant (Figure 4B and D), suggesting thatPam3CSK4 is causing an inflammatory reaction when combined with the highly immunogenic Formulation 4. An increase in in the progression of indurations from mild to moderate was observed in both groups exposed to formulations containing Pam3CSK4 after the boost vaccination (Figure 4A and 4C) further supporting this conclusion. However, these are preliminary formulations and it is possible that different formulations and/or different combinations of peptides will show different effects of adjuvants.

[00298] In summary, all of the formulations containing the selected SARS-CoV-2 B cell epitopes in the water-free formulation were well tolerated in the CD-1 mouse model, regardless of the peptide antigens contained in the formulation. Observations of induration and erythema at the SOI were deemed acceptable for an oil-based product, and all mice appeared healthy for the duration of recorded observations. The Pam3CSK4 adjuvant was active in the formulation as indicated by the inflammatory response observed in one of the formulations.

[00299] Peptide-specific antibody titers in serum were determined by indirect ELISA. Indirect ELISA was performed to detect serum antigen specific antibody titers. Briefly, 96-well EIA/RIA Clear Flat Bottom Polystyrene High Bind Microplates (Corning®) were coated with 1 μg/mL of individual peptides in coating buffer (NaHCO₃, NazCO₃) overnight at 4°C. Plates were washed five times with TBS-T (0.2%) and blocked with warmed 3% Gelatin (BioRad) for 30-45 minutes at 37 °C. Plates were washed with TBS-T and incubated overnight at 4°C with sera at an initial starting dilution of 1:100. After TBS-T washes, bound antibodies were detected by incubation of alkaline phosphatase conjugated Protein G (EMD Millipore) with high affinity binding for IgG for 1 hour at 37°C and subsequent development with chromogenic alkaline phosphatase substrate. Optical density was measured at 405 nm within 1 hour of initial substrate addition on a plate reader.

[00300] Indirect ELISA results were expressed as end point log (10) titers, which was defined as the reciprocal of the highest dilution that gives a positive reaction. To determine whether a reaction is positive or negative, an absorbance cutoff value was defined. Readings above the cutoff were considered positive while readings at or below the cutoff were negative. (Frey et al, 1998). Titers below cut-off were assigned values of 10^1·85 which is Log 10 (LLOQ/√2) (Croghan et al. 2003).

[00301] Each serum sample was diluted at 1:100 as a starting dilution on SD14 and further diluted to 7 additional dilutions at 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400 and 1:12800. Starting dilutions for SD21, SD28, and SD42 were then determined based on prior titers to ensure the endpoint titers falls within prepared dilutions. The ability of the different formulations to elicit immune responses were determined based on increase in average log(10) endpoint titers. Average group responses were assessed for immunogenicity compared to background response for each group. Naive CD-1 sera were used to establish baseline cut-off.

[00302] In some cases, the endpoint titers exceeded the highest prepared dilution on the ELISA plate. The highest prepared dilution was then used to determine the corresponding titer and used for further downstream analysis. As per serum availability, repeats were performed to determine end-point titers for these samples.

[00303] Functional binding capacity of the antigen-specific antibodies was determined using the VaxArray Coronavirus SeroAssay (InDevR Inc, Boulder, Colorado), a multiplexed immunoassay for detection of antibodies against SARS-CoV-2, SARS-CoV, and MERS, as well as the endemic coronaviruses CoV HKU1, CoV OC43, CoV 229E, and CoV NL63. The assay allows for detection of antibody binding to the full SARS-CoV-2 Spike protein as well as to the S1 and S2 subunits. [00304] Serum samples were collected and stored at -20°C and shipped to InDevR Inc. on dry ice with temperature monitoring. Analysis was performed by InDevR Inc. according to the kit instructions. Briefly, test samples were serially diluted two-fold, starting at a 100-fold dilution. Standard VaxArray assay protocols were utilized for all testing. The serum samples were diluted in Protein Blocking Buffer and incubated on the VaxArray slide for 1 hour at 80 rpm. Wash Buffer 1 was applied to the slide, after the samples were removed, and then the microarrays were incubated with an anti-mouse IgG label for 30 minutes at 80 rpm. Label was removed and the slides were washed, dried, and imaged. Serum samples from the control group vaccinated with Formulation A were used as an irrelevant control to confirm assay specificity. VaxArray Coronavirus SeroAssay signals were reported as Signal/Background (S/B) ratio and relative fluorescent units (RFU). The maximum resolvable signal on the VaxArray Imaging System is 65,535 RFU. Endpoint titer values were defined as the highest dilution factor with S/B >1.5.

[00305] A pseudo-particle neutralizing assay (PNA) was performed using HEK293T/ACE2 cells which stably express human ACE2 (Creative Diagnostics) and pseudotyped GFP rSARs- CoV-2 Spike protein viruses (Creative Diagnostics). Serum samples collected on SD42 from mice vaccinated with Formulation 4 containing Pam3CSK4 adjuvant, from one mouse vaccinated with Formulation 3, from one mouse vaccinated with Formulation 6 and from mice vaccinated with Formulation A irrelevant control were heat inactivated at 56°C for 15 min. 2X serial dilutions of sera were mixed with pseudoviruses, incubated for 1 hour at 37°C and then mixed with HEK293T/ACE2 cells. Samples were incubated at 5% CO₂, 37°C humidified incubator. Media was replaced after 24 hours of incubation and samples were returned to 5% CO2, 37°C humidified incubator for additional 48 hours. Non-transduced 293T cells were infected with the pseudoviruses to confirm that pseudoviruses entry is mediated via ACE2 receptor on HEK293T/ACE2 cells. As expected, no GFP expression was detected in HEK293T. After incubation for 72 hours, the cells were harvested and analyzed for GFP expression by flow cytometry using BD FACSCelesta™ analyser and FlowJo™ software (BD Biosciences, San Jose, CA). Mean fluorescent intensity (MFI) was used to quantify level of infection of HEK293T/ACE2 and HEK293T cells by pseudoviruses.

[00306] Neutralizing activity was quantified using dose response curves evaluating the correlation between MFI and viral load in HEK293T-ACE2 cells infected with serial dilutions of pseudo-virus without addition of sera using the experimental procedure described herein. All experiments performed reported a strong linear relationship between MFI and viral load: (R² >0.9). Neutralization activity was reported if viral load was reduced by at least 50%.

[00307] Formulation 1 Results.

[00308] Table 7. Formulation 1 details.

00309] These peptides are located in separate regions of the S protein and target either the RBD or the fusion domain for potential neutralization antibody generation. COV2B-S494 falls into a region in which a mAb was detected in SARS-CoV patient sera that blocked SARS-CoV infection in vitro ( Wang et al. 2016). COV2B-S807 overlaps with one of two peptides known today that bind SARS-CoV-2 nAbs (Poh et al. 2020, Two linear epitopes on the SARS-CoV-2 spike protein that elicit neutralising antibodies in COVID-19 patients, Nature Communications volume 11, Article number: 2806). These peptides were tested with and without Pam3CSK4 adjuvant.

[00310] Table 8. Formulation 1 Solubility.

[00311] The immunogenicity of the two peptides were measured over 42 days. Both of the peptides were found to be poorly immunogenic, without and with the Pam3CSK4 adjuvant (Figure 5A and 5B, respectively and Table 9).

[00312] Table 9. Immunogenicity of Formulation 1.

[00313] Formulation 2 Results.

[00314] Table 10. Formulation 2 details.

[00315] These peptides are located in separate regions of the S protein and target either the RBD or the fusion domain. COV2B-S555 is part of one of two peptides known today that bind SARS-CoV-2 nAbs (Poh et al. 2020).

[00316] Table 11. Formulation 2 Solubility.

[00317] A reduced recovery of COV2B-S327 peptide was observed due to cysteine oxidation. Based on experience with RSV, a forced cysteine oxidation method was explored for the feasibility of making the peptide stock in fully oxidized manner (to form dimers) for formulation in a water-free oil-based composition. Details of this method are found in U.S. Patent Publication No. 2019/0224312, incorporated herein. The COV2B-S327 peptide was found to be soluble in 10% DMSO in 0.5% acetic acid and was 98% oxidized upon incubation at 37°C for 24 hrs.

[00318] The immunogenicity of the three peptides were measured over 42 days. All of the peptides were found to be poorly immunogenic (Figure 6 and Table 12).

[00319] Table 12. Formulation 2 immunogenicity.

[00320] Formulation 3 Results.

[00321] Table 13. Formulation 3 details.

[00322] These peptide targets are located in the RBD of the S protein and the immediately surrounding region. COV2B-S461 partially falls into region in which a mAb was detected in SARS-CoV patient sera that blocked SARS-CoV infection in vitro (Wang et al. 2016).

[00323] Table 14. Formulation 3 stability.

[00324] A reduced recovery of the COV2B-S461 peptide was observed due to cysteine oxidation. A forced cysteine oxidation method was explored for the feasibility of making the peptide stock in fully oxidized manner (to form dimers) for formulation in a water-free oil-based composition. Details of this method are found in U.S. Patent Publication No. 2019/0224312, incorporated herein. The recovery of COV2B-S461 was improved in 10% DMSO in 0.5% acetic acid to 95.5% upon incubation at 37°C for 24 hrs. [00325] The immunogenicity of the three peptides were measured over 42 days. While peptides COV2B-S496 and COV2B-S516 peptides were poorly immunogenic, the COV2B-S461 peptide was found to be immunogenic and was determined to be a possible candidate (Figure 7, Table 15).

[00326] Table 15. Formulation 3 immunogenicity.

[00327] Formulation 4 Results.

[00328] Table 16. Formulation 4 details.

[00329] These peptides are located in separate regions of the S protein, which target the RBD and the fusion domain. COV2B-S616 falls and COV2B-S1157 partially falls into a region in which a mAh was detected in SARS-CoV patient sera that blocked SARS-CoV infection in vitro (Wang et al. 2016). COV2B-S821 is based on demonstrated functionality in SARS-Cov- 1 as an immunodominant linear neutralization domain (Zhang et al. 2004), and also overlaps with one of two peptides known today, that bind SARS-CoV-2 nAbs (Poh et al. 2020).

[00330] Table 17. Formulation 4 solubility.

[00331] Reduced recovery of the COV2B-S369, COV2B-S616 and COV2B-S821 peptides were observed due to cysteine oxidation. Different diluents were tested with each of these peptides in order to improve recovery in the oxidized form. COV2B-S369 was found to have improved recovery (82.8%) in 10% DMSO in 0.5% Acetic add upon incubation at 37°C for 24 hrs. COV2B- S616 had improved recovery (89.32%) in 100% DMSO but was degraded in 10% DMSO in 25mM NaOH and 25% DMSO in 50mMNaHCO₃pH 11. COV2B-S821 was degraded in 50mMNaHCO₃ pH 11. However, COV2B-S616 recovery was increased to 90.95% in 10% DMSO in 0.125M ammonium bicarbonate with incubation at 24°C for 48 hrs with 100 RPM shaking. COV2B-S821 showed 76.62% recovery in 10% DMSO in 10 mM NaOH when incubated at 24°C with shaking at 100 RPM for the first 24 hrs and moved to 37 °C for the rest up to 65 hrs.

[00332] The immunogenicity of the four peptides was measured over 42 days, without and with the adjuvant Pam3CSK4 (Figure 8A and 8B, respectively; Table 18A and 18B, respectively). COV2B-S1157 was found to be poorly immunogenic, but COV2B-S369, COV2B-S616, and COV2B-S821 were immunogenic (Figure 8 A). The adjuvant Pam3CSK4 did not significantly increase the development of antibodies to any of the peptides in formulation 4 (Figure 8B). [00333] Table 18 A. Formulation 4 immunogenicity (without Pam3CSK4).

[00334] Table 18B . F ormulation 4 immunogenicity (with Pam3 C SK4).

[00335] Formulation 5 Results.

[00336] Table 19. Formulation 5 details

[00337] These peptides are located in separate regions of the S protein, which target the RBD and the fusion domain. COV2B-S1165 falls into a region in which a mAb was detected in SARS-CoV patient sera that blocked SARS-CoV infection in vitro (Wang et al. 2016.).

[00338] Table 20. Formulation 5 solubility.

[00339] Reduced recovery of COV2B-S524 was observed due to cysteine oxidation, however, a forced cysteine oxidation method has not yet been explored.

[00340] The immunogenicity of the three peptides was measured over 42 days. COV2B- S250 and COV2B-S524 are poorly immunogenic, but COV2B-S1165 is immunogenic and was determined to be a potential vaccine candidate (Figure 9 and Table 21).

[00341] Table 21. Formulation 5 immunogenicity.

[00342] Formulation 6 Results.

[00343] Table 22. Formulation 6 details

[00344] The peptide targets are located in separate regions of the S protein, two of which target the RBD for potential neutralizing antibody generation.

[00345] Table 23. Formulation 6 solubility.

[00346] Reduced recovery of COV2B-S373, COV2B-S431 and COV2B-S1252 peptides were observed due to cysteine oxidation. Recovery of COV2B-S373 and COV2B-S1252 peptides was improved by using 10% DMSO in 0.5% Acetic add as diluent. COV2B-S373 showed 97.77% recovery in 10% DMSO in 0.5% Acetic add when incubated at 24°C with shaking at 100 RPM for 48 hrs. However, use of the reducing agents tris (2-carboxy ethyl) phosphine hydrochloride (TCEP) and ethy lenedi aminetetraaceti c acid (EDTA) did not improve recovery of COV2B-S431. Also, noted the recovery of the other three peptides increased when COV2B-S431 was not added to the formulation. Hence, removed the COV2B-S431 peptide from formulation 6 peptide combination for the booster dose formulation.

[00347] The immunogenicity of the four peptides was measured over 42 days. COV2B-

S1252, COV2B-S431 and COV2B-S672 peptides were poorly immunogenic, but COV2B-S373 was found to be immunogenic (Figure 10, Table 24).

[00348] Table 24. Immunogenicity of Formulation 6.

[00349] ts.

[00350] Table 25. Formulation 7 details

[00351] These peptide targets are located in separate regions of S protein, one of which targets the RBD for potential neutralization antibody generation.

[00352] Table 26. Formulation 7 solubility

[00353] Reduced recovery of the COV2B-S329 peptide was observed due to cysteine oxidation. The diluent 10% DMSO in 0.5% Acetic acid was found to improve recovery (84.37%) with incubation at 37 °C 24 hrs.

[00354] The immunogenicity of the two peptides was measured over 42 days. The COV2B-

S634 peptide was poorly immunogenic but the COV2B-S329 peptide was strongly immunogenic (Figure 11, Table 27).

[00355] Table 27. Formulation 7 immunogenicity.

[00356] Formulation 8 Results.

[00357] Table 28. Formulation 8 details.

[00358] The peptide targets are located in separate regions of S protein; one of which targets

RBD for potential neutralizing antibody generation and another in HR2 which is involved in virus fusion with cell membrane. Both Abs are based on the regions in which a mAb was detected in SARS-CoV patient sera that blocked SARS-CoV infection in vitro (Wang et al. 2016).

[00359] Table 29. Formulation 8 solubility

[00360] Reduced recovery of COV2B-S1182 peptide was observed due to cysteine oxidation, however a forced cysteine oxidation has not yet been tested.

[00361] The immunogenicity of the two peptides was measured over 42 days. The COV2B-

S1182 peptide was poorly immunogenic but the COV2B-S486 peptide was immunogenic (Figure 12, Table 30).

[00362] Table 30. Formulation 8 immunogenicity.

[00363] Formulation 9 Results.

[00364] Table 31. Formulation 9 details.

[00365] These peptides are from newly published functional SARS-CoV-2 data from Poh et. al., 2020 who showed that COV2B-S553 and COV2B-S809 sequences are recognized by neutralizing Abs in the sera of COVID-19 convalescent patient.

[00366] Table 32. Formulation 9 solubility

[00367] The immunogenicity of the two peptides was measured over 42 days. The COV2B-

S553 peptide was poorly immunogenic but the COV2B-S809 peptide was immunogenic (Figure 13 Table 33).

[00368] Table 33. Formulation 9 immunogenicity.

[00369] Of the 25 candidate B cell peptide epitopes evaluated, nine peptides elicitec moderate to strong humoral responses. These peptides include COV2B-S461, COV2B-S369, COV2B-S821, COV2B-S1165, COV2B-S373, COV2B-S329, COV2B-S616, COV2B-S809, and COV2B-S486. In an attempt to gain insight into the relative immunogenicity of each peptide candidate, antibody titers were compared amongst all 9 peptides to that of the positive B cell epitope found in Formulation A, which was used as a positive control reference formulation (Figure 14). The results indicate that COV2B-S329 and COV2B-S369 were significantly more immunogenic than the positive control B cell epitope (p=0.048, p=0.02, SD42), whereas peptides COV2B-S821, COV2B-S616, COV2B-S373, COV2B-S1165, COV2B-S486, COV2B-S809, and COV2B-S461 were similarly immunogenic when compared to the positive control B cell epitope. Importantly, none of the selected peptides were significantly less immunogenic than the positive control B cell epitope peptide at SD21, 28 and 42. Previous work with Formulation A shows that positive control B cell epitope is able to generate a strong and sustained immune response in older adults that is comparable to that found in convalescent subjects (Langley et al. 2018). Therefore, if preclinical data translates to clinical observations, it is possible that any of the nine candidate peptides listed above can induce antibody responses in older adults. The nine peptides (COV2B- S329, COV2B-S369, COV2B-S373, COV2B-S461, COV2B-S486, COV2B-S616, COV2B-S821, COV2B-S809, and COV2B-S1165) that elicited humoral responses similar to or exceeding those achieved with the positive control, Formulation A showed the potency of the candidate epitope immunizations in mounting a peptide-specific antibody response (p<0.05 in comparison to time matched Formulation A titers by two-tailed impaired t-test). The specificity of the response was highlighted by the variability in antibody titers induced by different peptide epitopes within the same formulation (e.g., Formulation 4, Figure 8A), indicating that the observed responses are unlikely to be due to the actual water-free oil-based formulation or defined by interactions between peptides in the formulation, but rather may rely on immunogenic properties of each peptide separately. All peptides that demonstrated an immune response at SD14 showed improved titers on SD21 and SD28, after administration of the booster. The trend was most noticeable with peptides COV2B-S821, COV2B-S616, COV2B-S373, and COV2B-S1165.

[00370] To evaluate whether peptide-specific antibodies elicited by eight formulations (1- 8) can recognize and bind to the full SARS-COV-2 S protein, as well as its S1 or S2 subunits, Vax Array Coronavirus SeroAssay was performed on selected serum samples collected from mice exposed to each formulation over the course of the studies. Sera extracted from mice vaccinated with Formulation A served as the negative control and was used to confirm assay specificity. As expected, sera from mice vaccinated with Formulation A tested negative for binding to all coronaviruses including in the assay. Two pooled serum samples from mice vaccinated with Formulation 1 were tested at SD21 and SD28, and one pooled serum sample from mice vaccinated with Formulation 2 was tested at SD14. All three samples did not elicit peptide-specific immune responses as detected by indirect ELISA. As expected, all three samples failed to demonstrate any SARS-CoV-2 S-specific antibody binding capacity. Collectively, these data support the conclusion that Formulations 1 and 2 are poorly immunogenic.

[00371] Twelve selected serum samples from mice vaccinated with six immunogenic formulations (Formulations 3-8) were tested at SD42. The data suggests that five of the eight candidate epitopes identified in Formulations 3-8 as being immunogenic by indirect ELISA bind to the relevant subunit of the SARS-CoV-2 S protein (Table 34), with binding capacity roughly proportional to antibody titer. These peptides include COV2B-S461, COV2B-S616, COV2B- S821, COV2B-S373, COV2B-S329, and COV2B-S486. Binding ability of S616-specific antibodies to S1 subunit could not be tested due to assay limitation.

[00372] Candidates COV2B-S369 and COV2B-S1165, while strongly immunogenic, did not exhibit any propensity to bind to the relevant subunit of S protein. To confirm these findings, pooled serum samples from multiple mice vaccinated with either Formulations 4 or 5 were tested at earlier timepoint (SD21), with the identical results obtained, suggesting that COV2B-S369 and COV2B-S1165 peptides should not be included in the final formulation. Alternatively, higher titers of COV2B-S369- and COV2B-S1165-specific antibodies are necessary to demonstrate binding to the SARS-CoV-2 S protein.

[00373] As shown in Table 34 the binding capacities of antibodies in SeroAssay were restricted to the epitope-specific subunit of S protein with no binding detected to the alternative subunit (e.g., serum samples exposed to COV2B-S461, COV2B-S373, COV2B-S329 exhibit a propensity to bind to S1 subunit, which is consistent with the location of all 3 peptides in RBD of SARS-CoV-2 S1). As expected, all samples tested negative for binding capacity against MERS or any of the endemic coronavirus included in the assay (CoV HKU1, CoV OC43, CoV 229E, CoVNL63). This strongly suggests that Formulation X formulations elicit humoral responses that are species-specific and are restricted to the area of the epitope target on SARS-CoV-2 S protein.

[00374] Table 34. Peptide-specific antibodies can recognize and bind to the SARS-CoV-2 S protein.

[00375] Outbred CD-1 mice were vaccinated with Formulation X containing SARS-CoV- 2 S peptides on SDO and were boosted on SD14. On SD42, presence of peptide-specific antibodies was evaluated in serum samples by indirect ELISA. Capacity of the peptide-specific antibodies in each formulation to recognize and bind to the full SARS-CoV-2 S protein as well as to the subunits S1 and S2 was evaluated in VaxArray Coronavirus SeroAssay. Note: no immunogenic peptides were detected in formulations 1 and 2 (not shown). *binding ability of S616-specific antibodies to S1 subunit cannot be tested as sequence of the S1 subunit includes 319-541 aa only. The ability of the mouse sera collected from mice vaccinated with SARS-CoV-2 S peptides formulated in water- free oil-based compositions to inhibit vims entry into the cells that ACE2 receptor was evaluated in the pseudotype virus neutralization assay using HEK293T/ACE2 cells which stably express human ACE2 and pseudotyped GFP rSARs-CoV-2 Spike protein viruses. Serum samples collected from mice exposed to three formulations (Formulations 3, 4 and 6) were selected for this analysis: two serum samples collected on SD42 from mice vaccinated with Formulation 4 containing Pam3CSK4 adjuvant, from one mouse vaccinated with Formulation 3, from one mouse vaccinated with Formulation 6. One irrelevant control sample collected on SD42 from mice vaccinated with irrelevant peptide, Formulation A, was also included. All samples elicited strong peptide-specific immune responses as detected by indirect ELISA. Neutralization activity was found in antisera to Formulation 4 (containing immunogenic peptides COV2B-S616 /-S821/ -S369 and Pam3CSK4) and Formulation 6 (containing immunogenic peptide COV2B-S373). Anti-sera to Formulation 3 (containing immunogenic peptide COV2B-S461) neutralized <50% of the virus, however only one sample was examined (Figure 15).

[00376] To evaluate the impact of the Pam3CSK4 adjuvant on immune response elicited by the water-free formulation containing SARS-CoV-2 B cell epitopes, two formulations (Formulation 1 and Formulation 4) were evaluated with and without the addition of adjuvant (Pam3CSK4) to the formulation. The peptides in Formulation 1 were not found to induce antibody responses, and the inclusion of Pam3CSK4 in the formulation did not improve this response. Pam3CSK4 had no appreciable increase in antibody responses toward the 3 B cell epitopes in Formulation 4 that could induce antibody responses, and when tested in the pseudovirus neutralization assay the antigen-specific antibodies induced by Formulation 4+Pam3CSK4 demonstrated neutralization activity. For the fourth peptide (S1157) in formulation 4, antibody titers were below the limit of detection with and without Pam3CSK4. These data suggest that Pam3CSK4 cannot increase antibody responses to B cell epitopes that are not immunogenic, but does not reduce responses of immunogenic peptides.

[00377] Example 2 — Animal Testing of water-free formulation containing 4 immunogenic SARS-CoV-2 B cell epitopes (Confirmatory immunogenicity testing of Formulation X)

[00378] This Example focuses on one (1) homogeneous water-free formulation (DPX) containing 4 B cell epitopes from SARS-CoV-2 spike protein identified as immunogenic as described in the Example 1, this formulation is herein referred to as Formulation X. Ability of the multi-antigen containing Fonnulation X to elicit a humoral immune response was tested in CD1 mice.

[00379] Formulation X contained four peptides COV2B-S373, COV2B-S461, COV2B- S616, COV2B-S821. All four peptides contain cysteine residues and are capable of dimerization. To avoid uncontrolled dimerization during formulation, peptide dimers were prepared from monomers and then used to produce the test vaccine, Formulation X.

[00380] Due to global urgency to develop a SARS-CoV-2 vaccine, and while waiting for the synthesis of commercial dimer peptides, in-house-made dimers were prepared from the research grade monomer peptides and were used to test a non-clinical Formulation X in a first confirmatory study (Study 1). A second confirmatory study, Study 2, compared the immunogenicity of commercial dimers formulated in homogeneous water-free formulation at two different strengths 25 μg/dose and 10 μg/dose.

[00381] Table 35: Overview of Confirmatory Studies

[00382] Formulation A (positive control) is a homogeneous water-free formulation (DPX) containing SHeA antigen (Seq ID 41); and Formulation Z is a homogeneous water-free formulation (DPX) containing no peptides.

[00383] The selection of these four peptides was based on three major criteria: (1) the induction of antigen-specific immune responses, (2) the ability of the targets to span the primary areas of function within the SARS-CoV-2 spike protein, and (3) the ability of the peptides to be appropriately formulated together in the homogeneous water-free platform (DPX) for rapid vaccine development.

[00384] The objectives of these studies were: (1) to assess and confirm the immunogenicity of pre-clinical Formulation X; (2) to evaluate functionality of peptide-specific antibodies; and (3) to evaluate changes in peripheral cytokine profiles induced by treatment.

[00385] Mice (outbred CD-1, female) were vaccinated with Formulation X containing four peptides COV2B-S373D, COV2B-S461D, COV2B-S616D, and COV2B-S821D. Vaccines were administered through two intramuscular injections. Mice received a total dose volume of 50 μL split into equal portions delivered to the right and left caudal thigh. Mice were treated with the vaccine on study day 0 and 14 and blood was collected at study day 14, 21, 28 , 42/43, 56 (study 2 only), 84, 112, and 140 for immunogenicity assessment At day 21 (Study 1) or 43 (Study 2), 3 animals per group were sacrificed and sera analyzed for cytokine levels and functional activity. Mouse health status and site of injection reactions in response to the treatment with Formulation X were monitored throughout the study.

[00386] Materials

[00387] The formulations administered to the treatment groups were based on the homogeneous water-free platform (DPX) platform technologies discussed herein. Specific formulations are as follows.

Table 36. Vaccine Formulations

[00388] The following B cell epitopes were tested.

Table 37. Epitope Sequences in Formulation X and Formulation A

[00389] Injection sites on the mice were swabbed with alcohol prior to injection. Immunization start time and end time was noted. Animals were anesthetized by isoflurane (2-4%, lL/min O2) for vaccine treatment on Study Day (SD) 0 and 14. Each mouse received a vaccine injection (I.M.: intramuscular); 25 μL dose in both caudal thigh muscles (50 μL total dose).

[00390] Peptide-specific antibody titers in serum were determined by indirect enzyme- linked immunosorbent assays (ELISA). Indirect ELISA was performed to detect serum antigen specific antibody titers. Briefly, 96-well EIA/RIA Clear Flat Bottom Polystyrene High Bind Microplates (Corning®) were coated with 1 μg/mL of individual peptides in coating buffer (NaHCO₃, Na₂CO₃) overnight at 4°C. Plates were washed five times with TBS-T (0.2%) and blocked with warmed 3% Gelatin (BioRad) for 30-45 minutes at 37 °C. Plates were washed with TBS-T and incubated overnight at 4°C with sera at an initial starting dilution of 1:100. After TBS- T washes, bound antibodies were detected by incubation of alkaline phosphatase conjugated Protein G (EMD Millipore) with high affinity binding for IgG for 1 hour at 37°C and subsequent development with chromogenic alkaline phosphatase substrate. Optical density was measured at 405 nm within 1 hour of initial substrate addition on a plate reader.

[00391] Indirect enzyme-linked immunosorbent assays (ELISA) were performed to detect serum SARS-CoV-2 S-protein-specific antibody titers. Briefly, 96-well EIA/RIA Clear Flat- Bottom Polystyrene High Bind Microplates (Corning®) were coated with 1 μg/mL of S protein (S1+S2 ECD, His tag, Sino Biologicals) diluted in manufacturer’s recommended coating buffer (136.9 mM NaCl, 10.1 mM Na₂HPO₄, 2.7 mM KC1, 1.8 mM KH₂PO₄, pH 7.4 and incubated overnight at 4°C. Plates were washed five times with TBS-T (0.2%) and blocked with warmed 3% Gelatin (BioRad) for 30-45 minutes at 37°C. Plates were washed with TBS-T and incubated overnight at 4°C with sera at an initial starting dilution of 1:100. After TBS-T washes, bound antibodies were detected by incubation of alkaline phosphatase conjugated Protein G (EMD Millipore-Sigma) with high affinity binding for IgG for 1 hour at 37°C and subsequent development with chromogenic alkaline phosphatase substrate. Optical density was measured at 405 nm within 1 hour of initial substrate addition on a plate reader. ELISA results were expressed as end point Log10 titers using a calculation method described by Frey et al. (Frey et al. 1998).

[00392] ELISA results were expressed as end point log (10) titers, which was defined as the reciprocal of the highest dilution that gives a positive reaction. To determine whether a reaction is positive or negative, an absorbance cutoff value was defined. Readings above the cutoff were considered positive while readings at or below the cutoff were negative. (Frey et al, 1998). Naive group sera were used to establish the baseline cut-off.

[00393] Each serum sample was diluted at 1:100 as a starting dilution on SD14 and further diluted to 7 additional dilutions at 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400 and 1:12800. Starting dilutions were then determined based on prior titers to ensure the endpoint titers falls within prepared dilutions.

[00394] The ability of the different formulations to elicit immune responses were determined based on increase in average log(10) endpoint titers. Average group responses were assessed for immunogenicity compared to background response for each group. In some cases, the endpoint titers exceeded the highest prepared dilution on the ELISA plate. The highest prepared dilution was then used to determine the corresponding titer and used for further downstream analysis. Since serum availability was limited, no repeats were performed to determine end-point titers for these samples. An immunogenic response definition of a peptide-specific antibody titer ≥Log₁₀=2 was used to determine the number of responders within each group at each time point.

[00395] Functional binding capacity of the antigen-specific antibodies was determined using the VaxArray Coronavirus SeroAssay (InDevR Inc, Boulder, Colorado), a multiplexed immunoassay for detection of antibodies against SARS-CoV-2, SARS-CoV, MERS, as well as the endemic coronaviruses CoV HKU1, CoV OC43, CoV 229E, and CoV NL63 in serum. The assay allows for detection of antibody binding to the full SARS-CoV-2 Spike protein as well as to the S1 and S2 subunits.

[00396] Serum samples were collected and stored at -20°C, and shipped to InDevR Inc on dry ice with temperature monitoring. Analysis was performed by InDevR Inc according to the kit instructions. Briefly, for analysis samples were serially diluted two-fold, starting at a 100-fold dilution. Standard VaxArray assay protocols were utilized for all testing. The serum samples were diluted in Protein Blocking Buffer and incubated on the VaxArray slide for 1 hour at 80 rpm. Wash Buffer 1 was applied to the slide, after the samples were removed, and then the microarrays were incubated with an anti-mouse IgG label for 30 minutes at 80 rpm. Label was removed and the slides were washed, dried, and imaged. VaxArray Coronavirus SeroAssay signals were reported as Signal/Background (S/B) ratio and relative fluorescent units (RFU). The maximum resolvable signal on the VaxArray Imaging System is 65,535 RFU. Endpoint titer values were defined as the highest dilution factor with S/B >1.5.

[00397] Cytokine analysis was performed externally by Eve Technologies (Calgary, AB) and was primarily focused on assessing biomarkers of T cell response (Th1/Th2) and inflammation, including: GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12 (p70), IL-13, IL-17A, KC/CXCL1, LIX, MCP-1, MIP-2, and TNF-α. Samples were analyzed in duplicate by multiplex cytokine array (Mouse High Sensitivity 18-Plex Discovery Assay).

[00398] Results.

[00399] Both Study 1 and Study 2 confirmatory studies allowed the evaluation of the immunogenicity of Formulation X for a period of 20 weeks after prime vaccination and presented in Figure 16 and 17 and Table 38 and Figure 17 and Table 39, respectively).

[00400] The magnitude of antigen-specific humoral responses to Formulation X antigens following vaccinations were similar in both confirmatory studies, suggesting that use of in-house or GMP manufactured peptide dimers to formulate Formulation X had no strong effect on immunogenic property of Formulation X vaccine (see Figures 16A and 17A). In both studies, peptide-specific antibody responses were detected as early as SDH (two weeks after prime vaccinations, and prior to boosting), with further increase in antibody titers and number of responding animals one or two weeks after boost vaccination at SD21 or SD28 (peptides COV2B- S373, COV2B-S461 and COV2B-S821, one-way ANOVA with Tukey’s post hoc). Slower kinetics of immune response was observed in response to COV2B-S616, with significant increase in antibody titers detected four weeks after boost vaccination, at SD42. Study 2 evaluates two strengths of Formulation X, 10 μg/dose and 25 μg/dose (Figure 17A). A significant difference in the magnitude of the response to two doses of COV2B-S461 were observed at earlier time points (SDH, SD21 and SD28, t-test p<0.05), with both doses elicited similar responses after SD28. Additionally, significant difference in the magnitude of the response to two doses of COV2B-S373 was observed at SD56. No statistical differences were detected in response to two strengths of COV2B-S616 and COV2B-S821 at any time point examined.

[00401] Importantly, strong peptide-specific IgG antibody responses to all four peptides were elicited in both studies by SD42/SD43 in nearly all vaccinated mice, regardless of the dose of Formulation X (Study 2; 10 μg/dose and 25 μg/dose; see Table 38 and Table 39). Sera from mice vaccinated with Formulation Z were assessed for production of COV2B-S373-, COV2B- S461-, COV2B-S616- and COV2B-S821 -specific antibodies. As shown in Figures 16B and 17B, minimal background antibody responses were detected in mice vaccinated with Formulation Z. This indicated that peptides formulated in homogeneous water-free formulation (DPX) are key components to generation of antigen-specific immune responses.

[00402] Immune responses were maintained up to at least SD140. There were no significant decreases in peptide based immune responses detected, except for the response to peptide COV2B- S373 on SD112 (when compared to the immune response at SD56) in mice vaccinated with Formulation X, 25μg/dose. This data demonstrated the ability of this vaccine formulation to induce robust and sustained immune responses in mice. The Formulation X tested here may not have a ‘typical’ vaccine induced immune response with a distinct maximal immune response peak after a bolus vaccine injection and a decline to a protective level, rather the formulation has an immune response that is a sustained immune response induced over time and correlated to the unique way in which the water-free platform presents vaccine components directly to regional lymphoid organs. [00403] Table 38. Endpoint titer ranges and total number of responders in CD-1 mice vaccinated with Formulation X at 25 μg dose strength prepared using in house dimers (Study 1).

* Tabulated Logic endpoint antibody titers (min-max) and number of responders for each peptide. An immunogenic response definition of a peptide-specific antibody titer ≥Log₁₀(100)=2 was used to determine the number of responders within each group at each timepoint.

[00404] Table 39. Endpoint titer ranges and total number of responders in CD-1 mice vaccinated with Formulation X at 25 μg dose strength prepared using commercial dimers (Study

2)·

[00405] * Tabulated Log₁₀ endpoint antibody titers (min-max) and number of responders for each peptide. An immunogenic response definition of a peptide-specific antibody titer ≥Log₁₀(100)=2 was used to determine the number of responders within each group at each timepoint. Note: 3 mice per group were terminated on SD43 for an additional analysis of serum samples.

[00406] The binding ability of antibodies elicited by Formulation X vaccine to the SARS CoV-2 spike protein was evaluated in the Vax Array Coronavirus SeroAssay. Selected serum samples collected from mice on SD21 or SD28 were tested using the VaxArray Coronavirus SeroAssay to evaluate whether peptide-specific antibodies elicited by Formulation X can recognize and bind to the full SARS-CoV-2 S protein, as well as its S1 or S2 subunits. This assay is capable of detecting binding of COV2B-S373- and COV2B-S461-specific antibodies to S1 subunit and to the full spike, as well as binding of COV2B-S821 -specific antibodies to the S2 subunit and to the full spike. Binding of COV2B-S616-specific antibodies to the S1 subunit was difficult to test because of assay limitations. The construct used to assess S1 binding encompassed amino acids 319-514 and S2 binding amino acids 686-1213. Thus, any spike binding antibodies directed to peptide COV2B-S616 (an epitope originating at amino acid 616) would only be demonstrated in binding the complete spike molecule. In addition to the detection of SARS-CoV- 2 S antibodies, this assay detected antibodies to SARS-CoV S protein, MERS S protein, as well as the endemic coronaviruses CoV HKU1, CoV OC43, CoV 229E, CoVNL63.

[00407] Five serum samples from mice vaccinated with Formulation X were tested at SD21 or at SD28 as shown in Table 40. Binding to either full S, S1 or S2 subunit of spike protein was detected in all 5 tested samples. As expected, all samples tested negative for binding capacity against MERS or any of the endemic coronavirus included in the assay (CoV HKU1, CoV OC43, CoV 229E, CoV NL63). Interestingly, the propensity to bind to SARS-CoV S was detected in four out of 5 tested samples. At least one peptide in Formulation X (COV2B-S821) has a high degree of similarity to the known functional sequence of SARS-CoV S, with the SARS-CoV-2 sequence different from SARS-CoV by 2 amino acids. The homologous SARS-CoV peptide (Leu803- A1a828) was able to induce the antisera with binding ability to the native S protein and neutralizing activity to the SARS-CoV pseudovirus (Zhang et al., 2004). [00408] Additionally, binding of Formulation X anti-sera to the full spike protein was also evaluated in indirect ELISA (referred to as S-ELISA) with plates coated using commercially available SARS CoV-2 spike protein (Table 40). Consistently with the results of VaxArray, anti- spike IgG antibodies were detected in all five tested samples with both assays generally show similar trends in binding affinities.

[00409] Table 40. Peptide-specific antibodies can recognize and bind to the SARS-CoV-2 S protein by VaxArray and indirect ELISA. (Study 1)

Ό0410] VaxArray titers: Data was acquired using the 21CFR Part 11 compliant VaxArray Software v2.1.118.0. Data was analyzed for signal response of serial dilutions of the sample. Samples were serially diluted two-fold starting at a 100-fold dilution. Vax array signals were reported as Signal/Background (S/B) based on relative fluorescent units (RFU). The maximum resolvable signal on the VaxArray Imaging System is 65535 RFU. Vax array end point titer values were defined as the highest dilution factor for which S/B > 1.5. Table provides Log₁₀ end point titer values for the samples where S/B > 1.5. Samples where S/B was below 1.5 at the starting 100-fold dilution were marked as <2.00 (Log₁₀(100)=2). Peptide and spike titers: Log₁₀ peptide- specific and S-specific antibody titers determined by indirect ELISA in the same serum samples are also shown. ELISA titers values were defined as the inverse of the greatest dilution above the assay cutoff determined using a calculation method described by Frey et al. (Frey et al. 1998). [00411] The relationship amongst the peptide-specific antibodies induced by four Formulation X epitopes as well as the relationship between these antibodies and the spike-binding capacity of anti-sera were evaluated in seventeen samples collected from mice vaccinated with Formulation X at 25 μg at five different time points (SD21- SD140) (Tables 41 and 42). Strong positive correlations were detected amongst all four peptide-specific antibodies (r=0.8, p<0.001) (Table 41), suggesting that immune responses induced by one of the peptides in the formulation does not hinder immune responses induced by other peptides in Formulation X and therefore supporting the approach of formulating four peptides together. Positive correlations were also observed between titers induced by each of four Formulation X peptides and SARS-CoV-2 spike- binding IgG antibody titers. This observation is consistent with the results obtained in VaxArray showing that antibody induced by each of four Formulation X peptides can recognize and bind to the full spike protein or its subunits. It is noteworthy, that amongst four Formulation X antibodies the strongest correlation with anti-spike titers were observed for anti-CO V2B-S461 (R=0.7; p=0.002). The COV2B-S461 sequence is located within RBD of S protein and covers amino acids from the proposed ACE2 direct binding site of the RBD, and as such COV2B-S461-specific antibodies may compete with the ACE2 receptor for binding to the virus, which could be one of the most potent mechanisms of viral neutralization.

[00412] Table 41. Positive correlations amongst Formulation X induced epitope-specific antibody titers and SARS CoV-2 binding antibody titers in sera samples of mice vaccinated with Formulation X (Study 1).

[00413] correlation is significant at the 0.01 level; * correlation is significant at the 0.05 level (2-tailed).

[00414] Table 42. Peptide-specific and S-specific antibody titers determined by indirect ELISA in serum samples collected from mice vaccinated with Formulation X (Study 1).

[00415] Data in Table 42 were used to evaluate correlations presented in Table 41. Table 42 presents Log₁₀ peptide-specific and S-specific antibody titers detected by indirect ELISA. ELISA titer values were defined as the inverse of the greatest dilution above the assay cutoff determined using a calculation method described by Frey et al. (Frey et al. 1998). Samples with titers below or at the assay cut off at starting 100-fold dilution were marked as <2.00 (Log₁₀100=2.00). [00416] In summary, results of functional analyses indicated that peptide-specific antibodies induced by Formulation X can recognize and bind to the SARS-CoV-2 spike protein.

[00417] To evaluate whether Formulation X vaccines could induce systemic inflammatory immune responses, cytokine and chemokine levels were assessed in the mouse sera at two timepoints: 21 days post prime vaccination (Study 1) and 43 days post prime vaccination (Study 2). Peripheral cytokine levels in mice vaccinated with Formulation X were compared to those in mice vaccinated with Formulation A, Formulation Z, or to those in naive mice that did not receive any treatment. Cytokine analysis was performed independently by a multiplex array (Eve Technologies, Calgary AB) and was primarily focused on assessing biomarkers of T cell response (Th1/Th2) and inflammation, including: GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL- 7, IL-10, IL-12 (p70), IL-13, IL-17A, KC/CXCL1, LIX, MCP-1, MIP-2, TNF-α.

[00418] There were no statistical differences in the peripheral levels of any of 18 cytokines/ chemokines evaluated among the different treatment groups at SD21 (Study 1). There were no statistical differences in the peripheral levels of 17 of 18 cytokines/chemokines evaluated among the different treatment groups at SD43 (Study 2). A slight increase in IL-6 level was detected in two of three examined animals in group vaccinated with Formulation X administered at 10 μg of each peptide per dose at SD43 (Study 2). This increase was not observed in animals vaccinated with higher dose (25 μg of each peptide) of Formulation X and therefore unlikely linked to the peripheral effect of Formulation X. These results (Figures 18 and 19), suggest that vaccination with Formulation X had no effect on the levels of systemic cytokines. This finding was consistent with other non-clinical studies with the homogenous water-free formulation (Brewer et al, 2018) that indicated that compared to other similar depot-based formulation, inflammation was relatively limited, and that in response to vaccination, only a small subset of CD4⁺ T cells that produce IFN- γ were found in the blood of vaccinated older adults. Without wishing to be bound by theory, it is suggested that Formulation X is unlikely to generate unwanted inflammatory immune responses after vaccination.

[00419] As shown herein, Formulation X can generate robust and durable antibody responses. These antibodies are capable of binding SARS-CoV-2 spike protein and/or its subunits. Formulation X does not induce a systemic cytokine response. [00420] Discussion.

[00421] Of the twenty-five B-cell peptides assessed (Figure 20), nine peptides were able to generate an immune response that was comparable to that elicited by the positive control B cell epitope in Formulation A. As shown herein, the nine down-selected candidates spanned different functional areas of the SARS-CoV-2 spike protein and have potential to induce antibodies that interfere with the infection at multiple steps. It was also found that five of the nine candidate epitopes bound to the relevant subunit of the SARS-CoV-2 S protein. The final selection of the four peptides for the formulation in Formulation X was guided by peptide compatibility, scalability, and stability. In addition, preference was given to the peptide candidates that comprise different S areas and induce antibodies with potentially different mechanisms of targeting the S protein. Indeed, peptides COV2B-S373 and COV2B-S461 are located in different portions of the receptor binding domain, COV2B-S616 is located on the S 1 subunit in close proximity to the RBD, and COV2B-S821 targets the fusion peptide. Based on 3D modelling of four epitope sequences on the surface of the S protein, COV2B-S373, COV2B-S461, COV2B-S616, and COV2B-S821 are sterically separated and antibodies that recognize these sites may not compete for the binding.

[00422] Based on the location of the COV2B-S373 sequence within the RBD, antibodies elicited in response to vaccination with COV2B-S373 are thought to be unlikely to compete directly with ACE2 for binding to the virus but may indirectly hinder its attachment to the RBD. A similar mechanism was described for human monoclonal antibody 47D11 that neutralizes both SARS-CoV and SARS-CoV-2.

[00423] The COV2B-S461 sequence covers amino acids from the predicted ACE2 direct binding site of the RBD, and as such, COV2B-S461-specific antibodies may compete with the ACE2 receptor for binding to the vims which is generally one of the most potent mechanisms of viral neutralization. A recent serology analysis of 149 COVID-19 patients showed that only 1% of those tested had high titers of neutralizing antibodies, but that RBD-specific antibodies with potent antiviral activity were identified in all individuals, suggesting that humans are intrinsically capable of generating anti-RBD antibodies that potently neutralize SARS-CoV-2. Both the COV2B-S373 and COV2B-S461 peptides are located within the RBD region and were strongly immunogenic, which corroborates the inventors’ peptide-based approach to developing an effective vaccine against SARS-CoV-2.

[00424] Antibodies induced by COV2B-S616 may sterically hinder virion binding to the ACE2 receptor, or they may have allosteric effects on ACE binding, thereby inhibiting S protein binding and viral entry. It has been shown that a SARS-CoV peptide similar to COV2B-S616 elicited antibody responses capable of inhibiting SARS infection in non-human primates.

[00425] The sequence of peptide COV2B-S821 was based on a known functional SARS- CoV peptide, with the SARS-CoV-2 sequence differing from SARS-CoV by two amino acids. The homologous SARS peptide (Leu803-A1a828) was able to induce the antisera with binding ability to the native S protein and neutralizing activity to the SARS-CoV pseudovirus. In the experiments shown herein, the SARS-CoV-2 equivalent peptide COV2B-S821 demonstrated a strong immune response, and antibodies directed to this region may interfere with the fusion between the cell membrane and viral particle, thereby preventing viral entry. High homology between SARS-CoV- 2 S821 and SARS-COV (Leu803-A1a828) sequences may explain cross-reactivity of Formulation X anti-sera with the SARS-CoV spike protein detected in VaxArray Coronavirus SeroAssay.

[00426] It was shown that the Formulation X elicits peptide-specific antibodies to all four peptides in formulation, with the ability of antisera to recognize and bind to the full S protein. This multi-epitope approach allows for the generation of targeted immune responses to pre-selected highly immunogenic peptides without the need of whole-protein or whole-virus vaccination. This approach also leverages the benefits of synthetic peptide manufacture: it is rapid, highly scalable, and is without a biologic intermediate thereby eliminating the risk of pathogen transmission.

[00427] The use of the homogeneous water-free formulation, referred to herein as DPX or DPX™, has again proven effective in the delivery of antigenic peptides designed to induce targeted, robust, and sustained immune protection. Further, immunogenicity was achieved using small linear peptides without conjugation to an immunogenic carrier such as Keyhole Limpet Hemocyanin or bovine serum albumin. This immunogenicity speaks to the efficacy of the DPX™ platform as a true depot at the site of immunization, with encapsulation of all vaccine components such that antigens are not released from vaccine at the injection site. The inventors’ previous work has shown that DPX can also be formulated with small molecule adjuvants, such as TLR agonists, which can be used to enhance and shape the immune response, but the data presented here demonstrates that it is not specifically required in all cases to generate targeted immune responses. The DPX platform is versatile and it has been formulated with a variety of different antigens and adjuvants.

[00428] Importantly, all the peptides selected for further development provoked antibody titers similar to those achieved following vaccination with Formulation A (positive control), which contains an immunogenic B cell epitope and has been tested in human clinical trials, highlighting their potency in eliciting a peptide-specific antibody response. Further, antibody titers increased following administration of the second dose on Day 14 and were maintained for the remaining duration of the study. This is consistent with the results from the First-in-Human study of Formulation A), which demonstrated a robust response to both the initial and the booster dose, and antibody titers continuing to rise between Day 28 and Day 56 post-vaccination. Without wishing to be bound by theory, it is possible that this response may be due to sustained antigen retention at the injection site; MRI imaging has previously shown the injected peptides could be found in the site of injection and draining lymph nodes for months after administration.

[00429] The ability to induce robust and sustained immune responses in older adults and those with co-morbidities such as cancer is highly relevant to vaccine development for COVTD- 19. The case fatality ratio has been reported as much higher in adults over 60 years of age, and long-term care facilities have been a major source of outbreaks worldwide. The development of vaccines for this population has been challenging due to the immunosenescence that accompanies the aging process. For this reason, vaccine technologies that have demonstrated the ability to generate immune responses in this population may be required and may be an important part of the vaccine landscape being developed.

[00430] Not only were robust and reproducible immune responses achieved following vaccination with Formulation X and the formulations used in the preliminary evaluation, but there was evidence that the corresponding antibodies were specific to the epitopes presented. This is supported by the lack of any meaningful antibody response to Formulation Z (vehicle only) and the lack of SARS-CoV-2 spike binding capacity observed in mice vaccinated with Formulation A (irrelevant control). Without wishing to be bound by theory, while no obvious associations were observed between antibody titers detected by indirect ELISA and binding capacity to either full S, S1, or S2 subunits, it is hypothesized that this is likely a reflection of domination of different clonotypes with different affinities to the spike protein within the highly polyclonal antibody populations induced by the four strong antigens in the Formulation X.

[00431] A fundamental aspect of vaccine development is reactogenicity and safety of vaccines. While it is expected that some inflammatory response will occur consequent to a successful immune response towards SARS-CoV-2, the release of pro- and anti-inflammatory cytokines must be in a controlled state to prevent overexertion. It is generally known that the loss of this local control leads to systemic inflammation and potential deleterious consequences. Based on the cytokine analysis performed at SD21, vaccination with Formulation X did not induce a pattern of prolonged production of systemic cytokines, consistent with both the unique mechanism of action of DPX and the inventors’ previous analysis examining the cell types systemically induced in response to Formulation A vaccination.

[00432] There have been safety concerns about development of a SARS-CoV vaccine, given the observation of antibody-dependent enhancement (ADE) of SARS-CoV infection in vitro and in non-human primates that could, in theory, exacerbate disease symptoms. While this has not been observed to date with SARS-CoV-2, it remains a theoretical concern. An advantage of the present approach is the ability to avoid any implicated peptide sequences now or in the future, including the area of the S protein identified to mediate ADE in the SARS-CoV study. Further, inducing targeted immune responses as achieved using the DPX platform enables damaging epitope sequences to be identified in a timely manner, and before clinical testing is initiated.

[00433] Studies have shown the presence of antibodies generated against the nucleocapsid protein in patients infected with SARS-CoV, albeit not sustained in the long term. However, the versatility of both the approach and the platform described herein could allow for additional study to evaluate the inclusion of additional targets, should evidence of their immune functionality emerge. The approach could also facilitate the addition of targets specifically to induce T cell responses, or allow for addition of adjuvants if required. [00434] As a single-stranded RNA virus, the mutation rate in the SARS-CoV-2 genome has been estimated to be 0.80-2.38 × 10^-3 nucleotide substitutions per site per year which is a similar order of magnitude as other RNA viruses. This mutation rate does present the potential for spontaneous mutations in the region of the SARS-CoV-2 genome encoding the target peptides to enable immunologic escape. For example, the emergence of the D614G mutation to become the most prevalent form in the global pandemic has been attributed to increased infectivity compared with the wild-type, although some have disputed this. To address this, the presence of known mutations at the time of the study (March 24, 2020, GSAID Database) was evaluated at a threshold of two known SARS-CoV-2 sequences with the same mutation pattern and some peptides were rejected due to presence of genetic variants in the population. All considered epitopes continue to be monitored in real-time for future development. As such, judicious choice of target peptides may actually enable immunologic resilience to genetic drift.

[00435] The experiments presented herein have demonstrated proof of principle for exploiting an immunoinformatic strategy to rapidly identify, downselect, and synthesize potential peptide epitopes for formulation in water-free formulations and preclinical evaluation.

[00436] Example 3 - Phase 1 Human Vaccine Trial

[00437] Based on the ability of the water-free platform (DPX) to serve as an adjuvant in clinical studies of Respiratory Syncytial Vims (RSV) and other antigens, and the urgency of responding to the current global COVID-19 pandemic, this COVID-19 investigational vaccine will enter phase I testing.

[00438] This is a phase 1, First-in-Humans, randomized, observer-blind, placebo- controlled, dose ranging, multi-arm parallel-group clinical trial in healthy persons 18 to 55 and ≥56 years of age to assess the safety and immunogenicity of two dose levels of a novel vaccine formulation, DPX-COVID-19, consisting of a synthetic antigen based on B and T cell epitopes derived from the spike protein of SARS-CoV-2 formulated into the water-free platform DPX, in one or two doses, compared to a saline placebo control.

[00439] The study is randomized, placebo-controlled, and observer-blinded in order that allocation is concealed from the investigative team and the participant. [00440] The inclusion of a placebo comparator group allows for estimation of the attributable risk of adverse events. Since the study vaccines are not identical in appearance, an unblinded study nurse who has no other role in the study will administer the study vaccines.

[00441] Only adults in stable health will be eligible in order to minimize participant risk. To be eligible, females of childbearing age must be non-pregnant. Study holding rules and a safety evaluation by an independent Safety Review Committee (SRC) will be in place. The study will follow a staggered dose-escalation design for two dosage levels (Step 1 and Step 2). Step 1 will consist of four study groups (DPX-COVID-19 25 μg on a 2-dose schedule and placebo on a 2- dose schedule in two separate age groups) with dose administration on Day (D)0 and D56. At the beginning of Step 1, three participants at one study site will be randomized (2:1; Groups A and B) to the DPX-COVID-19 vaccine or placebo, a minimum of one hour apart. Once three participants have received treatment there will be a 72-hour waiting period, and if no holding rule is met then the remaining participants from Group A and B will be vaccinated, a minimum of 30 minutes apart.

[00442] An interim analysis of accumulating safety data will be conducted by the SRC of Day 0 to 6 post-immunization data in at least 75% of participants of Step 1 (Groups A and B), and any other safety data that is available. The timing of this SRC meeting is therefore dependent on enrollment, data collection, and analysis. At this meeting, the SRC will decide if: a) dose 2 can be given at Day 56 to 18- to 55-year-old adults in Step 1 (Study Groups A and

B); b) enrollment of 18- to 55-year-old adults in Step 2 can begin (Study Groups E, F, G, dose 1). [00443] A second SRC meeting will occur when at least 75% of participants in Groups A and B have completed their D28 visit. At this meeting the SRC will decide if enrollment of ≥ 56- year-old adults can begin in Step 1 (Groups C and D, dose 1).

[00444] Step 2 has six study groups (DPX-COVID-19 50 μg in a 2-dose schedule, DPX- COVID-19 50 μg one dose followed by a placebo dose, and placebo in a 2-dose schedule in two separate age groups) with dose administration on DO and D56. A maximum of 5 participants 18 to 55 years old will receive an intramuscular injection of a study vaccine or placebo (randomized 2:2: 1), a minimum of one hour apart, at one study site. After a 72-hour waiting period and provided no holding rule is met, subsequent participants in Groups E, F, and G will receive vaccine or placebo sequentially, a minimum of 30 minutes apart.

[00445] Interim analysis of accumulating safety data will be conducted by the SRC to determine if: a) dose 2 of study vaccine in Step 1 can be given to ≥ 56-year-old age group (Study Groups C, D); b) dose 2 of study vaccine in Step 2 can be given at D56 to the 18- to 55-year-old age group (Study Groups E, F, G); c) enrollment of ≥ 56-year-old adults can begin in Step 2 (Study Groups H, I, J, dose 1).

[00446] This analysis for items a and b above will summarize Day 0 to 6 post-immunization data in at least 75% of participants of the preceding dose groups, and any other safety data that is available. For item c, data will include completion of the D28 visit for 75% of participants in Groups E, F, and G. The SRC will also meet to review Day 0 to 7 safety data of the ≥ 56-year-old adults, to determine if the second dose of study vaccine can be given at D56 (Study Groups H, I,

J).

[00447] The primary objective of this Phase 1 clinical trial is to evaluate the safety and reactogenicity of the intramuscular DPX-COVID-19 vaccine at various doses up to 28 days after first injection. The safety and reactogenicity is evaluated by:

[00448] (i) Solicited adverse events (AE) within 0-6 days after each vaccination;

[00449] (ii) Unsolicited AE within 0-28 days after 1^st vaccination;

[00450] (iii) Serious adverse events (SAE), medically attended adverse events (MAE), and adverse events of special interest (AES1) in all groups throughout the study;

[00451] Secondary objectives for this trial include:

[00452] (i) To evaluate the solicited (within 0-6 days) and unsolicited AE (0 to 28 days) of a second dose of DPX-COVID-19 up to 28 days after vaccination on D56. [00453] (ii) To evaluate the immunogenicity and persistence of the immune response after one or two doses of DPX-COVID-19 as measured by antibody directed to the spike antigen at Days 0, 7, 28, 56, 63, 84, 236 and 421.

[00454] Exploratory objectives for this trial include:

[00455] (i) To explore the ability of antigen-specific immune responses to neutralize virus

[00456] (ii) To describe the cell-mediated immune responses to study vaccine

[00457] (iii) To explore the immunogenicity and cell mediated immune response at the optional blood collection timepoints (Days 3, 14, 21).

[00458] The study will be a Phase 1, randomized, placebo controlled, observer-blind, multicenter, study with dose escalation design in two steps. The study will last approximately 15 months. There will be 84 participants divided into groups as follows:

[00459] Step 1 : 25 μg of each SARS-CoV-2 spike protein antigen (low dose DPX-COVID- 19 vaccine) or placebo, n = 24.

[00460] Group A. DPX-COVID-19 (low dose): 8 healthy adults 18 to 55 years of age receive the low dose of DPX-COVID-19 vaccine on Day 0, followed by a second low dose of DPX-COVID-19 vaccine on Day 56.

[00461] Group B. Placebo control: 4 healthy adults 18 to 55 years of age receive a dose of normal saline (placebo) on Day 0 followed by a dose of normal saline (placebo) on Day 56.

[00462] Group C. DPX-COVID-19 (low dose): 8 healthy adults ≥ 56 years of age receive a low dose of DPX-COVID-19 vaccine on Day 0, followed by a second low dose of DPX-COVID- 19 vaccine on Day 56.

[00463] Group D. Placebo control. 4 healthy adults ≥ 56 years of age receive a dose of normal saline (placebo) on Day 0 followed by a dose of saline placebo on Day 56.

[00464] Step 2: 50 μg of each SARS-CoV-2 spike protein antigen (high dose of DPX- COVID-19) or placebo, n=60

[00465] Group E. DPX-COVID-19 (high dose): 12 healthy adults 18 to 55 years of age receive a high dose of DPX-COVID- 19 vaccine, followed by a second high dose of DPX-COVID- 19 vaccine on Day 56. [00466] Group F. DPX-COVID-19 (high dose): 12 healthy adults 18 to 55 years of age receive the high dose of DPX-COVID-19 vaccine, followed by a dose of normal saline (placebo) on Day 56.

[00467] Group G. Placebo control: 6 healthy adults 18 to 55 years of age receive a single dose of normal saline (placebo) on Day 0, followed by a dose of normal saline on Day 56. [00468] Group H. DPX-COVID-19 (high dose): 12 healthy adults ≥ 56 years of age receive the high dose of DPX-COVID-19 vaccine, followed by a second high dose of DPX- COVID-19 vaccine on Day 56.

[00469] Group L DPX-COVID-19 (high dose): 12 healthy adults ≥ 56 years of age receive the high dose of DPX-COVID-19 vaccine, followed by a dose of normal saline (placebo) on Day

56.

[00470] Group J. Placebo control: 6 healthy adults ≥ 56 years of age receive a single dose of normal saline (placebo) on Day 0, followed by a dose of normal saline on Day 56.

[00471] To be eligible for the study, each participant must satisfy ALL of the following criteria: (i) Age 18 years or older, (ii) Good general health status, as determined by history and physical examination no greater than 30 days prior to administration of the test article, (iii) Body Mass Index (BMI) 18.0 to 35.0, inclusive (iv) Participants who, in the opinion of the investigator, can and will comply with the requirements of the protocol (e.g. completion of Diary Cards, return for follow-up visits), (v) Written informed consent obtained from the participant, (vi) If female of child-bearing potential and heterosexually active, has practiced adequate contraception for 30 days prior to injection, has a negative pregnancy test on the day of injection, and has agreed to continue adequate contraception until 180 days after injection.

[00472] Participants with any of the following criteria will be excluded: (i) Prior laboratory confirmed infection with SARS-CoV-2 as reported by participant, (ii) Use of any investigational or non-registered product (drug or vaccine) other than the study product within 30 days preceding the dose of study product or planned use during the study period, (iii) Concurrently participating in another clinical study, at any time during the study period, in which the participant has been or will be exposed to an investigational or a non-investigational vaccine/product (pharmaceutical product or device), (iv). Planned administration/ administration of a vaccine/product not foreseen by the study protocol within the period starting 14 days before injection of a study vaccine 14 days after (e.g. influenza vaccine or other recommended vaccine), (v) Administration of immunoglobulins and/or any blood products within the 3 months preceding the dose of study product or planned administration during the study period, (vi) Any confirmed or suspected immunosuppressive or immunodeficient condition, based on medical history and physical examination. (Laboratory testing for HIV, Hepatitis C and Hepatitis B will be performed during the screening visit), (vii) Chronic administration (defined as more than 14 days in total) of immunosuppressants or other immune-modifying drug within 6 months prior to the product dose (for corticosteroids, this will mean prednisone ≥ 20 mg/day, or equivalent). Inhaled and topical steroids are allowed, (viii) Family history of congenital or hereditary immunodeficiency, (ix) History of or current autoimmune disease, (x) Known or suspected hypersensitivity to any ingredient in the formulation or component of the container, (xi) Pregnant or lactating female, (xii) Female planning to become pregnant or planning to discontinue contraceptive precautions within 180 days of study vaccine receipt, (xiii) ≥ Grade 2 hematological (hemoglobin level, white blood cell [WBC], lymphocyte or neutrophil count, and platelet count) or biochemical (alanine aminotransferase [ALT], aspartate aminotransferase [AST], blood urea nitrogen [BUN] and creatinine) abnormality, as per the local laboratory values. Grade 1 abnormalities considered not clinically significant by the treating investigator are not exclusionary. Grade 1 clinically significant laboratory abnormalities may be rescreened, and the participant will be deemed eligible if the laboratory repeat test is normal as per local laboratory normal values or deemed not clinically significant by the investigator assessment, (xiv) Any acute or chronic, clinically significant disease, as determined by physical examination or laboratory screening tests, (xv) Malignancies within previous 5 years (excluding non-melanomous skin cancer) and lymphoproliferative disorders, (xvi) Current alcoholism and/or drag abuse, (xvii) Acute disease and/or fever at the time of Screening ≥38.0°C. Fever is defined as temperature ≥ 38.0°C /100.4°F by any route; the preferred route for recording temperature in this study will be oral. Participants with a minor illness (such as mild diarrhea, mild upper respiratory infection) without fever may be enrolled at the discretion of the investigator. Participants with acute disease and/ or fever at the time of Screening may be rescreened at a later date, (xviii) Planned move to a location that will prohibit participating in the trial until study end. (xix) Any other condition that the investigator judges may interfere with study procedures (e.g. drawing blood) or findings (e.g. immune response). [00473] Primary endpoints for the Phase 1 trial include: Occurrence of AEs from first injection to Day 28 following each injection, in all participants, in all groups: (i) The occurrence of each solicited local and general AE, during the 7-day follow-up period after injection (i.e. the day of 1st injection and 6 subsequent days), (ii) The occurrence of any unsolicited AE through to Day 28. (iii) The occurrence of any hematological (hemoglobin level, WBC, lymphocyte, neutrophil, eosinophil and platelet count) and biochemical (ALT, AST, BUN and creatinine) laboratory abnormality though to Day 28. (iv) The occurrence of any SAE, MAE or adverse event of special interest disease through to Day 421.

[00474] Secondary endpoints for the Phase 1 trial include: Occurrence of AEs from second injection to Day 84 (28 days post injection) in all participants in all groups: i) The occurrence of each solicited local and general AE, during the 7-day follow-up period after injection (i.e. the day of the 2^nd injection and 6 subsequent days), (ii) The occurrence of any unsolicited AE through to 28 days following the second injection, (iii) The occurrence of any hematological (hemoglobin level, WBC, lymphocyte, neutrophil, eosinophil and platelet count) and biochemical (ALT, AST, BUN and creatinine) laboratory abnormality through to 28 days after the second dose of study vaccine.

[00475] Another secondary endpoint is the immune response to the study vaccine(s), as measured by antibody (e.g. IgG and other isotypes) directed to spike antigen pre-injection (Day 0) and post-injection(s) (Days 7, 28, 56, 63, 84, 236, 421), in all participants, in all groups.

[00476] The exploratory endpoints for the Phase 1 trial are (i) the ability of antigen specific immune responses to neutralize virus in either a direct virus neutralization assay and/or a pseudotype-based assay, (ii) the immune response to study vaccine(s) as measured by cell- mediated immune responses to antigen pre-injection and post-injection, (iii) the immune response to the study vaccine(s), as measured by antibody (e.g. IgG and other isotypes) directed to spike antigen at optional sampling time points Days 3, 14, and 21 (optional for participants in Step 2 only).

[00477] The study groups and treatments are shown in Table 43 below.

[00478] Table 43. Phase 1 Study groups and treatments.

[00479] EXAMPLE 4 - Native Antibody Responses to Identified B cell Epitopes

Detected in Human Sera from Confirmed SARS-CoV-2 Infected Subjects

[00480] A study was performed to explore the level of peptide -specific antibody responses in human serum samples. The study utilized commercially available human serum samples from two groups. The first was a set of pre-COVID-19 pandemic healthy serum samples (collected between 2007-2011) to explore any pre-existing antibody based immune reactivity to SARS-CoV- 2 B cell epitopes included in Formulation X (Example 2). This control group could facilitate understanding of potential background in the planned clinical indirect ELISA assay and facilitate the selection of an appropriate set of negative control samples for this assay, to be used in the assessment of immune responses in the Phase 1/2 clinical trial discussed in Example 3. The second set of sera was collected from SARS-CoV-2 positive subjects, taken at least 14 days post infection confirmation, to evaluate the peptide specific antibody responses in a set of convalescent serum samples. This analysis of the test group could facilitate investigation of peptide specific immune responses induced by SARS-CoV-2 infection, as well as identify individual serum samples that could be utilized as positive controls in the analysis of human samples post Formulation X vaccination.

[00481] Study Outcomes

[00482] A set of 15 healthy subject, pre-COVID-19 pandemic, serum samples were assessed. In addition, a cohort of 35 serum samples from individuals drawn at least 14 days post SARS-CoV-2 diagnosis were also assessed. The assay used was adapted from the indirect ELISA used in previous assays with DPX-RSV(A) to assess peptide specific immune responses to the viral peptide epitope SHe(A) (Langley et al, 2018) and may be optimized and qualified internally for the assessment of Formulation X specific immune responses in the Phase 1/2 clinical trial discussed in Example 3. The inventors tested for antibodies to the 4 immunogenic SARS-CoV-2 B cell epitopes identified in Examples 1 & 2: COV2B-S373, -S461, -S616 and -S821.

[00483] The data collected from this study is presented in Figure 21A-21B. In healthy human subjects (Figure 21 A) used as negative control in this study, there was very little antibody to any of the four SARS-CoV-2 B cell epitopes tested, with most of the samples giving reading near the background of the assay. Statistical analysis (Mann-Whitney test) was performed to compare the response between healthy (Figure 21 A) and convalescent (Figure 21B) serum samples for each of the peptides. A significant difference was observed between healthy and convalescent samples (higher response) with the COV2B-S461D and COV2B-S616D peptides. A significant difference with a lower response in convalescent serum samples compared to healthy sera was observed with the COV2B-S821D peptide. No significant difference comparing healthy and convalescent sera with COV2B-S373D peptide was demonstrated in this study.

[00484] These data are supportive of the immunogenicity and relevance of two of the four identified immunogenic B cell epitopes (COV2B-S461D and COV2B-S616D), demonstrating that a subset of human subjects may naturally develop antibodies to these specific areas of the SARS- CoV-2 protein in response to infection. While no increase in antibody responses to COV2-S821D or COV2B-S373D was detected in SARS-CoV-2 infected subjects, it does not invalidate their potential utility as relevant targets for a vaccine.

[00485] EXAMPLE 5. Immune mechanisms of action of Formulation X in CD-1 mice.

[00486] Previous studies have been focused on the antibody mediated immune response stimulated in mice, yet more recent studies with SARS-CoV-2 and other coronaviruses have indicated that T cell responses may also be important in protection against COVID-19. The peptide epitopes selected for inclusion in Formulation X are long peptides (17 to 27 amino acids) and were not predicted to contain T cell epitopes in mice or humans. However, it is possible that predictive algorithms may not capture all epitopes and T cell epitopes are contained within these sequences (Chaves et al, 2012). The present study offers an analysis outlining some of the potential immune mediated mechanisms of action of Formulation X in CD-1 mice. To ensure immune profiles are evaluated during an active phase of the immune response induced by Formulation X, the study was executed on SD21, one week following the SD14 booster vaccination.

[00487] The objectives of the study were to assess the potential immune-based mechanisms of action of Formulation X by SD21 based on: (1) immune cell profiles in spleens and vaccine draining lymph nodes using immunofluorescence staining, (2) cytokine production by splenocytes after in vitro stimulation with the Formulation X peptides, (3) T cell-specific IFN-γ production by splenocytes using ELISPOT assay, (4) peptide-specific and spike-specific antibody production by indirect ELISA and (5) isotyping of peptide-specific antibodies by an immunofluorescence-based bead assay.

[00488] Mice (outbred CD-1, N=10 female) were vaccinated as per the table below with

Formulation X containing 25 μg per dose of each of four peptides COV2B-S373D, COV2B - S461D, COV2B-S616D, and COV2B-S821D. All four peptides contain cysteine residues and are capable of dimerization. To avoid uncontrolled dimerization during formulation, peptide dimers were prepared commercially (Group 1). Both Formulation Z (the homogeneous water-free formulation (DPX) containing no peptide epitopes) and unvaccinated mice were used as controls.

[00489] Table 44. Summary of vaccination groups.

00490] The immune assessment was performed on SD21, when all mice were euthanizec and blood, spleen and vaccine-draining lymph nodes collected. Splenocytes were used in the IFN- γ ELISPOT assay, immunophenotyping using immunofluorescence staining (IMF) and cultured overnight with Formulation X peptides to measure stimulated cytokine secretion. Lymph node cells were used in immunofluorescence staining and blood was utilized to assess antibody responses via peptide-specific and spike-specific indirect ELISA, and to determine peptide- specific antibody isotypes using IMF-based bead assay.

[00491] An objective of this study was both to bridge and extend the data on the antibody responses induced by Formulation X. Production of anti-COV2B-S373, -S461, S616 and -S821 antibodies in response to Formulation X vaccinations was first explored in the peptide ELISA where plates were coated with each individual COV2B-S373, -S461, -S616 and -S821 peptide (Figure 22). The data indicated that mice in this study had similar levels of peptide-specific antibody responses at SD21 to those described in previous confirmatory studies shown in Example

2. [00492] Indirect ELISA was also performed to demonstrate capacity of the peptide-specific antibodies induced by Formulation X vaccination to recognize and to bind to the full-length SARS CoV-2 S protein, S1 subunit or to the receptor-binding domain (RBD) of the spike protein (Figure 23). These data confirmed that in vaccinated mice used in this analysis, peptide-induced immune responses to the immunizing peptides of Formulation X were able to recognize SARS-CoV-2 spike protein and its purified subunits.

[00493] To determine the scope of antibody-mediated responses to Formulation X, the inventors measured four immunoglobulin isotypes (IgG, IgM, IgA, IgE) using peptide-conjugated beads and secondary antibodies specific to each isotype. Results showed that IgG was the dominant isotype in sera of mice vaccinated with Formulation X and predictably was not present in mice vaccinated with the Formulation Z or naive mice. All four peptides of Formulation X elicited IgG antibodies (Figure 24A-24D). IgA, IgE and IgM did not show any increases in response to Formulation X vaccination compared to the levels of IgA, IgE and IgM in untreated mice or in mice treated with Formulation Z at the examined SD21 timepoint.

[00494] In addition to assessing the humoral immune response, this study also examined cell-mediated response to Formulation X using an IFN-γ ELISPOT assay (Figure 25). Splenocytes collected from mice vaccinated with Formulation X or Formulation Z and from naive mice were stimulated with media, irrelevant peptide (COV2B-S 1157, one of 23 SARS-CoV-2 spike peptides that was used in peptide selection studies in Example 1, but did not elicit immune response and therefore was not used in the final Formulation X) as well as with individual or pooled Formulation X peptides and IFN-γ production was assessed using anti-1FN-γ capture antibody. Results revealed a strong IFN-γ response in splenocytes stimulated with COV2B-S616 that was also evident in the samples stimulated with peptide pool. Stimulation by COV2B-S373 and -S821 produced a low level of IFN-γ while COV2B-S461 appeared to induce little to no IFN-γ production by splenocytes (Figure 25). Without wishing to be bound by theory, it is suggested that in CD-1 mice, COV2B- S616 may be responsible for the IFN-γ-mediated cellular responses to Formulation X vaccine.

[00495] To investigate immune cell populations within immune sites of activation, which may be impacted by Formulation X treatment, cells were harvested from the vaccine-draining lymph nodes and spleens of vaccinated and naive mice and the immunophenotype of those cells was determined using immunofluorescence staining procedures. Two immune phenotyping panels were evaluated: 1) a generic immune cell panel identifying the major immune cell types; and 2) a B cell-specific panel characterizing the different subsets of B cells.

[00496] No significant differences were observed in the overall frequency of B cells, CD4 or CDS T cells, dendritic cells, macrophages, neutrophils, or monocytes within splenic and lymph node lymphocyte (CD45+) populations in response to Formulation X vaccination. These data are supportive of the targeted mechanism of action induced by the water-free oil-based formulation platform. Formulation X vaccination thus can be directed to highly specific subsets of immune cells with no substantial changes in frequencies of many other immune populations. In the examination of global cell types with these organs, there were measurable differences within only two cell subsets, NK cells and subsets of activated B cells.

[00497] A statistically significant reduction of NK cells within the spleen was noted in the Formulation X treated animals compared to the naive control group (Figure 26 A). The frequency of NK cells in spleen were also reduced in the group treated with Formulation Z compared to the naive control group, however this difference was not statistically significant. Without wishing to be bound by theory, it is suggested that the observed changes in the frequency of NK cells may indicate that NK cells contribute to the immune response to water-free oil-based formulation treatment and may be redistributed to other organs such as the liver.

[00498] A moderate reduction in frequency of plasmablasts within B cell population (Figure

26B) was noted in water-free oil-based formulation treatment groups (F ormulation Z / Formulation X) compared to the naive control group in lymph nodes. Similarly, frequency of plasmablasts within splenic B cell population was also decreased in group treated with Formulation X, but did not reach significant threshold. The mature B cell population (Figure 26C) within the splenic B cells of the Formulation X group was significantly elevated over the naive control groups (but not the Formulation Z group). No significant changes were observed within the follicular and marginal zone subpopulations of mature B cells in either the spleen or lymph nodes. No significant changes were observed within the immature/transitional B cell T1/T2/T3 sub populations within the lymph nodes, however, significant reductions of T1 and T2 transitional populations were observed within the spleens of Formulation X-treated animals compared to naive controls. Local increases in mature B cells and decrease in plasmablasts were supportive of induction of antibody response to Formulation X vaccination. These data are also supportive of the potential specific and targeted immune responses induced within lymphoid organs to Formulation X.

[00499] Expression of MHC class II on B cells can influence their activation and capacity to present antigens of neighboring immune cells, fostering immune responses. Nearly all B cells are intrinsically positive for MHC class II, therefore, the mean fluorescence intensity of MHC class II on each subset of B cells was measured (Figure 27A-27B). Within the spleen, a slight elevation of MHC class II expression was observed in all mice treated with water-free oil-based formulation (either Formulation Z or Formulation X), however, only mature marginal zone B cells from Formulation X-treated mice had significantly elevated MHC class II expression compared to the naive control group (Figure 27 A). In contrast, within the lymph nodes, Formulation X-treated mice had significantly elevated MHC class II expression compared to the naive controls in: plasmablast, memory B cell, follicular zone B cell, marginal zone B cell, transitional T1 and T3 B cells (Figure 27B). Similar trends were noted when comparing Formulation Z-treated mice to naive controls, however, the only statistically significant changes were observed within the mature B cell population. No significant differences were noted between the Formulation Z and Formulation X groups, suggesting the MHC class II upregulation may be formulation-dependent and significant to the mechanism of action by which this platform stimulates immune responses to peptide antigens. Again, the finding is more pronounced within the vaccine draining lymph node; consistent with the inventors’ previous data and suggesting that the water-free oil-based formulation is not released in a systemic manner, and instead delivers target antigens in an active way to antigen presenting cells who then move the antigen into the draining lymph node to induce the targeted immune response (Brewer et al, 2014; Brewer et al 2018). Without wishing to be bound by theory, it is possible that the formulation can induce detectable and local changes within the immune organs and yet not induce measurable systemic inflammatory responses.

[00500] It was previously shown in Example 2 that vaccination with Formulation X had no effect on the levels of systemic cytokines. To evaluate whether each of four peptides in Formulation X can induce local secretion of cytokines and chemokines by immune cells within the secondary lymphoid organs, supernatants were collected from splenocytes stimulated with individual COV2B-S373, -S461, -S616 and -S821 peptides and with pool of four peptides. Cytokine analysis was perfonned independently by a multiplex array (Eve Technologies, Calgary AB) and was primarily focused on assessing biomarkers of T cell response (Th1/Th2) and inflammation, including: GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12 (p70), IL-13, IL-17A, KC/CXCL1, LIX, MCP-1, MIP-2, TNF-α (Figure 28A-28R).

[00501] When stimulated with peptide pool, splenocytes from Formulation X-vaccinated mice demonstrated a significant increase in secretion of Th1 and Th2 cytokines IL-2, IL-4, IL-5, IL-6, IL-17A, GM-CSF, IFN-γ, and TNF-α, as well as chemokines CCL-2, and CXCL-2 when compared to the cytokine/chemokine secretion in splenocytes from Formulation Z and naive groups. The peptide pool also significantly induced secretion of CXCL-1 in splenocytes of Formulation X group compared to the CXCL-1 secretion in the naive group, but not to the CXCL- 1 secretion in the Formulation Z group. An increase in secretion of GM-CSF, IFN-γ, and TNF-α in splenocytes positively correlated with responses detected in IFN-γ ELISPOT in this group (Pearson correlations r=0.8-0.9; p≤0.003), suggesting that Formulation X may induce Th1 responses in CD-1 mice which may be likely mediated by COV2B-S616 peptide.

[00502] No significant changes were detected in cytokines IL-13 (Figure 28 J), CXCL-5 (Figure 28Q) and IL-10 (Figure 28H) in response to any of the examined stimulations. However, a trend of increased IL-10 secretion was observed in peptide pool- and COV2B-S616-stimulated splenocytes from Formulation X-vaccinated mice compared to the splenocytes from Formulation Z and naive mice (Figure 28H). These data suggested that epitope directed immune response could be generated with immune organs and show a balanced Th1/Th2 immune profile.

[00503] Exploring the cytokine profile induced by individual peptides after splenocyte stimulation in culture can give some potential mechanistic insights into the types of immune responses induced by each of the peptides and can be correlated to the other antibody or T cell- based data generated elsewhere in the study. As subjects would not be exposed to these peptides individually, it is suggested that the data with the pooled peptides may more accurately represent the overall immune response induced in immune organs by Formulation X.

[00504] Analysis of cytokine profiles secreted in response to stimulations with individual peptides suggested that three of four Formulation X peptides can induce unique cytokine profile in splenocytes of vaccinated mice at SD21 time point. [00505] Stimulations of splenocytes from Formulation X-treated mice with COV2B-S616 peptide significantly induced secretion of IFN-γ, IL-17A, IL-6, IL-4, CCL-2 and TNF-α compared to the secretion of these cytokines in COV2B-S616-stimulated splenocytes from Formulation Z or naive mice. Increased secretion of IL-2, GM-CSF and IL-10 in COV2B-S616-stimulated splenocytes from Formulation X group was also observed, though these changes were not significant. This is consistent with the finding that COV2B-S616 can induce IFN-γ secretion from T cells from the spleen in the ELISPOT assay.

[00506] Stimulation of splenocytes from Formulation X-treated mice with peptide COV2B- S373 alone induced IL-5 and IL-4 and IL-6, indicative of induction of a primarily humoral response to this antigen. Stimulation of splenocytes with peptide COV2B-S821 induced a significant increase in secretion of CCL-2 in the Formulation X-vaccinated group compared to the CCL-2 secretion in COV2B-S821-stimulated splenocytes from Formulation Z group (but not from the naive group). There was also a trend of increased secretion of IL-4, IL-5, IL-6 and IFN-γ in COV2B-S821 -stimulated splenocytes from Formulation X group, though these changes were not statistically significant but considering this consistent finding across multiple cytokines, it may represent biological relevance. It was noticed that cytokine secretion (IL- 1α, IL-1β, IL-7, IL-12p70 and CXCL-2) in COV2B-S821-stimulated splenocytes from Formulation X group was reduced when compared to the cytokine secretion in COV2B-S821 -stimulated splenocytes from naive mice. These changes may be partially explained by high variation in secretion of these cytokines amongst individual samples in naive group (%RSD: IL-1a 88% for naive vs 24% and 14% for Formulation Z/X groups, IL-1β 11% for naive vs 5% and 3% for Formulation Z/X groups, IL-7 47% for naive vs 11% and 21% for Formulation Z/X groups, IL-12p70 73% for naive vs 18% and 9% for Formulation Z/X groups, CXCL-2 113% for naive vs 70% and 40% for Formulation Z/X groups).

[00507] Stimulation of splenocytes with irrelevant peptide (COV2B-S1157, one of 23 SARS-CoV-2 spike peptides that was used in peptide selection studies but did not elicit immune response and therefore was not used in the final Formulation X) and COV2B-S461 did not significantly change the secretion of any of the cytokine assessed in any of the treatment groups. While there were no specific cytokine profiles induced in response to COV2B-S461 peptide, levels of COV2B-S461 -specific antibodies positively correlated with levels of RBD-specific antibodies in sera (Figures 25 and 26), defining a possible functional importance of this peptide in the formulation. Interestingly, strong/moderate COV2B-S461 -specific antibody responses can be detected as early as 14 days post vaccination (before boost) which is one week earlier that the detection of responses of the same magnitude to other peptides in Formulation X, indicating that kinetics of humoral response to COV2B-S461 peptide may be somewhat different than the response to COV2B-S373, -S616, -S821 and therefore may not be accurately reflected at SD21 timepoint.

[00508] Importantly, there were no correlations between the induction of Th2/humoral responses (upregulation of Th2 cytokines, peptide-specific and S-specific antibody responses by ELISA) and Th1/cellular responses (IFN-γ production by ELISPOT, Th1 cytokine upregulation), suggesting that the induced immune response by Formulation X is balanced and neither enhancing nor suppressing individually induced responses. In summary, a combination of four Formulation X peptides can induce balanced cytokine responses that can be detected in the secondary lymphoid organs but not in circulation.

[00509] The findings of the study as a whole offer additional insight into the immunological footprint of the Formulation X vaccine as early as one week after the boost administration. Results of this study indicate that Formulation X vaccine can induce balanced humoral and cellular immune responses. Antibody responses are induced by immune cell specific secretion Th2 cytokines, increases in mature B cells, reduction in plasmablasts, and increased MHC class II expression on B cells detected in the secondary lymphoid organs. This results in strong IgG antibody responses to all four peptides in formulation detected in circulation. In addition, there is the induction Th1 cytokines and reduction (likely due to redistribution to other organs) of NK cells observed in the secondary lymphoid organs. There is also the secretion of epitope specific IFN-γ by T cells induced specifically by one of the epitopes, indicative of a targeted T cell response. This data gives an insight into additional mechanisms by which an immune response induced by Formulation X could protect a vaccinated subject from severe COVID-19 infection.

[00510] Evaluation of humoral and cell mediated responses one week after the boost immunization with Formulation X revealed: Formulation X can induce peptide-specific IgG antibodies capable of binding to the spike protein. In addition, T cell responses were detected using IFN-γ ELISPOT. Formulation X can induce a balanced immune response within immune organs that can be detected using immune cell production of cytokines post peptide stimulation in vitro.

[00511] Methods.

[00512] Indirect ELISA.

[00513] Indirect enzyme-linked immunosorbent assays (ELISA) were performed to detect serum peptide-specific antibody titers and S-spedfic antibody titers. For the peptide ELISA, 96- well EIA/RIA Clear Flat-Bottom Polystyrene High Bind Microplates (Corning®) were coated with 1 μg/mL of individual peptides in coating buffer (NaHCO₃, Na₂CO₃) overnight at 4°C. Research grade monomer peptides were used for coating. Plates were washed five times with TBS-T (0.2%) and blocked with warmed 3% Gelatin (BioRad) for 30-45 minutes at 37°C. Plates were washed with TBS-T and incubated overnight at 4°C with sera at an initial starting dilution of 1 : 100.

[00514] For the S ELISA, pre-coated plates were obtained from AcrobioSy stems (pre- coated S1 and pre-coated RBD, cat no. SP-12 and RP-13, respectively) or coated internally with commercially available S protein (S1+S2 ECD, His tag, Sino Biologicals).

[00515] The S1+S2 ECD (Sino Biologicals) was constructed from a DNA sequence encoding the SARS-CoV-2 Spike protdn (YP 009724390.1) (Val16-Pro1213) and was expressed with a poly-histidine tag at the C-terminus. For coating, S1+S2 ECD was diluted in manufacturer’s recommended coating buffer (136.9 mM NaCl, 10.1 mM Na₂HPO₄, 2.7 mM KC1, 1.8 mM KH2PO4, pH 7.4) for a final concentration of 1 μg/mL.

[00516] S1 protein (AcrobioSystems) that was used on pre-coated plates contains amino acids 16-685 of SARS-CoV-2 spike protein. RBD domain (AcrobioSystems) that was used on pre- coated plates contains amino adds 319-541 of SARS-CoV-2 spike protdn. Prior to procurement, the Acrobiosystems microplates were coated with 100 μL of Streptavidin tetramer, blocked with 300 μL of 2% BSA Blocking Buffer and captured 0.1 μg/well of biotinylated SARS-CoV-2 Spike (Acrobiosystems). [00517] The pre-coated plate protocol started with the addition of serum samples. TBS-T with 0.5% bovine serum albumin (BSA) was used to prepare serum dilutions starting at 1:100. Sera were subsequently incubated overnight at 4°C. For all ELISAs, serum was removed with five TBS-T washes, and bound antibodies were detected by incubation of alkaline phosphatase conjugated Protein G (EMD Millipore-Sigma) with high affinity binding for IgG for 1 hour at 37oC and subsequent development with chromogenic alkaline phosphatase substrate. Optical density was measured at 405 nm within 1 hour of initial substrate addition on a spectrophotometer plate reader. ELISA results were expressed as end point Log₁₀ titers using a calculation method described by Frey et al. (Frey et al. 1998).

[00518] An immunogenic response definition of a peptide-specific antibody titer ≥Log₁₀=2 was used to determine the number of responders within each group. Samples with non-detectable titers were assigned a value of 1.85. Data were considered significantly different at the level of p<0.05 using either one-way ANOVA with post-hoc Tukey HSD, or two-tailed unpaired t test as indicated in the figure legends.

[00519] Immunofluorescence staining.

[00520] Immunofluorescence (IMF) staining was performed on cells isolated from spleens, popliteal and inguinal lymph nodes collected on SD21 to characterize immune cell phenotype. Two panels were designed: 1) a generic immune cell panel identifying the major immune cell types; 2) a B cell-specific panel characterizing the different subsets of B cells (Tables 45 and 46).

[00521] Spleens and lymph nodes were processed and cell concentrations were adjusted to 1-2x10⁶ cells per tube. Staining for splenocytes/lymph node cells followed antibodies in panel 1 and panel 2 outlined below using basic surface IMF staining protocol.

[00522] Table 45. Panel 1: Generic immune cell profiles

[00524] Stained samples were acquired on a BD FACSCelesta™ flow cytometer and analysis was done using FlowJo software (vlO). Panels 1 and 2 were analyzed based on a gating strategy and represented as the percentage of marker-positive cells.

[00525] Splenocyte ELISPOT

[00526] To measure IFN-γ production from splenocytes, an enzyme-linked immune absorbent spot (ELISPOT) assay was performed on SD21. Splenocytes were stimulated in duplicate with media, COV2B peptides (individually and pooled), and irrelevant control peptide COV2B-S1157.

[00527] Table 47. Stimulation of groups in IFN-γ ELISPOT.

[00528] Multiplex cytokine profiling

[00529] A set of splenocyte samples was used to assess stimulated cytokine release. 1.5 x 10⁶ splenocytes were cultured in complete RPMI in a deep 96-well u-bottom plate and stimulated with the COV2B peptides individually, or as a pool (COV2B-S373, -S461, -S616, -S821), each at a final concentration of 10 μg/mL. Controls included unstimulated cells and cells stimulated with the irrelevant peptide COV2B-S1157. Supernatants were collected ~18 hrs post stimulation and stored immediately at -20°C and shipped on dry ice to Eve Technologies (Calgary, AB) for multiplex cytokine profiling

[00530] Samples were analyzed in duplicate using a Mouse High Sensitivity 18-Plex Discovery Assay, independently by Eve Technologies. This multiplex assay is primarily focused on T-cell biomarkers and includes quantification of the following cytokines: GM-CSF, IFN-γ, IL- 1α, IL-1β, IL-2, EL-4, IL-5, IL-6, IL-7, IL-10, IL-12 (p70), IL-13, IL-17A, KC/CXCL1, LIX, MCP-1, MIP-2, and TNF-α. For quantification and interpretation of the dataset, Eve Technologies applied a point-to-point semi-log regression model that extrapolates concentrations for any fluorescence intensity value in the analyser Bio-Plex Manager Software. Fold-change in the level of each cytokine in the peptide stimulated samples was calculated relative to the level of the same cytokine in the corresponding non-stimulated media only sample. Productions of cytokines by splenocytes in mice vaccinated with Formulation X were compared to those in mice vaccinated with Formulation Z (vehicle only), or to those in naive mice that did not receive any treatment. Data were considered significantly different at the level of p<0.05 using two-way ANOVA with post-hoc Tukey HSD. [00531] Isotyping of Formulation X antibodies

[00532] To assess isotypes of immunoglobulins induced in response to Formulation X vaccination, the inventors utilized peptide-conjugated beads (Bangs Laboratories Inc., Indiana, USA) to capture four antibody subtypes (IgG, IgM, IgA and IgE).

[00533] Preparation of carboxyl beads

[00534] 10⁷ beads were mixed with 400 μL of coupling buffer (0.1 M phosphate buffer pH

7.4 (Sigma, Cat no. P5244), pH 6.0) into Tube A. Beads in Tube A were centrifuged at 13 800 xg for 3 minutes to pellet beads. Supernatants were collected into a fresh tube (Tube B) and centrifuged (13 800 xg, 2 minutes). Supernatants were discarded and beads were combined (from Tubes A and B) to maximize bead recovery. Beads were washed with coupling buffer two more times with the same Tube B recovery method. Beads were finally resuspended in 160 μL coupling buffer in preparation for activation.

[00535] Bead activation with EDC and Sulfo-NHS

[00536] To activate the beads, 50 mg/mL solution of EDC (Thermo Fisher Cat no. 22980) and Sulfo-NHS (Thermo Fisher Cat no. 24510) were prepared and 20 μL of each solution were added to beads and incubated for 20 minutes at room temperature with agitation (1000 rpm). Beads were then centrifuged at 13 800 xg for 3 minutes and a coupling buffer wash was performed three times (as described above). The beads were resuspended in 180 μL of coupling buffer after the last wash.

[00537] Peptide coupling to beads

[00538] Peptide dimers (CPC Scientific, California, USA, see Table 48 below) were coupled to the beads as follows: peptides were added to the activated beads at 10 μg of each peptide per 10⁶ beads and reaction volume was adjusted with coupling buffer to 20 μL. The beads were incubated for 2 hours at room temperature with agitation (1000 rpm) protected from the light. Beads were then centrifuged (13 800 xg, 3 minutes), resuspended in 400 μL blocking buffer (IX PBS, 1% BSA, pH 7.4) and incubated for 1 hour at room temperature with agitation (1000 rpm). After the incubation, beads were centrifuged (13 800 xg, 3 minutes) and resuspended in 200 μL storage buffer (IX PBS, 0.01% Tween 20, 0.05% NaN3, 0.1% BSA, pH 7.4).

[00539] Table 48. Concentration and reconstitution of the four Formulation X dimeric peptides.

Ό0540] Serum incubation with peptide-coated beads and immunofluorescent staining.

[00541] For each reaction, 2.5 μL of peptide-conjugated beads and 7.5 μL of binding buffer

(IX PBS, 10% FBS, 0.2% BSA) were mixed and incubated with 10 μL of serum for 2 hours at 2- 8 °C with vortexing at the 1-hour timepoint. Samples were washed twice with 500-1000 μL of wash buffer (1X PBS, 0.2% BSA) and centrifuged for 5 minutes at 250 xg. To detect the four antibody isotypes, samples were incubated with 50 μL of a master mix of secondary antibodies (Table 49) for 30 minutes at 2-8 °C. Samples were then washed three times with 500-1000 μL of IMF buffer (10XDPBS, 1% sodium azide, 5% BSA). Beads were resuspended in 150 μL of DPBS. Samples were acquired on the BD FACSCelesta™ and analysis was completed using the FlowJo software (V10.6.0).

[00542] Table 49. Isotyping secondary antibodies

[00543] EXAMPLE 6 Cross-sectional assessment of immune responses to Formulation X in CD-1 mice

[00544] Data from both confirmatory in vivo studies Study 1 and Study 2 described in Example 2 demonstrated the ability of the Formulation X to induce robust and sustained immune responses in mice using lots manufactured during the clinical process development. In these confirmatory studies, the immunological response to Formulation X was evaluated through various assays including indirect ELISA to detect peptide-specific and spike-specific IgG antibodies, VaxArray Coronavirus SeroAssay to detect antibody binding to the full spike protein or its subunits, as well as cytokine profiling in sera.

[00545] In Example 5, the potential immune-based mechanisms of action of Formulation X were evaluated at one early timepoint (SD21) during an active phase of the immune response. Further assessment of immune mechanisms of action of Formulation X were performed in the present Example. In this study, CD-1 mice were immunized with the Formulation X clinical GMP lot at SD14 and boosted at SD 14. Immune responses were evaluated at SD56 using a combination of assays that offered a more complete picture of the immunological underpinning of the immune response to Formulation X. Table 50 provides the study design.

[00546] Study objectives were:

[00547] to perform an extensive cross-sectional evaluation of the immune responses to Formulation X in comparison to the homogeneous water-free formulation (DPX) containing no peptide epitopes. This formulation is hereafter referred to as Formulation Z. Evaluation was performed in CD-1 mice using several different readouts (peptide-specific and spike-specific IgG titers by indirect ELISA;

[00548] to evaluate the neutralizing activity of anti-sera by pseudoparticle neutralization assay (PNA) [00549] to measure IFN-γ production by splenocytes using ELISPOT;

[00550] Perform additional functional analysis of elicited antibodies.

[00551] Table 50: Overview of the cross-sectional study

[00552] The main objective of this study was to assess and confirm the immunogenicity of Formulation X (clinical lot) in CD-1 mice at a later time point (SD56, six weeks post boost vaccination) when antibody responses should be at maximum levels. Humoral immune responses were first evaluated by measuring peptide-specific IgG antibody titers using indirect ELISA.

[00553] Consistent with the results obtained in the confirmatory studies, peptide-specific IgG responses for all four peptides were elicited in most mice vaccinated with Formulation X (Figure 29 and Table 51). [00554] In addition to assessing peptide-specific IgG titers, S-protein-specific antibody responses were also measured to determine whether the Formulation X anti-sera can recognize the SARS CoV-2 spike protein. All mice (10/10 mice) generated antibodies capable of binding to the spike protein. (Figure 30 and Table 51).

[00555] Consistent with the data in Example 2, minimal to no peptide-specific or -S-protein- specific responses were detected in all samples collected from mice vaccinated with Formulation Z (Figure 29 and Figure 30), suggesting that peptides formulated in the homogeneous water-free formulation are key components to generation of antigen-specific immune responses.

[00556] Table 51. Peptide-specific and spike-specific IgG titer ranges and total number of responders in CD-1 mice vaccinated with Formulation X GMP clinical lot.

* Tabulated Logic endpoint antibody titers (min-max) and number of responders for each peptide/S protein. An immunogenic response definition of a peptide-specific or spike-specific antibody titer ≥Log₁₀=2 was used to determine the number of responders within each group at each timepoint.

[00557] To demonstrate the capacity of Formulation X-induced antibodies to neutralize virus in vitro, a pseudoparticle neutralization assay was performed on all serum samples. Neutralization activity in these samples was evaluated using SARS-CoV-2 pseudotyped virus expressing the SARS-CoV-2 spike protein and HEK293T cells expressing the human SARS-CoV- 2 receptor ACE2 and serine protease TMPRSS2, a protein responsible for cleaving the S protein, leading to membrane fusion. [00558] Neutralization activity was detected in all animals vaccinated with Formulation X (Figure 31 and Table 52). There was a significant increase in neutralization activity in the samples collected from Formulation X vaccinated animals compared to the samples collected from mice in Formulation Z and naive control groups.

[00559] Table 52. Neutralization titer ranges and number of responders in CD-1 mice vaccinated with Formulation X GMP clinical lot.

00560] Tabulated Log2 IC50 titers (min-max) and number of responders. A response definition of a titer ≥Log2=2 was used to determine the number of responders within each group.* IC50 titer values were defined as the inverse of the greatest dilution of sera providing 50% viral load reduction relative to virus-only infection.

[00561] Blocking the ability for a virus to enter cells through induction of neutralizing antibodies is important to the overall efficacy of a vaccine, however, antibodies can provide additional protection by stimulating other cell processes that promote viral clearance and further activation of virus-specific immunity. It has been shown by others that multifunctional antibodies are often induced in response to COVID-19 vaccination and in individuals who recovered from SARS-CoV2 infection (Barrett, 2021 and Atyeo, 2020).

[00562] Fc receptor dependent phagocytosis is one potential mechanism by which Formulation- X -induced antibodies can activate the innate immune response to eliminate antibody-opsonized targets. This study assessed whether antibody-containing sera from Formulation X-vaccinated mice were able to bind S-peptide-coated fluorescent beads and facilitate phagocytosis by M-CSF- differentiated macrophages.

[00563] Results presented in Figure 32 indicate that antibodies elicited by Formulation X vaccination were capable of binding S-protein coated beads and increasing their uptake by phagocytic cells. The phagocytic scores generated by Formulation X sera were significantly higher than that by sera from Formulation Z (p-value < 0.0001) and PBS -treated group (p-value < 0.0001). No phagocytic activity above assay background was observed in samples collected from Formulation Z or naive animals.

[00564] These results suggest that Formulation X can induce viral clearance, possibly via enhancing antibody-dependent phagocytosis by macrophages or potentially other phagocytic cells.

[00565] To assess cell-mediated response that may be induced by Formulation X, IFN-γ ELISPOT assays were performed using splenocytes collected from mice vaccinated with Formulation X or with Formulation Z (Figure 33). Splenocytes were stimulated with media, irrelevant peptide as well as with individual or pooled Formulation X peptides and IFN-γ production was assessed using anti-1FN-γ capture antibody.

[00566] Consistent with the results observed in Example 5, stimulation of splenocytes of Formulation X-vaccinated mice with the COV2B-S616 and peptide pool resulted in elevated IFN- γ ELISPOT responses above media and irrelevant peptide controls, indicating that COV2B-S616 is the primary driver of cell mediated response. IFN-γ represents a key mediator of anti-viral immune responses and can enhance additional effector functions such as T cell polarization to Th1, NK cell activation, increased antigen presentation and cytolytic responses.

[00567] Minor responses were observed in other peptide stimulations; COV2B-S461 and -S821 which contribute slightly to the overall responses observed in the peptide-pool stimulations. As expected, no responses were observed in either of the naive or Formulation Z vaccinated control groups.

[00568] In summary, strong peptide-specific IgG antibodies capable of recognizing and binding to the spike protein of SARS CoV-2 were elicited by Formulation X in all or almost all mice, demonstrating the ability of this vaccine formulation to induce robust humoral responses. Results of this study are supported by the results presented in Example 2 which demonstrated sustained IgG antibody response until SD140. Moreover, this study reveals that Formulation X can elicit multifunctional antibody responses that can inhibit viral infection, possibly directly by neutralization and/or indirectly by antibody-dependent Fc-mediated phagocytosis.

[00569] Although Formulation X is primarily designed to elicit potent humoral responses, this vaccine has the potential to drive T cell mediated response from the short linear peptides within formulation. Consistent with the results of the analysis of immune response at SD21 in Example 5, sustained and durable cell mediated responses were detected in mice vaccinated with Formulation X in this study. It is possible that a balanced approach between both B and T cells may give the best overall outcome and could provide multiple mechanisms to reduce the chances of viral escape.

[00570] Methods

[00571] Indirect ELISA

[00572] Indirect enzyme-linked immunosorbent assays (ELISA) were performed to detect serum peptide- or S-protein- specific antibody titers. Briefly, 96-well EIAZRIA Clear Flat-Bottom Polystyrene High Bind Microplates (Corning®) were coated with 1 μg/mL of individual peptides in coating buffer (NaHCO₃, Na₂CO₃) or with S protein (S1+S2 ECD, His tag, Sino Biologicals) diluted in manufacturer’s recommended coating buffer (136.9 mMNaCl, 10.1 Na₂HPO₄ , 2.7 mM KC1, 1.8 mM KH₂PO₄, pH 7.4) and incubated overnight at 4°C. Research grade monomer peptides were used for coating. Plates were washed five times with TBS-T (0.2%) and blocked with warmed 3% Gelatin (BioRad) for 30-45 minutes at 37°C. Plates were washed with TBS-T and incubated overnight at 4°C with sera at an initial starting dilution of 1:100. After TBS-T washes, bound antibodies were detected by incubation of alkaline phosphatase conjugated Protein G (EMD Millipore-Sigma) with high affinity binding for IgG for 1 hour at 37°C and subsequent development with chromogenic alkaline phosphatase substrate. Optical density was measured at 405 nm within 1 hour of initial substrate addition on a spectrophotometer plate reader. ELISA results were expressed as end point Log 10 titers using a calculation method described by Frey et al. (Frey et al. 1998). Each serum sample was diluted at 1 :100 as a starting dilution. Titers that did not fall under the calculated cut-off at the highest dilution, or titers that resulted from a high background were repeated to ensure the endpoint titers fall within prepared dilutions and remove any background signal, respectively.

[00573] An immunogenic response definition of a peptide-specific antibody titer ≥Log10=2 was used to determine the number of responders within each group at each timepoint.

[00574] Neutralization Assay [00575] Neutralization capacity was assessed in serum samples from Formulation X- vaccinated mice. A PNA assay was performed as discussed herein. A positive control of mouse monoclonal anti-SARS-CoV-2 spike neutralizing antibody (clone NN54) was included as reference. Briefly, serum samples were heat inactivated at 56°C for 15 min. 2X serial dilutions of sera were mixed with GFP-rSARS-CoV-2 pseudovirus, and the samples were incubated for 1 hour at 37°C. Following incubation, the samples were mixed with HEK293T-ACE2/TMPRSS2 cells, which were then incubated for 24 hr at 5% C02, 37°C in a humidified incubator. After a media change, the samples were incubated at 5% C02, 37°C for an additional 48 hours in a humidified incubator.

[00576] Following incubation, the cells were harvested and analyzed for GFP expression by flow cytometry using BD FACSCelesta™ and FlowJo software. Mean fluorescent intensity (MFI) was used to quantify the level of infection (viral load) of HEK293 T- ACE2/TMPRS S2 cells by the pseudovirus. Data were considered significantly different at the level of p<0.05 using two-way ANOVA with post-hoc Tukey HSD.

[00577] Phagocytosis Assay

[00578] Antibody-containing sera from Formulation X-vaccinated mice were assessed for the ability to bind peptide-coated fluorescent beads and mediate Fc-receptor-mediated phagocytosis by M-CSF-differentiated macrophages. In this assay, fluorescent neutravidin fluorophore beads coupled to a trimer of His-biotin S protein. Formulation X mouse serum was added to the beads and mixed. The mixture was then added to either 96-well plates. This procedure was adapted from a published phagocytosis assay protocol (Ackerman et al. (2011). J Immunol Methods. PubMed ID: 21192942) and is as follows.

[00579] Murine macrophages were isolated and cultured as described below. Briefly, long bones were isolated from naive CD1 mice and flushed to collect progenitor cells. RBCs were lysed and cells were cultured at 2.5x 10⁵ cells/mL in 5 mL of complete RPMI media (cRPMI) [RPMI 1640 (Hyclone) + 10% FBS (Hyclone), 2% penicillin/ streptomycin (Gibco), 2mM L-glutamine (Gibco), 50 mM β-mercaptoethanol (Sigma-Aldrich), and 5mM HEPES buffer (Gibco)] supplemented with 30 ng/ mL of murine of M-CSF.

[00580] Bead-Protein Coupling and Addition to Serum [00581] Yellow-green fluorescent NeutrAvidin™ beads-505/515 (Thermo Fisher, F8776) were incubated at two bead: S-protein ratios: 1 :0 ratio (uncoated) and 3 : 1 (coated) in low-binding tubes (Low-binding microcentrifuge tubes, Coming CLS3207 or equivalent). The S-protein is a biotinylated trimeric spike protein capable of binding the NeutrAvidin beads (Biotinylated Recombinant SARS-CoV-2 Spike, trimeric) His-tag, R&D Systems, BT10549-050). 0.1 μL of beads (10 μL after dilution) was found to be optimal for one sample. 0.0667 μL of S-protein (stock 500 μg/mL, 50 μg reconstituted in 100 μL PBS) was found to be equivalent to the 0.1 μL ofbeads if they were to be added at a 1 : 1 ratio. The bead: protein tubes were incubated in a 37°C water bath for two hours, protected from light. Beads were then washed 2X with 500 μL PBS w/v 5%-BSA (5% BSA in PBS) and centrifuged at 10,000 rpm x 3 min between washes. Bead: protein tubes were resuspended based on volume of beads added at a final dilution of 1 : 100 in PBS w/v 0.1%- BSA for subsequent steps.

[00582] 10 μL of the appropriate beads: protein mixture were added to each FACS tube, followed by 10 μL of the corresponding serum sample and 30 μL of 0.1% BSA-PBS for a final volume of 50 μL No-serum controls were included with both 1:0 and 3:1 ratios; in this case, 40 μL of 0.1% BSA-PBS was needed for the final volume of 50 μL. The bead: protein +semm reaction tubes were centrifuged briefly at 1000 rpm (233 xg) for 3 min to ensure all solutions are the bottom of the FACS tubes. The latter was then briefly vortexed (2-3 seconds per tube) and wrapped with parafilm, and then incubated at 37 °C for two hours, protected from light. After incubation, beads were washed with 1000 μL PBS w/v 0.1%-BSA, then centrifuged at 10,000 rpm x 3 min using available tube inserts. Tubes were partially aspirated and a small volume of solution was left (~ 50μL).

[00583] Culture media (cRPMI) was removed from the 96-well plate by inverting and tapping the plate into a waste container and allowed to briefly drain on a Kim Wipe. Each bead: protein: serum reaction (~ 50μL) was gently vortexed, then added to one well of the cultured macrophages. An additional 50 μL of complete media was then added for a final volume of ~ 100μL. Macrophages were then incubated under standard tissue culture conditions (37 °C and 5% CO2) for 4 hours before flow cytometry acquisition. At the end of the four-hour incubation period, culture media was removed from the 96-well plate by inverting and tapping the plate into a waste container and allowed to briefly drain on a Kim Wipe. Cells were then washed four times with PBS to remove any unbound beads. 50 μL of 0.25% Tryspin (8-10min at 37 °C) was then added promote cell detachment. Cells were then collected in complete media (400 μL, 2X 200 μL collections) by vigorous pipetting (~10 times) using a multi-channel pipette into new FACS tubes. Wells were checked under the microscope to ensure the majority of cells were collected. Tubes were then topped up with IMF buffer and centrifuged at 1000rpm (233g) for 6 min. An additional IMF buffer wash (1000 μL) was then performed, and cells were eventually resuspended in 100- 200 μL of PBS and subject to flow cytometric acquisition on the FACS Celesta (QC-042; BD FACS Celesta and FACS Diva Use). Analysis was performed using the FlowJo software. The percentage of positive beads, MFI (mean fluorescent intensity) of the positive population was used to measure and compare bead uptake. In addition, a phagocytic score was calculated as follows: Phagocytic score = % bead positive × MFI bead positive.

[00584] An IFN-γ ELISPOT was performed as described below. Single cell suspensions of splenocytes were prepared by lysing RBCs with ammonium-chloride-potassium solution and resuspended at 5 x 10⁶ cells/mL in complete RPMI media. A 100 μL volume of cells was added into ELISPOT plates (BD Bioscience) and stimulated with 100 μL of complete RPMI containing no peptide (background control), 10 μg/mL of COV2B peptide (individually and pooled) or with irrelevant peptide in duplicate. ELISPOT plates were incubated overnight at 37°C, 5% C02. Plates were developed the next day as per the manufacturer’s instructions using AEC substrate (Sigma- Aldrich). Spots were enumerated using an ImmunoSpot Analyzer (C.T.L. Ltd, Shaker Heights, OH) as the number of spot-forming units (SFU) per well.

[00585] EXAMPLE 7. Specificity of Formulation X antibodies to mutated SARS-CoV-2 Spike protein

[00586] Several new variants of the original SARS-COV-2 virus have been independently emerged in different geographic regions of the world. These Variants of Concern (VOC) include: United Kingdom variant B.1.1.7 (Alpha), South Africa variant B.1.351 (Beta), Brazil variant P.1.(Gamma), US variant B.1.427, and US variant B .1.429 (collectively Epsilon). Another rapidly emerging variant of interest is the India variant B.1.617 (Delta).

[00587] S-protein mutations in each variant include:

[00588] B.l.1.7; 201/501 Y. V1 (del69-70, dell44, E484K*, S494P*, N501Y, A570D, D614G, P681H, T761I, S982A, D1118H, K1191N*)

[00589] B.1.351; 20H/501.V2 (D80A, D215G, del241-243, K417N, E484K, N501Y, D614G,

A701V)

[00590] P.l; 20J/501Y.V3 (L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I)

[00591] B.1.427; 20C/S:452R (L452R, D614G)

[00592] B.1.429; 20C.S:452R (S13I, W152C, L452R, D614G)

[00593] B.1.617.1; 20A/S:154K (T95I*, G142D, E154K, L452R, E484Q, D614G, P681R, Q1071H

[00594] B.1.617.2; 20A/S:478K (T19R, G142D*, dell56-157, R158G, L452R, T478K, D614G, P681R, D950N)

[00595] B.1.617.3; 20A (T19R, G142D, L452R, E484Q, D614G, P681R, D950N)

[00596] (*) = detected in some sequences but not all (www.cdc.gov/coronavims/2019- ncov/cases-updates/variant-surveillance/vari ant-info.html)

[00597] The specificity of Formulation X antibodies to mutated SARS-CoV-2 Spike protein was tested using indirect ELISA. Formulation X contains four peptide dimers COV2B-S373D, - S461D, -S616D, -S821D derived from different non-overlapping regions of S protein of SARS- CoV-2 as indicated below:

[00598] COV2B-S373D 373-390aa

[00599] COV2B-S461D 461-487aa

[00600] COV2B-S616D 616-632aa

[00601] COV2B-S821D 821-846aa

[00602] Based on the alignment of four Formulation X epitopes with the locations of known spike mutations, only binding of one of the four Formulation X antibodies may be directly impacted by known spike mutations: binding of the COV2B-S461D antibodies to its epitope that contains point spike mutation E484K in B.1.351 and PI variants (and occasionally B.1.1.7), mutation E484Q in B.l.617.1 /B.1.617.3 variants and mutation T478K in B.l.617.2 variant. However, it is also possible that mutations may affect antibody binding indirectly by altering spatial conformation of the spike trimers, making them less or more accessible to the antibodies.

[00603] It has been shown in Examples 2, 5, 6 that Formulation X can elicit strong peptide- specific IgG antibodies that are capable of recognizing and binding to the full spike protein of the non-mutated SARS-CoV-2. The objectives of the study described in this Example were to evaluate capability of antibodies elicited by Formulation X to recognize and bind to the mutated variants of SARS-CoV-2 S protein.

[00604] Commercially available mutated and non-mutated S proteins were used to assess the effects of mutation on antibody binding by indirect ELISA. The assessment was performed for two commercially available mutated spike proteins that represent Variants of Concern (VOC) B.l.1.7 and B.1.351, and contain the E484K mutation.

[00605] CD-1 mice (n=10) were vaccinated with Formulation X on SD0 and were boosted on SD14. Vaccinations were performed as described in Example 6. Serum samples were collected on SD56 and profiled by indirect ELISA with plates coated with mutated and non-mutated S1 subunit of S protein and with mutated (E484K) and non-mutated COV2B-S461 peptide in parallel (Figure 34A-34B).

[00606] Results of this experiment indicated that mutations in the S1 subunit of spike protein commonly associated with the Variants of Concern B.1.1.7 andB.1.351, including E484K mutation do not decrease binding capacity of Formulation X antibodies (Figure 34A-34B).

[00607] Method: Indirect ELISA

[00608] Indirect enzyme-linked immunosorbent assays (ELISA) were performed to detect serum peptide- or protein-specific antibody titers. Briefly, 96-well EIA/RIA Clear Flat-Bottom Polystyrene High Bind Microplates (Corning®) were coated with 1 μg/rnL of mutated and non- mutated peptide COV2B-S461 in coating buffer (18.9 mM NaHCO₃, 27.5 Na₂CO₃, 3.08 mM NaN₃, pH 9.5) or with mutated and non-mutated S1 protein diluted in manufacturer’s recommended coating buffer (136.9 mM NaCl, 10.1 mM Na₂HPO₄, 2.7 mM KC1, 1.8 mM KH₂PO₄, pH 7.4) for a final concentration of 1 μg/mL and incubated overnight at 4°C. Plates were washed five times with TBS-T (0.2%) and blocked with warmed 3% Gelatin (BioRad) for 30-45 minutes at 37°C. Plates were washed with TBS-T and incubated overnight at 4°C with sera at an initial starting dilution of 1:100. After TBS-T washes, bound antibodies were detected by incubation of alkaline phosphatase conjugated Protein G (EMD Millipore-Sigma) with high affinity binding for IgG for 1 hour at 37°C and subsequent development with chromqgenic alkaline phosphatase substrate. Optical density was measured at 405 nm within 1 hour of initial substrate addition on a spectrophotometer plate reader. ELISA results were expressed as end point Log 10 titers using a calculation method described by Frey et al. (Frey et al. 1998). Each serum sample was diluted at 1 : 100 as a starting dilution. Titers that did not fall under the calculated cut-off at the highest dilution, or titers that resulted from a high background were repeated to ensure the endpoint titers fall within prepared dilutions and remove any background signal, respectively.

[00609] An immunogenic response definition of a peptide-specific antibody titer ≥Log 10=2 was used to determine the number of responders within each group at each timepoint.

[00610] EXAMPLE 8. Toxicology Studies.

[00611] A Formulation X repeated dose intramuscular GLP Toxicity Study was conducted in Sprague-Dawley Rats to evaluate the potential local and/or systemic toxic effects of the test item, Formulation X, following 3 intramuscular injection administrations to Sprague-Dawley rats and to assess the persistence, delayed onset or reversibility of any changes following a 28-day recovery period.

[00612] The test articles, vehicle control (Formulation Z) and PBS-control items were administered to groups of rats on Days 0, 14 and 28 by intramuscular injection as described in Table 53.

Table 53. Study Design.

a PBS -control animals received the control item, PBS, alone. b Vehicle control animals received the vehicle control item, Formulation Z.

[00613] Parameters monitored during this study included mortality, clinical observations, body weight, dermal changes (using a modified Draize scoring scheme), hematology, coagulation, clinical chemistry, organ weights, macroscopic and microscopic examinations. In addition, blood samples were collected at termination (Day 30 Main animals and Day 56 Recovery animals) to quantify levels of T3, T4 and cytokines as well as for an assessment of immunogenicity.

[00614] There were no Formulation X-related deaths, clinical observations, or effects on body weight, hematology, coagulation, T3 and T4 concentrations and organ weights at doses up to 0.050 mg/dose.

[00615] The administration of Formulation Z vehicle control and Formulation X at 0.025 and 0.050 mg/dose resulted in erythema ranging from very slight (barely perceptible) to moderate to severe, edema ranging from very slight (barely perceptible) to well-defined/slight, increased induration, desquamation and presence of focal red spot in a few animals at the end of the treatment period. In general, there was no difference in severity/incidence between the two dosing sites. Following the 28-day recovery period, local skin reactions were still noted in the 0.025 and 0.050 mg/dose Formulation X groups, however, the severity/incidence was lower than at the end of the treatment period indicative of a partial, ongoing recovery.

[00616] When compared to PBS-controls, increases in creatine kinase (CK) and lactate dehydrogenase (LDH) were noted on Day 30 in the Formulation Z vehicle control females and the Formulation X females. Following the 28-day recovery period, there were no changes in any of the clinical chemistry parameters, indicating complete recovery.

[00617] At the end of treatment (Day 30), microscopic findings at the dosing site included accumulation of foreign material (considered to be Montanide® ISA 51 VG) which was noted in the dosing sites of Formulation Z-vehicle control rats and rats dosed with 0.025 and 0.050 mg/dose of Formulation X. The foreign material was surrounded by macrophage infiltrate in rats dosed with the Formulation Z-vehicle control and rats dosed with 0.025 and 0.050 mg/dose of Formulation X.

[00618] Other microscopic changes were aligned along the periphery of the foreign material and mixed with the macrophage infiltrate and characterized by various cell infiltrates including: lymphoplasmacytic infiltrate, mixed cell infiltrate, and fibrosis in rats dosed with 0.025 and 0.050 mg/dose of Formulation X. Myofiber degeneration was noted at the dosing sites of rats from all treatment groups, including the PBS and Formulation Z-vehicle control group. Hematoma with concurrent hemosiderin pigment were noted in the dosing sites of three female rats dosed with of 0.050 mg/dose of Formulation X, correlating with macroscopic findings. Microscopic changes in the inguinal and popliteal lymph nodes included increased cellularity of the follicular and paracortical lymphocytes and medullary cord plasma cells and sinus histiocytosis with similar incidence and severity in rats dosed with 0.025 and 0.050 mg/dose of Formulation X. The lymph node findings were considered to be associated, predominantly with the inflammatory changes in the dosing sites. Increased lymphocyte and plasma cell cellularity were noted occasionally in Formulation Z-vehicle control females suggesting these findings were, at least partially, vehicle- related. Microscopic findings at the end of the recovery period were similar to the ones noted at the end of the treatment period however they were at a lower incidence and/or severity indicating incomplete but progressive on-going reversal following a 28-day recovery period.

[00619] There were no major differences observed in the cytokines and chemokines examined and no dose-dependent effects were observed with Formulation X at 2 days post vaccination, at the end of the treatment period, and following the 28-day recovery period.

[00620] In conclusion, the intramuscular injection of Formulation X on Days 0, 14 and 28 to Sprague-Dawley rats resulted in local skin reactions, however, as the severity and incidence of the dermal findings were in general similar between the Formulation X dose groups and the Formulation Z vehicle control group, it is suggested that the local reactions were most probably a result of the use of the lipid-based formulation (Montanide® ISA 51 VG) rather than the antigen. Partial, ongoing recovery was noted in the 0.025 and 0.050 mg/dose Formulation X animals following the 28-day recovery period. In addition, an increase in creatine kinase and lactate dehydrogenase was noted in the females of the Formulation Z vehicle control and the Formulation X dose groups.

[00621] Although the increase was noted in the Formulation Z (vehicle control group), the magnitude of the effect appeared to be slightly more elevated in the 0.050 mg/dose Formulation X group. The increases in creatine kinase and lactate dehydrogenase were suggestive of muscle damage and inflammation as they correlated with the local lesions (erythema and edema) as well as macroscopic and microscopic lesions observed at the dosing sites. Complete recovery of the effect on creatine kinase and lactate dehydrogenase was noted following the 28-day recovery period. At the end of the treatment period, macroscopic and microscopic findings related to the test item, Formulation X, were noted at the dosing site of the caudal thigh muscle and the inguinal and popliteal lymph nodes at doses of 0.025 and 0.050 mg/dose. Similar findings were noted following the 28-day recovery period, however at a lower incidence and/or severity indicating incomplete but progressive on-going reversal.

[00622] Formulation X at both 0.025 and 0.050 mg/dose did not appear to induce the systemic release of cytokines and chemokines involved in inflammation and T cell polarization at the end of the treatment period and following the 28-day recovery period.

[00623] Immunogenicity results.

[00624] The antibody response to each of the 4 peptide antigens in Formulation X (COV2B- S616D, COV2B-S821D, COV2B-S373D, and COV2B-S461D) was assessed by indirect ELISA (see Figure 35A-35B and Table 54).

[00625] Table 54. Endpoint titer ranges and total number of responders in group vaccinated with Formulation X L (25 μg) and H (50 μg) dose strength.

[00626] Tabulated Log₁₀ endpoint antibody titers (min-max) and number of responders for each peptide. An immunogenic response definition of a peptide-specific antibody titer ≥Log₁₀ 400=2.60 was used to determine the number of responders within each group at each timepoint.

[00627] Sera from rats in groups 2, 3 and 4 were tested in triplicate for the presence of antigen specific IgG antibodies using an indirect ELISA method with rat sera tested at sera dilution levels ranging from 1:400 to 1:64000 to cover the range of responses seen in rat sera against the 4 peptides (COV2B-S616D, COV2B-S461D, COV2B-S373D and COV2B-S821D).

[00628] At the end of treatment, SD 30, a similar immune response was generated in rats vaccinated with the Formulation X drug products with both 0.025 and 0.050 mg/dose against all 4 peptide antigens in formulation (COV2B-S616D, COV2B-S821D, COV2B-S373D, and COV2B- S461D). The immune response generated was specific to the peptide antigens as minimal immune response was generated in the rats vaccinated with the Formulation Z vehicle control .

[00629] Following the 28-day recovery period, SD 56, similar immune response was generated in rats vaccinated with the Formulation X drug products with both 0.025 and 0.050 mg/dose against 3 peptide antigens in formulation (COV2B-S821D, COV2B-S373D, and COV2BS461D).

[00630] A statistically significant difference (Student T-test p-value = 0.02) was seen when comparing immune responses generated by the 0.25 mg/dose and 0.050 mg/dose of Formulation X against COV2B-S616D peptide antigen. The immune response generated was specific to the peptide antigens as minimal immune response was generated in the rats vaccinated with the Formulation Z vehicle control.

[00631] In general, more rats appeared to generate a measurable immune response upon vaccination with the 50 μg/dose, but the magnitude of these responses after three vaccinations was not significantly different.

[00632] Additional Antibody Response Characterization

[00633] To further characterize the functionality of the antibody response elicited by Formulation X in the rat model using sera obtained from the GLP toxicology study, the inventors evaluated the ability of antibodies induced by Formulation X to bind the SARS-CoV-2 spike protein in indirect ELISA using a commercially available S protein. The data are presented in Figure 36. It was observed that rats vaccinated with the higher dose of Formulation X (50 μg/dose) generated stronger spike binding antibodies than those vaccinated with the lower dose and this immune response was increased at the later timepoint, which may indicate some antibody maturation over time.

[00634] The data presented in Examples 1, 2 and 5-6 demonstrate the highly targeted nature of Formulation X based immune activation. The ability of Formulation X to generate a balanced Th1/Th2 immune responses is demonstrated by the peptide simulation of splenocytes and yet the finding that none of these cytokines are found to be elevated systemically (in either mice or rats) underlines that these immune responses appear to be specifically generated in immune organs. This is further shown by the increases in MHC class II expression by B cell subsets, particularly in the vaccine draining lymph nodes of vaccinated animals. The active uptake of Formulation X components by antigen presenting cells and delivery directly to regional lymph nodes is evident in this specific impact on antigen presentation within these organs.

[00635] The GLP toxicology study extends this safety profile, with the major finding being inflammation at the injection site, consistent with the delivery of Montanide® carrier.

[00636] In conclusion, Formulation X can induce specific and targeted immune responses to key areas of the SARS-CoV-2 spike protein that is durable in animal models.

[00637] Methods: Indirect ELISA

[00638] For peptide-specific antibody titers, in brief, 96-well MaxiSorp ELISA Microplates (Biolegend) were coated with each Formulation X peptide in coating buffer (phosphate buffered saline, PBS, pH 6.2) for a final concentration of 5 μg/mL and incubated overnight at 4°C. Plates were washed five times with TBS-T (0.05%) and blocked with 1×casein in TBS (Surmodics) for 1 hour at 37°C, with 300 rpm shaking. Plates were washed with TBS-T and incubated with 2-fold serial dilutions of sera in lxcasein for 1.5-2.0 hours at 37°C, with 500 rpm shaking. After TBS-T washes, bound antibodies were detected by incubation of horseradish peroxidase conjugated Protein G (Thermofisher) for 1 hour at 37°C, with 500 rpm shaking, and subsequent development with Super Sensitive TMB substrate (Surmodics). Optical density was measured at 450 nm within 10 minutes of initial adding the stop solution on a spectrophotometer plate reader. ELISA results were expressed as end point LoglO titers using a calculation method described by Frey et al. (Frey et al. 1998). Each serum sample was diluted at 1:400 as a starting dilution. Endpoint titer values are defined as the inverse of the greatest dilution above the assay cutoff (mean naive OD + 2SD).

[00639] For S-protein specific-antibody titers, in brief, 96-well EIA/RIA Clear Flat-Bottom Polystyrene High Bind Microplates (Corning®) were coated with 1 μg/mL of Spike S1+S2 ECD- His Recombinant Protein in coating buffer: 136.9 mM NaCl, 10.1 mM Na₂HPO₄, 2.7 mM KC1, 1.8 mM KH2PO4, pH 7.4, overnight at 4°C. Plates were washed five times with TBS-T (0.2%) and blocked with commercial Chondrex ChonBlock, 150 μL/well for 1 to 1.5 hours at room temperature. Serum samples were diluted 2-fold in Chonblock solution starting at an initial dilution of 1:100 and incubated overnight on plates at 4°C. After TBS-T washes, bound antibodies were detected by incubation with alkaline phosphatase conjugated anti-rat IgG detection antibody (Sigma A6066), diluted 1:10000 in ChonBlock for 1 hour at room temperature and subsequent development with chromogenic alkaline phosphatase substrate. Optical density was measured at 405 nm within 1 hour of initial substrate addition on a spectrophotometer plate reader. ELISA results were expressed as end point LoglO titers using a calculation method described by Frey et al. (Frey et al. 1998).

[00640] EXAMPLE 9. Formulation with B and T cell Epitopes

[00641] After identifying the four B cell peptides that elicited the highest immune response (SEQ ID NOs: 5, 7, 14 and 19), T cell epitopes were also studied to obtain a vaccine formulation that could elicit both humoral and cellular immune responses.

[00642] HLA-A1, -A2, -A3, -A1l, -A24 restricted and promiscuous MHC-II T cell SARS CoV2 epitopes were selected based on bioinformatics and literature. In vitro and in vivo testing were conducted to select the T cell epitopes to combine with the four B cell epitopes (SEQ ID NOs: 5, 7, 14 and 19) included in the Formulation X.

[00643] Based on the bioinformatics and literature data, fourteen T cell epitopes were selected, listed in Table 55. [00644] First binding of the peptide to the HLA-A1.-A2, and -A3 alleles was tested in vitro using Flex-T HLA Binding Assay (BioLegend) according to the manufacturer instructions. Results of the in vitro binding assay are listed in Table 55.

[00645] Table 55. Selected T cell epitopes and bioinformatics.

[00646] Next, immimogenicity of HLA-A1, -A2 and -A24 peptides were tested in vivo in three transgenic mouse strains: (1) mouse strain that expresses human HLA-A1; (2) mouse strain that expresses human HLA-A2; and (3) mouse strain that expresses human HLA-A24. Mouse strains to test immunogenicity of HLA- A3 and HLA-A11 restricted peptides were not available for the analysis. [00647] Mice (n=8 per treatment) were immunized by a subcutaneous (SC) inj ection in the right flank. Treatment groups are described in Table 56. Mice were terminated 8 days post treatment when spleens were collected to assess peptide-specific IFN-γ production by ELISPOT.

[00648] Table 56 Study design

* Formulations contain no adjuvant or T helper.

[00649] Results are shown in Table 57 and Figure 37 (A-C). Detectable immune responses were elicited by peptides COV2T-N222, COV2T-S1220, COV2T-ORF1AB-4163, COV2T-S444, COV2T-ORF1AB-5299.

[00650] Table 57. Peptide-specific immune responses in spleens eight days post vaccination with homogeneous water-free formulation (DPX) containing listed SARS-CoV-2 peptides.

[00651] Based on the results of these initial screening studies and on the results of formulation compatibility studies, the peptides COV2T-S1220 (SEQ ID NO: 28), -S444 (SEQ ID NO: 34), and -ORF1 AB-5299 (SEQ ID NO: 38) were selected for further development.

[00652] COV2T-S1220, -S444, and -ORFlAB-5299 were combined in one homogeneous water-free formulation (DPX) together with COV2B-S373D, -S461D, -S616D and -S821D B cell peptides from Formulation X. This new formulation, named Formulation Y, also contained one MHC-n restricted SARS-CoV-2 peptide (COV2T-ORF1AB-3906, EAFEKMVSLLSVLLS, SEQ ID NO: 43), an additional T cell helper peptide A16L (tetanus toxin universal T-helper peptide TT_830-843 (AQYIKANSKFIGITEL (SEQ ID NO: 42)), and a dI:dC adjuvant. The final candidate Formulation Y contained fourB cell peptides dimer COV2B-S373D, -S461D, -S616D and -S821D each at 0.5 mgZmL concentration; four T cell peptides monomer COV2T-S1220, -S444, - ORF1 AB-3906 and -ORAFlAB-5299 each at 1 mg/mL; a T-helper peptide A16L at 0.25 mgZmL; and a DNA based polynucleotide dI:dC adjuvant at 0.4 mg/mL. Briefly, Formulation Y was prepared as follows: peptide stocks were prepared first in pools A and B. Peptide pool A contained COV2T-S1220, -S444, -ORF1AB-3906 and COV2B-S821D peptides dissolved in 65 mM sodium acetate pH 12.5 solution. Peptide pool B contained COV2T-ORAF1 AB-5299, COV2B-S373D and -616D peptides dissolved in 65 mM sodium acetate pH 8.0 solution. A16L peptide stock was prepared in 0.125% acetic add and COV2B-461D peptide stock was prepared in 0.5% acetic acid. Polynucleotide dI:dC adjuvant stock was prepared in sterile water. Formulation Y was prepared by adding peptide Pool A to previously sized DOPC/Chol lipid nanoparticles (<100 nm, pdi<0.1), followed by the sequential addition of A16L peptide, 46 ID peptide, peptide Pool B and polynucleotide dI:dC adjuvant to it. The mixture was then aseptically filled into 3 raL vials, lyophilized and stored at -20°C, until reconstituted in Montanide ISA 51 VG (Seppic, France) oil diluent for animal administration. [00653] In vivo assay to evaluate cell-mediated and humoral immune responses elicited by Formulation Y in transgenic mice using IFN-γ ELISPOT was performed.

[00654] HLA-A1, -A2 and -A24 transgenic mice were anesthetized for vaccine treatment on study Day 0 and 14. Each mouse received a vaccine injection (I.M: intramuscular); 25 μL dose in both caudal thigh muscles (50 μL total dose). Mice were terminated on SD42 and spleens were collected to assess peptide-specific IFN-γ production by ELISPOT. Blood was collected and processed for sera. Serum samples were used to perform indirect ELISA for detection of peptide- specific antibodies. Test groups are shown in Table 58.

[00655] Table 58. Treatment groups

Ό0656] Results are shown in Table 59 and Figure 38 (A-C). Strong and robust immune responses were elicited by all three T cell epitopes (COV2T-S1220, -S444, and -ORF1AB-5299) in Formulation Y 42 days after prime vaccination with Formulation Y, that were also evident in the samples stimulated with peptide pool.

[00657] Table 59. Peptide-specific immune responses in spleens of transgenic mice elicited by Formulation Y.

00658] As expected, no peptide-specific immune responses were detected in mice vaccinatet with Formulation Z (homogeneous water-free formulation (DPX) containing no peptide antigens). This indicated that peptides formulated in DPX are key components to generation of antigen- specific immune responses.

[00659] Peptide-specific immune responses induced by Formulation Y were drastically stronger than the immune responses to the same peptide induced by Formulations A1, A2 and A3 (Table 60). Differently from Formulation Y, Formulations A1, A2 and A3 did not contain T helper peptide or an adjuvant and were administered once. This suggests that inclusion of T helper and an adjuvant in the formulation and boost vaccination strongly increases peptide-specific immune responses.

[00660] Table 60. Peptide-spedfic immune responses in spleens of transgenic mice elidted by Formulation Y (containing T helper peptide and an adjuvant) and by Formulations A1, A2, and A3 (containing no T helper peptide or an adjuvant).

[00661] Indirect ELISA was used to measure peptide-spedfic antibody responses to Formulation Y. [00662] As shown in Figure 39, antibody responses to the COV2B-S821 peptide were detected in 7/10 HLA-A2 transgenic mice vaccinated with Formulation Y. No antibody responses were elicited by other peptides in Formulation Y in HLA-A2 mice. HLA-A2 transgenic mice express human HLA-A2 and HLA-DRl alleles, but do not express mouse MHC-I or MHC-II. This suggests that COV2B-S821 peptide may be presented by human HLA-DRl allele and other peptides in the Formulation Y are not specific to the human HLA-DRl allele.

[00663] Similar to the results obtained in CD-1 mice vaccinated with Formulation X

(Examples 2, 5,6), Formulation Y induces COV2B-S373 and -S461 antibody responses in HLA-

A1 or -A24 transgenic mice. HLA-A 1 transgenic mice express human HLA-A 1 allele and HLA- A24 transgenic mice express human HLA-A24 allele. These mice do not express human MHC-II alleles, but express limited mouse MHC-II alleles.

[00664] As shown in this Example, the Formulation Y is capable of inducing peptide- specific humoral and cellular immune responses. Strong, robust and sustained cellular responses to all three HLA-A peptides in the formulation are detectable up to four weeks after a boost vaccination.

[00665] Methods

[00666] Indirect ELISA

[00667] Indirect enzyme-linked immunosorbent assays (ELISA) were performed to detect serum peptide-specific antibody titers. Briefly, 96-well EIA/RIA Clear Flat-Bottom Polystyrene High Bind Microplates (Corning®) were coated with 1 μg/mL of individual peptides in coating buffer (NaHCO₃, Na₂CO₃) and incubated overnight at 4°C. Research grade monomer peptides were used for coating. Plates were washed five times with TB S-T (0.2%) and blocked with warmed 3% Gelatin (BioRad) for 30-45 minutes at 37°C. Plates were washed with TBS-T and incubated overnight at 4°C with sera at an initial starting dilution of 1:100. After TBS-T washes, bound antibodies were detected by incubation of alkaline phosphatase conjugated Protein G (EMD Millipore-Sigma) with high affinity binding for IgG for 1 hour at 37°C and subsequent development with chromogenic alkaline phosphatase substrate. Optical density was measured at 405 ran within 1 hour of initial substrate addition on a spectrophotometer plate reader. ELISA results were expressed as end point Log 10 titers using a calculation method described by Frey et al. (Frey et al. 1998). Each serum sample was diluted at 1 :100 as a starting dilution. Titers that did not fall under the calculated cut-off at the highest dilution, or titers that resulted from a high background were repeated to ensure the endpoint titers fall within prepared dilutions and remove any background signal, respectively.

[00668] An IFN-γ ELISPOT was performed as described below. Single cell suspensions of splenocytes were prepared by lysing RBCs with ammonium-chloride-potassium solution and resuspended at 5 x 10⁶ cells/mL in complete RPMI media. A 100 μL volume of cells was added into ELISPOT plates (BD Bioscience) and stimulated with 100 μL of complete RPMI containing no peptide (background control), 10 μg/mL of COV2 peptide (individually and pooled) or with irrelevant peptide in duplicate. ELISPOT plates were incubated overnight at 37°C, 5% C02. Plates were developed the next day as per the manufacturer’s instructions using AEC substrate (Sigma- Aldrich). Spots were enumerated using an ImmunoSpot Analyzer (C.T.L. Ltd, Shaker Heights, OH) as the number of spot-forming units (SFU) per well.

[00669] Non-exclusive list of items.

1. A composition comprising: at least one B cell epitope of SARS-CoV-2; a carrier; liposomes; optionally at least one T cell epitope; and optionally an adjuvant.

2. The composition of item 1, wherein the at least one B cell epitope is a peptide present in the SARS-CoV-2 spike protein.

3. The composition of items 1 or 2, wherein the at least one B cell epitope comprises an amino acid sequence from the SARS-CoV-2 spike protein (SEQ ID NO: 1) that is capable of eliciting neutralizing antibodies (NAbs) in a subject. 4. The composition of any of items 1-3, wherein the at least one B cell epitope comprises at least one of amino acid sequences

a nucleic acid molecule encoding said epitope, optionally wherein the at least one B cell epitope is in the form of a dimer.

5. The composition of item 1, wherein the at least one B cell epitope comprises the amino acid sequence of SEQ ID NOs: 5, 7, 14 or 19, optionally wherein the at least one B cell epitope is in the form of a dimer.

6. The composition of any of items 1-5, wherein the carrier comprises a continuous phase of a hydrophobic substance.

7. The composition of any of items 1-6, wherein the carrier is a hydrophobic substance such as a vegetable oil, nut oil, or mineral oil.

8. The composition of item any of items 1-7, wherein the carrier is mineral oil or is a mannide oleate in a mineral oil solution.

9. The composition of item any of items 1-8, wherein the carrier is Montanide® ISA 51.

10. The composition of any of items 1-9, wherein the liposomes comprise 1 ,2-Dioleoyl - sn-glycero- 3-phosphocholine (DOPC) and cholesterol. 11. The composition of any of items 1-10, wherein the optional adjuvant is a lipid-based adjuvant, a lipopolynucleotide-based adjuvant, and/or a polynucleotide-based adjuvant.

12. The composition of item 11, wherein the lipid-based adjuvant is PAM3CSK4, and/or the polynucleotide-based adjuvant is poly(dI:dC).

13. The composition of any of items 1-12, wherein the at least one B cell epitope is administered at a concentration of about 0.2 mg/ml to about 1 mg/ml for each B cell peptide, including about 0.2 mg/ml to about 0.5 mg/ml and 0.5 mg/ml to 1 mg/ml.

14. The composition of any of items 1-12, wherein the at least one B cell epitope is administered at a dose of about 0.01 ml to about 1 ml.

15. The composition of any of items 1-13, wherein the at least one B cell epitope is administered in at least one dose or in at least two doses.

16. The composition of any of items 1-15, wherein the composition is an immunogenic composition.

17. The composition of item 16, wherein the composition elicits an immune response against SARS-CoV-2 in a subject.

18. The composition of any of items 1-16, wherein the composition comprises a mixture of at least four B cell epitopes comprising the amino acid sequences of SEQ ID NOs: 5, 7, 14 and 19.

19. The composition of any of items 1-16, wherein the composition comprises a B cell epitope selected from the group consisting of SEQ ID NOs: 5, 7, 14 and 19, and combinations thereof.

20. The composition of any of items 1-19, wherein the B cell epitope has an amino add sequence with at least one mutation relative to the amino add sequence of SEQ ID NOs: 5, 7, 14 and/or 19.

21. A method of administering an immunogenic composition to a subject in need thereof, the method comprising: injecting the immunogenic composition into the subject, wherein the immunogenic composition comprises: at least one B cell epitope of SARS-CoV-2; a carrier; liposomes; optionally at least one T cell epitope; and optionally an adjuvant.

22. The method of item 21, wherein the injection is a subcutaneous or intramuscular injection.

23. The method of items 21 or 22, wherein the at least one B cell epitope is a peptide present in the SARS-CoV-2 spike protein.

24. The method of any of items 21-23, wherein the at least one B cell epitope comprises an amino acid sequence from the SARS-CoV-2 spike protein (SEQ ID NO: 1) that is capable of eliciting neutralizing antibodies (NAbs) in a subject.

25. The method of any of items 21-24, wherein the at least one B cell epitope comprises at least one of amino acid sequences SYGFQPTNGVGYQPY (SEQ ID NO: 2); GDEVRQIAPGQTGKIADYNYKLP (SEQ ID NO: 3); VRFPNITNLCPFGE (SEQ ID NO: 4); LLFNKVTLADAGFIKQYGDCLGDIAA (SEQ ID NO: 5); GCVIAWNSNNLDSKVGG (SEQ ID NO: 6); LKPFERDISTEIYQAGSTPCNGVEGFN (SEQ ID NO: 7); GFQPTNGVGY QPY (SEQ ID NO: 8); ELLHAPATVCGPKKSTNLVKN (SEQ ID NO: 9); RVYSTGSNVFQ (SEQ ID N: 10); DLGDISGINASWNIQK (SEQ ID NO: 11); VCGPKKSTNLVKN (SEQ ID NO: 12); KNHTSPDVDLGDISGIN (SEQ ID NO: 13); NCTEVPVAIHADQLTPT (SEQ ID NO: 14); SCCKFDEDDSEPVLKG (SEQ ID NO: 15); ASYQTQTNSPRRARSVASQ (SEQ ID NO: 16); YNSASFSTFKCYGVSPTKLNDLCFT (SEQ ID NO: 17); TPGDSSSGWTA (SEQ ID NO: 18); SFSTFKCYGVSPTKLNDL (SEQ ID NO: 19); SNKKFLPF (SEQ ID NO: 20); PDPSKPSK (SEQ ID NO: 21); EIDRLNEVAKNLNESLIDLQELGKYEQY (SEQ ID NO: 22); FNCYFPLQS YGFQPTNGV GY QPYRVWLSFE (SEQ ID NO: 23);

FPNITNLCPFGEVFNATRFASVYAWNRKRISNCVA (SEQ ID NO: 24);

TESNKKFLPFQQFGRDIA (SEQ ID NO:25); PSKPSKRSFIEDLLFNKV (SEQ ID NO:26), or a nucleic acid molecule encoding said epitope, optionally wherein the at least one B cell epitope is in the form of a dimer.

26. The method of any of items 21-25, wherein the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 5, 7, 14, or 19. 27. The method of any of items 21-26, wherein the carrier comprises a continuous phase of a hydrophobic substance.

28. The method of any of items 21-27, wherein the carrier is a hydrophobic substance such as a vegetable oil, nut oil, or mineral oil.

29. The method of any of items 21-28, wherein the carrier is mineral oil or is a mannide oleate in a mineral oil solution.

30. The method of any of items 21-29, wherein the carrier is Montanide® ISA 51.

31. The method of any of items 21-30, wherein the liposomes comprise 1,2-Dioleoyl-sn-glycero- 3-phosphocholine (DOPC) and cholesterol.

32. The method of any of items 21-31, wherein the optional adjuvant is a lipid-based adjuvant, a lipopolynucleotide-based adjuvant, and/or a polynucleotide-based adjuvant.

33. The method of item 32, wherein the lipid-based adjuvant is PAM3CSK4, and/or the polynucleotide-based adjuvant is poly(dI:dC).

34. The method of any of items 21-33, wherein the at least one B cell epitope is administered at a concentration of about 0.2 mg/ml to about 1 mg/ml for each B cell peptide, including about 0.2 mg/ml to about 0.5 mg/ml and 0.5 mg/ml to 1 mg/ml.

35. The method of any of items 21-33, wherein the at least one B cell epitope is administered at a dose of about 0.01 ml to about 1 ml.

36. The method of any of items 21-35, wherein the at least one B cell epitope is administered in at least one dose or in at least two doses.

37. The method of any of items 21-36, wherein the composition elicits an immune response against SARS-CoV-2 in a subject.

38. The method of any of items 21-37, wherein the composition comprises a mixture of at least four B cell epitopes comprising the amino acid sequences of SEQ ID NOs: 5, 7, 14 and 19.

39. The method of any of items 21-37, wherein the composition comprises a B cell epitope selected from the group consisting of SEQ ID NOs: 5, 7, 14 and 19, and combinations thereof. 40. The method of any of items 21-39, wherein the B cell epitope has an amino acid sequence with at least one mutation relative to the amino acid sequence of SEQ ID NOs: 5, 7, 14 and/or 19.

41. A method of eliciting an immune response against SARS-CoV-2 in a subject in need thereof, the method comprising: injecting the immunogenic composition into the subject, wherein the immunogenic composition comprises: at least one B cell epitope of SARS-CoV-2; a carrier; liposomes; optionally at least one T cell epitope; and optionally an adjuvant.

42. The method of item 41, wherein the injection is a subcutaneous or intramuscular injection.

43. The method of items 41 or 42, wherein the at least one B cell epitope is a peptide present in the SARS-CoV-2 spike protein.

44. The method of any of items 41-43, wherein the at least one B cell epitope comprises an amino acid sequence from the SARS-CoV-2 spike protein (SEQ ID NO: 1) that is capable of eliciting neutralizing antibodies (NAbs) in a subject.

45. The method of any of items 41-44, wherein the at least one B cell epitope comprises at least one of amino acid sequences SYGFQPTNGVGYQPY (SEQ ID NO: 2); GDEVRQIAPGQTGKIADYNYKLP (SEQ ID NO: 3); VRFPNITNLCPF GE (SEQ ID NO: 4); LLFNKVTLADAGFIKQYGDCLGDIAA (SEQ ID NO: 5); GCVIAWNSNNLDSKVGG (SEQ ID NO: 6); LKPFERDISTEIYQAGSTPCNGVEGFN (SEQ ID NO: 7); GFQPTNGVGYQPY (SEQ ID NO: 8); ELLHAPATVCGPKKSTNLVKN (SEQ ID NO: 9); RVYSTGSNVFQ (SEQ ID N: 10); DLGDISGINASWNIQK (SEQ ID NO: 11); VCGPKKSTNLVKN (SEQ ID NO: 12); KNHTSPDVDLGDISGIN (SEQ ID NO: 13); NCTEVPVAIHADQLTPT (SEQ ID NO: 14); SCCKFDEDDSEPVLKG (SEQ ID NO: 15); ASYQTQTNSPRRARSVASQ (SEQ ID NO: 16); YNSASFSTFKCYGVSPTKLNDLCFT (SEQ ID NO: 17); TPGDSSSGWTA (SEQ ID NO: 18); SFSTFKCYGVSPTKLNDL (SEQ ID NO: 19); SNKKFLPF (SEQ ID NO: 20); PDPSKPSK (SEQ ID NO: 21); EIDRLNEVAKNLNESLIDLQELGKYEQY (SEQ ID NO: 22); FNCYFPLQS YGFQPTNGV GY QPYRVWLSFE (SEQ ID NO: 23); FPNITNLCPFGEVFNATRFASVYAWNRKRISNCVA (SEQ ID NO: 24);

46. The method of any of items 41-45, wherein the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 5, 7, 14, or 19.

47. The method of any of items 41-46, wherein the carrier comprises a continuous phase of a hydrophobic substance.

48. The method of any of items 41-47, wherein the carrier is a hydrophobic substance such as a vegetable oil, nut oil, or mineral oil.

49. The method of any of items 41-48, wherein the carrier is mineral oil or is a mannide oleate in a mineral oil solution.

50. The method of any of items 41-49, wherein the carrier is Montanide® ISA 51.

51. The method of any of items 41-50, wherein the liposomes comprise 1 ,2-Dioleoyl-sn-glycero- 3 -phosphocholine (DOPC) and cholesterol.

52. The method of any of items 41-51, wherein the optional adjuvant is a lipid-based adjuvant, a lipopolynucleotide-based adjuvant, and/or a polynucleotide-based adjuvant.

53. The method of item 52, wherein the lipid-based adjuvant is PAM3CSK4, and/or the polynucleotide-based adjuvant is poly(dI:dC).

54. The method of any of items 41-53, wherein the at least one B cell epitope is administered at a concentration of about 0.2 mg/ml to about 1 mg/ml for each B cell peptide, including about 0.2 mg/ml to about 0.5 mg/ml and 0.5 mg/ml to 1 mg/ml.

55. The method of any of items 41-54, wherein the at least one B cell epitope is administered at a dose of about 0.01 ml to about 1 ml.

56. The method of any of items 41-55, wherein the at least one B cell epitope is administered in at least one dose or in at least two doses. 57. The method of any of items 41-56, wherein the composition comprises a mixture of at least four B cell epitopes comprising the amino acid sequences of SEQ ID NOs: 5, 7, 14 and 19.

58. The method of any of items 41-56, wherein the composition comprises a B cell epitope selected from the group consisting of SEQ ID NOs: 5, 7, 14 and 19, and combinations thereof.

59. The method of any of items 41-58, wherein the B cell epitope has an amino acid sequence with at least one mutation relative to the amino acid sequence of SEQ ID NOs: 5, 7, 14 and/or 19.

60. A method of making a composition, the composition comprising at least one B cell epitope of SARS-CoV-2, a carrier, liposomes, and optionally an adjuvant and/or at least one T cell epitope of SARS-CoV-2, wherein the method comprises:

(a) forming liposomes by solubilizing a phospholipid in an organic solvent, followed by filtering the resulting solution and then drying the solution to remove the solvents and form the liposomes;(b) introducing the at least one B cell epitope and the optional adjuvant and/or T cell epitope into an aqueous solution after formation of the lipid bilayers of the liposomes;

(c) lyophilizing the preparation of the at least one B cell epitope, optional adjuvant and/or T cell epitope, and liposomes; and

(d) reconstituting the preparation of the at least one B cell epitope, optional adjuvant and/or T cell epitope, and liposomes in a hydrophobic carrier.

61. The composition of any of items 1-20, wherein the composition further comprises at least one T cell epitope of SARS-CoV-2.

62. The composition of any of items 1-20 and 61, wherein the at least one T cell comprises at least one of SEQ ID NOs: 27-40 and/or 42-43, ), or a nucleic acid molecule encoding said epitope, optionally wherein the at least one T cell epitope is in the form of a dimer.

63. The composition of any of items 1 -20 and 61 -62, wherein the composition comprises a mixture of at least two T cell epitopes comprising the amino acid sequences of SEQ ID NOs: 27, 28, 32, 34, 38, 42 and/or 43.

64. The composition of any of items 1-20 and 61-63, wherein the composition comprises a T cell epitope selected from the group consisting of SEQ ID NOs: 27, 28, 32, 34, 38, 42, 43, and combinations thereof. 65. The composition of any of items 1-20 and 61-64, wherein the T cell epitope has an amino acid sequence with at least one mutation relative to the amino acid sequence of SEQ ID NOs: 27, 28, 32, 34, 38, 42 and/or 43.

66. The method of any of items 21-40, wherein the composition further comprises at least one T cell epitope of SARS-CoV-2.

67. The method of any of items 21-40 and 66, wherein the at least one T cell epitope comprises at least one of SEQ ID NOs: 27-40 and/or 42-43, or a nucleic acid molecule encoding said epitope, optionally wherein the at least one T cell epitope is in the form of a dimer.

68. The method of any of items 21-40 and 66-67, wherein the composition comprises a mixture of at least two T cell epitopes comprising the amino acid sequences of SEQ ID NOs: 27, 28, 32, 34, 38, 42 and/or 43.

69. The method of any of items 21-40 and 66-68, wherein the composition comprises a T cell epitope selected from the group consisting of SEQ ID NOs: 27, 28, 32, 34, 38, 42, 43, and combinations thereof.

70. The method of any of claims 21-40 and 66-69, wherein the T cell epitope has an amino acid sequence with at least one mutation relative to the amino acid sequence of SEQ ID NOs: 27, 28, 32, 34, 38, 42 and/or 43.

71. The method of any of items 41-59, wherein the composition further comprises at least one T cell epitope of SARS-CoV-2.

72. The method of any of items 41-59 and 71, wherein the at least one T cell epitope comprises at least one of SEQ ID NOs: 27-40 and/or 42-43, or a nucleic acid molecule encoding said epitope, optionally wherein the at least one T cell epitope is in the form of a dimer.

73. The method of any of items 41-59 and 71-72, wherein the composition comprises a mixture of at least two T cell epitopes comprising the amino acid sequences of SEQ ID NOs: 27, 28, 32, 34, 38, 42 and/or 43.

74. The method of any of items 41-59 and 71-73, wherein the composition comprises a T cell epitope selected from the group consisting of SEQ ID NOs: 27, 28, 32, 34, 38, 42, 43, and combinations thereof. 75. The method of any of items 41-59 and 71-74, wherein the T cell epitope has an amino acid sequence with at least one mutation relative to the amino acid sequence of SEQ ID NOs: 27, 28, 32, 34, 38, 42 and/or 43.

76. The composition of any of items 1-75, wherein the mutation relative to the amino acid sequences of SEQ ID NOs: 5, 7, 14, 17, 27, 28, 32, 34, 38, 42, and/or 43 is a substitution, an insertion, or a deletion.

77. A composition comprising: at least one B cell epitope selected from the group consisting of SEQ ID NOs: 5, 7, 14, 17, 27, and combinations thereof; at least one T cell epitope selected from the group consisting of SEQ ID NOs: 27, 28, 32, 34, 38, 42, and/or 43; a earner; liposomes; and an adjuvant.

78. The composition of item 77, wherein the at least one T cell epitope is selected from the group consisting of SEQ ID NOs: 28, 34, 38, 42, 43, and combinations thereof.

79. The composition of any of items 77-78, wherein the carrier comprises a continuous phase of a hydrophobic substance.

80. The composition of any of items 77-79, wherein the carrier is a hydrophobic substance such as a vegetable oil, nut oil, or mineral oil.

81. The composition of any of items 77-80, wherein the carrier is mineral oil or is a mannide oleate in a mineral oil solution.

82. The composition of item 81, wherein the carrier is Montanide® ISA 51.

83. The composition of any of items 77-82, wherein the liposomes comprise 1,2-Dioleoyl-sn- glycero-3-phosphocholine (DOPC) and cholesterol.

84. The composition of any of items 77-83, wherein the adjuvant is a lipid-based adjuvant, a lipopolynucleotide-based adjuvant, and/or a polynucleotide-based adjuvant. 85. The composition of item 84, wherein the polynucleotide adjuvant is poly(dI:dC).

86. The composition of item 84, wherein the lipid-based adjuvant is PAM3CSK4.

87. The composition of item 1, wherein the at least one B cell epitope comprises at least one of SEQ ID NOs: 2-25 and the at least one T cell epitope comprises at least one of SEQ ID NOs: 27- 40 and/or 42-43.

88. The method of item 21, wherein the at least one B cell epitope comprises at least one of SEQ ID NOs: 2-25 and the at least one T cell epitope comprises at least one of SEQ ID NOs: 27-40 and/or 42-43.

89. The method of item 41, wherein the at least one B cell epitope comprises at least one of SEQ ID NOs: 2-25 and the at least one T cell epitope comprises at least one of SEQ ID NOs: 27-40 and/or 42-43.

90. The composition of any of items 1-20 and 61-65, wherein the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 5.

91. The composition of any of items 1-20 and 61-65, wherein the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 7.

92. The composition of any of items 1-20 and 61-65, wherein the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 14.

93. The composition of any of items 1-20 and 61-65, wherein the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 19.

94. The method of any of items 21-40 and 66-70, wherein the at least one B cell epitope comprises the amino add sequence of SEQ ID NO: 5.

95. The method of any of items 21-40 and 66-70, wherein the at least one B cell epitope comprises the amino add sequence of SEQ ID NO: 7.

96. The method of any of items 21-40 and 66-70, wherein the at least one B cell epitope comprises the amino add sequence of SEQ ID NO: 14.

97. The method of any of items 21-40 and 66-70, wherein the at least one B cell epitope comprises the amino add sequence of SEQ ID NO: 19. 98. The method of any of items 41-59 and 71-75, wherein the at least one B cell epitope comprises the amino add sequence of SEQ ID NO: 5.

99. The method of any of items 41-59 and 71-75, wherein the at least one B cell epitope comprises the amino add sequence of SEQ ID NO: 7.

100. The method of any of items 41-59 and 71-75, wherein the at least one B cell epitope comprises the amino add sequence of SEQ ID NO: 14.

101. The method of any of items 41-59 and 71-75, wherein the at least one B cell epitope comprises the amino add sequence of SEQ ID NO: 19.

[00670] Sequence listing table

[00671] References.

[00672] Ahmed, S.F.; Quadeer, A. A; McKay, M.R. Preliminary Identification of Potential Vaccine Targets for the COVID-19 Coronavirus (SARS-CoV-2) Based on SARS-CoV Immunological Studies. Viruses. 2020, 12, 254.

[00673] Atyeo C, Fischinger S, Zohar T, Slein MD, Burke J, Loos C, McCulloch DJ, Newman KL, Wolf C, Yu J, Shuey K, Feldman J, Hauser BM, Caradonna T, Schmidt AG, Suscovich TJ, Linde C, Cai Y, Barouch D, Ryan ET, Charles RC, Lauffenburger D, Chu H, Alter G. Distinct Early Serological Signatures Track with SARS-CoV-2 Survival. Immunity. 2020 Sep 15;53(3):524-532.e4. doi: 10.1016/j .immuni.2020.07.020. Epub 2020 Jul 30. PMID: 32783920; PMCID: PMC7392190.

[00674] Barrett JR, Belij-Rammerstorfer S, Dold C, Ewer KJ, Folegatti PM, Gilbride C, Halkerston R, Hill J, Jenkin D, Stockdale L, Verheul MK, Aley PK, Angus B, Bellamy D, Berrie E, Bibi S, Bittaye M, Carroll MW, Cavell B, Clutterbuck EA, Edwards N, Flaxman A, Fuskova M, Gorringe A, Hallis B, Kerridge S, Lawrie AM, Linder A, Liu X, Madhavan M, Makinson R, Mellors J, Minassian A, Moore M, Mujadidi Y, Plested E, Poulton I, Ramasamy MN, Robinson H, Rollier CS, Song R, Snape MD, Tarrant R, Taylor S, Thomas KM, Voysey M, Watson MEE, Wright D, Douglas AD, Green CM, Hill AVS, Lambe T, Gilbert S, Pollard AJ; Oxford COVID Vaccine Trial Group. Phase 1/2 trial of SARS-CoV-2 vaccine ChAdOxl nCoV-19 with a booster dose induces multifunctional antibody responses. Nat Med. 2021 Feb;27(2):279-288. doi: 10.1038/s41591-020-01179-4. Epub 2020 Dec 17. Erratum in: Nat Med. 2021 May 6;: PMID: 33335322.

[00675] Baruah, V, Bose, S. Immunoinformatics-aided identification of T cell and B cell epitopes in the surface glycoprotein of 2019-nCoV. J Med Virol. 2020; 92: 495- 500.

[00676] Bhattacharya, M, Sharma, AR, Patra, P, et al. Development of epitope-based peptide vaccine against novel coronavirus 2019 (SARS-COV-2): Immunoinformatics approach. J Med Virol. 2020; 92: 618- 631.

[00677] Brewer KD, Lake K, Pelot N, Stanford MM, DeBay DR, Penwell A, Weir GM, Karkada M, Mansour M, Bowen CV. Clearance of depot vaccine SPIO-labeled antigen and substrate visualized using MRI. Vaccine. 2014 Dec 5;32(51):6956-6962. doi: 10.1016/j.vaccine.2014.10.058. Epub 2014 Nov 4. PMID: 25444822.

[00678] Brewer KD, Weir GM, Dude I, Davis C, Parsons C, Penwell A, Rajagopalan R, Sammatur L, Bowen CV, Stanford MM. Unique depot formed by an oil based vaccine facilitates active antigen uptake and provides effective tumour control. J Biomed Sci. 2018 Jan 27;25(1):7. doi: 10.1186/S12929-018-0413-9. PMID: 29374458; PMCID: PMC5787234.

[00679] Chaves FA, Lee AH, Nayak JL, Richards KA, Sant AJ. The utility and limitations of current Web-available algorithms to predict peptides recognized by CD4 T cells in response to pathogen infection. J Immunol. 2012 May l;188(9):4235-48. doi: 10.4049/j immunol .1103640. Epub 2012 Mar 30. PMID: 22467652; PMCID: PMC3331894.

[00680] Covián C, Retamal-Díaz A, Bueno SM, Kalergis AM. Could BCG Vaccination Induce Protective Trained Immunity for SARS-CoV-2? Front Immunol. 2020 May 8; 11:970. doi: 10.3389/fimmu.2020.00970. PMID: 32574258; PMCID: PMC7227382.

[00681] Croghan, C, and Egeghy, PP. Methods of Dealing with Values Below the Limit of Detection using SAS. Presented at Southeastern SAS User Group, St. Petersburg, FL, September 22-24, 2003.

[00682] Fast E, Altman RB, Chen B. Potential T-cell and B-cell Epitopes of 2019-nCoV. bioRxiv 2020.02.19.955484; doi: https://doi.org/10.1101/2020.02.19.955484.

[00683] Frey, A, di Canzio, J, Zurabowski, D. A statistically defined endpoint titer determination method for immunoassays. Journal of Immunological Methods; 221 (1998)1 35-41.

[00684] Grifoni, A, Weiskopf, D, Ramirez, S1, Mateus, J, Dan, JM, Moderbacher, CR, Rawlings, SA, Sutherland, A. Targets of T Cell Responses to SAR.S-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals. Cell 2020; 181:1-13

[00685] Koiber B, Fischer WM, Gnanakaran S, Yoon H, Theiler J, Abfalterer W, Hengartner N, Giorgi EE, Bhattacharya T, Foley B, Hastie KM, Parker MD, Partridge DG, Evans CM, Freeman TM, de Silva TI; Sheffield COVID-19 Genomics Group, McDanal C, Perez LG, Tang H, Moon-Walker A, Whelan SP, LaBranche CC, Saphire EO, Montefiori DC. Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus. Cell. 2020 Aug 20;182(4):812-827.el9. doi: 10.1016/j.cell.2020.06.043. Epub 2020 Jul 3. PMID: 32697968; PMCID: PMC7332439.

[00686] Langley JM, MacDonald L, Weir GM, MacKinnon-Cameron D, Ye L, McNeil S, Schepens B, Saelens X, Stanford MM, Halperin SA. A respiratory syncytial virus vaccine based on the small hydrophobic protein ectodomain presented with a novel lipid-based formulation in highly immunogenic and safe in adults: A first in humans study. JID 2018, 218 (3), 378-387).

[00687] McAuley AJ, Kuiper MJ, Durr PA, Bruce MP, Barr J, Todd S, Au GG, Blasdell K, Tachedjian M, Lowther S, Marsh GA, Edwards S, Poole T, Layton R, Riddell SJ, Drew TW, Druce JD, Smith TRF, Broderick KE, Vasan SS. Experimental and in silico evidence suggests vaccines are unlikely to be affected by D614G mutation in SAR.S-CoV-2 spike protein. NPJ Vaccines. 2020 Oct 8;5:96. doi: 10.1038/s41541-020-00246-8. PMID: 33083031; PMCID: PMC7546614.

[00688] Munoz FM, Cramer JP, Dekker CL, Dudley MZ, Graham BS, Gurwith M, Law B, Perlman S, Polack FP, Spergel JM, Van Braeckel E, Ward BJ, Didierlaurent AM, Lambert PH. Vaccine-associated Enhanced Disease: Case Definition and Guidelines for Data Collection, Analysis, and Presentation of Immunization Safety Data. JVAC. 2020 [00689] Munoz-Fontela, C., Dowling, W.E., Funnell, S.G.P. et al. Animal models for COVID- 19. Nature 586, 509-515 (2020). https://doi.org/10.1038/s41586-020-2787-6

[00690] Nie J, Li Q, Wu J, Zhao C, Hao H, Liu H, Zhang L, Nie L, Qin H, Wang M, Lu Q, Li X, Sun Q, Liu J, Fan C, Huang W, Xu M, Wang Y. Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2. Emerg Microbes Infect. 2020 Dec;9(l):680-686. doi: 10.1080/22221751.2020.1743767. PMID: 32207377; PMCID: PMC7144318.

[00691] Plotkin SA. Correlates of Protection Induced by Vaccination. Clin Vaccine Immunol. 2010; 17(7): 1055-1065; DOI: 10.1128/CVI.00131-10

[00692] Rasheed, M.A.; Raza, S.; Zohaib, A.; Yaqub, T.; Rabbani, M.; Riaz, M.I.; Awais, M.; Afzal, A. In Silico Identification of Novel B Cell and T Cell Epitopes of Wuhan Coronavirus (2019-nCoV) for Effective Multi Epitope-Based Peptide Vaccine Production. Preprints 2020, 2020020359 (doi: 10.20944/preprints202002.0359.vl).

[00693] Torrey HL, Kaliaperumal V, Bramhecha Y, Weir GM, Falsey AR, Walsh EE, Langley JM, Schepens B, Saelens X, Stanford MM. (2020) Evaluation of the protective potential of antibody and T cell responses elicited by a novel preventative vaccine towards respiratory syncytial virus small hydrophobic protein, Human Vaccines & Immunotherapeutics, 16:9, 2007- 2017, DOI: 10.1080/21645515.2020.1756671

[00694] Wang C, Liu Z, Chen Z, et al. The establishment of reference sequence for SARS-CoV- 2 and variation analysis. J Med Virol. 2020; 1-8. https://doi.org/10.1002/jmv.25762

[00695] Wang Q, Zhang L, Kuwahara K, et al. Immunodominant SARS Coronavirus Epitopes in Humans Elicited both Enhancing and Neutralizing Effects on Infection in Non-human Primates [published correction appears in ACS Infect Dis. 2020 May 8;6(5): 1284-1285], ACS Infect Dis. 2016;2(5):361-376. doi:10.1021/acsinfecdis.6b00006

[00696] Yu J. etal., Science 10.1126/science.abc6284 (2020).

[00697] Zhang H., G. Wang, J. Li, Y. Nie, X. Shi, G. Lian, W. Wang, X. Yin, Y. Zhao, X. Qu, M. Ding, H Deng. Identification of an antigenic determinant on the S2 domain of the severe acute respiratory syndrome coronavirus spike glycoprotein capable of inducing neutralizing antibodies. J. Virol., 78 (2004), pp. 6938-6945. [00698] All publications and patent applications cited in this specification are herein incorporated by reference in their entirety as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[00699] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

2. The composition of claim 1, wherein the at least one B cell epitope comprises a peptide present in the SARS-CoV-2 spike protein.

3. The composition of claims 1 or 2, wherein the at least one B cell epitope comprises an amino acid sequence from the SARS-CoV-2 spike protein (SEQ ID NO: 1) that is capable of eliciting antibodies in a subject.

4. The composition of any of claims 1-3, wherein the at least one B cell epitope is a peptide antigen comprising at least one of amino acid sequences SYGFQPTNGVGYQPY (SEQ ID NO: 2); GDEVRQIAPGQTGKIADYNYKLP (SEQ ID NO: 3); VRFPNITNLCPF GE (SEQ ID NO: 4); LLFNKVTLADAGFIKQYGDCLGDIAA (SEQ ID NO: 5); GCVIAWNSNNLDSKVGG (SEQ ID NO: 6); LKPFERDISTEIYQAGSTPCNGVEGFN (SEQ ID NO: 7); GFQPTNGVGY QPY (SEQ ID NO: 8); ELLHAPATVCGPKKSTNLVKN (SEQ ID NO: 9); RVYSTGSNVFQ (SEQ ID N: 10); DLGDISGINASWNIQK (SEQ ID NO: 11); VCGPKKSTNLVKN (SEQ ID NO: 12); KNHTSPDVDLGDISGIN (SEQ ID NO: 13); NCTEVPVAIHADQLTPT (SEQ ID NO: 14); SCCKFDEDDSEPVLKG (SEQ ID NO: 15); ASYQTQTNSPRRARSVASQ (SEQ ID NO: 16); YNSASFSTFKCYGVSPTKLNDLCFT (SEQ ID NO: 17); TPGDSSSGWTA (SEQ ID NO: 18); SFSTFKCYGVSPTKLNDL (SEQ ID NO: 19); SNKKFLPF (SEQ ID NO: 20); PDPSKPSK (SEQ ID NO: 21); EIDRLNEVAKNLNESLIDLQELGKYEQY (SEQ ID NO: 22); FNCYFPLQS YGFQPTNGV GY QPYRVWLSFE (SEQ ID NO: 23);

FPNITNLCPFGEVFNATRFASVYAWNRKRISNCVA (SEQ ID NO: 24);

TESNKKFLPFQQFGRDIA (SEQ ID NO:25); PSKPSKRSFIEDLLFNKV (SEQ ID NO:26), or a nucleic acid molecule encoding said peptide antigen, optionally wherein the at least one B cell epitope is present in the form of a dimer.

5. The composition of claim 1, wherein the at least one B cell epitope comprises the amino acid sequence of SEQ ID NOs: 5, 7, 14 or 17, optionally wherein the at least one B cell epitope is present in the form of a dimer.

6. The composition of any of claims 1-5, wherein the carrier comprises a continuous phase of a hydrophobic substance.

7. The composition of any of claims 1-6, wherein the carrier is a hydrophobic substance such as a vegetable oil, nut oil, or mineral oil.

8. The composition of any of claims 1-7, wherein the carrier is mineral oil or is a mannide oleate in a mineral oil solution.

9. The composition of any of claims 1-8, wherein the carrier is Montanide® ISA 51.

10. The composition of any of claims 1-9, wherein the liposomes comprise 1,2-Dioleoyl-sn- glycero-3-phosphocholine (DOPC) and cholesterol.

11. The composition of any of claims 1-10, wherein the optional adjuvant is a lipid-based adjuvant, a lipopolynucleotide-based adjuvant, and/or a polynucleotide-based adjuvant.

12. The composition of claim 11, wherein the lipid-based adjuvant is PAM3CSK4, and/or the polynucleotide-based adjuvant is poly(dI:dC).

13. The composition of any of claims 1-12, wherein the at least one B cell epitope is administered at a concentration of about 0.2 mg/ml to about 1 mg/ml for each B cell peptide, including about 0.2 mg/ml to about 0.5 mg/ml and 0.5 mg/ml to 1 mg/ml.

14. The composition of any of claims 1-12, wherein the at least one B cell epitope is administered at a dose of about 0.01 ml to about 1 ml.

15. The composition of any of claims 1-13, wherein the at least one B cell epitope is administered in at least one dose or in at least two doses.

16. The composition of any of claims 1-15, wherein the composition is an immunogenic composition.

17. The composition of claim 16, wherein the composition elicits an immune response against SARS-CoV-2 in a subject.

18. The composition of any of claims 1-16, wherein the composition comprises a mixture of at least four B cell epitopes comprising the amino acid sequences of SEQ ID NOs: 5, 7, 14 and 19.

19. The composition of any of claims 1-16, wherein the composition comprises a B cell epitope selected from the group consisting of SEQ ID NOs: 5, 7, 14 and 19, and combinations thereof.

20. The composition of any of claims 1-19, wherein the B cell epitope has an amino acid sequence with at least one mutation relative to the amino add sequence of SEQ ID NOs: 5, 7, 14 and/or 19.

21. A method of administering an immunogenic composition to a subject in need thereof, the method comprising: injecting the immunogenic composition into the subject, wherein the immunogenic composition comprises: at least one B cell epitope of SARS-CoV-2; a earner; liposomes; optionally at least one T cell epitope of SARS-CoV-2; and optionally an adjuvant.

22. The method of claim 21, wherein the injection is a subcutaneous or intramuscular injection.

23. The method of claims 21 or 22, wherein the at least one B cell epitope is a peptide present in the SARS-CoV-2 spike protein.

24. The method of any of claims 21-23, wherein the at least one B cell epitope comprises an amino acid sequence from the SARS-CoV-2 spike protein (SEQ ID NO: 1) that is capable of eliciting antibodies in a subject.

25. The method of any of claims 21-24, wherein the at least one B cell epitope is a peptide antigen comprising at least one of amino acid sequences SYGFQPTNGVGYQPY (SEQ ID NO: 2); GDEVRQIAPGQTGKIADYNYKLP (SEQ ID NO: 3); VRFPNITNLCPFGE (SEQ ID NO: 4); LLFNKVTLADAGFIKQYGDCLGDIAA (SEQ ID NO: 5); GCVIAWNSNNLDSKVGG (SEQ ID NO: 6); LKPFERDISTEIYQAGSTPCNGVEGFN (SEQ ID NO: 7); GFQPTNGVGYQPY (SEQ ID NO: 8); ELLHAPATVCGPKKSTNLVKN (SEQ ID NO: 9); RVYSTGSNVFQ (SEQ ID N: 10); DLGDISGINASWNIQK (SEQ ID NO: 11); VCGPKKSTNLVKN (SEQ ID NO: 12); KNHTSPDVDLGDISGIN (SEQ ID NO: 13); NCTEVPVAIHADQLTPT (SEQ ID NO: 14); SCCKFDEDDSEPVLKG (SEQ ID NO: 15); ASYQTQTNSPRRARSVASQ (SEQ ID NO: 16); YNSASFSTFKCYGVSPTKLNDLCFT (SEQ ID NO: 17); TPGDSSSGWTA (SEQ ID NO: 18); SFSTFKCYGVSPTKLNDL (SEQ ID NO: 19); SNKKFLPF (SEQ ID NO: 20); PDPSKPSK (SEQ ID NO: 21); EIDRLNEVAKNLNESLIDLQELGKYEQY (SEQ ID NO: 22); FNCYFPLQS YGFQPINGV GY QPYRVVVLSFE (SEQ ID NO: 23);

FPNITNLCPFGEVFNATRFASVYAWNRKRISNCVA (SEQ ID NO: 24);

26. The method of any of claims 21-25, wherein the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 5, 7, 14, or 19.

27. The method of any of claims 21-26, wherein the carrier comprises a continuous phase of a hydrophobic substance.

28. The method of any of claims 21-27, wherein the carrier is a hydrophobic substance such as a vegetable oil, nut oil, or mineral oil.

29. The method of any of claims 21-28, wherein the carrier is mineral oil or is a mannide oleate in a mineral oil solution.

30. The method of any of claims 21-29, wherein the carrier is Montanide® ISA 51.

31. The method of any of claims 21-30, wherein the liposomes comprise 1,2-Dioleoyl-sn-glycero- 3 -phosphocholine (DOPC) and cholesterol.

32. The method of any of claims 21-31, wherein the optional adjuvant is a lipid-based adjuvant, a lipopolynucleotide-based adjuvant, and/or a polynucleotide-based adjuvant.

33. The method of claim 32, wherein the lipid-based adjuvant is PAM3CSK4, and/or the polynucleotide-based adjuvant is poly(dI:dC).

34. The method of any of claims 21-33, wherein the at least one B cell epitope is administered at a concentration of about 0.2 mg/ml to about 1 mg/ml for each B cell peptide, including about 0.2 mg/ml to about 0.5 mg/ml and 0.5 mg/ml to 1 mg/ml.

35. The method of any of claims 21-33, wherein the at least one B cell epitope is administered at a dose of about 0.01 ml to about 1 ml.

36. The method of any of claims 21-35, wherein the at least one B cell epitope is administered in at least one dose or in at least two doses.

37. The method of any of claims 21-36, wherein the composition elicits an immune response against SARS-CoV-2 in a subject.

38. The method of any of claims 21-37, wherein the composition comprises a mixture of at least four B cell epitopes comprising the amino acid sequences of SEQ ID NOs: 5, 7, 14 and 19.

39. The method of any of claims 21-37, wherein the composition comprises a B cell epitope selected from the group consisting of SEQ ID NOs: 5, 7, 14 and 19, and combinations thereof.

40. The method of any of claims 21-39, wherein the B cell epitope has an amino acid sequence with at least one mutation relative to the amino add sequence of SEQ ID NOs: 5, 7, 14 and/or 19.

41. A method of elidting an immune response against SARS-CoV-2 in a subject in need thereof, the method comprising: injecting the immunogenic composition into the subject, wherein the immunogenic composition comprises: at least one B cell epitope of SARS-CoV-2; a carrier; liposomes; optionally at least one T cell epitope of SARS-CoV-2; and optionally an adjuvant.

42. The method of claim 41, wherein the injection is a subcutaneous or intramuscular injection.

43. The method of claims 41 or 42, wherdn the at least one B cell epitope is a peptide present in the SARS-CoV-2 spike protein.

44. The method of any of claims 41-43, wherein the at least one B cell epitope comprises an amino acid sequence from the SARS-CoV-2 spike protein (SEQ ID NO: 1) that is capable of eliciting antibodies in a subject.

45. The method of any of claims 41-44, wherein the at least one B cell epitope is a peptide antigen comprising at least one of amino acid sequences SYGFQPTNGVGYQPY (SEQ ID NO: 2); GDEVRQIAPGQTGKIADYNYKLP (SEQ ID NO: 3); VRFPNITNLCPF GE (SEQ ID NO: 4); LLFNKVTLADAGFIKQYGDCLGDIAA (SEQ ID NO: 5); GCVIAWNSNNLDSKVGG (SEQ ID NO: 6); LKPFERDISTEIYQAGSTPCNGVEGFN (SEQ ID NO: 7); GFQPTNGVGY QPY (SEQ ID NO: 8); ELLHAPATVCGPKKSTNLVKN (SEQ ID NO: 9); RVYSTGSNVF Q (SEQ ID N: 10); DLGDISGINASWNIQK (SEQ ID NO: 11); VCGPKKSTNLVKN (SEQ ID NO: 12); KNHTSPDVDLGDISGIN (SEQ ID NO: 13); NCTEVPVAIHADQLTPT (SEQ ID NO: 14); SCCKFDEDDSEPVLKG (SEQ ID NO: 15); ASYQTQTNSPRRARSVASQ (SEQ ID NO: 16); YNSASFSTFKCYGVSPTKLNDLCFT (SEQ ID NO: 17); TPGDSSSGWTA (SEQ ID NO: 18); SFSTFKCYGVSPTKLNDL (SEQ ID NO: 19); SNKKFLPF (SEQ ID NO: 20); PDPSKPSK (SEQ ID NO: 21); EIDRLNEVAKNLNESLIDLQELGKYEQY (SEQ ID NO: 22); FNCYFPLQS YGFQPTNGV GY QPYRVWLSFE (SEQ ID NO: 23);

FPNITNLCPFGEVFNATRFASVYAWNRKRISNCVA (SEQ ID NO: 24);

46. The method of any of claims 41-45, wherein the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 5, 7, 14, or 19.

47. The method of any of claims 41-46, wherein the carrier comprises a continuous phase of a hydrophobic substance.

48. The method of any of claims 41-47, wherein the carrier is a hydrophobic substance such as a vegetable oil, nut oil, or mineral oil.

49. The method of any of claims 41-48, wherein the carrier is mineral oil or is a mannide oleate in a mineral oil solution.

50. The method of any of claims 41-49, wherein the carrier is Montanide® ISA 51.

51. The method of any of claims 41-50, wherein the liposomes comprise 1,2-Dioleoyl-sn-glycero- 3-phosphocholine (DOPC) and cholesterol.

52. The method of any of claims 41-51, wherein the optional adjuvant is a lipid-based adjuvant, a lipopolynucleotide-based adjuvant, and/or a polynucleotide-based adjuvant.

53. The method of claim 52, wherein the lipid-based adjuvant is PAM3CSK4, and/or the polynucleotide-based adjuvant is poly(dI:dC).

54. The method of any of claims 41-53, wherein the at least one B cell epitope is administered at a concentration of about 0.2 mg/ml to about 1 mg/ml for each B cell peptide, including about 0.2 mg/ml to about 0.5 mg/ml and 0.5 mg/ml to 1 mg/ml.

55. The method of any of claims 41-54, wherein the at least one B cell epitope is administered at a dose of about 0.01 ml to about 1 ml.

56. The method of any of claims 41-55, wherein the at least one B cell epitope is administered in at least one dose or in at least two doses.

57. The method of any of claims 41-56, wherein the composition comprises a mixture of at least four B cell epitopes comprising the amino acid sequences of SEQ ID NOs: 5, 7, 14 and 19.

58. The method of any of claims 41-56, wherein the composition comprises a B cell epitope selected from the group consisting of SEQ ID NOs: 5, 7, 14 and 19, and combinations thereof.

59. The method of any of claims 41-58, wherein the B cell epitope has an amino acid sequence with at least one mutation relative to the amino add sequence of SEQ ID NOs: 5, 7, 14 and/or 19.

(c) lyophilizing the preparation of the at least one B cell epitope, optional adjuvant and/or T cell epitope, and liposomes; and (d) reconstituting the preparation of the at least one B cell epitope, optional adjuvant and/or T cell epitope, and liposomes in a hydrophobic carrier.

61. The composition of any of claims 1-20, wherein the composition further comprises at least one T cell epitope of SARS-CoV-2.

62. The composition of any of claims 1-20 and 61, wherein the at least one T cell epitope comprises at least one of SEQ ID NOs: 27-40 and/or 42-43, or a nucleic acid molecule encoding said epitope, optionally wherein the at least one T cell epitope is present in the form of a dimer.

63. The composition of any of claims 1-20 and 61-62, wherein the composition comprises a mixture of at least two T cell epitopes comprising the amino acid sequences of SEQ ID NOs: 27, 28, 32, 34, 38, 42 and/or 43.

64. The composition of any of claims 1-20 and 61-63, wherein the composition comprises at least one T cell epitope selected from the group consisting of SEQ ID NOs: 27, 28, 32, 34, 38, 42, 43, and combinations thereof.

65. The composition of any of claims 1-20 and 61-64, wherein the T cell epitope has an amino acid sequence with at least one mutation relative to the amino add sequence of SEQ ID NOs: 27, 28, 32, 34, 38, 42 and/or 43.

66. The method of any of claims 21-40, wherein the composition further comprises at least one T cell epitope of SARS-CoV-2.

67. The method of any of claims 21-40 and 66, wherein the at least one T cell epitope comprises at least one of SEQ ID NOs: 27-40 and/or 42-43, or a nucleic acid molecule encoding said epitope, optionally wherein the at least one T cell epitope is present in the form of a dimer.

68. The method of any of claims 21-40 and 66-67, wherein the composition comprises a mixture of at least two T cell epitopes comprising the amino acid sequences of SEQ ID NOs: 27, 28, 32, 34, 38, 42 and/or 43.

69. The method of any of claims 21-40 and 66-68, wherein the composition comprises at least one T cell epitope selected from the group consisting of SEQ ID NOs: 27, 28, 32, 34, 38, 42, 43, and combinations thereof.

71. The method of any of claims 41-59, wherein the composition further comprises at least one T cell epitope of SARS-CoV-2.

72. The method of any of claims 41-59 and 71, wherein the at least one T cell epitope comprises at least one of SEQ ID NOs: 27-40 and/or 42-43, or a nucleic acid molecule encoding said epitope, optionally wherein the at least one T cell epitope is present in the form of a dimer.

73. The method of any of claims 41-59 and 71-72, wherein the composition comprises a mixture of at least two T cell epitopes comprising the amino acid sequences of SEQ ID NOs: 27, 28, 32, 34, 38, 42 and/or 43.

74. The method of any of claims 41-59 and 71-73, wherein the composition comprises a T cell epitope selected from the group consisting of SEQ ID NOs: 27, 28, 32, 34, 38, 42, 43, and combinations thereof.

75. The method of any of claims 41-59 and 71-74, wherein the T cell epitope has an amino acid sequence with at least one mutation relative to the amino acid sequence of SEQ ID NOs: 27, 28, 32, 34, 38, 42 and/or 43.

76. The composition of any of claims 1-75 wherein the mutation relative to the amino acid sequences of SEQ ID NOs: 5, 7, 14, 17, 27, 28, 32, 34, 38, 42, and/or 43 is a substitution, an insertion, or a deletion.

77. A composition comprising: at least one B cell epitope selected from the group consisting of SEQ ID NOs: 5, 7, 14, 17, 27, and combinations thereof, optionally in the form of a dimer; at least one T cell epitope selected from the group consisting of SEQ ID NOs: 27, 28, 32, 34, 38, 42, and/or 43, optionally in the form of a dimer; a earner; liposomes; and an adjuvant.

78. The composition of claim 77, wherein the at least one T cell epitope is selected from the group consisting of SEQ ID NOs: 28, 34, 38, 42, 43, and combinations thereof.

79. The composition of any of claims 77-78, wherein the carrier comprises a continuous phase of a hydrophobic substance.

80. The composition of any of claims 77-79, wherein the carrier is a hydrophobic substance such as a vegetable oil, nut oil, or mineral oil.

81. The composition of any of claims 77-80, wherein the carrier is mineral oil or is a mannide oleate in a mineral oil solution.

82. The composition of claim 81, wherein the carrier is Montanide® ISA 51.

83. The composition of any of claims 77-82, wherein the liposomes comprise 1,2-Dioleoyl-sn- glycero-3-phosphocholine (DOPC) and cholesterol.

84. The composition of any of claims 77-83, wherein the adjuvant is a lipid-based adjuvant, a lipopolynucleotide-based adjuvant, and/or a polynucleotide-based adjuvant.

85. The composition of claim 84, wherein the polynucleotide adjuvant is poly(dI:dC).

86. The composition of claim 84, wherein the lipid-based adjuvant is PAM3CSK4.

87. The composition of claim 1, wherein the at least one B cell epitope comprises at least one of SEQ ID NOs: 2-25 and the at least one T cell epitope comprises at least one of SEQ ID NOs: 27- 40 and/or 42-43.

88. The method of claim 21, wherein the at least one B cell epitope comprises at least one of SEQ ID NOs: 2-25 and the at least one T cell epitope comprises at least one of SEQ ID NOs: 27-40 and/or 42-43.

89. The method of claim 41, wherein the at least one B cell epitope comprises at least one of SEQ ID NOs: 2-25 and the at least one T cell epitope comprises at least one of SEQ ID NOs: 27-40 and/or 42-43.

90. The composition of any of claims 1-20 and 61-65, wherein the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 5.

91. The composition of any of claims 1-20 and 61-65, wherein the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 7.

92. The composition of any of claims 1-20 and 61-65, wherein the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 14.

93. The composition of any of claims 1-20 and 61-65, wherein the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 19.

94. The method of any of claims 21-40 and 66-70, wherein the at least one B cell epitope comprises the amino add sequence of SEQ ID NO: 5.

95. The method of any of claims 21-40 and 66-70, wherein the at least one B cell epitope comprises the amino add sequence of SEQ ID NO: 7.

96. The method of any of claims 21-40 and 66-70, wherein the at least one B cell epitope comprises the amino add sequence of SEQ ID NO: 14.

97. The method of any of claims 21-40 and 66-70, wherein the at least one B cell epitope comprises the amino add sequence of SEQ ID NO: 19.

98. The method of any of claims 41-59 and 71-75, wherein the at least one B cell epitope comprises the amino add sequence of SEQ ID NO: 5.

99. The method of any of claims 41-59 and 71-75, wherein the at least one B cell epitope comprises the amino add sequence of SEQ ID NO: 7.

100. The method of any of claims 41-59 and 71-75, wherein the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 14.

101. The method of any of claims 41-59 and 71-75, wherein the at least one B cell epitope comprises the amino acid sequence of SEQ ID NO: 19.