Coronavirus and influenza compositions and methods of using the same

Abstract

An immunogenic composition for inducing an immune response against both influenza and coronavirus comprises (a) coronavirus S (CoV S) glycoprotein in the form of surfactant core nanoparticles, wherein the surfactant is a nonionic surfactant; (b) at least three hemagglutinin (HA) glycoproteins, each HA glycoprotein derived from a different influenza strain; and (c) a pharmaceutically acceptable buffer. An immunogenic composition for inducing an immune response against influenza comprises (a) at least three hemagglutinin (HA) glycoproteins, each HA glycoprotein derived from a different influenza strain, wherein 30 to 60 μg of HA is present in the composition for each strain; and (b) a pharmaceutically acceptable buffer.

Description

[Technical Field]

[0001] Electronic sequence list reference This application claims priority to U.S. Provisional Application No. 63 / 508,798, filed June 16, 2023, and U.S. Provisional Application No. 63 / 493,884, filed April 3, 2023. The entire contents of said Provisional Application are incorporated herein by reference.

[0002] This application includes a sequence listing, which is filed electronically in XML format and incorporated herein by reference in its entirety. The above XML copy, created on 3 April 2024, is named 1450_099WO1_Sequence_Listing_04_03_2024 and is 768,523 bytes in size.

[0003] This disclosure relates to compositions and methods for inducing an immune response against both influenza and coronavirus. [Background technology]

[0004] Influenza and COVID-19 are life-threatening illnesses caused by the influenza virus and the SARS-CoV-2 virus, respectively. The case fatality rate for patients diagnosed with influenza is approximately 0.1%, while the case fatality rate for patients diagnosed with COVID-19 ranges from 0.2% to 7.7%.

[0005] The development of vaccines to prevent or mitigate the severity of these life-threatening infectious diseases is highly desirable. However, the development of human vaccines remains challenging due to the highly sophisticated evasion mechanisms of pathogens and the difficulty in stabilizing vaccines. Optimally, vaccines need to both induce antibodies that block or neutralize infectious agents and maintain stability in various environments, including those where refrigeration is not possible. Combining two antigens from two pathogens in a single vaccine composition is particularly difficult because the antigens interact and interfere with a sufficient immune response to either pathogen. [Overview of the project] [Means for solving the problem]

[0006] This disclosure provides compositions and methods for inducing an immune response against both influenza and coronavirus.

[0007] This specification provides immunogenic compositions comprising: (a) coronavirus S (CoV S) glycoprotein in the form of surfactant core nanoparticles, wherein the surfactant is a nonionic surfactant; (b) at least three hemagglutinin (HA) glycoproteins, wherein each HA glycoprotein is derived from a different influenza strain; and (c) a pharmaceutically acceptable buffer. In the immunogenic compositions, the ratio of hemagglutinin per strain to coronavirus S glycoprotein is about 1.2:1, 1.3:1, about 1.5:1, about 1.7:1, about 2:1, about 2.2:1, about 2.4:1, about 2.8:1, about 2.6:1, or about 3:1.

[0008] This specification provides an immunogenic composition comprising (a) at least three hemagglutinin (HA) glycoproteins, each HA glycoprotein derived from a different influenza strain, with 30 to 60 μg of HA present in the composition for each strain, and (b) a pharmaceutically acceptable buffer.

[0009] In an embodiment, the immunogenic composition comprises an adjuvant. In an embodiment, the adjuvant comprises at least two ISCOM particles, wherein the first ISCOM particle comprises fraction A of Quillaja Saponaria Molina and does not comprise fraction C of Quillaja Saponaria Molina, and the second ISCOM particle comprises fraction C of Quillaja Saponaria Molina and does not comprise fraction A of Quillaja Saponaria Molina.

[0010] In an embodiment, provided herein is a method of stimulating an immune response against SARS-CoV-2, a heterologous SARS-CoV-2 strain, an influenza virus, or a combination thereof, comprising administering the immunogenic composition described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] [Figure 1] Shows an overview of the wild-type amino acid sequence of the SARS-CoV-2 spike (S) protein (SEQ ID NO: 1). The furin cleavage site RRAR (SEQ ID NO: 6) is highlighted in bold, and the signal peptide is underlined.

[0012] [Figure 2] Shows the purification of CoV S polypeptides BV2364, BV2365, BV2366, BV2367, BV2368, BV2369, BV2373, BV2374, and BV2375. These data demonstrate that BV2365 (SEQ ID NO: 4) and BV2373 (SEQ ID NO: 87), which have an inactive furin cleavage site with the amino acid sequence QQAQ (SEQ ID NO: 7), are expressed as single-chain (S0). In contrast, CoV S polypeptides containing an intact furin cleavage site (e.g., BV2364, BV2366, and BV2374) are cleaved, as evidenced by the presence of the cleavage product S2.

[0013] [Figure 3]This shows the primary structure of the CoV S polypeptide of BV2373, as well as modifications to the furin cleavage sites, K986P, and V987P.

[0014] [Figure 4] This shows the purification of wild-type CoV S polypeptides as well as CoV S polypeptides BV2365 and BV2373.

[0015] [Figure 5] This shows an overview of the coronavirus spike (S) protein (SEQ ID NO: 86) (BV2373). The furin cleavage site QQAQ (SEQ ID NO: 7) is underlined once, and the K986P and V987P mutations are double-underlined.

[0016] [Figure 6] Figure A shows the primary structure of the wild-type SARS-CoV-2 S polypeptide containing the signal peptide, and is numbered with respect to SEQ ID NO: 1. Figure B shows the primary structure of the wild-type SARS-CoV-2 S polypeptide without the signal peptide, and is numbered with respect to SEQ ID NO: 2.

[0017] [Figure 7] Examples of transmission electron microscope (TEM) images of influenza HA surfactant core nanoparticles alone (left), saponin adjuvant alone (center) (i.e., ISCOM matrix of 85% fraction A and 15% fraction C), and combinations of HA nanoparticles and saponin adjuvant forming hemagglutinin saponin matrix nanoparticles (HaSMaN) (right) are shown.

[0018] [Figure 8A]This study demonstrates the immune response to SARS-CoV-2 spike glycoprotein and influenza hemagglutinin in ferrets immunized with the qNIV / CoV2373 combination vaccine. Male and female ferrets (n=6 / group) were immunized with either 15 μg or 60 μg of hemagglutinin (HA) per strain, along with 5 μg of CoV2373 and 50 μg of saponin adjuvant. The control group was immunized with either 15 μg or 60 μg of HA per strain, or 5 μg of CoV22373 and 50 μg of saponin adjuvant. All groups were immunized twice, 21 days apart. Red triangles indicate serum collection dates. [Figure 8B] This graph shows the immune response to SARS-CoV-2 spike glycoprotein and influenza hemagglutinin in ferrets immunized with the qNIV / CoV2373 combination vaccine. It also shows the titer of human angiotensin-converting enzyme 2 (hACE2) receptor blocking antibody, and hemagglutinin inhibitor antibody (HAI) titers 21 days after the first dose and 14 days after the booster immunization (day 35 of the study). Bars represent geometric mean titers (GMT), and error bars represent 95% confidence intervals. [Figure 8C] This image shows the immune response to SARS-CoV-2 spike glycoprotein and influenza hemagglutinin in ferrets immunized with the qNIV / CoV2373 combination vaccine. A / Kansas / 14 / 2017. Bars represent geometric mean titer (GMT), and error bars represent 95% confidence intervals. [Figure 8D] This image shows the immune response to SARS-CoV-2 spike glycoprotein and influenza hemagglutinin in ferrets immunized with the qNIV / CoV2373 combination vaccine. A / Brisbane / 02 / 2018. Bars represent geometric mean titer (GMT), and error bars represent 95% confidence intervals. [Figure 8E] This image shows the immune response to SARS-CoV-2 spike glycoprotein and influenza hemagglutinin in ferrets immunized with the qNIV / CoV2373 combination vaccine. B / Phuket / 3070 / 2013. Bars represent geometric mean titer (GMT), and error bars represent 95% confidence intervals. [Figure 8F]This image shows the immune response to SARS-CoV-2 spike glycoprotein and influenza hemagglutinin in ferrets immunized with the qNIV / CoV2373 combination vaccine. B / Maryland / 15 / 2016. Bars represent geometric mean titer (GMT), and error bars represent 95% confidence intervals.

[0019] [Figure 9A] This study describes the immune response to SARS-CoV-2 spike glycoprotein and receptor blocking antibodies in hamsters immunized with a qNIV / CoV2373 combination vaccine. Male and female hamsters (n=5-6 / group) were immunized with 2.5 or 10 μg of hemagglutinin (HA) and 1 or 5 μg of CoV2373 and 15 μg of saponin adjuvant, depending on the strain. The control group was immunized with 2.5 or 10 μg of HA, or 1 or 5 μg of CoV2373 and 15 μg of saponin adjuvant, depending on the strain. Animals were immunized twice via the intramuscular route (IM) at 14-day intervals. The placebo group received formulation buffer. Serum was collected for analysis as indicated by the red triangles. Immunized and placebo animals were infected with 2.0 x 10⁴ pfu of SARS-CoV-2 via the intranasal (IN) route 21 days after the second immunization. Oral swabs were collected at 2, 4, and 7 days post-infection (dpi, blue triangles). Bronchoalveolar lavage and lung samples were collected at 7 dpi (black triangles). [Figure 9B] This graph shows the immune response to SARS-CoV-2 spike glycoprotein and receptor blocking antibodies in hamsters immunized with the qNIV / CoV2373 combination vaccine. Anti-spike IgG titers are shown 14 days after a single dose. Bars represent the geometric mean titer (GMT) of the group, and error bars represent the 95% confidence interval (95% CI). Values ​​for individual animals are indicated by colored symbols. The black horizontal dashed line indicates the limit of detection (LOD). [Figure 9C]This graph shows the immune response to SARS-CoV-2 spike glycoprotein and receptor blocking antibodies in hamsters immunized with the qNIV / CoV2373 combination vaccine. Anti-spike IgG titers are shown 14 days after booster immunization (day 28 of the study). Bars represent the geometric mean titer (GMT) of the group, and error bars represent the 95% confidence interval (95% CI). Values ​​for individual animals are indicated by colored symbols. The black horizontal dashed line indicates the limit of detection (LOD). [Figure 9D] This graph shows the immune response to SARS-CoV-2 spike glycoprotein and receptor blocking antibodies in hamsters immunized with the qNIV / CoV2373 combination vaccine. Human ACE-2 (hACE2) receptor blocking antibody titers are shown 14 days after a single dose. Bars represent the geometric mean titer (GMT) of the group, and error bars represent the 95% confidence interval (95% CI). Values ​​for individual animals are indicated by colored symbols. The black horizontal dashed line indicates the limit of detection (LOD). [Figure 9E] This graph shows the immune response to SARS-CoV-2 spike glycoprotein and receptor blocking antibodies in hamsters immunized with the qNIV / CoV2373 combination vaccine. Human ACE-2 (hACE2) receptor blocking antibody titers 14 days after booster immunization are shown. Bars represent the geometric mean titer (GMT) of the group, and error bars represent the 95% confidence interval (95% CI). Values ​​for individual animals are indicated by colored symbols. The black horizontal dashed line indicates the limit of detection (LOD).

[0020] [Figure 10A] This figure shows the immune response to influenza HA in hamsters immunized with the qNIV / CoV2373 combination vaccine. Hamsters were immunized with either the qNIV / CoV2373 vaccine combination or the component vaccines shown in Figure 8A. Serum hemagglutinin inhibitory antibody (HAI) titers were analyzed 14 days after a single dose. A / Kansas / 14 / 2017. Bars represent geometric mean titers, and error bars represent 95% confidence intervals. [Figure 10B]This figure shows the immune response to influenza HA in hamsters immunized with the qNIV / CoV2373 combination vaccine. The hamster group was immunized with either the qNIV / CoV2373 vaccine combination or the component vaccines shown in Figure 8A. Serum hemagglutinin inhibitory antibody (HAI) titers were analyzed 14 days after booster immunization (day 28 of the study). A / Kansas / 14 / 2017. Bars represent geometric mean titers, and error bars represent 95% confidence intervals. [Figure 10C] This figure shows the immune response to influenza HA in hamsters immunized with the qNIV / CoV2373 combination vaccine. Hamsters were immunized with either the qNIV / CoV2373 vaccine combination or the component vaccines shown in Figure 8A. Serum hemagglutinin inhibitory antibody (HAI) titers were analyzed 14 days after a single dose. A / Brisbane / 02 / 2018. Bars represent geometric mean titers, and error bars represent 95% confidence intervals. [Figure 10D] This figure shows the immune response to influenza HA in hamsters immunized with the qNIV / CoV2373 combination vaccine. Hamsters were immunized with either the qNIV / CoV2373 vaccine combination or the component vaccines shown in Figure 8A. Serum hemagglutinin inhibitory antibody (HAI) titers were analyzed 14 days after booster immunization (day 28 of the study). A / Brisbane / 02 / 2018. Bars represent geometric mean titers, and error bars represent 95% confidence intervals. [Figure 10E] This figure shows the immune response to influenza HA in hamsters immunized with the qNIV / CoV2373 combination vaccine. Hamsters were immunized with either the qNIV / CoV2373 vaccine combination or the component vaccines shown in Figure 8A. Serum hemagglutinin inhibitory antibody (HAI) titers were analyzed 14 days after a single dose. B / Phuket / 3070 / 2013. Bars represent geometric mean titers, and error bars represent 95% confidence intervals. [Figure 10F]This figure shows the immune response to influenza HA in hamsters immunized with the qNIV / CoV2373 combination vaccine. The hamster group was immunized with either the qNIV / CoV2373 vaccine combination or the component vaccines shown in Figure 8A. Serum hemagglutinin inhibitory antibody (HAI) titers were analyzed 14 days after booster immunization (day 28 of the study). B / Phuket / 3070 / 2013. Bars represent geometric mean titers, and error bars represent 95% confidence intervals. [Figure 10G] This figure shows the immune response to influenza HA in hamsters immunized with the qNIV / CoV2373 combination vaccine. Hamsters were immunized with either the qNIV / CoV2373 vaccine combination or the component vaccines shown in Figure 8A. Serum hemagglutinin inhibitory antibody (HAI) titers were analyzed 14 days after a single dose. B / Maryland / 15 / 2016. Bars represent geometric mean titers, and error bars represent 95% confidence intervals. [Figure 10H] This figure shows the immune response to influenza HA in hamsters immunized with the qNIV / CoV2373 combination vaccine. Hamsters were immunized with either the qNIV / CoV2373 vaccine combination or the component vaccines shown in Figure 8A. Serum hemagglutinin inhibitory antibody (HAI) titers were analyzed 14 days after booster immunization (day 28 of the study). B / Maryland / 15 / 2016. Bars represent geometric mean titers, and error bars represent 95% confidence intervals.

[0021] [Figure 11A] This figure shows influenza virus neutralizing antibodies in hamsters immunized with the qNIV / CoV2373 combination vaccine. The hamster group was immunized with either the qNIV / CoV2373 combination or the component vaccines shown in Figure 8A. Virus neutralizing titers in response to A / Kansas / 14 / 2017 were measured 14 days after a single dose. Bars represent geometric mean titers (GMT), and error bars represent 95% confidence intervals. Horizontal dashed lines indicate the limit of detection (LOD). [Figure 11B]This figure shows influenza virus neutralizing antibodies in hamsters immunized with the qNIV / CoV2373 combination vaccine. The hamster group was immunized with either the qNIV / CoV2373 combination or the component vaccine shown in Figure 8A. Virus neutralizing titers in response to A / Kansas / 14 / 2017 were measured 14 days after booster immunization (day 28 of the study). Bars represent geometric mean titers (GMT), and error bars represent 95% confidence intervals. The horizontal dashed line indicates the limit of detection (LOD). [Figure 11C] This figure shows influenza virus neutralizing antibodies in hamsters immunized with the qNIV / CoV2373 combination vaccine. The hamster group was immunized with either the qNIV / CoV2373 combination or the component vaccines shown in Figure 8A. Virus neutralizing titers in response to A / Brisbane / 02 / 2018 were measured 14 days after a single dose. Bars represent geometric mean titers (GMT), and error bars represent 95% confidence intervals. The horizontal dashed line indicates the limit of detection (LOD). [Figure 11D] This figure shows influenza virus neutralizing antibodies in hamsters immunized with the qNIV / CoV2373 combination vaccine. The hamster group was immunized with either the qNIV / CoV2373 combination or the component vaccine shown in Figure 8A. Virus neutralizing titers in response to A / Brisbane / 02 / 2018 were measured 14 days after booster immunization (day 28 of the study). Bars represent geometric mean titers (GMT), and error bars represent 95% confidence intervals. The horizontal dashed line indicates the limit of detection (LOD). [Figure 11E] This figure shows influenza virus neutralizing antibodies in hamsters immunized with the qNIV / CoV2373 combination vaccine. The hamster group was immunized with either the qNIV / CoV2373 combination or the component vaccines shown in Figure 8A. Viral neutralizing titers in response to B / Phuket / 3073 / 2013 were measured 14 days after a single dose. Bars represent geometric mean titers (GMT), and error bars represent 95% confidence intervals. Horizontal dashed lines indicate the limit of detection (LOD). [Figure 11F]This figure shows influenza virus neutralizing antibodies in hamsters immunized with the qNIV / CoV2373 combination vaccine. The hamster group was immunized with either the qNIV / CoV2373 combination or the component vaccine shown in Figure 8A. Virus neutralizing titers in response to B / Phuket / 3073 / 2013 were measured 14 days after booster immunization (day 28 of the study). Bars represent geometric mean titers (GMT), and error bars represent 95% confidence intervals. The horizontal dashed line indicates the limit of detection (LOD). [Figure 11G] This figure shows influenza virus neutralizing antibodies in hamsters immunized with the qNIV / CoV2373 combination vaccine. The hamster group was immunized with either the qNIV / CoV2373 combination or the component vaccine shown in Figure 8A. Virus neutralizing titers in response to B / Maryland / 15 / 2016 were measured 14 days after a single dose. Bars represent geometric mean titers (GMT), and error bars represent 95% confidence intervals. Horizontal dashed lines indicate the limit of detection (LOD). [Figure 11H] This figure shows influenza virus neutralizing antibodies in hamsters immunized with the qNIV / CoV2373 combination vaccine. The hamster group was immunized with either the qNIV / CoV2373 combination or the component vaccine shown in Figure 8A. Virus neutralizing titers in response to B / Maryland / 15 / 2016 were measured 14 days after booster immunization (day 28 of the study). Bars represent geometric mean titers (GMT), and error bars represent 95% confidence intervals. The horizontal dashed line indicates the limit of detection (LOD).

[0022] [Figure 12A]This study demonstrates that the mixed qNIV / CoV2373 vaccine induces antibodies that bind to a highly conserved latent epitope in the receptor-binding domain (RBD) of SARS-CoV-2, as measured by biolayer interferometry (BLI). The specificity of antibodies induced by the qNIV / CoV2373 combination or monovalent CoV2373 was measured by competitive antibody binding of immunoserum with receptor-site-specific neutralizing monoclonal antibodies using BLI. Horizontal bars represent the geometric mean of the group, and error bars represent the 95% confidence interval. Colored symbols indicate values ​​for individual animals. Antibodies against SARS-CoV-2 US-WA RBD are shown. [Figure 12B] This study demonstrates that the mixed qNIV / CoV2373 vaccine induces antibodies that bind to a highly conserved latent epitope in the receptor-binding domain (RBD) of SARS-CoV-2, as measured by biolayer interferometry (BLI). The specificity of antibodies induced by the qNIV / CoV2373 combination or monovalent CoV2373 was measured by competitive antibody binding of immunoserum with receptor-site-specific neutralizing monoclonal antibodies using BLI. Horizontal bars represent the geometric mean of the group, and error bars represent the 95% confidence interval. Colored symbols indicate values ​​for individual animals. Antibodies against SARS-CoV-2 US-WA RBD are shown. [Figure 12C] This study demonstrates that the mixed qNIV / CoV2373 vaccine induces antibodies that bind to a highly conserved latent epitope in the receptor-binding domain (RBD) of SARS-CoV-2, as measured by biolayer interferometry (BLI). The specificity of antibodies induced by the qNIV / CoV2373 combination or monovalent CoV2373 was measured by competitive antibody binding of immunoserum with receptor-site-specific neutralizing monoclonal antibodies using BLI. Horizontal bars represent the geometric mean of the group, and error bars represent the 95% confidence interval. Colored symbols indicate values ​​for individual animals. Antibodies against SARS-CoV-2 US-WA RBD are shown. [Figure 12D]This study demonstrates that the mixed qNIV / CoV2373 vaccine induces antibodies that bind to a highly conserved latent epitope in the receptor-binding domain (RBD) of SARS-CoV-2, as measured by biolayer interferometry (BLI). The specificity of antibodies induced by the qNIV / CoV2373 combination or monovalent CoV2373 was measured by competitive antibody binding of immunoserum with receptor-site-specific neutralizing monoclonal antibodies using BLI. Horizontal bars represent the geometric mean of the group, and error bars represent the 95% confidence interval. Colored symbols indicate values ​​for individual animals. Antibodies against the SARS-CoV-2 B.1.351 South African RBD are shown. [Figure 12E] This study demonstrates that the mixed qNIV / CoV2373 vaccine induces antibodies that bind to a highly conserved latent epitope in the receptor-binding domain (RBD) of SARS-CoV-2, as measured by biolayer interferometry (BLI). The specificity of antibodies induced by the qNIV / CoV2373 combination or monovalent CoV2373 was measured by competitive antibody binding of immunoserum with receptor-site-specific neutralizing monoclonal antibodies using BLI. Horizontal bars represent the geometric mean of the group, and error bars represent the 95% confidence interval. Colored symbols indicate values ​​for individual animals. Antibodies against the SARS-CoV-2 B.1.351 South African RBD are shown.

[0023] [Figure 13A] This study demonstrates weight changes and protection against SARS-CoV-2 upper and lower respiratory tract infections in hamsters immunized with the qNIV / CoV2373 combination vaccine. Male and female hamsters (n=5-6 / group) were immunized with either qNIV / CoV2373 or a prime / boost regimen of the component vaccine at 14-day intervals. Three weeks after the second immunization (day 35 of the study), the animals were administered 2.0 x 10⁴ pfu of antigen via the intranasal route. Weight changes up to 7 days post-infection (dpi) (percentage change after SARS-CoV-2 antigen administration) are shown. Data are mean ± SEM for the vaccinated, placebo, and Siamese groups. [Figure 13B]This study demonstrates weight changes and protection against SARS-CoV-2 upper and lower respiratory tract infections in hamsters immunized with the qNIV / CoV2373 combination vaccine. Male and female hamsters (n=5-6 / group) were immunized with either qNIV / CoV2373 or a prime / boost regimen of the component vaccine at 14-day intervals. Three weeks after the second immunization (day 35 of the study), the animals were administered 2.0 x 10⁴ pfu of antigen via the intranasal route. Weight changes up to 7 days post-infection (dpi) (percentage change after SARS-CoV-2 antigen administration) are shown. Data are mean ± SEM for the vaccinated, placebo, and Siamese groups. [Figure 13C] This study demonstrates weight changes and protection against upper and lower respiratory tract infections caused by SARS-CoV-2 in hamsters immunized with the qNIV / CoV2373 combination vaccine. Groups of male and female hamsters (n=5-6 / group) were immunized with qNIV / CoV2373 or a prime / boost regimen of the component vaccine at 14-day intervals. Three weeks after the second immunization (day 35 of the study), the animals were administered 2.0 x 10⁴ pfu of antigen via the intranasal route. Subgenomic (sg) RNA was collected from oral swabs at 2, 4, and 7 days post-infection (dpi), and sg RNA was analyzed by qRT-PCR. [Figure 13D] This study demonstrates weight changes and protection against upper and lower respiratory tract infections caused by SARS-CoV-2 in hamsters immunized with the qNIV / CoV2373 combination vaccine. Groups of male and female hamsters (n=5-6 / group) were immunized with qNIV / CoV2373 or a prime / boost regimen of the component vaccine at 14-day intervals. Three weeks after the second immunization (day 35 of the study), the animals were administered 2.0 x 10⁴ pfu of antigen via the intranasal route. Subgenomic (sg) RNA was collected from oral swabs at 2, 4, and 7 days post-infection (dpi), and sg RNA was analyzed by qRT-PCR. [Figure 13E]This study demonstrates weight changes and protection against upper and lower respiratory tract infections caused by SARS-CoV-2 in hamsters immunized with the qNIV / CoV2373 combination vaccine. Groups of male and female hamsters (n=5-6 / group) were immunized with qNIV / CoV2373 or a prime / boost regimen of the component vaccine at 14-day intervals. Three weeks after the second immunization (day 35 of the study), the animals were administered 2.0 x 10⁴ pfu of antigen via the intranasal route. The viral load of sgRNA in bronchoalveolar lavage fluid collected seven days post-infection (dpi) is shown. [Figure 13F] This study demonstrates weight changes and protection against upper and lower respiratory tract infections caused by SARS-CoV-2 in hamsters immunized with the qNIV / CoV2373 combination vaccine. Groups of male and female hamsters (n=5-6 / group) were immunized with qNIV / CoV2373 or a prime / boost regimen of the component vaccine at 14-day intervals. Three weeks after the second immunization (day 35 of the study), animals were administered 2.0 x 10⁴ pfu of antigen via the intranasal route. The viral load of sgRNA in lung homogenates collected at 7 dpi is shown. In this box plot, the median is shown by the horizontal line, the top and bottom of the box indicate the interquartile range, and the whiskers indicate the minimum and maximum values ​​for each experimental group (n=5-6 / group). Values ​​for individual animals are indicated by colored symbols. The dashed line indicates the limit of detection (LOD) of the assay. Student's t-test (paired, two-tailed) was used to identify significant differences in viral sgRNA levels at 2, 4, and 7 dpi between placebo-treated and immunized animals. Not significant (ns), ***p ≤ 0.001, ****p ≤ 0.0001. [Figure 13G]This study demonstrates weight changes and protection against upper and lower respiratory tract infections caused by SARS-CoV-2 in hamsters immunized with the qNIV / CoV2373 combination vaccine. Groups of male and female hamsters (n=5-6 / group) were immunized with qNIV / CoV2373 or a prime / boost regimen of the component vaccine at 14-day intervals. Three weeks after the second immunization (day 35 of the study), animals were administered 2.0 x 10⁴ pfu of antigen via the intranasal route. Lung weights collected at 7 dpi from vaccinated, placebo-administered, and untreated Siamese hamsters are shown. Bars represent the mean, and error bars represent ± standard deviation (SD). Individual animal values ​​are indicated by colored symbols. Student's t-test (paired, two-sided) was used to identify significant differences in lung weight between the paired groups shown.

[0024] [Figure 14]A-J show histopathological changes in the lungs of hamsters immunized with the qNIV / CoV2373 combination vaccine and administered SARS-CoV-2 antigen. Male and female hamsters were immunized with qNIV / CoV2373 in combination with 2.5 μg or 10 μg of hemagglutinin (HA) per strain and 1 μg or 5 μg of CoV2373 adjuvanted with a saponin adjuvant. The control group was immunized with 2.5 μg or 10 μg of HA per strain, or 1 μg or 5 μg of recombinant CoV2373 spike adjuvanted with 15 μg of saponin adjuvant per strain. All groups were immunized twice at 14-day intervals. The placebo group was administered a drug buffer. Three weeks after the second immunization (day 35 of the study), all animals were administered 2.0 x 10⁴ pfu of SARS-CoV-2 antigen intranasally, and lung tissue was collected at 7 dpi. Histological images were stained with hematoxylin-eosin (H&E). A is an image of a sham control showing a normal lung. B shows microscopic findings of the lungs of placebo-treated animals, indicating that the airways were consolidated by bronchioloalveolar hyperplasia (arrow) with mixed alveolar inflammation. Mononuclear inflammatory cells surround blood vessels (arrowheads) with edema spreading into the surrounding tissue (arrowheads). C-D show microscopic findings of the lungs of qNIV-immunized animals, showing similar histological changes to the placebo group, including extensive bronchioloalveolar hyperplasia (arrow) and vascular wall dilation (arrowhead) with mixed alveolar inflammation and perivasculitis. Images E-F show lung images of animals immunized with monovalent CoV2373, indicating no significant microscopic findings. Images G-J show lung images of animals immunized with qNIV / CoV2373 combined vaccine, indicating no significant microscopic findings. Magnification: 10x.

[0025] [Figure 15] The titers of the anti-spike IgG antibody are shown as a function of the doses of hemagglutinin and CoV S polypeptide from day 0 to day 28 post-immunization according to Example 5.

[0026] [Figure 16]The geometric mean titer of HAI against A / Brisbane H1N1 is shown as a function of hemagglutinin and CoV S polypeptide doses from day 0 to day 28 post-immunization according to Example 5.

[0027] [Figure 17] The geometric mean titer of HAI against A / Kansas H3N2 is shown as a function of hemagglutinin and CoV S polypeptide doses from day 0 to day 28 post-immunization according to Example 5.

[0028] [Figure 18] The geometric mean titer of HAI against B / Maryland (Vic) is shown as a function of hemagglutinin and CoV S polypeptide doses from day 0 to day 28 post-immunization according to Example 5.

[0029] [Figure 19] The geometric mean titer of HAI against B / Phuket (Yam) is shown as a function of hemagglutinin and CoV S polypeptide doses from day 0 to day 28 post-immunization according to Example 5.

[0030] [Figure 20] Five quadratic models of Example 6 are shown.

[0031] [Figure 21] The geometric mean titer of HAI against A / Brisbane H1N1 is shown as a function of hemagglutinin and CoV S polypeptide doses from day 0 to day 182 post-immunization according to Example 5.

[0032] [Figure 22] The titers of the anti-spike IgG antibody (in geometric mean ELISA units) are shown as a function of the doses of hemagglutinin and CoV S polypeptide from day 0 to day 182 post-immunization according to Example 5.

[0033] [Figure 23]The amount of CD4+ T cells producing dual cytokines is shown as a function of the doses of hemagglutinin and CoV S polypeptide from day 0 to day 7 post-immunization according to Example 5 (SARS-CoV-2 parental strain (allogeneic)).

[0034] [Figure 24] The geometric mean titer of HAI against A / Hong Kong H3N2 (heterogeneic) is shown as a function of hemagglutinin and CoV S polypeptide doses from day 0 to day 28 post-immunization according to Example 5.

[0035] [Figure 25] The titers of anti-spike IgG antibodies against SARS-CoV-2 omicron BA.1 (heterogeneous strain) are shown as a function of hemagglutinin and CoV S polypeptide doses from day 0 to day 28 post-immunization according to Example 5.

[0036] [Figure 26] The amount of CD4+ T cells producing dual cytokines is shown as a function of hemagglutinin and CoV S polypeptide doses from day 0 to day 7 (A / Cambodia (heterologous H3N2)).

[0037] [Figure 27] The images show serum from patients administered with the composition described in Example 8, which includes hemagglutinin, CoV S polypeptide, and saponin adjuvant, and which contains anti-spike IgG antibody and antibody to neutralize SARS-CoV-2. "M50" refers to a dose of 50 μg of saponin adjuvant. "M75" refers to a dose of 75 μg of saponin adjuvant. "HA30" refers to a dose of 30 μg of hemagglutinin per strain. "HA45" refers to a dose of 45 μg of hemagglutinin per strain. "HA60" refers to a dose of 60 μg of hemagglutinin per strain. "rS15" refers to a dose of 15 μg of CoV S polypeptide. "rS25" refers to a dose of 25 μg of CoV S polypeptide. "rS35" refers to a dose of 35 μg of CoV S polypeptide.

[0038] [Figure 28A] The geometric mean titers of HAI against influenza strains A / Wisconsin (H1N1) and B / Victoria are shown for patients administered the composition of Example 8. "M50" refers to a dose of 50 μg of saponin adjuvant. "M75" refers to a dose of 75 μg of saponin adjuvant. "HA30" refers to a dose of 30 μg of hemagglutinin per strain. "HA45" refers to a dose of 45 μg of hemagglutinin per strain. "HA60" refers to a dose of 60 μg of hemagglutinin per strain. "rS15" refers to a dose of 15 μg of CoV S polypeptide. "rS25" is [Figure 28B] The geometric mean titers of HAI against influenza strains A / Darwin (H3N2) and B / Yamagata are shown for patients administered the composition of Example 8. "M50" refers to a dose of 50 μg of saponin adjuvant. "M75" refers to a dose of 75 μg of saponin adjuvant. "HA30" refers to a dose of 30 μg of hemagglutinin per strain. "HA45" refers to a dose of 45 μg of hemagglutinin per strain. "HA60" refers to a dose of 60 μg of hemagglutinin per strain. "rS15" refers to a dose of 15 μg of CoV S polypeptide. "rS25" is

[0039] [Figure 29] The titers (in geometric mean ELISA units) of anti-spike IgG and neutralizing antibodies for compositions F and K of Example 8 are shown. Composition F contained 30 μg of HA, 25 μg of CoV S polypeptide, and 75 μg of saponin adjuvant per strain. Composition K contained 60 μg of HA, 35 μg of CoV S polypeptide, and 75 μg of saponin adjuvant per strain. These compositions contain the CoV S polypeptide of SEQ ID NO: 87 (NVX-CoV2373) and induce titers comparable to compositions that do not contain hemagglutinin protein.

[0040] [Figure 30]The geometric mean titers of HAI against influenza strains A / Wisconsin (H1N1), A / Darwin (H3N2), B / Victoria, and B / Yamagata in the serum of patients administered compositions F and K of Example 8 are shown in comparison to those from the serum of patients administered with commercially available influenza vaccines (FLUZONE® HD or FLUAD®). Composition F contained 30 μg of HA, 25 μg of CoV S polypeptide, and 75 μg of saponin adjuvant per strain. Composition K contained 60 μg of HA, 35 μg of CoV S polypeptide, and 75 μg of saponin adjuvant per strain. These compositions contain the CoV S polypeptide of SEQ ID NO: 87 (NVX-CoV2373) and induce titers comparable to those of compositions without hemagglutinin.

[0041] [Figure 31] The HAI titers are shown by the commercially available influenza vaccines FLUZONE®HD (Composition S of Example 8) and FLUAD® (Composition T of Example 8), as well as by compositions L, M, and N of Example 8, which contain HA glycoprotein and saponin adjuvant but do not contain CoV S polypeptide. [Modes for carrying out the invention]

[0042] definition As used herein and in the appended claims, the singular nouns “a,” “an,” and “the” include multiple references unless the context clearly indicates otherwise. Thus, for example, a reference to “a protein” may refer to one protein or a mixture of such proteins, and a reference to “the method” may include multiple equivalent steps and / or methods known to those skilled in the art, and so on.

[0043] As used herein, the term "adjuvant" refers to a compound that, when used in combination with an immunogen, amplifies, or otherwise alters or modifies, the immune response induced to an immunogen. Modification of the immune response may include enhancing or expanding the specificity of one or both of the antibody immune response and the cellular immune response.

[0044] As used herein, the terms “about” or “approximately” when preceding a number indicate a range of plus or minus 10% of that number. For example, “about 100” includes 90 and 110.

[0045] As used herein, the terms “immunogen,” “antigen,” and “epitope” refer to substances that can induce an immune response, such as proteins and peptides, including glycoproteins.

[0046] As used herein, “immunogenic composition” means a composition comprising an antigen, wherein administration of the composition to a subject induces a humoral and / or cellular immune response to the antigen.

[0047] As used herein, a “subunit” composition, such as a vaccine, contains one or more selected antigens, but not all, of the antigens derived from a pathogen. Such compositions are substantially free of intact viruses or lysates of such cells or particles, and are typically prepared from immunogenic polypeptides that are at least partially, and often substantially, purified from the pathogen. The antigens in the subunit compositions disclosed herein are typically prepared by recombinant means, often using baculovirus systems.

[0048] As used herein, “substantially” means the isolation of a substance (e.g., a compound, polynucleotide, or polypeptide) such that the substance constitutes the majority of the sample in which it is contained. For example, in a sample, the substantially purified component constitutes 85%, preferably 85% to 90%, more preferably at least 95% to 99.5%, and most preferably at least 99% of the sample. If the component is substantially replaced, the amount remaining in the sample is about 0.5% to about 10% or less, preferably about 0.5% to less than 1.0%.

[0049] The terms “to treat,” “treatment,” and “to treat” as used herein refer to methods for obtaining beneficial or desired outcomes, such as clinical outcomes. For the purposes of this disclosure, beneficial or desired outcomes could include inhibiting or suppressing the onset or progression of an infection or disease, improving or reducing the occurrence of symptoms of an infection or disease, or a combination thereof.

[0050] "Prevention," as used herein, is interchangeable with "prevention" and may mean the complete prevention of an infection or disease, or the prevention of the onset of symptoms of such infection or disease; the delay of the onset of an infection or disease or symptoms of such infection or disease; or a reduction in the severity of any subsequent infection or disease or symptoms of such infection or disease.

[0051] As used herein, “effective dose” or “effective amount” refers to the amount of immunogen sufficient to induce an immune response that alleviates at least one symptom of a pathogenic infection. The effective dose or effective amount may be determined by measuring, for example, the amount of neutralizing secretion and / or serum antibodies by plaque neutralization, complement fixation, enzyme-linked immunosorbent assay (ELISA), or microneutralization assay.

[0052] As used herein, the term “vaccine” refers to an immunogenic composition, such as an immunogen derived from a pathogen, used to induce an immune response against the pathogen. Such immune response may include antibody formation and / or cellular responses. Depending on the context, the term “vaccine” may also refer to a suspension or solution of an immunogen administered to a subject to evoke an immune response. Preferably, the vaccine induces an immune response that is effective in preventing infection from SARS-CoV-2, its SARS-CoV-2 variants, influenza, or a combination thereof.

[0053] As used herein, the term “Subject” includes humans and other animals. Typically, the subject is human. For example, the subject may be an adult, a teenager, a child (2 to 14 years of age), an infant (birth to 2 years of age), or a newborn (up to 2 months of age). In certain embodiments, the subject may be up to 4 months of age or up to 6 months of age. In several embodiments, the adult may be approximately 65 years of age or older, or approximately 60 years of age or older. In several embodiments, the subject may be a pregnant woman or a woman intending to become pregnant. In other embodiments, the subject may not be human, but a non-human primate, such as a baboon, chimpanzee, gorilla, or macaque. In certain embodiments, the subject may be a pet, such as a dog or cat.

[0054] In several embodiments, the subject is immunocompromised. In several embodiments, the immunocompromised subject is administered an immunosuppressant drug. Non-limiting examples of immunosuppressant drugs include corticosteroids (e.g., prednisone), alkylating agents (e.g., cyclophosphamide), antimetabolites (e.g., azathioprine or 6-mercaptopurine), transplant-related immunosuppressants (e.g., cyclosporine, tacrolimus, sirolimus, or mycophenolate mofetil), mitoxantrone, chemotherapeutic agents, methotrexate, and tumor necrosis factor (TNF) blockers (e.g., etanercept, adalimumab, infliximab). In several embodiments, the immunocompromised subject is infected with a virus (e.g., human immunodeficiency virus or Epstein-Barr virus). In several embodiments, the virus is a respiratory virus, e.g., polynuclear respiratory virus, influenza, parainfluenza, adenovirus, or picornavirus. In multiple embodiments, the immunodeficiency subject has acquired immunodeficiency syndrome (AIDS). In multiple embodiments, the immunodeficiency subject is infected with human immunodeficiency virus (HIV). In multiple embodiments, the immunodeficiency subject is immunodeficiency due to a treatment regimen designed to prevent inflammation or graft rejection. In multiple embodiments, the immunodeficiency subject is a transplant recipient. In multiple embodiments, the immunodeficiency subject has undergone radiotherapy or splenectomy.In several embodiments, the immunodeficiency target is cancer, autoimmune disease, tuberculosis, substance use disorder (e.g., alcohol, opioid, or cocaine use disorder), stroke or cerebrovascular disease, parenchymal organ or hematopoietic stem cell transplantation, sickle cell disease, thalassemia, autoimmune lymphoproliferative syndrome (ALPS), polyglandular autoimmune syndrome type 1 (APS-1), B cell proliferation (BENTA) disorder with NF-κB and T cell anergy, caspase-8 deficiency (CEDS), chronic granulomatous disease (CGD), unclassified immunodeficiency (CVID), congenital neutropenia syndrome, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) deficiency, DOCK8 deficiency, GATA2 deficiency, glycosylation disorders with immunodeficiency, hyperimmuneglobulin E syndrome ( Diagnosed with HIES, hyperimmune globulin M syndrome, diabetes mellitus, type 1 diabetes mellitus, type 2 diabetes mellitus, interferon-gamma deficiency, interleukin-12 deficiency, interleukin-23 deficiency, leukocyte adhesion disorder, lipopolysaccharide-responsive beige-like anchor (LRBA) deficiency, PI3 kinase disorder, PLCG2-related antibody deficiency / immunodeficiency (PLAID), severe combined immunodeficiency (SCID), STAT3 dominant-negative disorder, STAT3 gain-of-function disorder, verrucae-hypogammaglobulinemia-infectious disease-myeloid cell retention (WHIM) syndrome, Wiscott-Aldrich syndrome (WAS), X-linked agammaglobulinemia (XLA), X-linked lymphoproliferative disorder (XLP), uremia, malnutrition, or X-Men disease. In multiple embodiments, the immunocompromised subject was either a current or former cigarette smoker. In several embodiments, the immunodeficiency subject has B cell deficiency, T cell deficiency, macrophage deficiency, cytokine deficiency, phagocyte deficiency, phagocyte dysfunction, complement deficiency, or a combination thereof.

[0055] In several embodiments, the subject is overweight or obese. In several embodiments, the overweight subject is 25 kg / m². 2 More than 30 kg / m 2 Having a body mass index (BMI) of less than 30 kg / m². In several embodiments, an obese subject is defined as having a BMI of less than 30 kg / m². 2The subject has a BMI of the above. In some embodiments, the subject has a mental disorder. In some embodiments, the mental disorder is depression, schizophrenia, or anxiety.

[0056] As used herein, the term “pharmaceutically acceptable” means that it is approved by a U.S. federal or state regulatory authority, or is listed in the United States Pharmacopeia, the European Pharmacopeia, or any other pharmacopoeia generally accepted for use in mammals, more specifically in humans. These compositions may be useful as vaccines and / or antigenic compositions for inducing a protective immune response in vertebrates.

[0057] As used herein, the term "approximately" means plus or minus 10% of the indicated value.

[0058] As used herein, the term "NVX-CoV2373" refers to a vaccine composition comprising the BV2373 spike glycoprotein (SEQ ID NO: 87) and the ISCOM matrix (e.g., MATRIX-M®) of fractions A and C.

[0059] As used herein, “Quad-NIV,” “QuadNIV,” “tetravalent nanoparticle influenza vaccine,” or “qNIV” refers to an influenza vaccine formulation containing antigens derived from four influenza virus strains.

[0060] As used herein, the term “modification” refers to a mutation, deletion, or addition of one or more amino acids in a CoV S polypeptide, when referring to a CoV S polypeptide. The location of a modification within a CoV S polypeptide can be identified by aligning the polypeptide sequence to SEQ ID NO: 1 (a CoV S polypeptide containing a signal peptide) or SEQ ID NO: 2 (a mature CoV S polypeptide lacking a signal peptide).

[0061] In this specification, the term SARS-CoV-2 "variant," used interchangeably with "heterogeneous SARS-CoV-2 strain," refers to a SARS-CoV-2 virus containing a CoV S polypeptide having one or more modifications compared to the SARS-CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. For example, a SARS-CoV-2 variant may have at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, or at least about 35 modifications compared to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. For example, a SARS-CoV-2 variant may have at least one and up to 2, up to 3, up to 4, up to 5, up to 5, up to 6, up to 65, up to 70, up to 75, up to 80, up to 85, up to 90, up to 95, or up to 100 modifications compared to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2.In several embodiments, the SARS-CoV-2 variant may have about 2 to about 35 modifications, about 5 to about 10 modifications, about 5 to about 20 modifications, about 10 to about 20 modifications, about 15 to about 25 modifications, about 20 to about 30 modifications, about 20 to about 40 modifications, about 25 to about 45 modifications, about 25 to about 100 modifications, about 25 to about 45 modifications, and about 35 to about 100 modifications compared to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2.

[0062] In one embodiment, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus containing a CoV S polypeptide having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with respect to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In another embodiment, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus containing a CoV S polypeptide having at least 70% to about 99.9% identity with respect to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In yet another embodiment, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus containing a CoV S polypeptide having at least 70% to about 99.5% identity with respect to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In one embodiment, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus containing a CoV S polypeptide having approximately 90% to approximately 99.9% identity with the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus containing a CoV S polypeptide having approximately 90% to approximately 99.8% identity with the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In yet another embodiment, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus containing a CoV S polypeptide having approximately 95% to approximately 99.9% identity with the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In yet another embodiment, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus containing a CoV S polypeptide having approximately 95% to approximately 99.8% identity with the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In this embodiment, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus containing a CoV S polypeptide that has approximately 95% to 99% identity with the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2.In embodiments, the heterologous SARS-CoV-2 strain has the World Health Organization label alpha, beta, gamma, delta, epsilon, eta, iota, kappa, zeta, mu, or omicron. In embodiments, the heterologous SARS-CoV-2 strain has a PANGO lineage selected from the group consisting of B.1.1.529, BA.1, BA.1.1, BA.2, BA.3, BA.4, BA.5, B.1.1.7, B.1.351, P.1, B.1.617.2, AY, B.1.427, B.1.429, B.1.525, B.1.526, B.1.617.1, B.1.617.3, P.2, B.1.621, or B.1.621.1. The following reference describes the designation of the PANGO lineage and is incorporated herein by reference in its entirety: O'Toole et al. BMC Genomics, 23, 121 (2022).

[0063] In one embodiment, the heterologous SARS-CoV-2 strain has the omicron labeled by the World Health Organization. In another embodiment, the heterologous SARS-CoV-2 strain having the omicron labeled by the World Health Organization has at least 35 modifications compared to the wild-type SARS-CoV-2 S polypeptide of SEQ ID NO: 2. In yet another embodiment, the heterologous SARS-CoV-2 strain having the omicron labeled by the World Health Organization has 35-55, 35-65, 35-75, 35-85, 35-95, or 35-105 modifications compared to the wild-type SARS-CoV-2 S polypeptide of SEQ ID NO: 2. In this embodiment, the modification applies to T6I, T6R, A14S, A54V, V70A, T82I, G129D, H133Q, K134E, W139R, E143G, F144L, Q170E, I197V, L199I, V200E, V200G, G239V, G244S, G326D, G326H, R333T, L355I, S 358F, S358L, S360P, S362F, T363A, D392N, R395S, K404N, N427K, K431T, V432P, G433S, L439R, L439Q, N447K, S464N, T465K, E471A, F473V, F473S, F477S, Q480R, G483S, Q485R, The following are selected from the group consisting of N488Y, Y492H, T534K, T591I, D601G, G626V, H642Y, N645S, N666K, P668H, S691L, N751K, D783Y, N843K, Q941H, N956K, L968F, D1186N, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, deletion of amino acid 57, deletion of amino acid 130, deletion of amino acid 131, deletion of amino acid 132, deletion of amino acid 144, deletion of amino acid 145, deletion of amino acid 198, insertion of a tripeptide having the amino acid sequence EPE between amino acids 214 and 215, and combinations thereof.

[0064] In the embodiment, the CoV S polypeptide of the variant includes a combination of modifications selected from the group consisting of: (i)A54V, T82I, G129D, L199I, G326D, S358L, S360P, S362F, K404N, N427K, G433S, S464 N, T465K, E471A, Q480R, G483S, Q485R, N488Y, Y492H, T534K, D601G, H642Y, N666K, P66 8H, N751K, D783Y, N843K, Q941H, N956K, L968F, deletion of amino acid 56, deletion of amino acid 57, deletion of amino acid 130, deletion of amino acid 131, deletion of amino acid 132, deletion of amino acid 198, and insertion of a tripeptide having amino acid sequence EPE between amino acids 214 and 215. (ii) T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, S464N, T465K, E471A, Q480R, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, and deletion of amino acid 13, (iii) T6R, A14S, T82I, G129D, E143G, L199I, G326D, S358L, S360P, K404N, N427K, G433S, S464N, T465K, E471A, Q480R, G483S, Q485R, N488Y, Y492H, T534K, D601G, H642Y, N666K, P668H, N751K, D783Y, N843K, Q941H, N956K, L968F, deletion of amino acid 144, deletion of amino acid 145, deletion of amino acid 198, and insertion of a tripeptide having amino acid sequence EPE between amino acids 214 and 215. (iv) T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, K404N, N427K, L439Q, S464N, T465K, E471A, Q480R, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, S691L, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, and deletion of amino acid 13, (v)T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, S464N, T465K, E471A, Q480R, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, and deletion of amino acid 13, (vi) T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, D601G, H642Y, N645S, N666K, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57, (vii) V3G, T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, G626V, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57, (viii) V3G, T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57, (ix) T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57, (x)T6I, A14S, G129D, K134E, W139R, F144L, I197V, V200G, G244S, G326H, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, G433S, N447K, S464N, T465K, E471A, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, and deletion of amino acid 13. (xi)T6I, A14S, G129D, K134E, W139R, F144L, I197V, V200G, G244S, G326H, R333T, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, G433S, L439R, N447K, S 464N, T465K, E471A, F473S, Q485R, N488Y, Y492H, T591I, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, D1186N, deletion of amino acid 11, deletion of amino acid 12, and deletion of amino acid 13, (xii)T6I, A14S, G129D, V200G, G326D, R333T, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N645S, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57, (xiii)T6I, A14S, G129D, V200G, G326D, R333T, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57, (xiv)T6I, A14S, V70A, G129D, H133Q, Q170E, V200E, G239V, G326H, R333T, L355I, S 358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, V432P, G433S, N447K, S46 4N, T465K, E471A, F473S, F477S, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, and deletion of amino acid 131, (xv)T6I, A14S, G129D, H133Q, Q170E, V200E, G326H, R333T, L355I, S358F, S360P, S36 2F, T363A, D392N, R395S, K404N, N427K, V432P, G433S, N447K, S464N, T465K, E471A, F 473S, F477S, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, deletion of amino acid 57, and deletion of amino acid 131, (xvi)T6I, A14S, G129D, V200G, G326D, R333T, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, K431T, L439R, N447K, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57, (xvii)T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, K431T, L439R, N447K, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57, and (xviii)T6I, A14S, G129D, V200G, G326D, R333T, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, deletion of amino acid 57, and deletion of amino acid 131, (xix) Deletion of amino acid 56, deletion of amino acid 57, and deletion of amino acid 131, N488Y, A557D, D601G, P668H or P668R, T703I, S969A, and D1105H, (xx)D67A, K404N, E471K, N488Y, D601G, and A688V, (xxi)D67A, D202G, L229H, K404N, E471K, N488Y, D601G, and A688V, (xxii)D67A, D202G, deletion of 1, 2, or 3 amino acids from amino acids 228-230, K404N, E471K, N488Y, D601G, and A688V, (xxiii) D67A, L229H, R233I, N488Y, K404N, E471K, D601G, and A688V, (xxiv) L5F, T7N, P13S, D125Y, R177S, K404T, E471K, N488Y, D601G, H642Y, T1014I, and V1163F, (xxv)W139C and L439, (xxvi) Deletion of amino acid 144, deletion of amino acid 145, T6R, E143G, L439R, T465K, D601G, P668R, and D937N, (xxvii) Deletion of amino acid 144, deletion of amino acid 145, T6R, G129D, E143G, L439R, T465K, D601G, P668R, and D937N, (xxviii) Deletion of amino acid 144, deletion of amino acid 145, T6R, T82I, G129D, Y132H, E143G, A209V, K404N, L439R, T465K, D601G, P668R, and D937N, (xxix) Deletion of amino acid 144, deletion of amino acid 145, T6R, G129D, E143G, W245I, K404N, N426K, L439R, T465K, E471K, N488Y, D601G, P668R, and D937N, (xxx) Deletion of amino acid 144, deletion of amino acid 145, T6R, W51H, H53W, G129D, E143G, D200V, L201R, W245I, K404N, N426K, L439R, T465K, E471K, N488Y, D601G, P668R, and D937N, (xxxi) Deletion of amino acid 144, deletion of amino acid 145, T6R, G129D, E143G, K404N, L439R, T465K, E471Q, D601G, P668R, and D937N, (xxxii) Deletion of Q39R, A54V, E471K, D601G, Q664H, F875L, and one, two, three, or four amino acids from 56, 57, 131, and 132. (xxxiii)T82I, D240G, E471K, D601G, and A688V, (xxxiv) L439R, E471Q, D601G, P668R, and Q1058H, (xxxv)G62V, T63I, R233N, L439Q, F477S, D601G, T846N, and deletions of 1, 2, 3, 4, 5, or 6 amino acids from 234 to 240. (xxxvi)T82I, Y131S, Y132N, R333K, E471K, N488Y, D601G, P668H, and D937N, and (xxxvii) G129D, G326D, S360P, S362F, K404N, N427K, T465K, E471A or E471K, Q480K or Q480R, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, and N953K, The amino acids of the CoV S glycoprotein are numbered relative to the polypeptide having the sequence of SEQ ID NO: 2.

[0065] A subject who tests "positive" for SARS-CoV-2 or its variants is defined as having tested positive for SARS-CoV-2 or its variants via PCR or serological testing. A positive PCR test detects genetic material from SARS-CoV-2 or its variants. A positive serological test indicates the presence of antibodies against the SARS-CoV-2 protein, usually the nucleocapsid protein derived from SARS-CoV-2 or its variants.

[0066] The term "asymptomatic" refers to individuals who test positive for SARS-CoV-2 or a SARS-CoV-2 variant but do not experience any symptoms of COVID-19.

[0067] The term “mild” refers, when referring to COVID-19, to a person who is positive for SARS-CoV-2 or a variant by PCR or serological test and has one or more of the following symptoms: (i) fever, (ii) new-onset cough, (iii) or two further COVID-19 symptoms selected from new-onset or worsening of shortness of breath or difficulty breathing, fatigue, generalized muscle aches or body aches, headache, loss of taste or smell, sore throat, congestion, or runny nose, or nausea, vomiting, or diarrhea.

[0068] The term "moderate" refers, when referring to COVID-19, to a person who is positive for SARS-CoV-2 or a variant by PCR or serological test and has one or more of the following symptoms: (i) a high fever of 38.4°C or higher for three days or more; (ii) (a) shortness of breath with or without exertion; (b) tachypnea (24-29 breaths per minute at rest); (c) SpO2 of 94-95%; (d) abnormal chest X-ray or computed tomography (CT) findings consistent with pneumonia or LRTI; or (e) findings of any of the following on lung auscultation: adventitious sounds (e.g., crackles / rales, dry rales, crackles, pleural friction rubs, wheezing).

[0069] The term "severe," when referring to COVID-19, means a person who is positive for SARS-CoV-2 or a variant by PCR or serological test and has one or more of the following symptoms: (i) tachypnea with a resting respiratory rate of 30 breaths per minute or more, (ii) a resting heart rate of 125 beats per minute or more, (iii) SpO2 of 93% or less or PaO2 / FiO2 of less than 300 mmHg, (iv) need for high-flow oxygen therapy or non-invasive ventilation, non-invasive positive pressure ventilation (e.g., continuous positive airway pressure (CPAP) or biphasic positive airway pressure (BiPAP)), (v) need for mechanical ventilation or extracorporeal membrane oxygenation (ECMO), (vi) (a) acute (b) acute respiratory failure, including respiratory distress syndrome (ARDS); (c) acute renal failure; (d) acute right or left heart failure; (e) sepsis or cardiac shock (with shock defined as systolic blood pressure (SBP) <90 mmHg or diastolic blood pressure (DBP) <60 mmHg); (f) acute stroke (ischemic or hemorrhagic); (g) acute thrombotic event, e.g., acute myocardial infarction (AMI), deep vein thrombosis (DVT), or pulmonary embolism (PE); (h) one or more major organ failures or impairments selected from the need for vasopressors, systemic corticosteroids, or hemodialysis; (vii) admission to the intensive care unit; or (viii) death.

[0070] As used herein, the terms “bedside mix,” “bedside preparation,” “bedside vaccine composition,” “bedside vial,” and “bedside vial preparation” refer to a vaccine preparation prepared immediately before administration. Such a vaccine preparation comprises a viral antigen and an adjuvant, which are stored separately in different containers and administered to the subject (for example, by administering two consecutive injections, or by combining the antigen and adjuvant into a single injection solution before administration).

[0071] As used herein, the terms “combination mix,” “combination,” “combination vaccine composition,” “filled syringe,” and “premix” refer to vaccine formulations prepared for short- to long-term storage prior to administration to a subject. Such vaccine formulations contain a combination of antigen and adjuvant in the same container and are prepared before administration. In embodiments, the formulation comprises hemagglutinin and an adjuvant (e.g., a saponin adjuvant) to form HaSMaN (hemagglutinin saponin matrix nanoparticles).

[0072] The term “effectiveness” of an immunogenic composition or vaccine composition as described herein refers to the percentage reduction in disease (e.g., COVID-19) in the group administered with the immunogenic composition compared to the group not administered with the immunogenic composition. In embodiments, effectiveness (E) is calculated using the following formula: E(%) = (1-RR) × 100 (wherein RR = relative risk of morbidity between the group administered with the immunogenic composition and the group not administered with the immunogenic composition). In embodiments, the immunogenic compositions described herein are effective against SARS-CoV-2 virus or heterologous SARS-CoV-2 strains by at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, and at least It has an effectiveness of approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, approximately 50% to approximately 99%, approximately 50% to approximately 98%, approximately 60% to approximately 99%, approximately 60% to approximately 98%, approximately 70% to approximately 98%, approximately 70% to approximately 95%, approximately 70% to approximately 99%, approximately 80% to approximately 99%, approximately 80% to approximately 98%, approximately 80% to approximately 95%, approximately 85% to approximately 99%, approximately 85% to approximately 98%, approximately 85% to approximately 95%, approximately 90% to approximately 95%, approximately 90% to approximately 98%, or approximately 90% to approximately 99%.

[0073] As used herein, the term “split virion” refers to a virus having a viral membrane disrupted with a surfactant (e.g., influenza virus or SARS-CoV-2 virus). Examples of surfactants are described throughout this disclosure. Split virions do not undergo further purification and therefore typically contain multiple viral proteins.

[0074] As used herein, the term “recombinant” refers to a protein (e.g., hemagglutinin) produced in a cell by the transcription and translation of a nucleic acid introduced into the cell. Nucleic acids may be introduced via a nucleic acid-coding vector or a virus.

[0075] As used herein, the term "whole influenza virus" refers to a virus including all of its components: the envelope, viral membrane, nucleocapsid, and genetic material. In several embodiments, the whole influenza virus is inactivated.

[0076] As used herein, the term "inactivated virus" refers to a virus that has been treated to substantially reduce or eliminate its pathogenicity compared to a wild-type virus.

[0077] immunogenic composition Immunogenic composition against influenza virus This specification provides immunogenic compositions and vaccine compositions comprising (i) at least three hemagglutinin (HA) glycoproteins, wherein the three hemagglutinin (HA) glycoproteins are derived from different influenza strains, and (ii) a pharmaceutically acceptable buffer. In embodiments, the at least three HA glycoproteins are in forms selected from the group consisting of (a) surfactant core nanoparticles containing hemagglutinin (HA), (b) HaSMaN (hemagglutinin saponin matrix nanoparticles), (c) inactivated whole influenza virus, (d) hemagglutinin compositions extracted from influenza viruses, optionally influenza split virion compositions or subunit influenza compositions, and any combination thereof. In embodiments, this specification provides methods for using the above immunogenic compositions and vaccine compositions to stimulate an immune response to influenza virus. In embodiments, the composition comprises four HA glycoproteins, each HA glycoprotein derived from a different strain.

[0078] In the embodiment, the composition contains approximately 25 μg to approximately 75 μg of hemagglutinin per strain. In the embodiment, the composition contains approximately 30 μg to approximately 60 μg of hemagglutinin per strain. In the embodiment, the composition contains approximately 30 μg, approximately 31 μg, approximately 32 μg, approximately 33 μg, approximately 34 μg, approximately 35 μg, approximately 36 μg, approximately 37 μg, approximately 38 μg, approximately 39 μg, approximately 40 μg, approximately 41 μg, approximately 42 μg, approximately 43 μg, approximately 44 μg, approximately 45 μg, approximately 46 μg, approximately 47 μg, approximately 48 μg, approximately 49 μg, approximately 50 μg, approximately 51 μg, approximately 52 μg, approximately 5 The composition includes 3 μg, approximately 54 μg, approximately 55 μg, approximately 56 μg, approximately 57 μg, approximately 58 μg, approximately 59 μg, approximately 60 μg, approximately 61 μg, approximately 62 μg, approximately 63 μg, approximately 64 μg, approximately 65 μg, approximately 66 μg, approximately 67 μg, approximately 68 μg, approximately 69 μg, approximately 70 μg, approximately 71 μg, approximately 72 μg, approximately 73 μg, approximately 74 μg, or approximately 75 μg, including all values ​​and partial ranges between them. In embodiments, the composition contains approximately 30 μg of hemagglutinin per strain. In embodiments, the composition contains approximately 45 μg of hemagglutinin per strain. In embodiments, the composition contains approximately 60 μg of hemagglutinin per strain.

[0079] In embodiments, the composition further comprises any pharmaceutically acceptable buffer as described herein. In embodiments, the composition further comprises any adjuvant as described herein. In embodiments, the composition comprises a saponin adjuvant. In embodiments, the saponin adjuvant comprises at least two iscom particles, the first iscom particle comprising fraction A of Quillaja Saponaria Molina but not fraction C of Quillaja Saponaria Molina, and the second iscom particle comprising fraction C of Quillaja Saponaria Molina but not fraction A of Quillaja Saponaria Molina. In embodiments, fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina account for about 92% by weight and about 8% by weight, respectively, of the total weight of fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina in the adjuvant. In the embodiment, fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina account for about 85% by weight and about 15% by weight, respectively, of the total weight of fractions A and C of Quillaja Saponaria Molina in the adjuvant. In the embodiment, fraction A of Quillaja Saponaria Molina accounts for at least 85% by weight of the adjuvant, and fraction C of Quillaja Saponaria accounts for the remainder of the weight of the adjuvant. In the embodiment, fraction A of Quillaja Saponaria Molina accounts for at least 70% by weight of the adjuvant, and fraction C of Quillaja Saponaria accounts for the remainder of the weight of the adjuvant. In the embodiment, the adjuvant is present in the composition in an amount of 30 μg to about 100 μg.In the embodiment, the composition is approximately 30 μg, approximately 31 μg, approximately 32 μg, approximately 33 μg, approximately 34 μg, approximately 35 μg, approximately 36 μg, approximately 37 μg, approximately 38 μg, approximately 39 μg, approximately 40 μg, approximately 41 μg, approximately 42 μg, approximately 43 μg, approximately 44 μg, approximately 45 μg, approximately 46 μg, approximately 47 μg, approximately 48 μg, approximately 49 μg, approximately 50 μg, approximately 51 μg, approximately 52 μg, approximately 53 μg, approximately 54 μg, approximately 55 μg, approximately 56 μg, approximately 57 μg, approximately 58 μg, approximately 59 μg, approximately 60 μg, approximately 61 μg, approximately 62 μg, approximately 63 μg, approximately 64 μg, approximately 65 The composition contains approximately 100 μg of adjuvant, about 66 μg, about 67 μg, about 68 μg, about 69 μg, about 70 μg, about 71 μg, about 72 μg, about 73 μg, about 74 μg, about 75 μg, about 76 μg, about 77 μg, about 78 μg, about 79 μg, about 80 μg, about 81 μg, about 82 μg, about 83 μg, about 84 μg, about 85 μg, about 86 μg, about 87 μg, about 88 μg, about 89 μg, about 90 μg, about 91 μg, about 92 μg, about 93 μg, about 94 μg, about 95 μg, about 96 μg, about 97 μg, about 98 μg, about 99 μg, or about 100 μg. In some embodiments, the composition contains about 50 μg or 75 μg of adjuvant.

[0080] Immunogenic compositions against coronavirus and influenza virus This specification provides immunogenic compositions and vaccine compositions comprising: (i) at least three hemagglutinin (HA) glycoproteins, wherein the three hemagglutinin (HA) glycoproteins are derived from different influenza strains; (ii) a CoV S polypeptide in the form of surfactant core nanoparticles, wherein the surfactant is a nonionic surfactant; and (iii) a pharmaceutically acceptable buffer. In embodiments, the at least three HA glycoproteins are in forms selected from the group consisting of (a) surfactant core nanoparticles containing hemagglutinin (HA); (b) HaSMaN (hemagglutinin saponin matrix nanoparticles); (c) inactivated whole influenza virus; (d) a hemagglutinin composition extracted from influenza virus; optionally, an influenza split virion composition or a subunit influenza composition; and any combination thereof. In embodiments, this specification provides methods for using the immunogenic compositions and vaccine compositions to stimulate an immune response to coronavirus, influenza virus, or a combination thereof.

[0081] Similarly, this specification provides a method for producing the above-mentioned nanoparticles and immunogenic compositions. Advantageously, the method provides nanoparticles that are substantially free from contamination by other proteins, such as proteins associated with the recombinant expression of proteins in insect cells. In embodiments, expression is carried out in a baculovirus / Sf9 system.

[0082] (i) non-spontaneously occurring CoV S polypeptides or nanoparticles The immunogenic compositions of this disclosure comprise non-spontaneously occurring CoV S polypeptides or nanoparticles containing them. The CoV S polypeptides may be derived from coronaviruses, including but not limited to SARS-CoV-2, and may be derived from, for example, SARS-CoV-2, MERS CoV, and SARS CoV. In embodiments, the immunogenic compositions of this disclosure comprise about 1 to about 15, about 2 to about 15, about 3 to about 15, about 3 to about 12, about 4 to about 6, about 3 to about 7, about 4 to about 12, about 5 to about 8, or about 6 to about 9 non-spontaneously occurring CoV S polypeptides.

[0083] In embodiments, the CoV S polypeptide is derived from a heterologous SARS-CoV-2 strain. In embodiments, the heterologous SARS-CoV-2 strain has a PANGO lineage selected from the group consisting of B.1.1.529, BA.1, BA.1.1, BA.2, BA.3, BA.4, BA.5, B.1.1.7, B.1.351, P.1, B.1.617.2, AY, B.1.427, B.1.429, B.1.525, B.1.526, B.1.617.1, B.1.617.3, P.2, B.1.621, or B.1.621.1. In embodiments, the heterologous SARS-CoV-2 strain has alpha, beta, gamma, delta, epsilon, eta, iota, kappa, zeta, mu, or omicron as labeled by the World Health Organization.

[0084] In the embodiment, the SARS-CoV-2 virus has a CoV S polypeptide having the amino acid sequence of SEQ ID NO: 1, and the variant of SARS-CoV-2 includes a CoV S polypeptide having 1 to 100 modifications compared to the CoV S polypeptide of SEQ ID NO: 1. In the embodiment, the SARS-CoV-2 virus has a CoV S polypeptide having the amino acid sequence of SEQ ID NO: 1The variant of SARS-CoV-2 having S-polypeptide is at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, and at least about 2 3, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, at least about 35, at least about 36, at least about 37, at least about 38, at least about 39, at least about 40, at least about 41, at least about 42, at least about 43, at least about 44, at least about 45, at least about 46, at least about 47, at least about 48, at least about 49, less Approximately 50, at least approximately 51, at least approximately 52, at least approximately 53, at least approximately 54, at least approximately 55, at least approximately 56, at least approximately 57, at least approximately 58, at least approximately 59, at least approximately 60, at least approximately 61, at least approximately 62, at least approximately 63, at least approximately 64, at least approximately 65, at least approximately 66, at least approximately 67, at least approximately 68, at least approximately 69, at least approximately 70, at least approximately 71, at least approximately 72, at least approximately 73, at least approximately 74, at least approximately 75, at least approximately 76 CoV having at least about 77, at least about 78, at least about 79, at least about 80, at least about 81, at least about 82, at least about 83, at least about 84, at least about 85, at least about 86, at least about 87, at least about 88, at least about 89, at least about 90, at least about 91, at least about 91, at least about 92, at least about 93, at least about 94, at least about 95, at least about 96, at least about 97, at least about 98, at least about 99, or at least 100 modificationsIt contains an S polypeptide. In one embodiment, the SARS-CoV-2 virus has a CoV S polypeptide having the amino acid sequence of SEQ ID NO: 1, and the variant of SARS-CoV-2 has at least 1 and up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 6, up to about 7, up to about 8, up to about 9, up to about 10, up to about 11, up to about 12, up to about 13, up to about 14, up to about 15, up to about 16, up to about 17, up to about 18, up to about 19, up to about 20, up to about 21 , maximum approximately 22, maximum approximately 23, maximum approximately 24, maximum approximately 25, maximum approximately 26, maximum approximately 27, maximum approximately 28, maximum approximately 29, maximum approximately 30, maximum approximately 31, maximum approximately 32, maximum approximately 33, maximum approximately 34, maximum approximately 35, maximum approximately 36, maximum approximately 37, maximum approximately 38, maximum approximately 39, maximum approximately 40, maximum approximately 41, maximum approximately 42, maximum approximately 43, maximum approximately 44, maximum approximately 45, maximum approximately 46, maximum approximately 47, maximum approximately 48, maximum Approximately 49, maximum approximately 50, maximum approximately 51, maximum approximately 52, maximum approximately 53, maximum approximately 54, maximum approximately 55, maximum approximately 56, maximum approximately 57, maximum approximately 58, maximum approximately 59, maximum approximately 60, maximum approximately 61, maximum approximately 62, 63 maximum, 64 maximum, 65 maximum, 66 maximum, 67 maximum, 68 maximum, 69 maximum, 70 maximum, 71 maximum, 72 maximum, 73 maximum, 74 maximum, 75 maximum, 76 maximum , containing CoV S polypeptide having up to approximately 77, up to approximately 78, up to approximately 79, up to approximately 80, up to approximately 81, up to approximately 82, up to approximately 83, up to approximately 84, up to approximately 85, up to approximately 86, up to approximately 87, up to approximately 88, up to approximately 89, up to approximately 90, up to approximately 91, up to approximately 91, up to approximately 92, up to approximately 93, up to approximately 94, up to approximately 95, up to approximately 96, up to approximately 97, up to approximately 98, up to approximately 99, or up to approximately 100 modifications.

[0085] In contrast to the SARS-CoV S protein, the SARS-CoV-2 S protein has four amino acid insertions at the S1 / S2 cleavage site, resulting in a polybasic RRAR furin-like cleavage motif. The SARS-CoV-2 S protein is synthesized as an inactive precursor (S0), which is proteolytically cleaved at the furin cleavage site into S1 and S2 subunits, which remain non-covalently linked to form a prefusion trimer. The S2 domain of the SARS-CoV-2 S protein contains a fusion peptide (FP), two 7-amino acid repeat sequences (HR1 and HR2), a transmembrane (TM) domain, and a cytoplasmic tail (CT). The S1 domain of the SARS-CoV-2 S protein folds into four separate domains: an N-terminal domain (NTD), and a C-terminal domain containing a receptor-binding domain (RBD) and two subdomains SD1 and SD2. The pre-fusion SARS-CoV-2 S protein trimer undergoes structural reconfiguration from its pre-fusion structure to its post-fusion structure upon binding to and cleavage of the S protein receptor.

[0086] In embodiments, the CoV S polypeptide is a glycoprotein for post-translational glycosylation. The glycoprotein comprises one or more of the following: a signal peptide, an S1 subunit, an S2 subunit, an NTD, an RBD, two subdomains (SD1 and SD2, shown as SD1 / 2 in Figures 6A-B and referred to herein as "SD1 / 2"), an intact or modified fusion peptide, an HR1 domain, an HR2 domain, TM, and CD. In embodiments, the amino acids of each domain are given in Figure 6A (shown based on Sequence ID No. 1) and Figure 6B (shown based on Sequence ID No. 2).

[0087] In embodiments, each domain may have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with respect to the domain-specific sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2. Each domain may have up to approximately 1, at least approximately 2, at least approximately 3, at least approximately 4, at least approximately 5, at least approximately 10, at least approximately 20, at least approximately 30, at least approximately 35, at least approximately 40, at least approximately 45, or at least approximately 50 amino acid deletions, insertions, or mutations compared to the amino acids shown in SEQ ID NO: 1 or SEQ ID NO: 2. Each domain may have deletions, insertions, or mutations of approximately 1–5 amino acids, 3–10 amino acids, 5–10 amino acids, 8–12 amino acids, 10–15 amino acids, 12–17 amino acids, 15–20 amino acids, 18–23 amino acids, 20–25 amino acids, 22–27 amino acids, or 25–30 amino acids compared to the amino acids shown in SEQ ID NO: 1 or SEQ ID NO: 2. Note that Figures 2 and 3 show the 13-amino acid N-terminal signal peptide not present in the mature peptide. The CoV S polypeptide may be used to stimulate an immune response against the native CoV spike (S) polypeptide.

[0088] In this embodiment, a naturally occurring CoV spike(S) polypeptide (SEQ ID NO: 2) is modified to obtain a non-naturally occurring CoV spike(S) polypeptide.

[0089] In one embodiment, a naturally occurring CoV spike(S) polypeptide (SEQ ID NO: 2) is modified to obtain a non-naturally occurring CoV spike(S) polypeptide (Figure 1). In this embodiment, the CoV spike(S) glycoprotein comprises an S1 subunit and an S2 subunit, the S1 subunit comprising NTD, RBD, SD1 / 2, and an inactive furin cleavage site (amino acids 669-672), and the S2 subunit comprising mutations at amino acids 973 and 974. The NTD (amino acids 1-318) optionally includes one or more modifications selected from the group consisting of: (a) Deletion of one or more amino acids selected from the group consisting of amino acids 56, 57, 131, 132, 144, 145, 228, 229, 230, 231, 234, 235, 236, 237, 238, 239, 240 and combinations thereof, (b) Insertion of 1, 2, 3, or 4 amino acids after amino acid 132, and (c) Mutations of one or more amino acids selected from the group consisting of amino acids 5, 6, 7, 13, 39, 51, 53, 54, 56, 57, 62, 63, 67, 82, 125, 129, 131, 132, 133, 139, 143, 144, 145, 177, 200, 201, 202, 209, 229, 233, 240, 245 and combinations thereof. The RBD optionally includes mutations in one or more amino acids selected from the group consisting of amino acids 333, 404, 419, 426, 439, 440, 464, 465, 471, 477, 481, 488 and combinations thereof. The SD1 / 2 domain optionally contains mutations in one or more amino acids selected from the group consisting of 557, 600, 601, 642, 664, 668, and combinations thereof. The S2 subunit optionally includes one or more modifications selected from the following group: (a) Deletion of one or more amino acids from 676-685, 676-702, 702-711, 775-793, 806-815 and combinations thereof, (b) Mutations of one or more amino acids selected from the group consisting of 688, 703, 846, 875, 937, 969, 973, 974, 1014, 1058, 1105, and 1163 and combinations thereof, and (c) Deletion of one or more amino acids from the transmembrane and cytoplasmic domains (TMCT) (amino acids 1201-1260), The amino acids of the CoV S glycoprotein are numbered relative to Sequence ID No. 2.

[0090] In embodiments, the CoV S polypeptide described herein is present in the pre-fusion structure. In embodiments, the CoV S polypeptide described herein includes a flexible HR2 domain. Unless otherwise noted, the flexibility of the domain is determined by transition electron microscopy (TEM) and 2D class averaging. A decrease in electron density corresponds to the flexible domain.

[0091] Modifications to the S1 subunit In the embodiment, the CoV S polypeptide comprises one or more modifications to the S1 subunit having the amino acid sequence of SEQ ID NO: 121.

[0092] The amino acid sequence of the S1 subunit (sequence number 121) is shown below. QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRAR

[0093] In embodiments, the CoV S polypeptide described herein comprises an S1 subunit having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with respect to the S1 subunit of SEQ ID NO: 1 or SEQ ID NO: 2. The S1 subunit may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acid deletions, insertions, or mutations compared to the amino acid sequence of the S1 subunit of SEQ ID NO: 1 or SEQ ID NO: 2. The S1 subunit may have deletions, insertions, or mutations of approximately 1 to 5 amino acids, 3 to 10 amino acids, 5 to 10 amino acids, 8 to 12 amino acids, 10 to 15 amino acids, 12 to 17 amino acids, 15 to 20 amino acids, 18 to 23 amino acids, 20 to 25 amino acids, 22 to 27 amino acids, or 25 to 30 amino acids compared to the S1 subunit of SEQ ID NO: 1 or SEQ ID NO: 2.

[0094] In the embodiment, the S1 subunit may include any combination of the modifications shown in Table 1A. [Table 1A-1] [Table 1A-2] [Table 1A-3] [Table 1A-4] [Table 1A-5] [Table 1A-6] [Table 1A-7] [Table 1A-8] [Table 1A-9]

[0095] Modifications to the S1 subunit - NTD In some embodiments, the CoV S polypeptide comprises one or more modifications to the NTD. In some embodiments, the NTD has the amino acid sequence of SEQ ID NO: 118, which corresponds to amino acids 14-305 of SEQ ID NO: 1 or amino acids 1-292 of SEQ ID NO: 2.

[0096] The amino acid sequence of NTD (sequence number 118) is shown below. [ka]

[0097] The underlined region in Sequence ID No. 118 represents the amino acids within the NTD that can be modified.

[0098] In this embodiment, the NTD has the amino acid sequence of SEQ ID NO: 45, which corresponds to amino acids 14-331 of SEQ ID NO: 1 or amino acids 1-318 of SEQ ID NO: 2. The amino acid sequence of the NTD (SEQ ID NO: 45) is shown below.

[0099] [ka]

[0100] In some embodiments, the NTD and RBD overlap by a maximum of approximately 1 amino acid, a maximum of approximately 5 amino acids, a maximum of approximately 10 amino acids, or a maximum of approximately 20 amino acids.

[0101] In embodiments, the NTDs provided herein may be elongated by up to 5, up to 10, up to 15, up to 20, up to 25, or up to 30 amino acids at the C-terminus.

[0102] In embodiments, the CoV S polypeptide described herein includes an NTD having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the NTD of SEQ ID NO: 1 or SEQ ID NO: 2. The NTD may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acid deletions, insertions, or mutations compared to the amino acid sequence of the NTD of SEQ ID NO: 1 or SEQ ID NO: 2. The NTD may have deletions, insertions, or mutations of approximately 1 to 5 amino acids, 3 to 10 amino acids, 5 to 10 amino acids, 8 to 12 amino acids, 10 to 15 amino acids, 12 to 17 amino acids, 15 to 20 amino acids, 18 to 23 amino acids, 20 to 25 amino acids, 22 to 27 amino acids, or 25 to 30 amino acids compared to the NTD of SEQ ID NO: 1 or SEQ ID NO: 2.

[0103] In embodiments, the CoV S polypeptide includes the deletion of one or more amino acids from the N-terminal domain (NTD) (corresponding to amino acids 1-292 of SEQ ID NO: 2). In embodiments, the CoV S polypeptide includes the deletion of up to approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 292 amino acids from the NTD.

[0104] In several embodiments, the CoV S polypeptide contains a deletion of one or more amino acids derived from an NTD (corresponding to amino acids 1-318 of SEQ ID NO: 2). In several embodiments, the CoV S polypeptide contains a deletion of amino acids 1-318 of the NTD of SEQ ID NO: 2. In several embodiments, the NTD deletion enhances the protein expression of the CoV spike (S) polypeptide. In several embodiments, the CoV S polypeptide having the NTD deletion has the amino acid sequence represented by SEQ ID NOs: 46, 48, 49, 51, 52, and 54. In several embodiments, the CoV S polypeptide having the NTD deletion is encoded by an isolated nucleic acid sequence selected from the group consisting of SEQ ID NOs: 47, SEQ ID NOs: 50, and SEQ ID NOs: 53.

[0105] In embodiments, the NTD may include any combination of the modifications shown in Table 1B. The modifications are shown with respect to Sequence ID No. 2, which is a mature S polypeptide sequence for reference. [Table 1B-1] [Table 1B-2] [Table 1B-3] [Table 1B-4] [Table 1B-5]

[0106] Modifications to the S1 subunit-RBD In the embodiment, the CoV S polypeptide includes one or more modifications to RBD.

[0107] In some embodiments, the RBD has the amino acid sequence of SEQ ID NO: 126, which corresponds to amino acids 331-527 of SEQ ID NO: 1 or amino acids 318-514 of SEQ ID NO: 2.

[0108] The amino acid sequence of RBD (SEQ ID NO: 126) is shown below: [ka]

[0109] The underlined region in Sequence ID No. 126 represents the amino acids within the modifiable RBD subunit.

[0110] In this embodiment, the RBD has the amino acid sequence of SEQ ID NO: 116, which corresponds to amino acids 335-530 of SEQ ID NO: 1 or amino acids 322-517 of SEQ ID NO: 2.

[0111] The amino acid sequence of RBD (sequence number 116) is shown below. LCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKS

[0112] In embodiments, the RBDs provided herein may be extended by up to 1 amino acid, up to 5 amino acids, up to 10 amino acids, up to 15 amino acids, up to 20 amino acids, up to 25 amino acids, or up to 30 amino acids at the N-terminus or C-terminus.

[0113] In embodiments, the CoV S polypeptide described herein comprises an RBD having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the RBD of SEQ ID NO: 1 or SEQ ID NO: 2. The RBD may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acid deletions, insertions, or mutations compared to the amino acid sequence of the RBD of SEQ ID NO: 1 or SEQ ID NO: 2. The RBD may have deletions, insertions, or mutations of approximately 1 to 5 amino acids, 3 to 10 amino acids, 5 to 10 amino acids, 8 to 12 amino acids, 10 to 15 amino acids, 12 to 17 amino acids, 15 to 20 amino acids, 18 to 23 amino acids, 20 to 25 amino acids, 22 to 27 amino acids, or 25 to 30 amino acids compared to the RBD of SEQ ID NO: 1 or SEQ ID NO: 2.

[0114] In one embodiment, the CoV S polypeptide has at least 1, at least 2, at least 3, at least 4, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 mutations in the RBD. In another embodiment, the RBD may include any combination of the modifications shown in Table 1C. [Table 1C-1] [Table 1C-2] [Table 1C-3]

[0115] Modifications to SD1 / 2 In the embodiment, the CoV S polypeptide comprises one or more modifications of SD1 / 2 having the amino acid sequence of SEQ ID NO: 122, which corresponds to amino acids 542-681 of SEQ ID NO: 1 or amino acids 529-668 of SEQ ID NO: 2.

[0116] The amino acid sequence of SD1 / 2 (sequence number 122) is shown below. NFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSP

[0117] In embodiments, the CoV S polypeptide described herein comprises an SD1 / 2 having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the SD1 / 2 of SEQ ID NO: 1 or SEQ ID NO: 2. The SD1 / 2 may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acid deletions, insertions, or mutations compared to the amino acid sequence of the SD1 / 2 of SEQ ID NO: 1 or SEQ ID NO: 2. The SD1 / 2 may have deletions, insertions, or mutations of approximately 1 to 5 amino acids, 3 to 10 amino acids, 5 to 10 amino acids, 8 to 12 amino acids, 10 to 15 amino acids, 12 to 17 amino acids, 15 to 20 amino acids, 18 to 23 amino acids, 20 to 25 amino acids, 22 to 27 amino acids, or 25 to 30 amino acids compared to the SD1 / 2 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0118] In one embodiment, the CoV S polypeptide has at least 1, at least 2, at least 3, at least 4, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 mutations in SD1 / 2. In one embodiment, the SD1 / 2 may include any combination of the modifications shown in Table 1D. [Table 1D-1] [Table 1D-2]

[0119] Modifications to the Fuhrin cleavage site In some embodiments, the CoV S polypeptide includes a furin site (RRAR) corresponding to amino acids 682-685 of SEQ ID NO: 1 or amino acids 669-672 of SEQ ID NO: 2, which is inactivated by one or more mutations. Inactivation of the furin cleavage site prevents furin from cleaving the CoV S polypeptide. In some embodiments, the CoV S polypeptide described herein, containing the inactivated furin cleavage site, is expressed as a single strand.

[0120] In some embodiments, one or more of the amino acids in the natural furin cleavage site are mutated to any natural amino acid. In some embodiments, the amino acid is an L-amino acid. Non-limiting examples of amino acids include alanine, arginine, glycine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, serine, threonine, histidine, lysine, methionine, proline, valine, isoleucine, leucine, tyrosine, tryptophan, and phenylalanine.

[0121] In several embodiments, one or more amino acids in the natural furin cleavage site are mutated to glutamine. In several embodiments, one, two, three, or four amino acids may be mutated to glutamine. In several embodiments, one of the arginines in the natural furin cleavage site is mutated to glutamine. In several embodiments, two of the arginines in the natural furin cleavage site are mutated to glutamine. In several embodiments, three of the arginines in the natural furin cleavage site are mutated to glutamine.

[0122] In several embodiments, one or more amino acids in the natural furin cleavage site are mutated to alanine. In several embodiments, one, two, three, or four amino acids may be mutated to alanine. In several embodiments, one of the arginine molecules in the natural furin cleavage site is mutated to alanine. In several embodiments, two of the arginine molecules in the natural furin cleavage site are mutated to alanine. In several embodiments, three of the arginine molecules in the natural furin cleavage site are mutated to alanine.

[0123] In several embodiments, one or more amino acids in the natural furin cleavage site are mutated to glycine. In several embodiments, one, two, three, or four amino acids may be mutated to glycine. In several embodiments, one of the arginines in the natural furin cleavage site is mutated to glycine. In several embodiments, two of the arginines in the natural furin cleavage site are mutated to glycine. In several embodiments, three of the arginines in the natural furin cleavage site are mutated to glycine.

[0124] In several embodiments, one or more amino acids in the natural furin cleavage site are mutated to asparagine. For example, 1, 2, 3, or 4 amino acids may be mutated to asparagine. In several embodiments, one of the arginine molecules in the natural furin cleavage site is mutated to asparagine. In several embodiments, two of the arginine molecules in the natural furin cleavage site are mutated to asparagine. In several embodiments, three of the arginine molecules in the natural furin cleavage site are mutated to asparagine.

[0125] Non-restrictive examples of amino acid sequences of inactivated furin sites contained in CoV S polypeptides can be found in Table 1E. [Table 1E]

[0126] In several embodiments, instead of an active furin cleavage site (SEQ ID NO: 6), the CoV S polypeptide described herein contains an inactivated furin cleavage site. In several embodiments, the amino acid sequence of the inactivated furin cleavage site is represented by one of SEQ ID NOs: 7-34 or SEQ ID NO: 97. In several embodiments, the amino acid sequence of the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7). In several embodiments, the amino acid sequence of the inactivated furin cleavage site is GSAS (SEQ ID NO: 97). In several embodiments, the amino acid sequence of the inactivated furin cleavage site is GSGA (SEQ ID NO: 111). In several embodiments, the amino acid sequence of the inactivated furin cleavage site is GG, GGG (SEQ ID NO: 127), GGGG (SEQ ID NO: 128), or GGGGG (SEQ ID NO: 129).

[0127] Modifications to the S2 subunit In the embodiment, the CoV S polypeptide includes one or more modifications to the S2 subunit having the amino acid sequence of SEQ ID NO: 120, which corresponds to amino acids 686-1273 of SEQ ID NO: 1 or amino acids 673-1260 of SEQ ID NO: 2.

[0128] The amino acid sequence of the S2 subunit (sequence number 120) is shown below. SVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAG FIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLND ILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNC DVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPLVLKGVKLHYT

[0129] In embodiments, the CoV S polypeptide described herein comprises an S2 subunit having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with respect to the S2 subunit of SEQ ID NO: 1 or SEQ ID NO: 2. The S2 subunit may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acid deletions, insertions, or mutations compared to the amino acid sequence of the S2 subunit of SEQ ID NO: 1 or SEQ ID NO: 2. The S2 subunit may have deletions, insertions, or mutations of approximately 1 to 5 amino acids, 3 to 10 amino acids, 5 to 10 amino acids, 8 to 12 amino acids, 10 to 15 amino acids, 12 to 17 amino acids, 15 to 20 amino acids, 18 to 23 amino acids, 20 to 25 amino acids, 22 to 27 amino acids, or 25 to 30 amino acids compared to the S2 subunit of SEQ ID NO: 1 or SEQ ID NO: 2.

[0130] In the embodiment, the S2 subunit may include any combination of the modifications shown in Table 1F. [Table 1F-1] [Table 1F-2] [Table 1F-3]

[0131] In one embodiment, the CoV S polypeptide contains deletions corresponding to one or more deletions within amino acids 676-685 of the natural CoV spike (SEQ ID NO: 2). In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from amino acids 676-685 of the natural CoV spike (SEQ ID NO: 2) are deleted. In one embodiment, the deletions of amino acids within amino acids 676-685 are contiguous, for example, amino acids 676 and 677 are deleted, or amino acids 680 and 681 are deleted. In one embodiment, the deletions of amino acids within amino acids 676-685 are discontinuous, for example, amino acids 676 and 680 are deleted, or amino acids 677 and 682 are deleted. In one embodiment, the CoV S polypeptide containing deletions corresponding to one or more deletions within amino acids 676-685 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 62 and 63.

[0132] In the embodiment, the CoV S polypeptide contains deletions corresponding to one or more deletions in amino acids 702-711 of the natural CoV spike (S) polypeptide (SEQ ID NO: 2). In the embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from amino acids 702-711 of the natural SARS-CoV-2 spike (S) polypeptide (SEQ ID NO: 2) are deleted. In the embodiment, the deletion of one or more amino acids in amino acids 702-711 is contiguous, for example, amino acids 702 and 703 are deleted, or amino acids 708 and 709 are deleted. In the embodiment, the deletion of amino acids in amino acids 702-711 is discontinuous, for example, amino acids 702 and 704 are deleted, or amino acids 707 and 710 are deleted. In the embodiment, the CoV S polypeptide containing deletions corresponding to one or more deletions among amino acids 702-711 has an amino acid sequence selected from the group consisting of SEQ ID NOs. 64 and 65.

[0133] In the embodiment, the CoV S polypeptide contains deletions corresponding to one or more deletions within amino acids 775-793 of the natural CoV S polypeptide (SEQ ID NO: 2). In the embodiment, up to approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids from amino acids 775-793 of the natural SARS-CoV-2 spike (SEQ ID NO: 2) are deleted. In the embodiment, the deletion of one or more amino acids within amino acids 775-793 is contiguous, for example, amino acids 776 and 777 are deleted, or amino acids 780 and 781 are deleted. In the embodiment, the deletion of amino acids within amino acids 775-793 is discontinuous, for example, amino acids 775 and 790 are deleted, or amino acids 777 and 781 are deleted.

[0134] In one embodiment, the CoV S polypeptide includes a deletion of a fusion peptide (SEQ ID NO: 104) corresponding to amino acids 806-815 of SEQ ID NO: 2. In another embodiment, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the fusion peptide of the CoV spike (S) polypeptide (SEQ ID NO: 2) are deleted. In one embodiment, the deletion of amino acids in the fusion peptide is contiguous, for example, amino acids 806 and 807 are deleted, or amino acids 809 and 810 are deleted. In another embodiment, the deletion of amino acids in the fusion peptide is discontinuous, for example, amino acids 806 and 808 are deleted, or amino acids 810 and 813 are deleted. In yet another embodiment, the CoV S polypeptide containing a deletion corresponding to one or more amino acids of the fusion peptide has an amino acid sequence selected from SEQ ID NOs: 66, 77, and 105-108.

[0135] In some embodiments, the CoV S polypeptide contains a mutation at Lys-973 of the natural CoV spike (S) polypeptide (SEQ ID NO: 2). In some embodiments, Lys-973 is mutated to any natural amino acid. In some embodiments, Lys-973 is mutated to proline. In some embodiments, Lys-973 is mutated to glycine. In some embodiments, the CoV S polypeptide containing the mutation at amino acid 973 is selected from the group consisting of SEQ ID NOs: 84-89, 105-106, and 109-110.

[0136] In several embodiments, the CoV S polypeptide contains a mutation at Val-974 of the natural CoV spike (S) polypeptide (SEQ ID NO: 2). In several embodiments, Val-974 is mutated to any natural amino acid. In several embodiments, Val-974 is mutated to proline. In several embodiments, Val-974 is mutated to glycine. In several embodiments, the CoV S polypeptide containing the mutation at amino acid 974 is selected from the group consisting of SEQ ID NOs: 84-89, 105-106, and 109-110.

[0137] In several embodiments, the CoV S polypeptide contains mutations at Lys-973 and Val-974 of the natural CoV spike (S) polypeptide (SEQ ID NO: 2). In several embodiments, Lys-973 and Val-974 are mutated to any natural amino acid. In several embodiments, Lys-973 and Val-974 are mutated to proline. In embodiments, the CoV S polypeptide containing the mutations at amino acids 973 and 974 is selected from SEQ ID NOs: 84-89, 105-106, and 109-110.

[0138] Modifications to the S2 subunit-HR1 domain In the embodiment, the CoV S polypeptide includes one or more modifications to the HR1 domain having the amino acid sequence of SEQ ID NO: 119, which corresponds to amino acids 912-984 of SEQ ID NO: 1 or amino acids 889-971 of SEQ ID NO: 2.

[0139] The amino acid sequence of the HR1 domain (SEQ ID NO: 119) is shown below. MAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRL

[0140] In embodiments, the CoV S polypeptide described herein comprises an HR1 domain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the HR1 domain of SEQ ID NO: 1 or SEQ ID NO: 2. The HR1 domain may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acid deletions, insertions, or mutations compared to the amino acid sequence of the HR1 domain of SEQ ID NO: 1 or SEQ ID NO: 2. The HR1 Domain may have deletions, insertions, or mutations of approximately 1 to 5 amino acids, 3 to 10 amino acids, 5 to 10 amino acids, 8 to 12 amino acids, 10 to 15 amino acids, 12 to 17 amino acids, 15 to 20 amino acids, 18 to 23 amino acids, 20 to 25 amino acids, 22 to 27 amino acids, or 25 to 30 amino acids compared to the HR1 Domain of SEQ ID NO: 1 or SEQ ID NO: 2.

[0141] In the embodiment, the HR1 domain may include any combination of the modifications shown in Table 1G. [Table 1G]

[0142] Modifications to the S2 subunit-HR2 domain In the embodiment, the CoV S polypeptide includes one or more modifications to the HR2 domain having the amino acid sequence of SEQ ID NO: 125, which corresponds to amino acids 1163-1213 of SEQ ID NO: 1 or amino acids 1150-1200 of SEQ ID NO: 2.

[0143] The amino acid sequence of the HR2 domain (SEQ ID NO: 125) is shown below. DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWP

[0144] In embodiments, the CoV S polypeptide described herein comprises an HR2 domain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the HR2 domain of SEQ ID NO: 1 or SEQ ID NO: 2. The HR2 domain may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acid deletions, insertions, or mutations compared to the amino acid sequence of the HR2 domain of SEQ ID NO: 1 or SEQ ID NO: 2. The HR2 Domain may have deletions, insertions, or mutations of approximately 1 to 5 amino acids, 3 to 10 amino acids, 5 to 10 amino acids, 8 to 12 amino acids, 10 to 15 amino acids, 12 to 17 amino acids, 15 to 20 amino acids, 18 to 23 amino acids, 20 to 25 amino acids, 22 to 27 amino acids, or 25 to 30 amino acids compared to the HR2 Domain of Sequence ID No. 1 or Sequence ID No. 2.

[0145] Modifications to the TM domain In the embodiment, the CoV S polypeptide includes one or more modifications to the TM domain having the amino acid sequence of SEQ ID NO: 123, which corresponds to amino acids 1214-1237 of SEQ ID NO: 1 or amino acids 1201-1224 of SEQ ID NO: 2.

[0146] The amino acid sequence of the TM domain (SEQ ID NO: 123) is shown below. WYIWLGFIAGLIAIVMVTIMLCCM

[0147] In embodiments, the CoV S polypeptide described herein comprises a TM domain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the TM domain of SEQ ID NO: 1 or SEQ ID NO: 2. The TM domain may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acid deletions, insertions, or mutations compared to the amino acid sequence of the TM domain of SEQ ID NO: 1 or SEQ ID NO: 2. The TM domain may have deletions, insertions, or mutations of approximately 1 to 5 amino acids, 3 to 10 amino acids, 5 to 10 amino acids, 8 to 12 amino acids, 10 to 15 amino acids, 12 to 17 amino acids, 15 to 20 amino acids, 18 to 23 amino acids, 20 to 25 amino acids, 22 to 27 amino acids, or 25 to 30 amino acids compared to the TM domain of SEQ ID NO: 1 or SEQ ID NO: 2.

[0148] In some embodiments, the CoV S polypeptide described herein lacks the entire TM domain. In some embodiments, the CoV S polypeptide includes the TM domain.

[0149] Modifications to the CT domain In the embodiment, the CoV S polypeptide comprises one or more modifications to the CT having the amino acid sequence of SEQ ID NO: 124, which corresponds to amino acids 1238-1273 of SEQ ID NO: 1 or amino acids 1225-1260 of SEQ ID NO: 2.

[0150] The amino acid sequence of CT (SEQ ID NO: 124) is shown below: TSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

[0151] In embodiments, the CoV S polypeptide described herein includes a CT having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the CT of SEQ ID NO: 1 or SEQ ID NO: 2. The CT may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acid deletions, insertions, or mutations compared to the amino acid sequence of the CT of SEQ ID NO: 1 or SEQ ID NO: 2. The CT may have about 1 to about 5 amino acids, about 3 to about 10 amino acids, about 5 to 10 amino acids, about 8 to 12 amino acids, about 10 to 15 amino acids, about 12 to 17 amino acids, about 15 to 20 amino acids, about 18 to 23 amino acids, about 20 to 25 amino acids, about 22 to about 27 amino acids, or about 25 to 30 amino acid deletions, insertions, or mutations compared to the CT of SEQ ID NO: 1 or SEQ ID NO: 2.

[0152] In some embodiments, the CoV S polypeptide described herein lacks CT. In some embodiments, the CoV S polypeptide includes CT.

[0153] In the embodiment, the CoV S polypeptide comprises TM and CT. In the embodiment, the CoV spike(S) polypeptide comprises one or more amino acid deletions derived from the transmembrane and cytoplasmic tail (TMCT) (corresponding to amino acids 1201-1260). The amino acid sequence of TMCT is represented by SEQ ID NO: 39. In the embodiment, the CoV S polypeptide having one or more deletions of TMCT has enhanced protein expression. In the embodiment, the CoV spike(S) polypeptide having one or more deletions from TMCT has an amino acid sequence selected from the group consisting of SEQ ID NOs: 40, 41, 42, 52, 54, 59, 61, 88, and 89. In the embodiment, the CoV S polypeptide having one or more deletions from TM-CT is encoded by an isolated nucleic acid sequence selected from the group consisting of SEQ ID NOs: 39, 43, 53, and 60.

[0154] Exemplary non-spontaneously occurring CoV S polypeptides or nanoparticles In this embodiment, the CoV S polypeptide comprises the deletion of amino acids 56 and 57 of the natural CoV spike (SEQ ID NO: 2) polypeptide.

[0155] In this embodiment, the CoV S polypeptide comprises deletions of amino acids 131 and 132 of the natural CoV spike (SEQ ID NO: 2) polypeptide.

[0156] In one embodiment, the CoV S polypeptide contains deletions of amino acids 56 and 131 of the natural CoV spike (S) polypeptide (SEQ ID NO: 2). In another embodiment, the CoV S polypeptide contains deletions of amino acids 57 and 131 of the natural CoV spike (S) polypeptide (SEQ ID NO: 2).

[0157] In this embodiment, the CoV S polypeptide comprises deletions of amino acids 56, 57, and 131 of the natural CoV spike (SEQ ID NO: 2) polypeptide.

[0158] In this embodiment, the CoV S polypeptide comprises deletions of amino acids 56 and 132 of the natural CoV spike (SEQ ID NO: 2) polypeptide.

[0159] In this embodiment, the CoV S polypeptide comprises deletions of amino acids 57 and 132 of the natural CoV spike (SEQ ID NO: 2) polypeptide.

[0160] In this embodiment, the CoV S polypeptide comprises deletions of amino acids 56, 57, and 132 of the natural CoV spike (SEQ ID NO: 2) polypeptide.

[0161] In this embodiment, the CoV S polypeptide comprises deletions of amino acids 56, 57, 131, and 132 of the natural CoV spike (SEQ ID NO: 2).

[0162] In embodiments, the CoV S polypeptide includes mutations that stabilize the pre-fusion structure of the CoV S polypeptide. In embodiments, the CoV S polypeptide includes proline or glycine substitutions that stabilize the pre-fusion structure. This strategy has been used to develop pre-fusion stabilized MERS-CoV S proteins, as described in the following literature, each incorporated herein by reference: Proc Natl Acad Sci USA. 2017 Aug 29;114(35):E7348-E7357, Sci Rep. 2018 Oct 24;8(1):15701, U.S. Publication No. 2020 / 0061185, and PCT Application No. PCT / US2017 / 058370.

[0163] In one embodiment, the CoV S polypeptide includes mutations at Lys-973 and Val-974, as well as an inactivating furin cleavage site. In another embodiment, the CoV S polypeptide includes mutations at Lys-973 and Val-974 to proline, as well as an inactivating furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96). An exemplary CoV S polypeptide including mutations at Lys-973 and Val-974 and an inactivating furin cleavage site is shown in Figure 3. In another embodiment, the CoV S polypeptide including mutations at Lys-973 and Val-974 to proline, as well as an inactivating furin cleavage site, has the amino acid sequence of SEQ ID NO: 86 or 87 and the nucleic acid sequence of SEQ ID NO: 96.

[0164] In one embodiment, the CoV S polypeptide comprises mutations at Lys-973 and Val-974, an inactivating furin cleavage site, and the deletion of one or more amino acids in the fusion peptide. In another embodiment, the CoV S polypeptide comprises mutations at Lys-973 and Val-974 to proline, an inactivating furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96), and the deletion of one or more amino acids in the fusion peptide. In yet another embodiment, the CoV S polypeptide comprising mutations at Lys-973 and Val-974 to proline, an inactivating furin cleavage site, and the deletion of one or more amino acids in the fusion peptide has the amino acid sequence of SEQ ID NO: 105 or 106. In the embodiment, the CoV S polypeptide includes, compared to the natural CoV spike (S) polypeptide (SEQ ID NO: 2), a mutation of Leu-5 to phenylalanine, a mutation of Thr-7 to asparagine, a mutation of Pro-13 to serine, a mutation of Asp-125 to tyrosine, a mutation of Arg-177 to serine, a mutation of Lys-404 to threonine, a mutation of Glu-471 to lysine, a mutation of Asn-488 to tyrosine, a mutation of His-642 to tyrosine, a mutation of Thr-1014 to isoleucine, a mutation of Lys-973 and Val-974 to proline, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96).

[0165] In one embodiment, the CoV S polypeptide includes, compared to the natural CoV spike (S) polypeptide (SEQ ID NO: 2), a mutation to cysteine ​​at Trp-139, a mutation to arginine at Leu-439, a mutation to proline at Lys-973 and Val-974, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96). In another embodiment, the CoV S polypeptide includes, compared to the natural CoV spike (S) polypeptide (SEQ ID NO: 1), a mutation to cysteine ​​at Trp-152, a mutation to arginine at Leu-452, a mutation to isoleucine at Ser-13, a mutation to proline at Lys-986 and Val-987, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96).

[0166] In the embodiment, the CoV S polypeptide includes, compared to the natural CoV spike (S) polypeptide (SEQ ID NO: 2), a mutation to threonine or asparagine at Lys-404, a mutation to lysine at Glu-471, a mutation to tyrosine at Asn-488, a mutation to phenylalanine at Leu-5, a mutation to alanine at Asp-67, a mutation to glycine at Asp-202, one or more deletions of amino acids 229-231, a mutation to isoleucine at Arg-233, mutations to proline at Lys-973 and Val-974, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96).

[0167] In one embodiment, the CoV S polypeptide includes a mutation to tyrosine at Asn-488, a mutation to proline at Lys-973 and Val-974, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96), compared to the natural CoV spike (S) polypeptide (SEQ ID NO: 2). In another embodiment, the CoV S polypeptide having the mutation to tyrosine at Asn-488, the mutation to proline at Lys-973 and Val-974, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96) includes the amino acid sequence of SEQ ID NO: 112.

[0168] In one embodiment, the CoV S polypeptide includes, compared to the natural CoV spike (S) polypeptide (SEQ ID NO: 2), a mutation to glycine at Asp-601, a mutation to tyrosine at Asn-488, a mutation to proline at Lys-973 and Val-974, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96). In another embodiment, the CoV S polypeptide having the mutation to tyrosine at Asn-488, the mutation to proline at Lys-973 and Val-974, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96) includes the amino acid sequence of SEQ ID NO: 113.

[0169] In this embodiment, the CoV S polypeptide includes, compared to the natural CoV spike (S) polypeptide (SEQ ID NO: 2), deletions of amino acids 56, 57, and 131, a mutation to tyrosine at Asn-488, a mutation to aspartic acid at Ala-557, a mutation to glycine at Asp-601, a mutation to histidine at Pro-668, a mutation to isoleucine at Thr-703, a mutation to alanine at Ser-969, a mutation to histidine at Asp-1105, mutations to proline at Lys-973 and Val-974, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7), GSAS (SEQ ID NO: 96), or GG. In this embodiment, the CoV S polypeptide having deletions of amino acids 56, 57, and 131, a mutation of Asn-488 to tyrosine, a mutation of Ala-557 to aspartic acid, a mutation of Asp-601 to glycine, a mutation of Pro-668 to histidine, a mutation of Thr-703 to isoleucine, a mutation of Ser-969 to alanine, a mutation of Asp-1105 to histidine, a mutation of Lys-973 and Val-974 to proline, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96) contains the amino acid sequence of SEQ ID NO: 114. In this embodiment, a CoV S polypeptide having deletions of amino acids 56, 57, and 131, a mutation of Asn-488 to tyrosine, a mutation of Ala-557 to aspartic acid, a mutation of Asp-601 to glycine, a mutation of Pro-668 to histidine, a mutation of Thr-703 to isoleucine, a mutation of Ser-969 to alanine, a mutation of Asp-1105 to histidine, a mutation of Lys-973 and Val-974 to proline, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96) or GG contains the amino acid sequence of SEQ ID NO: 136.In some embodiments, a CoV S polypeptide having deletions of amino acids 56, 57, and 131, a mutation of Asn-488 to tyrosine, a mutation of Ala-557 to aspartic acid, a mutation of Asp-601 to glycine, a mutation of Pro-668 to histidine, a mutation of Thr-703 to isoleucine, a mutation of Ser-969 to alanine, a mutation of Asp-1105 to histidine, mutations of Lys-973 and Val-974 to proline, and an inactivated furin cleavage site having the amino acid sequence GG comprises the amino acid sequence of SEQ ID NO: 137 or SEQ ID NO: 138. In some embodiments, the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 114 or SEQ ID NO: 136 is encoded by a nucleic acid having the nucleic acid sequence of SEQ ID NO: 135. In some embodiments, the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 137 or SEQ ID NO: 138 is encoded by a nucleic acid having the sequence of SEQ ID NO: 139.

[0170] In the embodiment, the CoV S polypeptide includes, compared to the natural CoV spike (S) polypeptide (SEQ ID NO: 2), deletions of amino acids 56, 57, and 132, a mutation of Asn-488 to tyrosine, a mutation of Ala-557 to aspartic acid, a mutation of Asp-601 to glycine, a mutation of Pro-668 to histidine, a mutation of Thr-703 to isoleucine, a mutation of Ser-969 to alanine, a mutation of Asp-1105 to histidine, a mutation of Lys-973 and Val-974 to proline, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96). In this embodiment, a CoV S polypeptide having deletions of amino acids 56, 57, and 132, a mutation of Asn-488 to tyrosine, a mutation of Ala-557 to aspartic acid, a mutation of Asp-601 to glycine, a mutation of Pro-668 to histidine, a mutation of Thr-703 to isoleucine, a mutation of Ser-969 to alanine, a mutation of Asp-1105 to histidine, a mutation of Lys-973 and Val-974 to proline, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96) contains the amino acid sequence of SEQ ID NO: 114.

[0171] In an embodiment, the CoV S polypeptide includes a mutation of Asn-488 to tyrosine, a mutation of Asp-67 to alanine, a mutation of Leu-229 to histidine, a mutation of Asp-202 to glycine, a mutation of Lys-404 to asparagine, a mutation of Glu-471 to lysine, a mutation of Ala-688 to valine, a mutation of Asp-601 to glycine, mutations of Lys-973 and Val-974 to proline, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96), with respect to the native CoV spike (S) polypeptide (SEQ ID NO: 2). In an embodiment, the CoV S polypeptide having the mutation of Asn-488 to tyrosine, the mutation of Asp-67 to alanine, the mutation of Leu-229 to histidine, the mutation of Asp-202 to glycine, the mutation of Lys-404 to asparagine, the mutation of Glu-471 to lysine, the mutation of Ala-688 to valine, the mutation of Asp-601 to glycine, mutations of Lys-973 and Val-974 to proline, and the inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96) includes the amino acid sequence of SEQ ID NO: 115.

[0172] In an embodiment, the CoV S polypeptide includes one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, a deletion of amino acid 56, a deletion of amino acid 57, a deletion of amino acid 131, N488Y, A557D, D601G, P668H, T703I, S969A, and D1105H, wherein the amino acids are numbered with respect to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In an embodiment, the inactivated furin cleavage site has the amino acid sequence QQAQ (SEQ ID NO: 7). In an embodiment, the inactivated furin cleavage site has the amino acid sequence GG.

[0173] In an embodiment, the CoV S polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, D67A, D202G, L229H, K404N, E471K, N488Y, D601G, and A688V, and the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In an embodiment, the inactivated furin cleavage site has the amino acid sequence QQAQ (SEQ ID NO: 7). In an embodiment, the inactivated furin cleavage site has the amino acid sequence GG.

[0174] In an embodiment, the CoV S polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, deletion of amino acids 229-231, D67A, D202G, K404N, E471K, N488Y, D601G, and A688V, and the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2.

[0175] In an embodiment, the CoV S polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7), deletion of amino acids 229-231, L5F, D67A, D202G, K404N, E471K, N488Y, D601G, and A688V, and the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In an embodiment, the CoV S polypeptide having one or more modifications selected from K973P, V974P, an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7), deletion of amino acids 229-231, L5F, D67A, D202G, K404N, E471K, N488Y, D601G, and A688V, and the amino acids being numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2 comprises the amino acid sequence of SEQ ID NO: 144. In an embodiment, the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 144 is encoded by a nucleic acid having the sequence of SEQ ID NO: 145.

[0176] In one embodiment, the CoV S polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site having the amino acid sequence GG, deletions of amino acids 229-231, L5F, D67A, D202G, K404N, E471K, N488Y, D601G, and A688V, wherein the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In another embodiment, the CoV S polypeptide comprises the amino acid sequence of SEQ ID NO: 144. In this embodiment, the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 144 is encoded by a nucleic acid having the sequence of SEQ ID NO: 145.

[0177] In the embodiment, the CoV S polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, L5F, T7N, P13S, D125Y, R177S, K404T, E471K, N488Y, D601G, H642Y, T1014I, and V1163F, wherein the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In the embodiment, the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 151 is encoded by a nucleic acid having the sequence of SEQ ID NO: 150.

[0178] In the embodiment, the CoV S polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, deletions of amino acids 229-231, L5F, D67A, D202G, L229H, K404N, E471K, N488Y, D601G, and A688V, wherein the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2.

[0179] In the embodiments, the CoV S polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, K404N, E471K, N488Y, L5F, D67A, D202G, L229H, D601G, A688V, and deletions of amino acids 229-231, the amino acids being numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In the embodiments, the inactivated furin cleavage site has the amino acid sequence QQAQ (SEQ ID NO: 7). In the embodiments, the inactivated furin cleavage site has the amino acid sequence GG.

[0180] In one embodiment, the CoV S polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, K404N, E471K, and N488K, and the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In another embodiment, the CoV S polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, K404N, E471K, and N488Y. In yet another embodiment, the CoV S polypeptide is an RBD of the CoV S polypeptide having one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, K404N, E471K, and N488K, and the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In the embodiment, the CoV S polypeptide is an RBD of the CoV S polypeptide having one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, K404N, E471K, and N488Y, wherein the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2.

[0181] In one embodiment, the CoV S polypeptide comprises one or more modifications selected from K973P, V974P, an inactivating furin cleavage site having the amino acid sequence GG, D601G, E404N, E471K, and N488Y. In another embodiment, the CoV S polypeptide comprises one or more modifications selected from mutations of K973P, V974P, an inactivating furin cleavage site having the amino acid sequence GG, and D601G, wherein the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In yet another embodiment, the CoV S polypeptide comprising modifications selected from mutations of K973P, V974P, an inactivating furin cleavage site having the amino acid sequence GG, and D601G has the amino acid sequence of SEQ ID NO: 133.

[0182] In the embodiment, the CoV S polypeptide comprises an inactivated furin cleavage site which is K973P, V974P, optionally QQAQ (SEQ ID NO: 7) or GG, one or more modifications selected from K404N, E471K, N488K, D67A, D202G, L229H, D601G, and A688V, wherein the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In the embodiment, the CoV S polypeptide comprising the inactivated furin cleavage site which is K973P, V974P, optionally QQAQ (SEQ ID NO: 7) or GG, one or more modifications selected from K404N, E471K, N488K, D67A, D202G, L229H, D601G, and A688V has the amino acid sequence of SEQ ID NO: 132 or SEQ ID NO: 141. In one embodiment, the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 132 is encoded by a nucleic acid having the nucleic acid sequence of SEQ ID NO: 131. In another embodiment, the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 132 is encoded by a nucleic acid having the nucleic acid sequence of SEQ ID NO: 142.

[0183] In one embodiment, the CoV S polypeptide comprises one or more modifications selected from K973P, V974P, an inactivating furin cleavage site, W139C, and L439R, the amino acids being numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In another embodiment, the CoV S polypeptide comprising the modifications of K973P, V974P, an inactivating furin cleavage site, W139C, and L439R is expressed together with a signal peptide having the amino acid sequence of SEQ ID NO: 117 or SEQ ID NO: 5. In yet another embodiment, the CoV S polypeptide comprises one or more modifications selected from K973P, V974P, an inactivating furin cleavage site, D601G, W139C, and L439R, the amino acids being numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In the embodiment, the CoV S polypeptide is expressed together with a signal peptide having the amino acid sequence of SEQ ID NO: 117 or SEQ ID NO: 5, and includes modifications of K973P, V974P, an inactivated furin cleavage site, D601G, W139C, and L439R.

[0184] In the embodiment, the CoV S polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, D601G, L5F, D67A, D202G, deletions of amino acids 229-231, R233I, K404N, E471K, N488Y, and A688V, wherein the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2.

[0185] In the embodiment, the CoV S polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site which is optionally QQAQ (SEQ ID NO: 7), W139C, S481P, D601G, and L439R, wherein the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In one embodiment, the CoV S polypeptide comprises one or more modifications selected from K973P, V974P, an inactivating furin cleavage site which is optionally QQAQ (SEQ ID NO: 7), W139C, S481P, and D601G, wherein the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In another embodiment, the CoV S polypeptide comprising one or more modifications selected from K973P, V974P, an inactivating furin cleavage site which is optionally QQAQ (SEQ ID NO: 7), W139C, S481P, D601G, and L439R has the amino acid sequence of SEQ ID NO: 153. In yet another embodiment, the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 153 comprises a signal peptide having the amino acid sequence of SEQ ID NO: 117. In yet another embodiment, the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 153 comprises a signal peptide having the amino acid sequence of SEQ ID NO: 5.

[0186] In one embodiment, the CoV S polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site which is optionally QQAQ (SEQ ID NO: 7), T82I, D240G, E471K, D601G, and A688V, wherein the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In another embodiment, the CoV S polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site which is optionally QQAQ (SEQ ID NO: 7), T82I, D240G, E471K, D601G, and A688V, wherein the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In the embodiment, the CoV S polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site which is optionally QQAQ (SEQ ID NO: 7), T82I, D240G, E471K, D601G, and A688V, wherein the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2, and the CoV S polypeptide comprises a signal peptide having the amino acid sequence of SEQ ID NO: 154 or SEQ ID NO: 5.

[0187] In one embodiment, the CoV S polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site which is optionally QQAQ (SEQ ID NO: 7), T82I, D240G, S464N, D601G, and A688V, wherein the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In another embodiment, the CoV S polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site which is optionally QQAQ (SEQ ID NO: 7), T82I, D240G, S464N, D601G, and A688V, wherein the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In the embodiment, the CoV S polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site which is optionally QQAQ (SEQ ID NO: 7), T82I, D240G, S464N, D601G, and A688V, wherein the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2, and includes the signal peptide of SEQ ID NO: 154.

[0188] In the embodiment, the CoV S polypeptide comprises one or more modifications selected from K973P, V974P, an inactivating furin cleavage site which is optionally QQAQ (SEQ ID NO: 7), a deletion of amino acid 56, a deletion of amino acid 57, a deletion of amino acid 131, a mutation of N488Y, a mutation of A557D, a mutation of D601G, a mutation of P668H, a mutation of T703I, a mutation of S969A, and a mutation of D1105H, wherein the CoV S polypeptide is numbered relative to the wild-type SARS-CoV-2 S polypeptide having the amino acid sequence of SEQ ID NO: 2. In the embodiment, the CoV S polypeptide comprises one or more modifications selected from K973P, V974P, an inactivating furin cleavage site which is optionally QQAQ (SEQ ID NO: 7), a deletion of amino acid 56, a deletion of amino acid 57, a deletion of amino acid 132, a mutation of N488Y, a mutation of A557D, a mutation of D601G, a mutation of P668H, a mutation of T703I, a mutation of S969A, and a mutation of D1105H, wherein the CoV S polypeptide is numbered relative to the wild-type SARS-CoV-2 S polypeptide having the amino acid sequence of SEQ ID NO: 2.

[0189] In the embodiment, the CoV S polypeptide comprises one or more modifications selected from the inactivating furin cleavage sites K973P, V974P, optionally QQAQ (SEQ ID NO: 7), the D67A mutation, the L229H mutation, the R233I mutation, the A688V mutation, the N488Y mutation, the K404N mutation, the E471K mutation, and the D601G mutation, and the CoV S polypeptide is numbered relative to the wild-type SARS-CoV-2 S polypeptide having the amino acid sequence of SEQ ID NO: 2.

[0190] In the embodiment, the CoV S polypeptide comprises one or more modifications selected from the following: an inactivating furin cleavage site which is K973P, V974P, optionally QQAQ (SEQ ID NO: 7), a mutation at L5F, a mutation at T7N, a mutation at P13S, a mutation at D125Y, a mutation at R177S, a mutation at K404T, a mutation at E471K, a mutation at N488Y, a mutation at D601G, a mutation at H642Y, a mutation at T1014I, and a mutation at T1163F, wherein the CoV S polypeptide is numbered relative to the wild-type SARS-CoV-2 S polypeptide having the amino acid sequence of SEQ ID NO: 2.

[0191] In one embodiment, the CoV S polypeptide comprises one or more modifications selected from the inactivating furin cleavage sites K986P, V987P, optionally QQAQ (SEQ ID NO: 7), the S13I mutation, the W152C mutation, and the L452R mutation, and the CoV S polypeptide is numbered relative to the wild-type SARS-CoV-2 S polypeptide having the amino acid sequence of SEQ ID NO: 1. In another embodiment, the CoV S polypeptide comprises one or more modifications selected from the inactivating furin cleavage sites K986P, V987P, optionally QQAQ (SEQ ID NO: 7), the S13I mutation, the W152C mutation, and the L452R mutation, and the CoV S polypeptide is numbered relative to the wild-type SARS-CoV-2 S polypeptide having the amino acid sequence of SEQ ID NO: 1 lacking the N-terminal signal peptide.

[0192] In embodiments, the CoV spike(S) polypeptide comprises a polypeptide linker. In embodiments, the polypeptide linker comprises glycine and serine. In embodiments, the linker has about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% glycine.

[0193] In the embodiment, the polypeptide linker is (SGGG) nIt has a repeating sequence (SEQ ID NO: 91), where n is an integer from 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50). In embodiments, the polypeptide linker has an amino acid sequence corresponding to SEQ ID NO: 90.

[0194] In the embodiment, the polypeptide linker is (GGGGS) n The sequence (sequence number 93) is repeated, where n is an integer from 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50).

[0195] In the embodiment, the polypeptide linker is (GGGS) n The sequence (sequence number 92) is repeated, where n is an integer from 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50).

[0196] In one embodiment, the polypeptide linker is a poly(Gly)n linker, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19, or 20. In other embodiments, the linker is selected from the group consisting of dipeptides, tripeptides, and quadrapeptides. In an embodiment, the linker is a dipeptide selected from the group consisting of alanine-serine (AS), leucine-glutamic acid (LE), and serine-arginine (SR).

[0197] In an embodiment, the polypeptide linker comprises 1 to 100 consecutive amino acids of a naturally occurring CoV S polypeptide or a CoV S polypeptide disclosed herein. In an embodiment, the polypeptide linker has an amino acid sequence corresponding to SEQ ID NO: 94.

[0198] In an embodiment, the CoV spike (S) polypeptide comprises a foldon. In an embodiment, the TMCT is replaced by a foldon. In an embodiment, the foldon causes trimerization of the CoV spike (S) polypeptide. In an embodiment, the foldon is an amino acid sequence known in the art. In an embodiment, the foldon has the amino acid sequence of SEQ ID NO: 68. In an embodiment, the foldon is a T4 fibritin trimerization motif. In an embodiment, the T4 fibritin trimerization domain has the amino acid sequence of SEQ ID NO: 103. In an embodiment, the foldon is separated from the CoV spike (S) polypeptide by an amino acid sequence by a polypeptide linker. Non-limiting examples of polypeptide linkers are found throughout the present disclosure.

[0199] In embodiments, the present disclosure provides a CoV S polypeptide containing a fragment of the coronavirus S protein, as well as nanoparticles and vaccines containing the same. In this embodiment, the coronavirus S protein fragment is about 10 to about 1500 amino acid lengths (for example, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1050, about 1100, about 1150, about 1200, about 1250, about 1300, about 1350, about 1400, about 1450, or about 1500 amino acid lengths). In the embodiment, the coronavirus S protein fragment is selected from the group consisting of a receptor-binding domain (RBD), subdomain 1, subdomain 2, upper helix, fusion peptide, linking region, 7-base repeat 1, central helix, 7-base repeat 2, NTD, and TMCT.

[0200] In the embodiment, the CoV S polypeptide comprises RBD and subdomain 1. In the embodiment, the CoV S polypeptide comprising RBD and subdomain 1 is amino acids 319-591 of SEQ ID NO: 1.

[0201] In the embodiment, the CoV S polypeptide comprises a fragment of the coronavirus S protein, the fragment of the coronavirus S protein being an RBD. Non-limiting examples of RBDs include the SARS-CoV-2 RBD (amino acid sequence = SEQ ID NO: 69), the SARS RBD (amino acid sequence = SEQ ID NO: 70), and the MERS RBD (amino acid sequence = SEQ ID NO: 71).

[0202] In the embodiment, the CoV S polypeptide comprises two or more RBDs linked by a polypeptide linker. In the embodiment, the polypeptide linker has the amino acid sequence of SEQ ID NO: 90 or SEQ ID NO: 94.

[0203] In the embodiment, the CoV S polypeptide contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 RBDs.

[0204] In some embodiments, the CoV S polypeptide comprises two or more SARS-CoV-2 RBDs linked by a polypeptide linker. In embodiments, the antigen comprising the two or more SARS-CoV-2 RBDs has an amino acid sequence corresponding to one of SEQ ID NOs. 72-75.

[0205] In one embodiment, the CoV S polypeptide comprises the RBD of SARS-CoV-2 and the RBD of SARS. In another embodiment, the CoV S polypeptide comprises the RBD of SARS-CoV-2 and the RBD of SARS, with each RBD being separated by a polypeptide linker. In yet another embodiment, the CoV S polypeptide comprising the RBD of SARS-CoV-2 and the RBD of SARS has an amino acid sequence selected from the group consisting of SEQ ID NOs. 76-79.

[0206] In one embodiment, the CoV S polypeptide comprises the RBD of SARS-CoV-2 and the RBD of MERS. In one embodiment, the CoV S polypeptide comprises the RBD of SARS-CoV-2 and the RBD of MERS, and each RBD is separated by a polypeptide linker.

[0207] In the embodiment, the CoV S polypeptide comprises SARS RBD and MERS RBD, with each RBD being separated by a polypeptide linker.

[0208] In the embodiment, the CoV S polypeptide comprises the RBD of SARS-CoV-2, the RBD of SARS, and the RBD of MERS. In the embodiment, the CoV S polypeptide comprises the RBD of SARS-CoV-2, the RBD of SARS, and the RBD of MERS, with each RBD being separated by a polypeptide linker. In the embodiment, the CoV S polypeptide comprising the RBD of SARS-CoV-2, the RBD of SARS, and the RBD of MERS has an amino acid sequence selected from the group consisting of SEQ ID NOs: 80-83.

[0209] In embodiments, the CoV S polypeptide described herein is expressed together with an N-terminal signal peptide. In embodiments, the N-terminal signal peptide has the amino acid sequence of SEQ ID NO: 5 (MFVFLVLLPLVSS). In some embodiments, the N-terminal signal peptide has the amino acid sequence of SEQ ID NO: 117 (MFVFLVLLPLVSI). In embodiments, the N-terminal signal peptide has the amino acid sequence of SEQ ID NO: 154 (MFVFFVLLPLVSS). In embodiments, the signal peptide may be replaced with any signal peptide that enables the expression of the CoV S protein. In embodiments, one or more amino acids of the CoV S protein signal peptide may be deleted or mutated. The initiating methionine residue is maintained to initiate expression. In embodiments, the CoV S polypeptide is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 35, 37, 95, 43, 47, 50, 53, 55, 57, 96, 60, 131, 135, 142, 145, 148, and 150. In embodiments, the N-terminal signal peptide of the CoV S polypeptide contains a mutation at Ser-13 compared to the native CoV spike (S) signal polypeptide (SEQ ID NO: 5). In embodiments, Ser-13 is mutated to any native amino acid. In several embodiments, Ser-13 is mutated to alanine, methionine, isoleucine, leucine, threonine, or valine. In embodiments, Ser-13 is mutated to isoleucine.

[0210] Following expression of the CoV S protein in host cells, the N-terminal signal peptide is cleaved to provide mature CoV protein sequences (SEQ ID NOs: 2, 4, 38, 41, 44, 48, 51, 54, 58, 61, 63, 65, 67, 73, 75, 78, 79, 82, 83, 85, 87, 89, 106, 110, 132, 133, 114, 138, 141, 144, 147, 151, 153, 156, 158, 174, 175, 176, 181-184, 186, 188, 190, 195, 217-228, 233-236, 243, 255-264, 273-280, and 298-302). In embodiments, the signal peptide is cleaved by a protease in the host cell. In one embodiment, the full-length protein may be isolated from the host cell, and then the signal peptide may be cleaved.

[0211] During the expression and purification process, SEQ ID NOs: 1, 3, 36, 40, 42, 46, 49, 52, 56, 59, 62, 64, 66, 72, 74, 76, 77, 80, 81, 84, 86, 87, 105, 107, 88, 109, 130, 134, 136, 137, 140, 143, 146, 149, 152, 155, 157, 159, 160, CoV spike(S) positories having amino acid sequences corresponding to 173, 177-180, 185, 189, 191, 194, 200, 203, 205, 207, 209, 211, 213, 215, 229-232, 242, 245-254, 265-272, 281-282, 285-286, 289-290, and 293-297 After cleavage of the signal peptide of the lipeptide, SEQ ID NOs: 2, 4, 38, 41, 44, 48, 51, 54, 58, 61, 63, 65, 67, 73, 75, 78, 79, 82, 83, 85, 106, 108, 89, and 110, 112-115, 132, 133, 114, 138, 141, 144, 147, 151, 153, 156, Mature polypeptides having amino acid sequences selected from the group consisting of 158, 174, 175, 176, 181-184, 186, 188, 190, 195, 217-228, 233-236, 243, 255-264, 273-280, 283, 284, 287, 288, 291, 292, and 298-302 are obtained and used for the production of CoV S nanoparticle vaccines or CoV S nanoparticles.

[0212] Advantageously, the CoV S polypeptides of this disclosure may exhibit enhanced protein expression and stability compared to the innate CoV spike (S) protein.

[0213] In embodiments, the CoV S polypeptide described herein includes further modifications from the natural coronavirus S protein (e.g., SEQ ID NO: 2). In embodiments, the coronavirus S protein described herein exhibits at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with respect to the natural coronavirus S protein. Those skilled in the art will be able to calculate the identity percentage of the recombinant coronavirus S protein to either the natural protein or the CoV S polypeptide described herein using known techniques. For example, the identity percentage can be calculated using the CLUSTALW2 tool, which is available online. The following default parameters may be used for CLUSTALW2 pairwise alignment: amino acid substitution matrix = Gonnet, gap start penalty = 10, gap continuation penalty = 0.1.

[0214] In embodiments, the amino acid sequence of the CoV S polypeptide described herein is at least 91%, at least 92%, at least 93%, at least 94%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to a CoV S polypeptide having one of the amino acid sequences of SEQ ID NOs. 87, SEQ ID NOs. 174, SEQ ID NOs. 175, SEQ ID NOs. 176, SEQ ID NOs. 181-184, SEQ ID NOs. 186, SEQ ID NOs. 188, SEQ ID NOs. 190, SEQ ID NOs. 195, SEQ ID NOs. 217-228, SEQ ID NOs. 233-236, SEQ ID NOs. 243, or SEQ ID NOs. 255-302. CoV S polypeptides may have up to approximately 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid deletions, insertions, or mutations compared to the amino acid sequence of a CoV S polypeptide having one of the amino acid sequences of SEQ ID NOs: 87, 174, 175, 176, 181-184, 186, 188, 190, 195, 217-228, 233-236, 243, or 255-302. Compared to a CoV S polypeptide having one of the amino acid sequences of SEQ ID NOs: 87, 174, 175, 176, 181-184, 186, 188, 190, 195, 217-228, 233-236, 243, or 255-302, the CoV S polypeptide may have deletions, insertions, or mutations of approximately 1-5 amino acids, 3-10 amino acids, 5-10 amino acids, 8-12 amino acids, 10-15 amino acids, 12-17 amino acids, 15-20 amino acids, 18-23 amino acids, 20-25 amino acids, 22-27 amino acids, 25-30 amino acids, 30-35 amino acids, 35-40 amino acids, 40-45 amino acids, or 45-50 amino acids.In embodiments, the CoV S polypeptides described herein include about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 24, compared to any one of the CoV S polypeptides of SEQ ID NOs: 87, 174, 175, 176, 181-184, 186, 188, 190, 195, 217-228, 233-236, 243, and 255-302.

[0215] In embodiments, the CoV S polypeptide described herein is at least 91%, at least 92%, at least 93%, at least 94%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the CoV S polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 2, 4, 38, 41, 44, 48, 51, 54, 58, 61, 63, 65, 67, 73, 75, 78, 79, 82, 83, 85, 106, 108, 89, and 110, 112-115, 132, 133, 114, 138, 141, 144, 147, 151, 153, 156, and 158, 164-168.

[0216] In several embodiments, the coronavirus S polypeptide is elongated at the N-terminus, C-terminus, or both the N-terminus and C-terminus. In several embodiments, the elongated portion is a tag useful for functions such as purification or detection. In several embodiments, the tag contains an epitope. For example, the tag may be a polyglutamate tag, FLAG tag, HA tag, polyHis tag (having about 5-10 histidines) (SEQ ID NO: 101), hexahistidine tag (SEQ ID NO: 100), 8×His tag (having 8 histidines) (SEQ ID NO: 102), Myc tag, glutathione-S-transferase tag, green fluorescent protein tag, maltose-binding protein tag, thioredoxin tag, or Fc tag. In other embodiments, the elongated portion may be an N-terminal signal peptide fused to a protein to enhance expression. Such signal peptides are often cleaved during expression in cells, but some nanoparticles may contain an antigen with a complete signal peptide. Therefore, if the nanoparticles contain an antigen, the antigen may contain an elongated portion and thus may be a fusion protein when incorporated into the nanoparticles. For the purpose of calculating identity to the sequence, the elongated portion is not included. In several embodiments, the tag is a protease cleavage site. Non-limiting examples of protease cleavage sites include the HRV3C protease cleavage site, chymotrypsin, trypsin, elastase, endopeptidase, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, enterokinase, factor Xa, granzyme B, TEV protease, and thrombin. In several embodiments, the protease cleavage site is the HRV3C protease cleavage site. In several embodiments, the protease cleavage site includes the amino acid sequence of SEQ ID NO: 98.

[0217] In several embodiments, the CoV S glycoprotein includes a fusion protein. In several embodiments, the CoV S glycoprotein includes an N-terminal fusion protein. In several embodiments, the CoV S glycoprotein includes a C-terminal fusion protein. In several embodiments, the fusion protein includes a tag useful for protein expression, purification, or detection. In several embodiments, the tag is a polyHis tag (having about 5-10 histidines), a Myc tag, a glutathione-S-transferase tag, a green fluorescent protein tag, a maltose-binding protein tag, a thioredoxin tag, a Strep tag, a Twin-Strep tag, or an Fc tag. In several embodiments, the tag is an Fc tag. In several embodiments, the Fc tag is a monomer, a dimer, or a trimer. In several embodiments, the tag is a hexahistidine tag, e.g., a polyHis tag (SEQ ID NO: 100) containing six histidines. In several embodiments, the tag is a Twin-Strep tag having the amino acid sequence of SEQ ID NO: 99.

[0218] In several embodiments, the CoV S polypeptide is a fusion protein containing another coronavirus protein. In several embodiments, the other coronavirus protein is derived from the same coronavirus. In several embodiments, the other coronavirus protein is derived from a different coronavirus.

[0219] In some embodiments, the CoV S glycoprotein may be truncated. For example, the N-terminus may be truncated by about 10, 30, 50, 75, 100, or 200 amino acids. The C-terminus may be truncated in place of or in addition to the N-terminus. For example, the C-terminus may be truncated by about 10, 30, 50, 75, 100, or 200 amino acids. For the purpose of calculating identity for a protein having truncation, identity is measured over the remainder of the protein.

[0220] Surfactant core nanoparticles containing CoV spike(S) polypeptide In embodiments, the compositions disclosed herein include surfactant core nanoparticles comprising a CoV S polypeptide associated with a surfactant core.

[0221] In embodiments, the nanoparticles of the Disclosure comprise a mature CoV S polypeptide. In embodiments, the nanoparticles of the Disclosure comprise a CoV S polypeptide associated with a surfactant core. The presence of the surfactant facilitates the formation of the nanoparticles by forming a core that organizes and presents the antigen. In embodiments, the nanoparticles may comprise a CoV S polypeptide constructed in a multi-oligomeric glycoprotein-surfactant (e.g., PS80) nanoparticle, in which a head region protrudes outward and a hydrophobic region and a PS80 surfactant form a central core surrounded by a glycoprotein. In embodiments, the CoV S polypeptide is essentially contained or configured to contain a transmembrane domain that facilitates the association of the protein to the surfactant core. In embodiments, the CoV S polypeptide comprises a head domain. Primarily, the transmembrane domain of the CoV S polypeptide trimer associates with the surfactant, but other parts of the polypeptide may also interact. Advantageously, the nanoparticles have improved resistance to environmental stress, resulting in enhanced stability and / or improved presentation to the immune system due to the organization of multiple copies of the protein around the surfactant.

[0222] In the embodiment, the surfactant core is a nonionic surfactant core. In the embodiment, the CoV S polypeptide associates with the nonionic surfactant core. In the embodiment, the surfactant is selected from the group consisting of polysorbate-20 (PS20), polysorbate-40 (PS40), polysorbate-60 (PS60), polysorbate-65 (PS65), and polysorbate-80 (PS80). In the embodiment, the surfactant is PS80.

[0223] In some embodiments, the CoV S polypeptide forms trimers. In some embodiments, the CoV S polypeptide nanoparticles consist of multiple polypeptide trimers surrounding a nonionic surfactant core. In some embodiments, the nanoparticles contain at least about one trimer or more trimers. In some embodiments, the nanoparticles contain at least about five trimers to about 30 trimer spike proteins. In some embodiments, each nanoparticle may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 15, 20, 25, or 30 trimers (including all values ​​and ranges between these). The compositions disclosed herein may contain nanoparticles having different numbers of trimers. For example, a composition may contain nanoparticles with a number of trimers ranging from 2 to 9, and in some embodiments, the nanoparticles in the composition may contain 2 to 6 trimers. In some embodiments, the composition contains a heterogeneous population of nanoparticles having 2 to 6 trimers per nanoparticle or 2 to 9 trimers per nanoparticle. In some embodiments, the composition may contain a substantially homogeneous population of nanoparticles. For example, the population may contain about 95% nanoparticles having 5 trimers.

[0224] The nanoparticles disclosed herein have varying particle sizes. In embodiments, the nanoparticles disclosed herein have particle sizes ranging from approximately 20 nm to approximately 60 nm, approximately 20 nm to approximately 50 nm, approximately 20 nm to approximately 45 nm, approximately 20 nm to approximately 35 nm, approximately 20 nm to approximately 30 nm, approximately 25 nm to approximately 35 nm, or approximately 25 nm to approximately 45 nm. The particle size (Z-mean) is measured by dynamic light scattering (DLS) using Zetasizer NanoZS (Malvern, UK) unless otherwise specified.

[0225] In embodiments, nanoparticles containing the CoV S polypeptide disclosed herein have a smaller particle size compared to nanoparticles containing wild-type CoV S polypeptide. In embodiments, the CoV S polypeptide has a particle size that is at least about 40% smaller, for example, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, or at least about 85% smaller.

[0226] Nanoparticles containing CoV S polypeptide disclosed herein are more uniform in size, shape, and mass than nanoparticles containing wild-type CoV S polypeptide. The polydispersity index (PDI), a measure of heterogeneity, is measured by dynamic light scattering using a Malvern Setasizer unless otherwise specified. In embodiments, the particles measured herein have a PDI of about 0.2 to about 0.45, for example, about 0.2, about 0.25, about 0.29, about 0.3, about 0.35, about 0.40, or about 0.45. In embodiments, the nanoparticles measured herein have a PDI that is at least about 25% lower than the PDI of nanoparticles containing wild-type CoV S polypeptide, for example, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, or at least about 60% lower.

[0227] The CoV S polypeptide and nanoparticles containing it have improved thermal stability compared to wild-type CoV S polypeptide or its nanoparticles. The thermal stability of the CoV S polypeptide is measured using differential scanning calorimetry (DSC) unless otherwise specified. The transition enthalpy (ΔHcal) is the energy required for the unfolding of the CoV S polypeptide. In embodiments, the CoV S polypeptide has an increased ΔHcal compared to wild-type CoV S polypeptide. In embodiments, the ΔHcal of the CoV S polypeptide is about 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater than the ΔHcal of wild-type CoV S polypeptide.

[0228] Several nanoparticle species may be included in the vaccine compositions disclosed herein. In several embodiments, the nanoparticle species are in the form of anisotropic rods, which may be dimers or monomers. In other embodiments, the nanoparticle species are spherical oligomers. In yet another embodiment, the nanoparticles may be described as intermediate nanoparticles having sedimentation properties intermediate between the first two species. The formation of the nanoparticle species can be controlled by controlling the surfactant and protein concentrations during the preparation process. The nanoparticle species can be determined by measuring the sedimentation coefficient.

[0229] (ii) At least three types of HA glycoproteins In some embodiments, the immunogenic composition of the Disclosure comprises at least three HA glycoproteins or at least four HA glycoproteins. In some embodiments, the immunogenic composition of the Disclosure comprises three or four HA glycoproteins. In some embodiments, each HA glycoprotein is derived from a different influenza strain. In some embodiments, three HA glycoproteins are derived from influenza A strains and one HA glycoprotein is derived from influenza B strains. In some embodiments, two HA glycoproteins are derived from influenza A strains and two HA glycoproteins are derived from influenza B strains. In some embodiments, two HA glycoproteins are derived from influenza A strains and one HA glycoprotein is derived from influenza B strains.

[0230] In some embodiments, each of at least three HA glycoproteins is isolated separately using an egg-based production method. In some embodiments, the egg-based production method includes (a) growing the influenza virus in an egg and (b) recovering the influenza virus.

[0231] In several embodiments, the influenza virus is a live virus. In several embodiments, the influenza virus is a weakened or “attenuated” virus. In several embodiments, the influenza virus is optimized to grow in an egg. In several embodiments, the optimized influenza virus lacks polynucleotide cleavage sites of hemagglutinin. The following paper details the development of an optimized influenza virus (referred to as a “vaccine candidate virus”), which is incorporated herein by reference in its entirety: Belser et al. Virology. 2017 Nov;511:135-141.

[0232] In several embodiments, the egg is a chicken egg. In several embodiments, the chicken egg is a fertilized chicken egg. In several embodiments, the egg is a pathogen-free egg. In several embodiments, the influenza virus is propagated by inoculating the allantois cavity of a fertilized chicken egg with the virus. The following paper describes an exemplary inoculation method, which is incorporated herein by reference in its entirety: Brauer et al. J Vis Exp. 2015;(97):52421.

[0233] In several embodiments, the egg-based production method includes purifying the influenza virus. In several embodiments, the influenza virus is purified using one of the following techniques: centrifugation, chromatography, precipitation, or nanofiltration. In several embodiments, the centrifugation technique is ultracentrifugation. In several embodiments, the virus is purified using zone centrifugation. In several embodiments, the zone centrifugation is continuous flow zone centrifugation.

[0234] In several embodiments, the egg-based manufacturing method includes inactivating (also referred to herein as “killing”) the influenza virus. In several embodiments, the influenza virus is inactivated using a low pH (e.g., pH about 3.5 to 5.5), heat, ethanol, ultraviolet light, exposure to a surfactant (e.g., octylphenol), or exposure to a chemical (e.g., 2-propanol, ethanol, povidone-iodine). In several embodiments, the purified virus is inactivated using ultraviolet light, beta-propiolactone, sodium deoxycholate, formaldehyde, or any combination thereof.

[0235] In several embodiments, the egg-based manufacturing method includes exposing the influenza virus to a surfactant. The surfactant may be sodium taurodeoxycholate, octylphenol ethoxylate (Triton®-X100), or cetyltrimethylammonium bromide. Exposure of the influenza virus to the surfactant results in the formation of influenza split virions. In several embodiments, at least three HA glycoproteins are in the form of influenza split virions.

[0236] In several embodiments, the egg-based manufacturing method includes purifying the influenza antigen (e.g., hemagglutinin) from the virus.

[0237] The following U.S. Food and Drug Administration (FDA) approved influenza vaccines are manufactured using egg-based manufacturing methods: AFLURIA® tetravalent, FLUARIX® tetravalent, FLULAVAL tetravalent, FLUZONE® tetravalent, FLUZONE® High-Dose tetravalent, FLUMIST® tetravalent, FLUAD®, and FLUAD® tetravalent.

[0238] In several embodiments, each of at least three HA glycoproteins is isolated using a cell culture-based method. In several embodiments, the cell culture-based method includes (i) growing influenza virus in cells and (ii) recovering the virus from the cells. In several embodiments, the cell culture-based method includes (i) transfecting cells with a vector containing hemagglutinin and (ii) recovering hemagglutinin from the cells. In several embodiments, the cell culture-based method includes (i) transducing cells with a virus encoding hemagglutinin and (ii) recovering hemagglutinin from the cells. In several embodiments, the recovered hemagglutinin is recombinant hemagglutinin.

[0239] In some embodiments, the cells are animal cells, bacterial cells, insect cells, or fungal cells. In some embodiments, the animals are humans, birds (e.g., chickens), dogs, reptiles, goats, pigs, mice, rabbits, or rats.

[0240] In several embodiments, the virus encoding hemagglutinin is a baculovirus, lentivirus, or adeno-associated virus.

[0241] In several embodiments, influenza viruses produced using cell culture-based methods are purified. Any of the purification techniques described for purifying influenza viruses produced using egg-based production methods can be used to purify influenza viruses produced using cell culture-based methods.

[0242] In several embodiments, hemagglutinin produced using cell culture-based methods is purified. Purification techniques include chromatography, centrifugation, precipitation, and nanofiltration.

[0243] FLUCELVAX® tetravalent, an FDA-approved influenza vaccine, is produced using a cell culture-based method.

[0244] In several embodiments, each of at least three HA glycoproteins is recombinant hemagglutinin. FLUBLOK® tetravalent, an FDA-approved influenza vaccine, is recombinant hemagglutinin.

[0245] In several embodiments, at least three hemagglutinins are in the form of recombinant hemagglutinins. Recombinant hemagglutinins are isolated from hemagglutinin-producing cells. In several embodiments, hemagglutinin-producing cells are transfected with a hemagglutinin-encoding vector. In several embodiments, hemagglutinin-producing cells are transfected with a hemagglutinin-encoding virus.

[0246] (iia) Surfactant core nanoparticles containing at least three types of HA glycoprotein-hemagglutinin In embodiments, the compositions disclosed herein include surfactant core nanoparticles containing hemagglutinin derived from the influenza virus. The aforementioned surfactant core nanoparticles are described in detail in U.S. Patent No. 10,426,829, which is incorporated herein by reference in its entirety for all purposes.

[0247] The surfactant-core nanoparticles contain hemagglutinin derived from the influenza virus, associated with the surfactant core. In several embodiments, the hemagglutinin is a trimer. Each nanoparticle may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 trimers. In several embodiments, the nanoparticles contain about 2 to about 9, about 2 to about 6, or about 5 hemagglutinin trimers. The hemagglutinin associates with the nonionic surfactant-containing core of the nanoparticles. In several embodiments, the surfactant is selected from polysorbate-20 (PS20), polysorbate-40 (PS40), polysorbate-60 (PS60), polysorbate-65 (PS65), and polysorbate-80 (PS80). The presence of the surfactant facilitates the formation of nanoparticles by forming a core that organizes and presents the antigen. In several embodiments, the nanoparticles may contain an antigen constructed in multi-oligomeric glycoprotein-PS80 protein surfactant nanoparticles, in which the head region protrudes outward and the hydrophobic region and PS80 surfactant form a central core surrounded by the antigen.

[0248] The nanoparticles disclosed herein have a Z-average size in the range of approximately 20 nm to approximately 60 nm, approximately 20 nm to approximately 50 nm, approximately 20 nm to approximately 45 nm, or approximately 25 nm to approximately 45 nm.

[0249] In this embodiment, surfactant-core nanoparticles are produced in insect cells by expressing the HA protein using a baculovirus expression system and extracting the HA protein with a surfactant. During the purification process, the first surfactant is replaced with a second surfactant, usually a nonionic surfactant, to obtain nanoparticles having a nonionic surfactant core, in which the transmembrane domain of the trimer-shaped HA protein is embedded. Figure 7 (left panel) shows these structures as observed by electron microscopy.

[0250] The hemagglutinin contained in the surfactant core nanoparticles or HaSMaN described herein may be derived from any influenza virus strain. Human influenza A and B viruses cause seasonal outbreaks of the disease almost every winter in the United States.

[0251] The HA protein can be selected from subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and H18. Phylogenetically, influenza can be divided into several groups. For HA, Group 1 includes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18, and Group 2 includes H3, H4, H7, H10, H14, and H15.

[0252] In embodiments, the surfactant core nanoparticles or HaSMaN are trypsin-resistant nanoparticles produced using neutral pH purification. Trypsin resistance is achieved by a neutral pH range of greater than 6.9 to 8.5 during the purification and formulation of the HA nanoparticles. Trypsin-resistant influenza glycoproteins and trypsin-resistant influenza nanoparticles, as well as methods for producing them, are described in detail in U.S. Patent No. 10,426,829.

[0253] In several embodiments, the hemagglutinin of the surfactant core nanoparticles or HaSMaN described herein comprises a full-length wild-type hemagglutinin amino acid sequence. In several embodiments, the hemagglutinin is a hemagglutinin variant. In embodiments, the hemagglutinin exhibits at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the wild-type hemagglutinin protein. The percentage of identity can be calculated using the alignment program ClustalW2, available at www.ebi.ac.uk / Tools / msa / clustalw2 / . For pairwise alignment, the following default parameters may be used: amino acid substitution matrix = Gonnet, gap start penalty = 10, gap continuation penalty = 0.1.

[0254] HaSMaN is a homotrimer in which each monomer consists of approximately 550 amino acid residues. Each monomer of HaSMaN is conceptually divided into three domains: an external domain of approximately 515 residues constitutes the viral extracellular portion of the molecule, a single stretch of 27 residues defines the transmembrane (TM) domain, and approximately 10 residues constitute the cytoplasmic tail (CT). Although some modifications may be made to hemagglutinin, the complete transmembrane domain (TM) is required for the formation of both the surfactant core nanoparticles and HaSMaN. Therefore, in certain examples, the modified HaSMaN protein sequence may have 100% identity with the wild-type TM and CT domains, with some flexibility in the remaining external domain portion, where identity may be at least 90% or at least 95%. The domains can be identified by homology to the amino acid sequences of the TM and CT domains of Japan / 305 / 57 HA, as shown in Figure 1 of Melikyan et al. (Mol Biol Cell. 1999 Jun;10(6):1821-1836). However, it should be noted that the boundaries between the external domain, TM domain, and CT domain may differ by up to 3 amino acids for each HA protein.

[0255] (iib) Influenza vaccine - HaSMaN (Hemagglutinin saponin matrix nanoparticles) In embodiments, the immunogenic and vaccine compositions described herein include HaSMaN (hemagglutinin saponin matrix nanoparticles). Figure 7 (right panel) shows the HaSMaN structure as observed under an electron microscope. The HA glycoprotein coats a matrix cage-like structure. The HaSMaN structure is formed by preparing surfactant core nanoparticles containing hemagglutinin derived from influenza virus and then incubating them with ISCOM matrix adjuvant particles for a certain period of time. ISCOM matrix particles are shown in the center panel of Figure 7. Notably, HaSMaN is readily formed with influenza A HA protein but not with influenza B HA protein.

[0256] HaSMaN disclosed herein is prepared by incubating surfactant core nanoparticles with an ISCOM matrix adjuvant comprising a saponin fraction, cholesterol, and phospholipids. In some embodiments, HaSMaN is formed by incubating surfactant core nanoparticles with the ISCOM matrix adjuvant for about 24 to about 48 hours. For example, surfactant core nanoparticles may be incubated with the ISCOM matrix adjuvant for about 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 hours, or longer. In some embodiments, HaSMaN is formed by incubating surfactant core nanoparticles with an ISCOM matrix adjuvant for at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 37 hours, at least about 38 hours, at least about 39 hours, at least about 40 hours, at least about 41 hours, at least about 42 hours, at least about 43 hours, at least about 44 hours, at least about 45 hours, at least about 46 hours, at least about 47 hours, or at least about 48 hours. In some embodiments, HaSMaN is formed by incubating surfactant core nanoparticles with an ISCOM matrix adjuvant at a temperature of about 4°C to about 25°C. For example, the HaSMaN may be formed by incubating surfactant core nanoparticles with an ISCOM matrix adjuvant at a temperature of approximately 4°C, approximately 5°C, approximately 6°C, approximately 7°C, approximately 8°C, approximately 9°C, approximately 10°C, approximately 11°C, approximately 12°C, approximately 13°C, approximately 14°C, approximately 15°C, approximately 16°C, approximately 17°C, approximately 18°C, approximately 19°C, approximately 20°C, approximately 21°C, approximately 22°C, approximately 23°C, approximately 24°C, approximately 25°C, or higher.In embodiments, HaSMaN is formed by incubating surfactant core nanoparticles with an ISCOM matrix adjuvant at temperatures of at least about 4°C, at least about 5°C, at least about 6°C, at least about 7°C, at least about 8°C, at least about 9°C, at least about 10°C, at least about 11°C, at least about 12°C, at least about 13°C, at least about 14°C, at least about 15°C, at least about 16°C, at least about 17°C, at least about 18°C, at least about 19°C, at least about 20°C, at least about 21°C, at least about 22°C, at least about 23°C, at least about 24°C, and at least about 25°C. Typically, incubation at 4°C or 25°C for about 24–48 hours is required for formation. HaSMaN formation is promoted by higher temperatures. In embodiments, HaSMaN formation occurs by incubating surfactant core nanoparticles with an ISCOM matrix adjuvant at about 25°C for at least 24 hours. Mixing the surfactant core nanoparticles with the ISCOM matrix adjuvant immediately before administration to the subject, i.e., using a bedside mix, does not generate HaSMaN. Longer incubation periods do not adversely affect HaSMaN formation.

[0257] Manufacturing of surfactant core nanoparticles The nanoparticles of this disclosure are non-naturally occurring products, and their components do not exist together in nature. In embodiments, the methods disclosed herein employ a surfactant exchange approach. In this case, a first surfactant is used to isolate a protein, which is then exchanged for a second surfactant to form nanoparticles.

[0258] The antigens contained in the nanoparticles are typically produced by recombinant expression in host cells. Standard recombination techniques may be used. In embodiments, the CoV S polypeptide is expressed in insect host cells using a baculovirus system. In embodiments, the baculovirus is a cathepsin-L knockout baculovirus, a chitinase knockout baculovirus. Optionally, the baculovirus is a double knockout of both cathepsin-L and chitinase. High levels of expression can be obtained in insect cell expression systems. Non-limiting examples of insect cells include Spodoptera frugiperda (Sf) cells, e.g., Sf9, Sf21, Trichoplusiani cells, e.g., High Five cells, and Drosophila S2 cells. In embodiments, the CoV S polypeptide or hemagglutinin described herein is produced in any suitable host cell. In embodiments, the host cell is an insect cell. In some embodiments, the insect cell is an Sf9 cell.

[0259] Typical transfection and cell growth methods can be used to culture cells. Vectors, such as those containing polynucleotides encoding fusion proteins, can be transfected into host cells according to methods well known in the art. For example, the introduction of nucleic acids into eukaryotic cells can be achieved by calcium phosphate coprecipitation, electroporation, microinjection, lipofection, and transfection using polyamine transfection reagents. In one embodiment, the vector is a recombinant baculovirus.

[0260] Methods for growing host cells include, but are not limited to, batch, batch feeding, continuous, and perfusion cell culture techniques. Cell culture refers to the growth and proliferation of cells in a bioreactor (fermentation chamber), where cells proliferate and express proteins (e.g., recombinant proteins) for purification and isolation. Typically, cell culture is carried out in a bioreactor under sterile and controlled temperature and atmospheric conditions. A bioreactor is a chamber used to culture cells, where environmental conditions such as temperature, atmosphere, agitation, and / or pH can be monitored. In one embodiment, the bioreactor is a stainless steel chamber. In another embodiment, the bioreactor is a sterile plastic bag (e.g., Cellbag®, Wave Biotech, Bridgewater, NJ). In yet another embodiment, the sterile plastic bag is approximately 50L to 3500L.

[0261] Extraction and purification of nanoparticles After host cell growth, the protein or nanoparticles can be collected from the host cells using a surfactant and purification protocol. After growing the host cells for 48–96 hours, the cells are isolated from the culture medium, and a surfactant-containing solution is added to solubilize the cell membrane and release the protein into the surfactant extract. Triton® X-100 and TERGITOL® nonylphenol ethoxylate, also known as NP-9, are preferred surfactants for extraction. The surfactant can be added up to a final concentration of about 0.1% to about 1.0%. For example, the concentration may be about 0.1%, about 0.2%, about 0.3%, about 0.5%, about 0.7%, about 0.8%, or about 1.0%. The range may be about 0.1% to about 0.3%. In embodiments, the concentration is about 0.5%.

[0262] In embodiments, various first surfactants may be used to isolate proteins from host cells. For example, the first surfactant may be bis(polyethylene glycol bis[imidazoylcarbonyl]), nonoxynol-9, bis(polyethylene glycol bis[imidazoylcarbonyl]), BRIJ® polyethylene glycol dodecyl ether 35, BRIJ® polyethylene glycol (3) cetyl ether 56, BRIJ® alcohol ethoxylate 72, BRIJ® polyoxyl 2 stearyl ether 76, BRIJ® polyethylene glycol monooleyl ether 92V, BRIJ® polyoxyethylene (10) oleyl ether 97, BRIJ® polyethylene glycol hexadecyl ether 58P, or CREMOPHOR® EL macrogol glycerol ricinoleate. Decaethylene glycol monododecyl ether, N-decanoyl-N-methylglucamine, n-decyl alpha-D-glucopyranoside, decyl beta-D-maltopyranoside, n-dodecanoyl-N-methylglucamide, n-dodecyl alpha-D-maltoside, n-dodecyl beta-D-maltoside, n-dodecyl beta-D-maltoside, heptaethylene glycol monodecyl ether, heptaethylene glycol monododecyl ether, heptaethylene glycol monotetradecyl ether, n-hexadecyl beta-D-maltoside, hexaethylene glycol monododecyl ether, hexaethylene glycol monohexadecyl ether, hexaethylene glycol monooctadecyl ether, hexaethylene glycol monotetradecyl ether, Igepal CA-630, Igepal CA-630, Methyl-6-0-(N-heptylcarbamoyl)-alpha-D-glucopyranoside, Nonaethylene glycol monododecyl ether, N-nonanoyl-N-methylglucamine, N-nonanoyl-N-methylglucamine, Octaethylene glycol monodecyl ether, Octaethylene glycol monododecyl ether, Octaethylene glycol monohexadecyl ether, Octaethylene glycol monooctadecyl ether, Octaethylene glycol monotetradecyl ether,Octyl-beta-D glucopyranoside, pentaethylene glycol monodecyl ether, pentaethylene glycol monododecyl ether, pentaethylene glycol monohexadecyl ether, pentaethylene glycol monohexyl ether, pentaethylene glycol monooctadecyl ether, pentaethylene glycol monooctyl ether, polyethylene glycol diglycidyl ether, polyethylene glycol ether W-1, polyoxyethylene 10 tridecyl ether, polyoxyethylene 100 stearate, polyoxyethylene 20 isohexadecyl ether, polyoxyethylene 20 oleyl ether, polyoxyethylene 40 stearate, polyoxyethylene 50 stearate, polyoxyethylene 8 stearate, polyoxyethylene bis(imidazolylcarbonyl), polyoxyethylene 25 propylene glycol stearate, saponin derived from Quillaja bark, SPAN® 20 sorbitan laurate, SPAN® 40 sorbitan monopalmitate, SPAN® 60 sorbitan stearate SPAN® 65 sorbitan tristearate, SPAN® 80 sorbitan monooleate, SPAN® 85 sorbitan trioleate, TERGITOL® secondary alcohol ethoxylate (type 15-S-12), TERGITOL® secondary alcohol ethoxylate (type 15-S-30), TERGITOL® secondary alcohol ethoxylate (type 15-S-5), TERGITOL® secondary alcohol ethoxylate (type 15-S- 7) TERGITOL® secondary alcohol ethoxylate (type 15-S-9), TERGITOL® nonylphenol ethoxylate (type NP-10), TERGITOL® nonylphenol ethoxylate (type NP-4), TERGITOL® nonylphenol ethoxylate (type NP-40), TERGITOL® nonylphenol ethoxylate (type NP-7), TERGITOL® nonylphenol ethoxylate (type NP-9),This may be TERGITOL® branched secondary alcohol ethoxylate (type TMN-10), TERGITOL® branched secondary alcohol ethoxylate (type TMN-6), TRITON® X-100 polyethylene glycol tert-octylphenyl ether, or a combination thereof.

[0263] The nanoparticles can then be isolated from the cell fragments using centrifugation. In several embodiments, gradient centrifugation using cesium chloride, sucrose, and iodixanol may be used. Other standard purification techniques, such as ion exchange chromatography, affinity chromatography, and gel filtration chromatography, may be used as alternatives or in addition.

[0264] For example, the first column may be an ion exchange chromatography resin such as FRACTOGEL® EMD methacrylate polymer beads TMAE (EMD Millipore), the second column may be a lentil (Lens culinaris) lectin affinity resin, and the third column may be a cation exchange column such as FRACTOGEL® EMD methacrylate polymer beads SO3 (EMD Millipore) resin. In other embodiments, the cation exchange column may be an MMC column or a Nuvia C Prime column (Bio-Rad Laboratories, Inc.). Preferably, the method disclosed herein does not use surfactant extraction columns, such as hydrophobic interaction columns. Such columns are often used to remove surfactants in the purification process, but they may adversely affect the method disclosed herein.

[0265] In the embodiment, a first surfactant used to extract proteins from host cells is substantially replaced with a second surfactant to obtain a nanoparticle structure. In the embodiment, the first surfactant is NP-9. Typically, the nanoparticles do not contain detectable NP-9 when measured by HPLC. The second surfactant is typically selected from the group consisting of PS20, PS40, PS60, PS65, and PS80. In the embodiment, the second surfactant is PS80.

[0266] In embodiments, surfactant exchange is carried out using affinity chromatography that binds to the glycoprotein via the carbohydrate moiety. For example, affinity chromatography may use a leguminous lectin column. Leguminous lectins are proteins originally identified in plants and found to interact specifically and reversibly with carbohydrate residues. See, for example, Sharon and Lis, “Legume lectins--a large family of homologous proteins,” FASEB J.1990 Nov;4(14):3198-208, and Liener, “The Lectins: Properties, Functions, and Applications in Biology and Medicine,” Elsevier, 2012. Suitable lectins include concanavalin A (con A), pea lectin, burdock lectin, and lentil lectin. Lentil lectin is a preferred column for surfactant exchange due to its binding properties. Lectin columns are commercially available; for example, Capto lentil lectin is available from GE Healthcare. In certain embodiments, the lentil lectin column may use recombinant lectin. At the molecular level, it is thought that the carbohydrate moiety binds to the lentil lectin, releasing amino acids from the protein and fusing them to the surfactant, resulting in the formation of a surfactant core that yields nanoparticles having a multi-copy antigen, which may be a glycoprotein oligomer, such as a dimer, trimer, or tetramer, immobilized on the surfactant. In embodiments, the CoV S polypeptide and / or hemagglutinin forms trimers. In embodiments, the CoV S polypeptide trimers and / or hemagglutinin are immobilized on the surfactant. In embodiments, each nanoparticle contains at least one trimer associated with a nonionic core.

[0267] The surfactant may be present in the early purification step at a maximum of approximately 0.1% (w / v) when incubated with the protein during the surfactant exchange process to form nanoparticles, and this amount is reduced to obtain final nanoparticles with optimal stability. For example, nonionic surfactants (e.g., PS80) may be present in amounts of approximately 0.005% (v / v) to approximately 0.1% (v / v), e.g., approximately 0.005% (v / v), approximately 0.006% (v / v), approximately 0.007% (v / v), approximately 0.008% (v / v), approximately 0.009% (v / v), approximately 0.01% (v / v), approximately 0.015% (v / v), approximately 0.02% (v / v), approximately 0.025% (v / v), approximately 0.03% (v / v), approximately 0 The PS80 content may be 0.035%(v / v), approximately 0.04%(v / v), approximately 0.045%(v / v), approximately 0.05%(v / v), approximately 0.055%(v / v), approximately 0.06%(v / v), approximately 0.065%(v / v), approximately 0.07%(v / v), approximately 0.075%(v / v), approximately 0.08%(v / v), approximately 0.085%(v / v), approximately 0.09%(v / v), approximately 0.095%(v / v), or approximately 0.1%(v / v)PS80. In several embodiments, the nanoparticles contain approximately 0.03% to approximately 0.05% PS80. In several embodiments, the nanoparticles contain approximately 0.01%(v / v)PS80.

[0268] In the embodiment, the purified CoV S polypeptide and / or hemagglutinin is dialyzed. In the embodiment, dialyzed is performed after purification. In the embodiment, the CoV S polypeptide and / or hemagglutinin is dialyzed in a solution containing sodium phosphate, NaCl, and PS80. In the embodiment, the dialyzed solution containing sodium phosphate contains about 5 mM to about 100 mM of sodium phosphate, for example, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM of sodium phosphate. In the embodiment, the pH of the solution containing sodium phosphate is about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5. In the embodiment, the dialysis solution containing sodium chloride is about 50 mM NaCl to about 500 mM NaCl, for example, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM Contains approximately 260 mM, 270 mM, 280 mM, 290 mM, 300 mM, 310 mM, 320 mM, 330 mM, 340 mM, 350 mM, 360 mM, 370 mM, 380 mM, 390 mM, 400 mM, 410 mM, 420 mM, 430 mM, 440 mM, 450 mM, 460 mM, 470 mM, 480 mM, 490 mM, or 500 mM NaCl.In this embodiment, the dialysis solution containing PS80 is approximately 0.005% (v / v), approximately 0.006% (v / v), approximately 0.007% (v / v), approximately 0.008% (v / v), approximately 0.009% (v / v), approximately 0.01% (v / v), approximately 0.015% (v / v), approximately 0.02% (v / v), approximately 0.025% (v / v), approximately 0.03% (v / v), approximately 0.035% (v / v), and approximately 0.04% The solution contains (v / v), approximately 0.045%(v / v), approximately 0.05%(v / v), approximately 0.055%(v / v), approximately 0.06%(v / v), approximately 0.065%(v / v), approximately 0.07%(v / v), approximately 0.075%(v / v), approximately 0.08%(v / v), approximately 0.085%(v / v), approximately 0.09%(v / v), approximately 0.095%(v / v), or approximately 0.1%(v / v) of PS80. In one embodiment, the dialysis solution contains approximately 25 mM sodium phosphate (pH 7.2), approximately 300 mM NaCl, and approximately 0.01%(v / v) of PS80.

[0269] Surfactant exchange can be carried out using proteins that have been purified as discussed above, frozen for storage, and then thawed for surfactant exchange.

[0270] The stability of the compositions disclosed herein can be measured by various means. One method may involve preparing peptide maps to determine the integrity of the antigen protein after various treatments designed to stress the nanoparticles by mimicking severe storage conditions. Thus, the measure of stability is the relative abundance of the antigen peptide in the stressed sample compared to a control sample. For example, the stability of nanoparticles containing the CoV S polypeptide can be evaluated by exposing the nanoparticles to various pH levels, proteases, salts, oxidizing agents including but not limited to hydrogen peroxide, various temperatures, freeze / thaw cycles, and stirring.

[0271] (iic) At least three types of hemagglutinin: inactivated whole influenza virus In the embodiments, at least three hemagglutinins are in the form of the whole influenza virus. The whole influenza virus includes its envelope, viral membrane, nucleocapsid, and genetic material. In the embodiments, the whole influenza virus is inactivated as described throughout this disclosure.

[0272] (iid) At least three types of hemagglutinin: Hemagglutinin composition extracted from influenza virus In the embodiment, at least three hemagglutinins are in the form of a hemagglutinin composition extracted from the influenza virus. In the embodiment, the hemagglutinin composition extracted from the influenza virus is a split virion composition or a subunit influenza composition of influenza.

[0273] Influenza split virions are influenza viruses having an influenza virus membrane destroyed by a surfactant. In embodiments, the surfactant is any surfactant described herein. In embodiments, the surfactant is sodium taurodeoxycholate, octylphenol ethoxylate (Triton®-X100), or cetyltrimethylammonium bromide. In embodiments, the influenza split virions are produced using egg-based or cell culture-based production methods. In embodiments, influenza split virions are produced by growing influenza viruses in Maidin Darby canine kidney (MDCK) cells, inactivating the viruses with β-propiolactone, exposing the viruses to the surfactant cetyltrimethylammonium bromide, and purifying the virions.

[0274] Similar to influenza split virions, subunit influenza compositions are produced by disrupting the influenza virus membrane with a surfactant. However, unlike influenza split virions, the subunit influenza compositions are subjected to further purification. In embodiments, the subunit influenza compositions are purified by removing the internal subviral core by different precipitation methods. In embodiments, the subunit influenza compositions are purified by centrifugation, chromatography, precipitation, or nanofiltration. The surfactant may be any surfactant described herein. In embodiments, the surfactant is sodium taurodeoxycholate, octylphenol ethoxylate (Triton®-X100), or cetyltrimethylammonium bromide. In embodiments, the subunit influenza vaccine is produced using an egg-based or cell culture-based production method.

[0275] In this embodiment, the hemagglutinin composition extracted from the virus contains hemagglutinin and excludes viral ribonucleoprotein (vRNP), matrix protein M1, and the viral envelope.

[0276] In the embodiment, the hemagglutinin composition extracted from the virus is obtained by (i) producing whole influenza virus using an egg-based production method, (ii) collecting and clarifying the whole influenza virus by centrifugation and filtration, (iii) inactivating the influenza virus with formaldehyde, (iv) concentrating and purifying the inactivated influenza virus by zone centrifugation, and (v) centrifugating the inactivated influenza virus in the presence of cetyltrimethylammonium bromide.

[0277] Immunogenic composition preparations This disclosure provides an immunogenic composition comprising: (i) at least three hemagglutinin (HA) glycoproteins, the three HA glycoproteins being derived from different influenza strains; (ii) a CoV S polypeptide in the form of surfactant core nanoparticles, the surfactant being a nonionic surfactant; and (iii) a pharmaceutically acceptable buffer. In embodiments, the at least three HA glycoproteins are in a form selected from the group consisting of (a) surfactant core nanoparticles containing hemagglutinin (HA); (b) HaSMaN (hemagglutinin saponin matrix nanoparticles); (c) influenza spirit virion; (d) whole influenza virus; (e) recombinant hemagglutinin; and (f) hemagglutinin compositions extracted from viruses. In embodiments, the immunogenic composition may comprise nanoparticles having antigens derived from multiple viral strains of the same species of virus.

[0278] In the embodiment, the immunogenic composition comprises (i) a CoV S polypeptide in the form of surfactant core nanoparticles, wherein the surfactant is a nonionic surfactant; and (ii) at least three hemagglutinin (HA) glycoproteins, wherein the three HA glycoproteins are derived from different influenza strains and are in the form of surfactant core nanoparticles containing HA glycoprotein and HaSMaN. In the embodiment, the immunogenic composition comprises (i) at least three hemagglutinin (HA) glycoproteins, wherein the three HA glycoproteins are derived from different influenza strains and are in the form of influenza split virions; and (ii) a CoV S polypeptide in the form of surfactant core nanoparticles, wherein the surfactant is a nonionic surfactant. In the embodiment, the immunogenic composition comprises (i) at least three hemagglutinin (HA) glycoproteins, wherein the three HA glycoproteins are derived from different influenza strains and are in the form of a hemagglutinin composition extracted from the virus, and (ii) a CoV S polypeptide in the form of surfactant core nanoparticles, wherein the surfactant is a nonionic surfactant.

[0279] In the embodiment, the composition comprises a total of about 1 to about 10 surfactant core nanoparticles containing hemagglutinin and HaSMaN, for example, a total of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nanoparticles. In the embodiment, each surfactant core nanoparticle and HaSMaN contains hemagglutinin derived from a different influenza strain. In the embodiment, each of the HaSMaN contains hemagglutinin derived from influenza A strain. In the embodiment, the hemagglutinin derived from each of the surfactant core nanoparticles is derived from influenza B strain. In the embodiment, each of the HaSMaN contains hemagglutinin derived from influenza B strain. In the embodiment, the hemagglutinin derived from each of the surfactant core nanoparticles is derived from influenza A strain. In the embodiment, the hemagglutinin derived from each of the surfactant core nanoparticles is derived from influenza B strain, and the hemagglutinin derived from each HaSMaN is derived from influenza A strain. In one embodiment, the composition comprises a total of four surfactant core nanoparticles containing hemagglutinin and HaSMaN. In another embodiment, the composition comprises a total of three surfactant core nanoparticles containing hemagglutinin and HaSMaN.

[0280] In another embodiment, the disclosure provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of an immunogenic composition.

[0281] The compositions disclosed herein may be used for either preventive or therapeutic purposes, but are typically used for preventive purposes. Accordingly, this disclosure includes methods for treating or preventing infectious diseases. The methods involve administering a therapeutic or preventive dose of the immunogenic composition of this disclosure to a subject. In embodiments, the pharmaceutical composition is a vaccine composition that provides a preventive effect. In embodiments, the preventive effect includes improvement of symptoms associated with infectious diseases in a certain proportion of an exposed population. For example, the composition may prevent or reduce symptoms of one or more viral diseases selected from symptoms of fever, fatigue, muscle pain, headache, sore throat, vomiting, diarrhea, rash, renal and hepatic dysfunction, internal and external bleeding, compared to an untreated subject.

[0282] Nanoparticles can be formulated for administration as vaccines in the presence of various excipients, buffers, etc. For example, a vaccine composition may contain sodium phosphate, sodium chloride, and / or histidine. Sodium phosphate may be present at about 10 mM to about 50 mM, about 15 mM to about 25 mM, or about 25 mM. In certain cases, about 22 mM sodium phosphate is present. Histidine may be present at about 0.1% (w / v), about 0.5% (w / v), about 0.7% (w / v), about 1% (w / v), about 1.5% (w / v), about 2% (w / v), or about 2.5% (w / v). Sodium chloride, if present, may be at about 150 mM. In certain compositions, sodium chloride may be present at higher concentrations, for example, about 200 mM to about 500 mM. In several embodiments, sodium chloride is present in high concentrations including, but not limited to, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, or about 500 mM.

[0283] In several embodiments, the nanoparticles described herein have improved stability at certain pH levels. In several embodiments, the nanoparticles are stable at slightly acidic pH levels. For example, nanoparticles stable at slightly acidic pH, e.g., pH 5.8 to pH 7.0. In several embodiments, nanoparticles and compositions containing nanoparticles may be stable at pH ranges of approximately pH 5.8 to approximately pH 7.0, including approximately pH 5.9 to approximately pH 6.8, approximately pH 6.0 to approximately pH 6.5, approximately pH 6.1 to approximately pH 6.4, approximately pH 6.1 to approximately pH 6.3, or approximately pH 6.2. In several embodiments, the nanoparticles and compositions described herein are stable at neutral pH, including approximately pH 7.0 to approximately pH 7.4. In several embodiments, the nanoparticles and compositions described herein are stable at slightly alkaline pH, e.g., approximately pH 7.0 to approximately pH 8.5, approximately pH 7.0 to approximately pH 8.0, or approximately pH 7.0 to approximately pH 7.5 (including all values ​​and ranges in between).

[0284] Adjuvant In certain embodiments, the compositions disclosed herein may be mixed with one or more adjuvants that enhance the immune response. In embodiments, the compositions are prepared without adjuvants and are therefore available for administration as adjuvant-free compositions. Advantageously, the adjuvant-free compositions disclosed herein may evoke a protective immune response when administered as a single dose. Adjuvant-free compositions that induce a potent immune response are particularly useful in adults about 60 years of age or older. In embodiments, the adjuvant is an oil-in-water emulsion comprising squalene as the oil phase, stabilized in citrate buffer with the surfactants polysorbate 80 and sorbitan trioleate.

[0285] Aluminum-based adjuvants In several embodiments, the adjuvant may be an alum (e.g., AlPO4 or Al(OH)3). Typically, the nanoparticles are substantially bound to the alum. For example, the nanoparticles may be bound to the alum by at least 80%, at least 85%, at least 90%, or at least 95%. In many cases, the nanoparticles are bound to the alum by 92% to 97% in the composition. The amount of alum present per dose is typically in the range of about 400 μg to about 1250 μg. For example, the alum may be present in amounts of about 300 μg to about 900 μg, about 400 μg to about 800 μg, about 500 μg to about 700 μg, about 400 μg to about 600 μg, or about 400 μg to about 500 μg per dose. Typically, there is about 400 μg of alum in a 120 μg dose of protein nanoparticles.

[0286] Saponin adjuvant Saponin-containing adjuvants may also be combined with the immunogens disclosed herein. Saponins are glycosides derived from the bark of the tree Quillaja saponaria Molina. Typically, saponins are prepared using multi-step purification processes that yield multiple fractions. As used herein, the term “saponin fraction derived from Quillaja saponaria Molina” is used comprehensively to describe a semi-purified or defined saponin fraction of Quillaja saponaria or a substantially pure fraction thereof.

[0287] Saponin fraction Several methods are preferred for preparing the saponin fractions. Fractions A, B, and C are described in U.S. Patent No. 6,352,697 and can be prepared as follows: The lipophilic fraction derived from Quil A, a crude aqueous extract of Quillaja saponaria Molina, is separated by chromatography and eluted with 70% acetonitrile in water to recover the lipophilic fraction. This lipophilic fraction is then separated by half-hole HPLC with elution using a gradient of 25% to 60% acetonitrile in acidic water. The fraction referred to herein as “Fraction A” or “QH-A” is or corresponds to the fraction eluted with approximately 39% acetonitrile. The fraction referred to herein as “Fraction B” or “QH-B” is or corresponds to the fraction eluted with approximately 47% acetonitrile. The fraction referred to herein as “Fraction C” or “QH-C” is or corresponds to the fraction eluted with approximately 49% acetonitrile. Further information regarding the purification of the fractions can be found in U.S. Patent No. 5,057,540. When prepared as described herein, fractions A, B, and C of Quillaja saponaria Molina represent a group or family of chemically closely related molecules with definable properties. The chromatographic conditions under which they are obtained are such that batch-to-batch reproducibility in terms of elution profiles and biological activity is highly consistent.

[0288] Other saponin fractions are listed. Fractions B3, B4, and B4b are listed in EP0436620. Fractions QA1 to QA22 are listed in EP03632279B2, Q-VAC (Nor-Feed, AS Denmark), and Quillaja saponaria Molina Spikoside (Isconova AB, Ultunaallen 2B, 756 51 Uppsala, Sweden). Fractions QA-1, QA-2, QA-3, QA-4, QA-5, QA-6, QA-7, QA-8, QA-9, QA-10, QA-11, QA-12, QA-13, QA-14, QA-15, QA-16, QA-17, QA-18, QA-19, QA-20, QA-21, and QA-22 of EP03632279B2, particularly QA-7, QA-17, QA-18, and QA-21, may be used. They are obtained as described in EP03632279B2, particularly on page 6, and in Example 1 on pages 8 and 9.

[0289] The saponin fractions used to form adjuvants as described herein are often substantially pure fractions, i.e., the fractions are substantially free from contamination from other materials. In certain embodiments, the substantially pure saponin fractions may contain up to 40% by weight, up to 30% by weight, up to 25% by weight, up to 20% by weight, up to 15% by weight, up to 10% by weight, up to 7% by weight, up to 5% by weight, up to 2% by weight, up to 1% by weight, up to 0.5% by weight, or up to 0.1% by weight of other compounds, such as other saponins or other adjuvant materials.

[0290] ISCOM structure The saponin fraction may be administered in the form of cage-like particles called ISCOM (Immune Stimulating Complex). ISCOM may be prepared as described in EP0109942B1, EP0242380B1 and EP0180546B1. In certain embodiments, transport and / or passenger antigens may be used as described in EP9600647-3 (PCT / SE97 / 00289).

[0291] Matrix Adjuvant In several embodiments, ISCOM is an ISCOM matrix complex. The ISCOM matrix complex comprises at least one saponin fraction and a lipid. The lipid is at least a sterol, such as cholesterol. In certain embodiments, the ISCOM matrix complex also contains phospholipids. The ISCOM matrix complex also may contain one or more other immunomodulatory (adjuvant-active) substances, which do not necessarily have to be glycosides, and the whole may be prepared as described in EP0436620B1, which is incorporated herein by reference.

[0292] In other embodiments, ISCOM is an ISCOM complex. The ISCOM complex contains at least one saponin, at least one lipid, and at least one antigen or epitope. The ISCOM complex contains an antigen associated by a surfactant treatment that incorporates a portion of the antigen into the particle. In contrast, the ISCOM matrix is ​​formulated as a miscion with an antigen, and the association of the ISCOM matrix particle with the antigen is mediated by electrostatic and / or hydrophobic interactions.

[0293] According to one embodiment, the ISCOM matrix complex or the saponin fraction incorporated into the ISCOM complex, or at least one further adjuvant (which is also incorporated into or mixed with ISCOM or the ISCOM matrix complex), is selected from fraction A, fraction B, or fraction C of Quillaja saponaria, a semi-purified preparation of Quillaja saponaria, a purified preparation of Quillaja saponaria, or a portion of any purified fraction, e.g., QA1 to QA21.

[0294] In certain embodiments, each ISCOM particle may contain at least two saponin fractions. Any combination of weight percent of different saponin fractions may be used. Any two fractions may be used in any combination of weight percent. For example, a particle may contain any weight percent of fraction A and any weight percent of another saponin fraction, such as a crude saponin fraction or fraction C. Accordingly, in certain embodiments, each ISCOM matrix particle or each ISCOM complex particle may contain one saponin fraction, e.g., fraction A, in amounts of 0.1–99.9 wt, 5–95 wt%, 10–90 wt%, 15–85 wt%, 20–80 wt%, 25–75 wt%, 30–70 wt%, 35–65 wt%, 40–60 wt%, 45–55 wt%, 40–60 wt%, or 50 wt%, and another saponin, e.g., any crude fraction or any other fraction, e.g., fraction C, up to 100% of the remaining amount in each case. The weight is calculated as the total weight of the saponin fractions. Examples of ISCOM matrix complexes and ISCOM complex adjuvants are disclosed in U.S. Publication Application No. 2013 / 0129770, which is incorporated herein by reference in its entirety.

[0295] In certain embodiments, the ISCOM matrix or ISCOM complex comprises one fraction, e.g., fraction A, in the weight of 5–99% and another fraction, e.g., crude saponin fraction or fraction C, in the weight of up to 100%. The weight is calculated as the total weight of the saponin fraction.

[0296] In another embodiment, the ISCOM matrix or ISCOM complex comprises one fraction, e.g., fraction A, in an amount of 40% to 99% by weight, and another fraction, e.g., crude saponin fraction or fraction C, in an amount of 1% to 60% by weight. The weight is calculated as the total weight of the saponin fraction.

[0297] In yet another embodiment, the ISCOM matrix or ISCOM complex comprises 70% to 95% by weight of one fraction, e.g., fraction A, and 30% to 5% by weight of another fraction, e.g., crude saponin fraction or fraction C. The weight is calculated as the total weight of the saponin fraction. In another embodiment, the saponin fraction derived from Quillaja saponaria Molina is selected from any one of QA1 to QA21.

[0298] In addition to particles containing a mixture of saponin fractions, ISCOM matrix particles and ISCOM complex particles may each be formed using only one saponin fraction. The compositions disclosed herein may contain multiple particles, each containing only one saponin fraction. That is, a particular composition may contain one or more different types of ISCOM matrix complex particles and / or one or more different types of ISCOM complex particles, each individual particle containing one saponin fraction derived from Quillaja saponaria Molina, where the saponin fraction in one complex is different from the saponin fraction in other complex particles.

[0299] In certain embodiments, one type of saponin fraction or crude saponin fraction may be incorporated into one ISCOM matrix complex or particle, and another type of substantially pure saponin fraction or crude saponin fraction may be incorporated into another ISCOM matrix complex or particle. The composition or vaccine may comprise at least two types of complexes or particles, each having one type of saponin incorporated into physically different particles.

[0300] In the composition, a mixture of ISCOM matrix complex particles and / or ISCOM complex particles may be used, in which one saponin fraction, Quillaja saponaria Molina, and another saponin fraction, Quillaja saponaria Molina, are separately incorporated into different ISCOM matrix complex particles and / or ISCOM complex particles.

[0301] ISCOM matrix or ISCOM complex particles, each containing one type of saponin fraction, may be present in the composition in any combination of weight percent. In certain embodiments, the composition may contain ISCOM matrix or complexes containing a first saponin fraction in amounts of 0.1% to 99.9% by weight, 5% to 95% by weight, 10% to 90% by weight, 15% to 85% by weight, 20% to 80% by weight, 25% to 75% by weight, 30% to 70% by weight, 35% to 65% by weight, 40% to 60% by weight, 45% to 55% by weight, 40% to 60% by weight, or 50% by weight, and the remainder consisting of ISCOM matrix or complexes containing different saponin fractions. In some embodiments, the remainder consists of one or more ISCOM matrix or complexes, where each matrix or complex particle contains only one type of saponin fraction. In other embodiments, the ISCOM matrix or complex particles may contain two or more saponin fractions.

[0302] In a particular composition, the sole saponin fraction in the first ISCOM matrix or ISCOM complex particles is fraction A, and the sole saponin fraction in the second ISCOM matrix or ISCOM complex particles is fraction C.

[0303] A preferred composition comprises a first ISCOM matrix containing fraction A and a second ISCOM matrix containing fraction C, where the fraction A ISCOM matrix constitutes about 70% by weight of the total saponin adjuvant and the fraction C ISCOM matrix constitutes about 30% by weight of the total saponin adjuvant. In another preferred composition, the fraction A ISCOM matrix constitutes about 85% by weight of the total saponin adjuvant and the fraction C ISCOM matrix constitutes about 15% by weight of the total saponin adjuvant. Thus, in a particular composition, the fraction A ISCOM matrix is ​​present in the range of about 70% to about 85% and the fraction C ISCOM matrix is ​​present in the range of about 15% to about 30% of the total weight of the saponin adjuvant in the composition. In several embodiments, the fraction A ISCOM matrix accounts for 50% to 96% by weight and the fraction C ISCOM matrix accounts for the remainder, respectively, of the combined weight of the fraction A ISCOM matrix and fraction C ISCOM in the adjuvant. In this specification, a particularly preferred composition referred to as MATRIX-M® contains fraction A ISCOM matrix at approximately 85% of the total weight of saponin adjuvants in the composition, and fraction C ISCOM matrix at approximately 15%. MATRIX-M® may be interchangeably referred to as Matrix-M1.

[0304] Exemplary QS-7 and QS-21 fractions, their preparation, and their use are described in U.S. Patents 5,057,540, 6,231,859, 6,352,697, 6,524,584, 6,846,489, 7,776,343, and 8,173,141, which are incorporated herein by reference.

[0305] In several embodiments, other adjuvants may be used additionally or as substitutes. The inclusion of any adjuvants described in Vogel et al., “A Compendium of Vaccine Adjuvants and Excipients (2nd Edition),” which is incorporated herein by reference in whole for all purposes, is assumed within the scope of this disclosure. Other adjuvants include complete Freund's adjuvant (a nonspecific stimulant of the immune response containing dead Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant. Other adjuvants include GMCSP, BCG, MDP compounds such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL), MF-59, and RIBI, which contains three components extracted from bacteria, namely MPL, trehalose dimycolic acid (TDM), and cell wall skeleton (CWS) in a 2% squalene / TWEEN® polysorbate 80 emulsion. In several embodiments, the adjuvant may be paucilamellar lipid vesicles, such as NOVASOMES®. NOVASOMES® are paucilamellar nonphospholipid vesicles in the range of about 100 nm to about 500 nm. These include BRIJ® alcohol ethoxylate 72, cholesterol, oleic acid, and squalene. NOVASOMES® has been shown to be an effective adjuvant (see U.S. Patents No. 5,629,021, 6,387,373, and 4,911,928).

[0306] Dosage and Administration In embodiments, this disclosure provides a method for inducing an immune response to one or more coronaviruses and / or influenza viruses. In embodiments, the response is to one or more of the SARS-CoV-2 virus, MERS, and SARS. In embodiments, the response is to heterologous SARS-CoV-2 strains. Non-limiting examples of heterologous SARS-CoV-2 strains include Cal.20C SARS-CoV-2 strain, P.1 SARS-CoV-2 strain, B.1.351 SARS-CoV-2 strain, and B.1.1.7 SARS-CoV-2 strain. The method comprises administering an immunologically effective amount of the immunogenic composition described herein to a subject. Advantageously, the proteins disclosed herein induce one or more particularly useful anti-coronavirus and / or anti-influenza responses.

[0307] In embodiments, the compositions described herein are administered together with an adjuvant. In embodiments, the compositions described herein are administered without an adjuvant. In some embodiments, the adjuvant may be bound to nanoparticles, for example, by non-covalent interactions. In other embodiments, the adjuvant is co-administered with the nanoparticles, but the adjuvant and the nanoparticles do not interact substantially.

[0308] In embodiments, the composition may be used for the prevention and / or treatment of one or more of the following: SARS-CoV-2 infection, heterologous SARS-CoV-2 strain infection, SARS infection, MERS infection, and influenza infection, or a combination thereof. Accordingly, this disclosure provides a method for inducing an immune response to one or more of the following: SARS-CoV-2 virus, heterologous SARS-CoV-2 virus, MERS, SARS, and influenza virus. The method comprises administering an immunologically effective amount of the composition described herein to a subject. Advantageously, the compositions disclosed herein induce particularly useful anti-coronavirus and / or anti-influenza responses.

[0309] In embodiments, the compositions described herein have efficacy against SARS-CoV-2 virus or heterologous SARS-CoV-2 strains of about 50% to about 99%, about 80% to about 99%, about 75% to about 99%, about 80% to about 95%, about 90% to about 98%, about 75% to about 95%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. In embodiments, the nanoparticles or CoV S polypeptides described herein have efficacy against Cal.20C SARS-CoV-2 strains of approximately 50% to approximately 99%, approximately 80% to approximately 99%, approximately 75% to approximately 99%, approximately 80% to approximately 95%, approximately 90% to approximately 98%, approximately 75% to approximately 95%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99%. In embodiments, the nanoparticles or CoV S polypeptides described herein have efficacy against P.1 SARS-CoV-2 strains of approximately 50% to approximately 99%, approximately 80% to approximately 99%, approximately 75% to approximately 99%, approximately 80% to approximately 95%, approximately 90% to approximately 98%, approximately 75% to approximately 95%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99%.In embodiments, the nanoparticles or CoV S polypeptides described herein have efficacy against strain B.1.351 SARS-CoV-2 of approximately 50% to approximately 99%, approximately 80% to approximately 99%, approximately 75% to approximately 99%, approximately 80% to approximately 95%, approximately 90% to approximately 98%, approximately 75% to approximately 95%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99%. In embodiments, the nanoparticles or CoV S polypeptides described herein have efficacy against the B.1.1.7 SARS-CoV-2 strain of approximately 50% to approximately 99%, approximately 80% to approximately 99%, approximately 75% to approximately 99%, approximately 80% to approximately 95%, approximately 90% to approximately 98%, approximately 75% to approximately 95%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99%.

[0310] In embodiments, the immunogenicity of the compositions described herein against influenza virus is determined by using an HAI assay or by measuring neutralizing antibodies. A method for performing an HAI assay is described in the following reference, incorporated herein by reference as a whole: Cowling et al. Clin Infect Dis. 2019;68(10):1713-1717. In embodiments, the immunogenicity of the nanoparticle influenza vaccine may be compared to a commercially available influenza vaccine composition. As used herein, “commercially available influenza vaccine composition” may be any influenza vaccine composition available for medical use. For example, the commercially available influenza vaccine composition may be formulated into a trivalent or tetravalent injectable formulation. In embodiments, the injectable formulation may contain an inactivated form of the virus. In embodiments, the commercially available influenza vaccine composition may be formulated for a nasal spray. In embodiments, the formulation for a nasal spray may contain an attenuated or weakened form of the virus. In embodiments, the compositions disclosed herein induce neutralizing antibodies that bind to influenza strains that have drifted (i.e., undergone minor mutations) against sequences used by viruses within the same subtype of influenza. In embodiments, one, two, three, four, or all of the strains used in the composition induce neutralizing antibodies against one drift strain, two drift strains, three drift strains, four drift strains, or five drift strains.

[0311] The compositions disclosed herein may be administered systemically, via mucosal, or transdermal routes, or directly to specific tissues. As used herein, the term “systemic administration” includes parenteral administration routes. Specifically, parenteral administration includes subcutaneous, intraperitoneal, intravenous, intra-arterial, intramuscular, or intrasternal injection, intravenous infusion techniques, or renal dialysis infusion techniques. Typically, systemic parenteral administration is intramuscular injection. As used herein, the term “mucosal administration” includes oral, intranasal, intravaginal, intrarectal, intratracheal, intestinal, and ophthalmic administration. Preferably, administration is intramuscular.

[0312] The composition may be administered in a single-dose schedule or a multi-dose schedule. Multi-dose administration may be used in a primary immunization schedule or a booster immunization schedule. In a multi-dose schedule, various doses may be administered via the same or different routes, e.g., parenteral prime and mucosal boost, mucosal prime and parenteral boost, etc. In some embodiments, the booster dose is administered approximately 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks after the previous dose. In some embodiments, the booster dose is administered 3 weeks after the previous dose. In some embodiments, the first dose is administered on day 0 and the booster dose is administered on day 21. In some embodiments, the first dose is administered on day 0 and the booster dose is administered on day 28.

[0313] In the embodiments, the dose measured in μg may be the total weight of the dose including the solute, the weight of the nanoparticles, or the weight of the protein in the nanoparticles (e.g., the weight of hemagglutinin or CoV S polypeptide). The dose is measured using either the A280 or ELISA protein concentration assay.

[0314] The dosage of CoV S polypeptide, including for pediatric administration, may range from approximately 1 μg to 25 μg, 3 μg to 25 μg, 5 μg to 25 μg, 5 μg to 50 μg, 1 μg to 300 μg, 90 μg to 270 μg, 100 μg to 160 μg, 110 μg to 150 μg, 120 μg to 140 μg, or 140 μg to 160 μg. In one embodiment, the dosage is approximately 120 μg and is administered with Alam. In another embodiment, the dosage is in the range of approximately 1 μg to 90 μg. In the embodiment, the dose of the CoV spike(S) polypeptide is approximately 1 μg, approximately 2 μg, approximately 3 μg, approximately 4 μg, approximately 5 μg, approximately 6 μg, approximately 7 μg, approximately 8 μg, approximately 9 μg, approximately 10 μg, approximately 11 μg, approximately 12 μg, approximately 13 μg, approximately 14 μg, approximately 15 μg, approximately 16 μg, approximately 17 μg, approximately 18 μg, approximately 19 μg, approximately 20 μg, approximately 21, approximately 22, approximately 23, approximately 24, approximately 25 μg, approximately 26 μg, approximately 27 μg, approximately 28 μg, approximately 29 μg, approximately 30 μg, approximately 4 The doses are 0 μg, approximately 50, approximately 60, approximately 70, approximately 80, approximately 90, approximately 100 μg, approximately 110 μg, approximately 120 μg, approximately 130 μg, approximately 140 μg, approximately 150 μg, approximately 160 μg, approximately 170 μg, approximately 180 μg, approximately 190 μg, approximately 200 μg, approximately 210 μg, approximately 220 μg, approximately 230 μg, approximately 240 μg, approximately 250 μg, approximately 260 μg, approximately 270 μg, approximately 280 μg, approximately 290 μg, or approximately 300 μg, and include all values ​​and ranges in between. In one embodiment, the dose of the CoV S polypeptide is approximately 3 μg. In another embodiment, the dose of the CoV S polypeptide is approximately 5 μg. In yet another embodiment, the dose of the CoV S polypeptide is approximately 25 μg. In one embodiment, the dose of the CoV S polypeptide is approximately 20 μg. In another embodiment, the dose of the CoV S polypeptide is approximately 35 μg.

[0315] The total amount of hemagglutinin in the immunogenic composition may range from about 25 μg to about 200 μg, about 30 μg to about 150 μg, about 50 μg to about 100 μg, about 45 μg to about 180 μg, about 60 μg to about 190 μg, or about 100 μg to about 200 μg. In certain embodiments, the amount of influenza HA protein in the immunogenic composition may range from about 5 μg to about 80 μg per strain, about 10 μg to about 75 μg per strain, about 15 μg to about 70 μg per strain, about 20 μg to about 65 μg per strain, about 25 μg to about 60 μg per strain, about 30 μg to about 55 μg per strain, about 35 μg to about 50 μg per strain, or about 15 μg to about 60 μg per strain. The dose per strain, for example, 10 μg per strain, refers to the dose of hemagglutinin derived from a specific influenza strain. In the embodiment, subjects are administered an immunogenic composition containing approximately 5 μg to approximately 100 μg of hemagglutinin per strain.For example, in the embodiment, the composition is approximately 5 μg, 6 μg, 7 μg, 7.5 μg, 8 μg, 9 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 16 μg, 17 μg, 18 μg, 19 μg, 20 μg, 21, 22, 23, 24, 25 μg, 26 μg, and 27 μg per strain. g, about 28μg, about 29μg, about 30μg, about 31μg, about 32μg, about 33μg, about 34μg, about 35μg, about 36μg, about 37μg, about 38μg, about 39μg, about 4 0μg, about 41μg, about 42μg, about 43μg, about 44μg, about 45μg, about 46μg, about 47μg, about 48μg, about 49μg, about 50μg, about 51μg, about 52μg, Approximately 53μg, approximately 54μg, approximately 55μg, approximately 56μg, approximately 57μg, approximately 58μg, approximately 59μg, approximately 60μg, approximately 61μg, approximately 62μg, approximately 63μg, approximately 64μg, approximately 65 μg, approximately 66 μg, approximately 67 μg, approximately 68 μg, approximately 69 μg, approximately 70 μg, approximately 71 μg, approximately 72 μg, approximately 73 μg, approximately 74 μg, approximately 75 μg, approximately 76 μg, approximately 77 μg, approximately The composition contains 78 μg, approximately 79 μg, approximately 80 μg, approximately 81 μg, approximately 82 μg, approximately 83 μg, approximately 84 μg, approximately 85 μg, approximately 86 μg, approximately 87 μg, approximately 88 μg, approximately 89 μg, approximately 90 μg, approximately 91 μg, approximately 92 μg, approximately 93 μg, approximately 94 μg, approximately 95 μg, approximately 96 μg, approximately 97 μg, approximately 98 μg, approximately 99 μg, or approximately 100 μg of hemagglutinin. In embodiments, the composition contains approximately 24 μg to approximately 40 μg of hemagglutinin per strain. In embodiments, the composition contains approximately 24 μg to approximately 75 μg of hemagglutinin per strain. In embodiments, the composition contains approximately 30 μg of hemagglutinin per strain. In embodiments, the composition contains approximately 60 μg of hemagglutinin per strain.

[0316] In one embodiment, the patient is administered an immunogenic composition containing approximately 5 to 60 μg of hemagglutinin and approximately 2.5 to 22.5 μg of CoV S polypeptide per strain. In another embodiment, the composition contains approximately 24 to 40 μg of hemagglutinin and 5 to approximately 25 μg of CoV S polypeptide per strain. In yet another embodiment, the immunogenic composition contains 5, 10, 35, or 60 μg of hemagglutinin per strain. In yet another embodiment, the immunogenic composition contains 2.5, 7.5, or 22.5 μg of CoV S polypeptide. In yet another embodiment, the immunogenic composition contains 30 μg to approximately 54 μg of hemagglutinin per strain. In yet another embodiment, the immunogenic composition contains 13 μg to approximately 25 μg of CoV S polypeptide. In yet another embodiment, the immunogenic composition contains hemagglutinin derived from three or four strains of influenza. In the embodiments, the immunogenic composition comprises about 40 μg of a saponin adjuvant, for example, MATRIX-M®. In the embodiments, the immunogenic composition comprises about 50 μg or 75 μg of a saponin adjuvant, for example, MATRIX-M®. In the embodiments, the doses of hemagglutinin and CoV S polypeptide per strain are found in Table 1H. In the embodiments, the patient is administered 30 μg of hemagglutinin and 25 μg of CoV S polypeptide per strain. In the embodiments, the patient is administered 60 μg of hemagglutinin and 35 μg of CoV S polypeptide per strain.

[0317] In the embodiment, the ratio of hemagglutinin to CoV S polypeptide per strain in the immunogenic composition is about 1:1 to about 5:1. In the embodiment, the ratio of hemagglutinin to CoV S polypeptide per strain in the immunogenic composition is about 1:1 to about 3:1. In the embodiment, the ratio of hemagglutinin to CoV S polypeptide per strain in the immunogenic composition is about 1.3:1 to about 3:1. In the embodiment, the ratio of hemagglutinin to CoV S polypeptide per strain in the immunogenic composition is about 1.3:1 to about 3:1. The ratios of S-polypeptides are approximately 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3.0:1, and 3. The ratios are approximately 0.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4.0:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, or 5:1.

[0318] In the embodiments, the patient is administered a boost dose of the immunogenic composition 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61 days after the initial dose of the immunogenic composition. In the embodiments, the patient is administered a boost dose of the immunogenic composition 56 days after the initial dose of the immunogenic composition. In the embodiments, the patient is administered a boost dose of the immunogenic composition 56 days (+5 days) after the initial dose of the immunogenic composition. In the embodiments, the boost dose contains the same amount of strain-specific hemagglutinin and CoV S polypeptide as the initial dose. In the embodiments, the boost dose contains a different amount of strain-specific hemagglutinin, CoV S polypeptide, or a combination thereof than the initial dose. In the embodiments, the amount of strain-specific hemagglutinin and CoV S polypeptide in the boost dose of the immunogenic composition is selected from Table 1H. [Table 1H-1]

Table 1H-2

[0319] In one embodiment, the patient is administered a first immunogenic composition containing about 5 to 60 μg of hemagglutinin per strain and a second immunogenic composition containing about 2.5 to 22.5 μg of CoV S polypeptide. In another embodiment, the patient is administered a first immunogenic composition containing about 20 to 60 μg of hemagglutinin per strain and a second immunogenic composition containing about 20 to 40 μg of CoV S polypeptide. In yet another embodiment, the patient is administered a first immunogenic composition containing 30 μg of hemagglutinin per strain and a second immunogenic composition containing 25 μg of CoV S polypeptide. In yet another embodiment, the patient is administered a first immunogenic composition containing 60 μg of hemagglutinin per strain and a second immunogenic composition containing 35 μg of CoV S polypeptide. In the embodiments, the first immunogenic composition contains about 5, about 10, about 35, or about 60 μg of hemagglutinin per strain. In the embodiments, the second immunogenic composition contains about 2.5 μg, about 7.5 μg, about 22.5 μg, or about 25 μg of CoV S polypeptide. In the embodiments, the first immunogenic composition contains hemagglutinin derived from three or four strains of influenza. In the embodiments, the amount of hemagglutinin per strain in the first immunogenic composition and the amount of CoV S polypeptide in the second immunogenic composition are shown in Table 1H. In the embodiments, the first immunogenic composition, the second immunogenic composition, or both, contain about 50 μg of a saponin adjuvant, for example, MATRIX-M®. In embodiments, the patient is administered a boost dose of the first immunogenic composition, the second immunogenic composition, or both, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61 days after the initial dose of the first immunogenic composition, the second immunogenic composition, or both. In embodiments, the patient is administered a boost dose of the immunogenic composition 56 days after the initial dose of the first immunogenic composition, the second immunogenic composition, or both. In embodiments, the patient is administered a boost dose of the immunogenic composition 56 days (+5 days) after the initial dose of the first immunogenic composition, the second immunogenic composition, or both. In embodiments, the boost dose contains the same amount of strain-specific hemagglutinin and CoV S polypeptide as the initial dose.In embodiments, the boost dose includes a different amount of strain-specific hemagglutinin, CoV S polypeptide, or a combination thereof, than the initial dose. In embodiments, the amounts of strain-specific hemagglutinin and CoV S polypeptide in the boost dose of the first or second immunogenic composition are selected from Table 1H. Certain populations may be administered with or without an adjuvant. In certain embodiments, the composition may not contain the adjuvant to be added. In such circumstances, the dose may be increased by approximately 10%.

[0320] In one embodiment, the patient is administered a dose of approximately 24 to 40 μg of hemagglutinin and more than 20 μg of CoV S polypeptide per strain. In another embodiment, the patient is administered a dose of approximately 24 to 40 μg of hemagglutinin and more than 25 μg of CoV S polypeptide per strain.

[0321] In the embodiment, the dose of the adjuvant is approximately 1 μg to approximately 100 μg, for example, approximately 1 μg, approximately 2 μg, approximately 3 μg, approximately 4 μg, approximately 5 μg, approximately 6 μg, approximately 7 μg, approximately 8 μg, approximately 9 μg, approximately 10 μg, approximately 11 μg, approximately 12 μg, approximately 13 μg, approximately 14 μg, approximately 15 μg, approximately 16 μg, approximately 17 μg, approximately 18 μg, approximately 19 μg, approximately 20 μg, approximately 21, approximately 22, approximately 23, About 24, about 25μg, about 26μg, about 27μg, about 28μg, about 29μg, about 30μg, about 31μg, about 32μg, about 33μg, about 34μg, about 35μg, about 36μg, about 37 μg, approximately 38 μg, approximately 39 μg, approximately 40 μg, approximately 41 μg, approximately 42 μg, approximately 43 μg, approximately 44 μg, approximately 45 μg, approximately 46 μg, approximately 47 μg, approximately 48 μg, approximately 49 μg, approximately 50 μg g, about 51μg, about 52μg, about 53μg, about 54μg, about 55μg, about 56μg, about 57μg, about 58μg, about 59μg, about 60μg, about 61μg, about 62μg, about 63μg , about 64μg, about 65μg, about 66μg, about 67μg, about 68μg, about 69μg, about 70μg, about 71μg, about 72μg, about 73μg, about 74μg, about 75μg, about 76μg, The adjuvant is approximately 77 μg, 78 μg, 79 μg, 80 μg, 81 μg, 82 μg, 83 μg, 84 μg, 85 μg, 86 μg, 87 μg, 88 μg, 89 μg, 90 μg, 91 μg, 92 μg, 93 μg, 94 μg, 95 μg, 96 μg, 97 μg, 98 μg, 99 μg, or 100 μg. In embodiments, the dose of the adjuvant is approximately 50 μg. In embodiments, the adjuvant is a saponin adjuvant, for example, MATRIX-M (trademark).

[0322] In several embodiments, the dose is administered in volumes of approximately 0.1 mL to approximately 1.5 mL, for example, approximately 0.1 mL, approximately 0.2 mL, approximately 0.25 mL, approximately 0.3 mL, approximately 0.4 mL, approximately 0.5 mL, approximately 0.6 mL, approximately 0.7 mL, approximately 0.8 mL, approximately 0.9 mL, approximately 1.0 mL, approximately 1.1 mL, approximately 1.2 mL, approximately 1.3 mL, approximately 1.4 mL, or approximately 1.5 mL. In several embodiments, the dose is administered in a volume of 0.25 mL. In several embodiments, the dose is administered in a volume of 0.5 mL. In several embodiments, the dose is administered in a volume of 0.6 mL.

[0323] In embodiments, the dose may contain CoV S polypeptide or hemagglutinin at concentrations of approximately 1 μg / mL to approximately 50 μg / mL, 10 μg / mL to approximately 100 μg / mL, approximately 10 μg / mL to approximately 50 μg / mL, approximately 175 μg / mL to approximately 325 μg / mL, approximately 200 μg / mL to approximately 300 μg / mL, approximately 220 μg / mL to approximately 280 μg / mL, or approximately 240 μg / mL to approximately 260 μg / mL.

[0324] In embodiments, the immunogenic compositions described herein are administered in combination with further immunogenic compositions. In embodiments, the further immunogenic compositions induce an immune response to SARS-CoV-2. In embodiments, the further immunogenic compositions are administered from the immunogenic compositions of this disclosure for approximately 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, and 20 hours. It is administered within approximately 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or 31 days. In embodiments, the further composition is administered together with a first dose of the composition described herein. In embodiments, the further composition is administered together with a boost dose of a composition comprising CoV S polypeptide or nanoparticles, surfactant core nanoparticles, HaSMaN, or a combination thereof. In embodiments, the further composition is administered together with a first immunogenic composition comprising (a)(i) a CoV S glycoprotein in the form of surfactant core nanoparticles, wherein the surfactant is a nonionic surfactant, and (ii) a pharmaceutically acceptable buffer; and (b)(i) at least three HA glycoproteins, wherein each HA glycoprotein is derived from a different influenza strain, and (ii) a pharmaceutically acceptable buffer. In embodiments, the further composition is administered together with an initial dose of the first immunogenic composition and / or the second immunogenic composition. In embodiments, the further composition is administered together with a boost dose of the first immunogenic composition and / or the second immunogenic composition.

[0325] In embodiments, disclosed herein are methods for inducing an immune response to one or more coronaviruses and / or influenza viruses, the methods comprising administering an immunogenic composition described herein. In embodiments, disclosed herein are methods for inducing an immune response to one or more coronaviruses and / or influenza viruses, the methods comprising (a) administering a first immunogenic composition comprising (i) a CoV S glycoprotein in the form of surfactant core nanoparticles, wherein the surfactant is a nonionic surfactant, and (ii) a pharmaceutically acceptable buffer; and (b) administering a second immunogenic composition comprising (i) at least three HA glycoproteins, each HA glycoprotein derived from a different influenza strain, and (ii) a pharmaceutically acceptable buffer. In this embodiment, the first immunogenic composition is administered approximately 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, and 1 day prior to the administration of the second immunogenic composition. In embodiments, the first immunogenic composition is administered approximately 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, and 1 day after the second immunogenic composition. Typically, the first and second immunogenic compositions are administered simultaneously (i.e., within 15 minutes of each other). In embodiments, the first immunogenic composition is administered intramuscularly. In embodiments, the second immunogenic composition is administered intramuscularly. In embodiments, the second immunogenic composition is administered intranasally. In this embodiment, the first immunogenic composition is administered intramuscularly, and the second immunogenic composition is administered intramuscularly.In one embodiment, the first immunogenic composition is administered intramuscularly, and the second immunogenic composition is administered intranasally. In another embodiment, the first and second immunogenic compositions are administered intramuscularly to the same arm. In yet another embodiment, the first and second immunogenic compositions are administered intramuscularly to different arms.

[0326] In the embodiment, the further immunogenic composition comprises mRNA encoding the SARS-CoV-2 spike glycoprotein, plasmid DNA encoding the SARS-CoV-2 spike glycoprotein, a viral vector encoding the SARS-CoV-2 spike glycoprotein, or an inactivated SARS-CoV-2 virus.

[0327] In an embodiment, the further immunogenic composition comprises mRNA encoding a CoV S polypeptide. In an embodiment, the mRNA encodes a CoV S polypeptide having proline substitutions at positions 986 and 987 of SEQ ID NO: 1. In an embodiment, the mRNA encodes a CoV S polypeptide having intact furin cleavage sites. In an embodiment, the mRNA encodes a CoV S polypeptide having proline substitutions and intact furin cleavage sites at positions 986 and 987 of SEQ ID NO: 1. In an embodiment, the mRNA encodes a CoV S polypeptide having proline substitutions and inactive furin cleavage sites at positions 986 and 987 of SEQ ID NO: 1. In an embodiment, the mRNA encodes a CoV S polypeptide having the amino acid sequence of SEQ ID NO: 87. In an embodiment, the mRNA encoding the CoV S polypeptide is encapsulated in lipid nanoparticles. Exemplary immunogenic compositions containing mRNA encoding the CoV S polypeptide are described in Jackson et al. N.Eng.J.Med.2020. An mRNA Vaccine against SARS-CoV-2 - preliminary report, which is incorporated herein by reference in whole. In embodiments, the composition containing mRNA encoding the CoV S polypeptide is administered in doses of 25 μg, 100 μg, or 250 μg.

[0328] In an embodiment, the further immunogenic composition comprises an adenovirus vector encoding a CoV S polypeptide. In an embodiment, the AAV vector encodes a wild-type CoV S polypeptide. In an embodiment, the AAV vector encodes a CoV S polypeptide having a proline substitution and an intact furin cleavage site at positions 986 and 987 of SEQ ID NO: 1. In an embodiment, the AAV vector encodes a CoV S polypeptide having a proline substitution and an inactive furin cleavage site at positions 986 and 987 of SEQ ID NO: 1. In an embodiment, the AAV vector encodes a CoV S polypeptide having the amino acid sequence of SEQ ID NO: 87. The following publications describe immunogenic compositions containing adenovirus vectors encoding CoV S polypeptides, each of which is incorporated herein by reference as a whole: van Doremalen N. et al. A single dose of ChAdOx1 MERS provides protective immunity in rhesus macaques. Science Advances, 2020; van Doremalen N. et al. ChAdOx1 nCoV-19 vaccination prevents SARS-CoV-2 pneumonia in rhesus macaques. bioRxiv, (2020).

[0329] In an embodiment, the further immunogenic composition comprises deoxyribonucleic acid (DNA). In an embodiment, the further immunogenic composition comprises plasmid DNA. In an embodiment, the plasmid DNA encodes a CoV S polypeptide. In an embodiment, the DNA encodes a CoV S polypeptide having a proline substitution and intact furin cleavage sites at positions 986 and 987 of SEQ ID NO: 1. In an embodiment, the DNA encodes a CoV S polypeptide having a proline substitution and inactive furin cleavage sites at positions 986 and 987 of SEQ ID NO: 1. In an embodiment, the DNA encodes a CoV S polypeptide having the amino acid sequence of SEQ ID NO: 87.

[0330] In this embodiment, the further immunogenic composition includes an inactivated viral vaccine.

[0331] In the embodiments, the immunogenic composition comprising the CoV S polypeptide or nanoparticles, surfactant core nanoparticles, and HaSMaN is administered to patients who have or have previously had a confirmed infection caused by SARS-CoV-2 or a heterologous SARS-CoV-2 strain. Infection caused by SARS-CoV-2 or a heterologous SARS-CoV-2 strain can be confirmed by nucleic acid amplification testing (e.g., polymerase chain reaction) or serological testing (e.g., testing for antibodies against SARS-CoV-2 viral antigen). In the embodiments, the composition comprising the CoV S polypeptide or nanoparticles, surfactant core nanoparticles, HaSMaN, or a combination thereof is administered to the patient at least about 3 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, and at least about 4 weeks after the patient is diagnosed with COVID-19. In embodiments, compositions comprising the CoV S polypeptide or nanoparticles, surfactant core nanoparticles, HaSMaN, or combinations thereof are administered to the patient 1 week to 1 year after the patient's diagnosis of COVID-19, for example, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 1 year after the patient's diagnosis of COVID-19. In embodiments, compositions comprising the CoV S polypeptide or nanoparticles, surfactant core nanoparticles, HaSMaN, or combinations thereof are administered to the patient 1 week to 20 years after the patient's diagnosis of COVID-19, for example, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, about 12 years, about 13 years, about 14 years, about 15 years, about 16 years, about 17 years, about 18 years, about 19 years, or about 20 years later.

[0332] In embodiments, a composition comprising the CoV S polypeptide or nanoparticles, surfactant core nanoparticles, HaSMaN, or a combination thereof is administered after the patient has been administered the first immunogenic composition. Non-limiting examples of the first immunogenic composition include SARS-CoV-2 spike glycoprotein, mRNA encoding SARS-CoV-2 spike glycoprotein, plasmid DNA encoding SARS-CoV-2 spike glycoprotein, a viral vector encoding SARS-CoV-2 spike glycoprotein, or an inactivated SARS-CoV-2 virus. In embodiments, the CoV S polypeptide or nanoparticles comprising the same are administered about 1 week to about 1 year, about 1 week to 1 month, about 3 weeks to 4 weeks, about 1 week to 5 years, about 1 year to about 5 years, about 1 year to about 3 years, about 3 years to about 5 years, about 5 years to about 10 years, about 1 year to about 10 years, or about 1 year to about 2 years after administration of the first immunogenic composition. In embodiments, the CoV S polypeptide or nanoparticles containing the same are administered about 1 week to about 1 year after administration of the first immunogenic composition, for example, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 1 year after administration of the first immunogenic composition.

[0333] In embodiments, compositions comprising the CoV S polypeptide or nanoparticles containing the CoV S polypeptide, surfactant core nanoparticles, HaSMaN, or combinations thereof are useful in preparing immunogenic compositions for stimulating an immune response that confers immunity or substantial immunity to one or more of MERS, SARS, SARS-CoV-2, and heterologous SARS-CoV-2 strains. Both mucosal and cellular immunity can contribute to immunity against infections and diseases. Antibodies secreted locally in the upper respiratory tract are a major factor in resistance to spontaneous infection. Secretory immunoglobulin A (sIgA) is involved in the protection of the upper respiratory tract, and serum IgG is involved in the protection of the lower respiratory tract. The immune response induced by infection prevents reinfection with the same virus or antigenically similar virus strains. Antibodies produced in the host after immunization with the nanoparticles disclosed herein can also be administered elsewhere, thereby resulting in passive administration to the subject.

[0334] In the embodiment, a composition comprising the CoV S polypeptide or nanoparticles, surfactant core nanoparticles, HaSMaN, or a combination thereof induces cross-neutralizing antibodies against the SARS-CoV-2 virus containing an S protein having one or more modifications selected from the following: (a) Deletion of one or more amino acids in an NTD, wherein the one or more amino acids are selected from the group consisting of amino acids 56, 57, 131, 132, 144, 145, 228, 229, 230, 231, 234, 235, 236, 237, 238, 239, 240, or combinations thereof. (b) A mutation in one or more amino acids of an NTD, wherein the one or more mutations are selected from the group consisting of amino acids 5, 6, 7, 13, 39, 51, 53, 54, 56, 57, 62, 63, 67, 82, 125, 129, 131, 132, 133, 139, 143, 144, 145, 177, 200, 201, 202, 209, 229, 233, 240, 245, or combinations thereof. (c) A mutation in one or more amino acids of RBD, wherein the one or more mutations are selected from the group consisting of amino acids 333, 404, 419, 426, 439, 440, 464, 465, 471, 477, 481, 488 and combinations thereof. (d) A mutation in one or more amino acids of SD1 / 2, where the one or more amino acids are selected from the group consisting of 557, 600, 601, 642, 664, 668 and combinations thereof. (e) Inactive furin cleavage sites (corresponding to one or more mutations in amino acids 669-672), (f) Deletion of one or more amino acids in the S2 subunit, wherein the amino acids are selected from the group consisting of 676-685, 676-702, 702-711, 775-793, 806-815, and combinations thereof. (g) A mutation in one or more amino acids of the S2 subunit, where the amino acid is selected from the group consisting of 688, 703, 846, 875, 937, 969, 973, 974, 1014, 1058, 1105, and 1163, and combinations thereof. (h) Deletion of one or more amino acids (amino acids 1201-1260) from TMCT, where the amino acids of the CoV S glycoprotein are numbered relative to SEQ ID NO: 2.

[0335] In embodiments, compositions comprising the CoV S polypeptide or nanoparticles containing the CoV S polypeptide, surfactant core nanoparticles, HaSMaN, or combinations thereof induce cross-neutralizing antibodies against the SARS-CoV-2 virus containing an S protein having one or more modifications selected from the following: deletion of amino acid 56, deletion of amino acid 57, deletion of amino acid 131, N488Y, A557D, D601G, P668H, T703I, S969A, D1105H, N426K, and Y440F, where the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2.

[0336] In embodiments, compositions comprising the CoV S polypeptide or nanoparticles containing the CoV S polypeptide, surfactant core nanoparticles, HaSMaN, or combinations thereof induce cross-neutralizing antibodies against the SARS-CoV-2 virus containing an S protein having one or more modifications selected from the following: deletion of amino acid 56, deletion of amino acid 57, deletion of amino acid 131, N488Y, A557D, D601G, P668H, T703I, S969A, and D1105H, where the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2.

[0337] In the embodiment, a composition comprising the CoV S polypeptide or nanoparticles containing the CoV S polypeptide, surfactant core nanoparticles, HaSMaN, or a combination thereof induces cross-neutralizing antibodies against the SARS-CoV-2 virus containing an S protein having one or more modifications selected from the following: D67A, D202G, L229H, K404N, E471K, N488Y, D601G, and A688V, where the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2.

[0338] In embodiments, compositions comprising the CoV S polypeptide or nanoparticles containing the CoV S polypeptide, surfactant core nanoparticles, HaSMaN, or combinations thereof induce cross-neutralizing antibodies against the SARS-CoV-2 virus containing an S protein having one or more modifications selected from the following: deletion of amino acids 229-231, D67A, D202G, K404N, E471K, N488Y, D601G, and A688V, where the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2.

[0339] In embodiments, compositions comprising the CoV S polypeptide or nanoparticles containing the CoV S polypeptide, surfactant core nanoparticles, HaSMaN, or combinations thereof induce cross-neutralizing antibodies against the SARS-CoV-2 virus containing an S protein having one or more modifications selected from the following: deletion of amino acids 229-231, L5F, D67A, D202G, K404N, E471K, N488Y, D601G, and A688V, where the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2.

[0340] In the embodiment, a composition comprising the CoV S polypeptide or nanoparticles containing the CoV S polypeptide, surfactant core nanoparticles, HaSMaN, or a combination thereof induces cross-neutralizing antibodies against the SARS-CoV-2 virus containing an S protein having one or more modifications selected from the following: L5F, T7N, P13S, D125Y, R177S, K404T, E471K, N488Y, D601G, H642Y, T1014I, and V1163F, where the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2.

[0341] In the embodiment, a composition comprising the CoV S polypeptide or nanoparticles containing the CoV S polypeptide, surfactant core nanoparticles, HaSMaN, or a combination thereof induces cross-neutralizing antibodies against the SARS-CoV-2 virus having an S protein comprising one or more modifications selected from the following: W139C and L439R, where the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In the embodiment, the CoV S protein comprising the modifications of W139C and L439R is expressed together with a signal peptide having the amino acid sequence of SEQ ID NO: 117 or SEQ ID NO: 5. In the embodiment, the CoV S protein or nanoparticles containing the CoV S protein induce cross-neutralizing antibodies against the SARS-CoV-2 virus having one or more mutations selected from the following: D601G, W139C, and L439R, where the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In the embodiment, the CoV S protein or nanoparticles containing the modifications of D601G, W139C, and L439R are expressed together with a signal peptide having the amino acid sequence of SEQ ID NO: 117 or SEQ ID NO: 5.

[0342] In embodiments, compositions comprising the CoV S polypeptide or nanoparticles containing the CoV S polypeptide, surfactant core nanoparticles, HaSMaN, or combinations thereof induce cross-neutralizing antibodies against SARS-CoV-2 virus having one or more modifications selected from the following: D601G, L5F, D67A, D202G, deletion of amino acids 229-231, R233I, K404N, E471K, N488Y, and A688V, where the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In embodiments, compositions comprising the CoV S polypeptide or nanoparticles, surfactant core nanoparticles, HaSMaN, or combinations thereof induce cross-neutralizing antibodies against SARS-CoV-2 virus having one or more modifications selected from the following: L5F, D67A, D202G, deletion of amino acids 229-231, R233I, K404N, E471K, N488Y, and A688V, where the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2.

[0343] In embodiments, compositions comprising the CoV S polypeptide or nanoparticles containing the CoV S polypeptide described herein, surfactant core nanoparticles, HaSMaN, or combinations thereof are used to contain the SARS-CoV-2 virus or heterologous SARS-CoV-2 strains (e.g., B.1.1.7 SARS-CoV-2 strain, B.1.351 SARS-CoV-2 strain, P.1 SARS-CoV-2 strain, B.1.617.2 SARS-CoV-2 strain, B.1.525 SARS-CoV-2 strain, B.1.526 SARS-CoV-2 strain, B.1.617.1 SARS-CoV-2 strain, C.37 SARS-CoV-2 strain, B.1.621 SARS-CoV-2 strain, B.1.1.529 SARS-CoV-2 strain, or Cal.20C In the prevention of COVID-19 from SARS-CoV-2 strain, from the last dose of the nanoparticles or CoV S polypeptide described herein, up to approximately 1 month, up to approximately 2 months, up to approximately 2.5 months, up to approximately 3 months, up to approximately 3.5 months, up to approximately 4 months, up to approximately 4.5 months, up to approximately 5 months, up to approximately 5.5 months, up to approximately 6 months, up to approximately 6.5 months, up to approximately 7 months, up to approximately 7.5 months, up to approximately 8 months, up to approximately 8.5 months, up to approximately 9 months, up to approximately 9.5 months, up to approximately 10 months, up to approximately 10.5 months, up to approximately 11 months, up to approximately 11.5 months, up to approximately 12 months, up to approximately 12.5 months, up to approximately 13 months, up to approximately 13.5 months, up to approximately 14 months, up to approximately 14.5 months. Months, up to approximately 15 months, up to approximately 15.5 months, up to approximately 16 months, up to approximately 16.5 months, up to approximately 17 months, up to approximately 17.5 months, up to approximately 18 months, up to approximately 18.5 months, up to approximately 19 months, up to approximately 19.5 months, up to approximately 20 months, up to approximately 20.5 months, up to approximately 21 months, up to approximately 21.5 months, up to approximately 22 months, up to approximately 22.5 months, up to approximately 23 months, up to approximately 23.5 months, up to approximately 24 months, up to approximately 2.1 years, up to approximately 2.2 years, up to approximately 2.3 years, up to approximately 2.4 years, up to approximately 2.5 years, up to approximately 2.6 years, up to approximately 2.7 years, up to approximately 2.8 years, up to approximately 2.It has efficacy of approximately 50% to approximately 99%, approximately 80% to approximately 99%, approximately 75% to approximately 99%, approximately 80% to approximately 95%, approximately 90% to approximately 98%, approximately 75% to approximately 95%, approximately 80% to approximately 90%, approximately 85% to approximately 95%, approximately 80% to approximately 95%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% over a period of 9 years, up to approximately 3 years, or longer. In this embodiment, the COVID-19 is moderate COVID-19. In this embodiment, the COVID-19 is severe COVID-19. In this embodiment, the COVID-19 is asymptomatic COVID-19.

[0344] In embodiments, this disclosure provides a method for producing one or more high-affinity anti-MERS-CoV, anti-SARS-CoV, anti-SARS-CoV-2, or anti-influenza virus antibodies. The high-affinity antibodies produced by immunization with nanoparticles disclosed herein are produced by administering an immunogenic composition comprising CoV S polypeptide or nanoparticles, surfactant core nanoparticles, HaSMaN, or a combination thereof to an animal, collecting serum and / or plasma from the animal, and purifying antibodies from the serum and / or plasma. In one embodiment, the animal is a human. In several embodiments, the animal is a chicken, mouse, guinea pig, rat, rabbit, goat, human, horse, sheep, or cattle. In one embodiment, the animal is a Bovidae or Equidae. In another embodiment, the Bovidae or Equidae is transgenic. In further embodiments, the transgenic Bovidae or Equidae produces human antibodies. In several embodiments, the animal produces monoclonal antibodies. In several embodiments, the animal produces polyclonal antibodies. In one embodiment, the method further comprises the administration of an adjuvant or immunostimulatory compound. In further embodiments, the purified high-affinity antibody is administered to a human subject. In one embodiment, the human subject is at risk of infection with one or more of the following viruses: MERS, SARS, SARS-CoV-2, and influenza.

[0345] In some embodiments, this disclosure provides formulation (i.e., pre-filled syringe or premix) strategies for immunogenic compositions containing nanoparticles. A typical vaccine administration strategy currently in use is the bedside mix formulation, where the vaccine composition and adjuvant are stored separately and mixed before administration. Premix, formulation, or pre-filled syringe strategies for vaccines are less common due to concerns about the stability of antigens (e.g., hemagglutinin and CoV S polypeptides) and their subsequent immunogenic performance. This disclosure provides immunogenic compositions that can be pre-mixed and stored in advance. The disclosed vaccination strategies and formulations can improve the efficiency of vaccination while maintaining overall safety and immunogenicity, and can reduce the risk of bedside mixing errors.

[0346] Various containers, including syringes and plastic ampoules for single-dose administration, can be used to store and transport premix formulations. In some cases, plastic ampoules can be manufactured using blow-fill-seal manufacturing techniques or methods. Generally, blow-fill-seal (BFS) manufacturing methods involve extruding a plastic material (e.g., resin) to form a parison, then placing it in a mold and cutting it to a predetermined size. The plastic is then inflated using a filling needle or mandrel, thereby obtaining a hollow ampoule that substantially conforms to the shape of the mold. Once inflated, the desired volume of liquid can be injected into the ampoule, the filling needle or mandrel can be removed, and the ampoule can be sealed. Thus, BFS can be an automated process that can be carried out in a sterile environment without direct human intervention.

[0347] In some cases, BFS-manufactured ampoules can be particularly well-suited to the pharmaceutical industry because they allow for the aseptic production of sterile ampoules containing the desired liquid. However, BFS technology is not compatible with all pharmaceutical liquids, products, etc. For example, some known BFS manufacturing methods involve placing the liquid or product into the ampoule while the plastic is still relatively hot, which can have adverse effects on temperature-sensitive liquids and / or products, such as vaccines and biologics. However, advances in low-temperature BFS technology have increased the variety of suitable products and liquids, making it possible to contain some vaccines, biologics, and / or other temperature-sensitive pharmaceuticals in BFS ampoules.

[0348] In some cases, a BFS ampoule may have a size, shape, and / or configuration that is at least partially based on a desired use and / or desired medicinal liquid or dosage, in which the ampoule is configured to contain it. For example, some known BFS ampoules may have a puncture head, a threaded head, a head with a male or female Luer connector, etc. Some known BFS ampoules may have a size and / or shape based on the volume or dosage of liquid configured to be placed therein. In addition, some known BFS ampoules may be manufactured as a series of multiple temporarily connected ampoules, which can improve manufacturing, packaging, and / or storage efficiency, etc.

[0349] In several embodiments, the immunogenic compositions described herein are provided in pre-filled syringes. When the immunogenic compositions are prepared in pre-filled syringes, the antigen and adjuvant are combined before administration. In embodiments, the pre-filled syringe contains hemagglutinin and CoV S polypeptide. In embodiments, the pre-filled syringe contains hemagglutinin but does not contain CoV S polypeptide. In embodiments, the pre-filled syringe contains CoV S polypeptide but does not contain hemagglutinin.

[0350] In one embodiment, the subject is administered an immunogenic composition from a pre-filled syringe. In another embodiment, the subject is administered an immunogenic composition containing both hemagglutinin and CoV S polypeptide in a single pre-filled syringe. In yet another embodiment, the subject is administered an immunogenic composition containing CoV S polypeptide but not hemagglutinin from a pre-filled syringe. In yet another embodiment, the subject is administered an immunogenic composition containing hemagglutinin but not CoV S polypeptide from a pre-filled syringe.

[0351] All patents, patent applications, references, and journal articles cited herein are expressly incorporated herein by reference in their entirety for any purpose. [Examples]

[0352] Example 1 Expression and purification of coronavirus spike (S) polypeptide nanoparticles CoV spike polypeptides having amino acid sequences corresponding to the natural coronavirus spike (S) polypeptides (SEQ ID NOs. 1 and 2) and SEQ ID NOs. 3, 4, 38, 41, 44, 48, 51, 54, 58, 61, 63, 65, 67, 73, 75, 78, 79, 82, 83, 85, 87, 106, 108, 89, 112-115, 132, 133, 114, 138, 141, 144, 147, 151, 153, 156, and 158 were expressed in a baculovirus expression system, and recombinant plaques expressing the coronavirus spike (S) polypeptide were collected and identified. In all cases, the signal peptide was SEQ ID NO. 5. Figures 2 and 4 show the successful purification of CoV spike polypeptides BV2364, BV2365, BV2366, BV2367, BV2368, BV2369, BV2373, BV2374, and BV2375. Table 2 shows the sequence characteristics of the aforementioned CoV spike polypeptides. [Table 2-1] [Table 2-2] [Table 2-3]

[0353] The wild-type BV2361 protein (SEQ ID NO: 2) binds to human angiotensin-converting enzyme 2 precursor (hACE2). CoV S polypeptide binding was evaluated using in vivo interferometry and ELISA.

[0354] Production of proteins and nanoparticles Recombinant virus is amplified by infecting Sf9 insect cells. Insect cell cultures are infected with baculovirus at approximately 3 MOI (infection multiplicity = virus ffu or pfu / cell). The culture and supernatant are collected 48-72 hours after infection. Approximately 30 mL of crude cell recovery is clarified by centrifugation at approximately 800 × g for 15 minutes. The resulting crude cell recovery containing coronavirus spike (S) protein is purified into nanoparticles as described below.

[0355] To produce nanoparticles, the nonionic surfactant TERGITOL® nonylphenol ethoxylate NP-9 is used in a membrane protein extraction protocol. The crude extract is further purified by anion exchange chromatography, lentil lectin affinity / HIC, and cation exchange chromatography. The washed cells are lysed with a surfactant and then subjected to a low pH treatment that results in the precipitation of BV and Sf9 host cell DNA and proteins. The neutralized low pH lysate is clarified and further purified by anion exchange and affinity chromatography, followed by a second low pH treatment.

[0356] Affinity chromatography is used to remove Sf9 / BV protein, DNA, and NP-9, and to concentrate the coronavirus spike (S) protein. Briefly, lentil lectin is a calcium and manganese-containing metalloprotein that reversibly binds to glycosylated proteins containing polysaccharides and glucose or mannose. The anion-exchange flow-through fraction containing coronavirus spike (S) protein is loaded into a lentil lectin affinity chromatography resin (Capto lentil lectin, GE Healthcare). Glycosylated coronavirus spike (S) protein selectively binds to the resin, while non-glycosylated proteins and DNA are removed in the column flow-through. Weakly bound glycoproteins are removed with a buffer containing high salt and low molar concentration methyl alpha-D-mannopyranoside (MMP).

[0357] Column washing is also used to replace the surfactant from NP-9 surfactant to the surfactant polysorbate 80 (PS80). Coronavirus spike (S) polypeptide is eluted from the lentil lectin column in a nanoparticle structure using a high concentration of MMP. After elution, the coronavirus spike (S) protein is constructed into nanoparticles composed of the coronavirus spike (S) protein trimer and PS80 contained in the surfactant core. Any CoV S polypeptide nanoparticles described herein are expressed and purified using the process described in this example.

[0358] Example 2 Expression and purification of influenza surfactant core nanoparticles HA proteins from a single strain were expressed in Sf9 cells via baculovirus infection and grown for 48–96 hours before harvesting. The HA proteins were then recovered by surfactant extraction and converted into surfactant core nanoparticles during the purification process. Briefly, a TMAE column was pre-equilibrated with a buffer consisting of 25 mM Tris, pH 8.0, 1.5 M sodium chloride, and 0.02% NP9. Samples were loaded at a rate of ≤90 cm / hour (residence time 24 minutes) and then washed with EQ buffer (25 mM Tris, pH 8.0, 50 mM sodium chloride or 81 mM sodium chloride (for strains A and B, respectively), and 0.02% NP-9). The purified samples were then eluted using 1.5 CV of EQ buffer.

[0359] For strain A, the product from the TMAE column was subjected to nanofiltration, and then added to a lentil lectin affinity chromatography column pre-equilibrated with a buffer consisting of 25 mM Tris, 50 mM and 107 mM sodium chloride (for strain A and strain B, respectively), 0.02% (w / v) NP-9, and pH 8.0 (flow rate: 150 cm / hour) for 3 CVs. The sample was loaded with a residence time of 4 minutes. After loading, washing was performed with 3 CVs of lentil lectin equilibration buffer. The product was eluted with 25 mM sodium phosphate, pH 7.5, 200 mM sodium chloride, 500 mM methyl-α-D-mannopyranoside, 0.01% (w / v) PS80, pH 7.5 by collecting 2 CVs with residence times of 75 cm / hour and 8 minutes.

[0360] For strain B, the TMAE column product was further purified using a Capto Blue column. The column was equilibrated with 25 mM Tris, pH 8.0, 107 mM sodium chloride, and 0.02% (w / v) NP-9. Subsequently, the TMAE product was loaded at a flow rate of 225 cm / hour with a residence time of 4 minutes and collected in 2 CV of equilibrium buffer. The product from the Capto Blue column was subjected to nanofiltration and then added to a lentil lectin affinity chromatography column pre-equilibriumized with a buffer consisting of 25 mM Tris, 50 mM and 107 mM sodium chloride (for strains A and B, respectively), 0.02% (w / v) NP-9, and pH 8.0 (flow rate: 150 cm / hour) for 3 CV. The sample was loaded with a residence time of 4 minutes. After loading, washing was performed with 3 CV of lentil lectin equilibrium buffer. The product was eluted with 25 mM sodium phosphate, pH 7.5, 200 mM sodium chloride, 500 mM methyl-α-D-mannopyranoside, and 0.01% (w / v) PS80, pH 7.5, by collecting 2CVs at a residence time of 75 cm / hour and 8 minutes.

[0361] Lentil lectin products from both strain A and strain B were concentrated to the target HA concentration, and then the buffer was exchanged for the final active pharmaceutical ingredient buffer. Concentration and buffer exchange were performed by ultrafiltration and diafiltration.

[0362] Example 3 HaSMaN formulations The surfactant core nanoparticles from Example 2 were mixed with a saponin adjuvant (i.e., 85 wt% of fraction A's ISCOM matrix and 15 wt% of fraction C's ISCOM matrix) and incubated for at least 24 hours. Figure 7 (right panel) shows electron microscope images of HaSMaN.

[0363] Example 4 Immunogenicity and efficacy of a combined vaccine against COVID-19 and influenza The immunogenicity and efficacy of a combined vaccine against COVID-19 and influenza, also known as "qNIV / CoV2373," were evaluated. This combined vaccine consists of (i) a first vaccine (referred to as "CoV2373") containing nanoparticles with CoV S polypeptide (SEQ ID NO: 87), and (ii) a second vaccine (referred to as "qNIV") containing nanoparticles with hemagglutinin (HA) derived from four different influenza strains. The nanoparticles containing influenza A particles form HaSMaN. The nanoparticles containing influenza A particles form surfactant core nanoparticles. The four different influenza strains are A / Kansas / 14 / 17, A / Brisbane / 02 / 016, B / Maryland / 15 / 16, and B / Phuket / 3073 / 13. The "standard" combined vaccine contained 5 μg of CoV S polypeptide and 15 μg of HA per strain. The "high-dose" combined vaccine contained 5 μg of CoV S polypeptide and 60 μg of HA per strain. These nanoparticles were pre-mixed with a saponin adjuvant (i.e., 85% of fraction A ISCOM matrix and 15% of fraction C ISCOM matrix).

[0364] Male and female animal groups were immunized with either a standard vaccine or a high-dose vaccine. The control group was immunized with a vaccine containing either (i) qNIV without CoV2373 (containing 15 μg of hemagglutinin per HA / strain), (ii) qNIV without CoV2373 (containing 50 μg of hemagglutinin per HA / strain), or (iii) CoV2373 without qNIV (5 μg of CoV S polypeptide). All vaccines contained 50 μg of saponin adjuvant (Figure 8A).

[0365] Levels of human ACE2 receptor inhibitory antibodies produced by the combination vaccine were compared with those of animals immunized with qNIV alone or CoV2373 alone. Animals immunized with qNIV / CoV2373 showed a slight increase in hACE2 receptor blocking antibody levels 2 weeks after a single dose (GMT=34-39) and a 3.2-7.3-fold increase 2 weeks after booster immunization (GMT=107-202). Human ACE2 inhibitory titers were comparable to those of animals immunized with nanoparticles containing 5 μg of CoV2373 (GMT=290). Animals immunized with qNIV alone did not have measurable hACE inhibitory antibodies (Figure 8B). Hemagglutination inhibition (HAI) antibody titers were compared between groups. HAI titers against influenza A and B strains increased 2 weeks after a single dose and increased 2-7 times 2 weeks after booster immunization. HAI titers generated by the qNIV / CoV2373 combination were comparable to those generated by immunization with low-dose or high-dose qNIV for all influenza A and B strains. Animals immunized with 5 μg of CoV2373 vaccine did not have measurable HAI antibodies against strain A or B (Figure 8C-F).

[0366] Immunogenicity of qNIV / CoV2373 combined vaccine in hamsters The inventors then evaluated the immunogenicity and protection provided by the qNIV / CoV2373 combined vaccine in hamsters antigen-administered with SARS-CoV-2, comparing it to a vaccine containing qNIV alone or a vaccine containing CoV2373 alone. The hamster groups were immunized with qNIV / CoV2373 consisting of 10 μg or 2.5 μg of HA / strain combined with 5 μg or 1 μg of CoV2373. The comparison groups were immunized with qNIV (10 μg or 2.5 μg of HA / strain) or CoV2373 (5 μg or 1 μg). All vaccine formulations were adjuvanted with 15 μg of saponin adjuvant. The placebo group was administered the formulation buffer (Figure 9A). Animals administered the qNIV / CoV2373 combination showed elevated anti-S IgG levels two weeks after initial immunization (GMT=9467~25,295) and a 15- to 30-fold increase two weeks after booster immunization (GMT=275,341~418,124) (Figures 9B and 9C). Anti-S IgG titers two weeks after initial administration of monovalent CoV2373 (GMT=4576~43,632) and two weeks after second administration (GMT=302,967~523,143) were comparable to those in animals administered the mixed vaccine (Figures 9B and 9C).

[0367] Human ACE2 receptor inhibitor antibody levels induced by the qNIV / CoV2373 combination compared to antibody levels induced by monovalent CoV2373. Hamsters immunized with qNIV / CoV2373 showed antibody levels (IC) that block spike binding to the hACE2 receptor after a single dose. 50 The ACE2 inhibitory titer increased (GMT=57-136). Receptor inhibitory titers increased 6.2-16.3 times after booster immunization (GMT=654-1086). Human ACE2 inhibition levels were similar to those of hamsters after single immunization with CoV2373 (GMT=24-230). Human ACE2 receptor inhibitory titers increased 7.7-68 times after booster immunization (GMT=1636-1769) (Figures 9D, 9E).

[0368] The immune response to influenza A and B strains induced by qNIV / CoV2373 was compared with immunization with qNIV. Hamsters immunized with this mixed vaccine had high HAI titers against A / Kansas H3N2 (GMT=113-202) and A / Brisbane H1N1 (GMT=143-226) after single immunization. The HAI titer for A / Kansas H3N2 increased 6.3-14.2 times after booster immunization (GMT=1280-1810), and the HAI titer for A / Brisbane H1N1 increased 5.7-10 times after booster immunization (GMT=1280-1437). (Figures 10A-D). Animals immunized with qNIV had HAI titers equivalent to those of strain A. After single immunization, A / Kansas (GMT=80-106) and A / Brisbane (GMT=121-211) showed elevated HAI titers. Following booster immunization, HAI titers for A / Kansas increased 24-fold (GMT=1940-2560), and HAI titers for A / Brisbane increased 8-12.1-fold (GMT=1470-1689). Animals immunized with this mixed vaccine showed elevated HAI titers against B / Phuket after single immunization (GMT=80-143), and increased 5-9-fold after a second immunization (GMT=570-718). Animals immunized with qNIV had similar HAI titers against B / Phuket (GMT=735-844) after prime / boost immunization (Figures 10E, 10F). Similarly, animals immunized twice with qNIV / CoV2373 had HAI titers (GMT=160-142) similar to those of B / Maryland, which corresponded to HAI titers (GMT=106-242) induced by immunization with qNIV (Figure 10G, Figure 10H).

[0369] Viral neutralizing antibody titers were comparable between groups of hamsters immunized with qNIV / CoV2373 compared to animals immunized with qNIV alone. Animals immunized with qNIV / CoV2373 prime / boost had high neutralizing titers against A / Kansas (GMT=15,693–16,916) and A / Brisbane (GMT=6,992–10,507). Immunization with qNIV induced similar neutralizing titers against A / Kansas (GMT=13,625–19,314) and A / Brisbane (GMT=20,146–22,146) (Figures 11A–D). Similarly, animals immunized with qNIV / CoV2373 or qNIV had comparable levels of B strain neutralizing titers (Figures 11E–H). Overall, these results demonstrate that the qNIV / CoV2373 combined vaccine is immunogenically equivalent to qNIV and monovalent CoV2373 in comparison, and that co-administration of qNIV and CoV2373 with saponin adjuvants did not interfere with the immune response.

[0370] Competitive polyclonal RBD antibodies against neutralizing mAbs Polyclonal antibodies were competitively induced in hamsters against the prototype US-WA spike RBD neutralizing mAbs CR3022, NVX.322.3, and NVX.239.12 (Table 3), despite vaccination with monovalent CoV2373 or mixed qNIV. Hamsters immunized with monovalent CoV2373 exhibited significantly higher polyclonal antibodies against US-WA RBD mAbs when immunized with 5 μg or 1 μg of monovalent CoV2373 (Figure 12A-C). [Table 3]

[0371] The combination of qNIV with 1 μg or 5 μg of CoV2373 rS somewhat reduced the levels of competitive polyclonal antibodies measured against US-WA RBD. A similar pattern of competitive polyclonal antibody response against the B.1.351 variant was induced in hamsters immunized with monovalent CoV2373 and qNIV / CoV2373 combined vaccines. However, combined vaccines with monovalent CoV2373 or qNIV induced significant polyclonal antibodies competing with CR3002 and NVX.322.3 (Figure 12D-E). NVX-239.12 did not bind to the B.1.351 variant RBD.

[0372] Clinical signals after antigen administration To evaluate the preventive effect of the combination vaccine, immunized and placebo-treated hamsters were administered SARS-CoV-2 antigen via the intranasal route 21 days after a second immunization with SARS-CoV-2 via the intranasal route (day 35 of the study). All animals survived the post-antigen administration phase until the scheduled necropsy (7 dpi). Animal body weight was observed daily throughout the post-antigen administration period. Animals treated with placebo or immunized with qNIV (2.5 μg or 10 μg HA / strain) lost 12.5%–15% of their body weight by 7 dpi. In contrast, animals immunized with 1 μg or 5 μg of CoV2373 maintained their body weight and gained 2.6%–5% of their body weight by 7 dpi (Figure 13A). Animals immunized with qNIV / CoV2373 maintained their body weight and gained 2.5% to 5% at 7 dpi, which was the same as the uninfected Siamese group (Figure 13B).

[0373] Subgenomic viral mRNA in oral swabs, bronchoalveolar lavage (BAL), and lung samples. To investigate the efficacy of the qNIV / CoV2373 vaccine, viral loads in the upper and lower respiratory tracts were determined using qRT-PCR designed to detect SARS-CoV-2 subgenomic (sg) SARS-CoV-2 nucleocapsid (N) RNA. Oral swabs were collected at 2, 4, and 7 days post-infection (dpi). The highest levels of sgRNA were observed in oral swabs from placebo-treated animals, with a median peak of 4.4 (range 2.7 - 5.1) log 10 RNA copies / mL -1 at 2 dpi. Viral levels remained elevated at 3.2 (range 2.9 - 4.2) log 10 RNA copies / mL -1 at 4 dpi and decreased to 3.0 log 10 (range 1.7 - 3.7) RNA copies / mL -1 [[ID=…]]at 7 dpi. Viral RNA levels were not significantly different in oral swabs from animals immunized with 10 μg or 2.5 μg of HA / strain, with the highest levels of sgRNA being 4.4 - 4.5 (range 3.9 - 4.9) log 10 RNA copies / mL -1 at 2 dpi, 3.2 - 3.4 (range 1.7 - 3.7) log 10 RNA copies / mL -1 at 4 dpi, and 2.5 - 3.4 (1.7 - 3.9) log 10 RNA copies / mL -1 at 7 dpi. Oral swabs from animals immunized with 5 μg or 1 μg of CoV2373 had detectable viral RNA at 2 dpi (2.9 - 3.6 log 10 copies / mL -1 ). Viral RNA was not detected in swabs from animals immunized with CoV2373 at 4 or 7 dpi (Figure 13C). Viral RNA (3.2 - 3.5 log 10 RNA copies / mL -1 ) was detected only in swabs collected at 2 dpi from animals immunized with qNIV / CoV2373, and replicating virus was not detected at 4 or 7 dpi (Figure 13D).

[0374] BAL lavage fluid was obtained at autopsy and analyzed for viral RNA. The placebo group and the groups immunized with 10 μg or 2.5 μg of HA / strain had the highest median levels of viral RNA. The aspirate from the placebo group had a median level of 5.6 (range 5.1-6.4) log. 10 sgRNA copy mL -1 The replicated viral load was also found in animals immunized with 10 μg of HA / strain, with a median of 5.6 (range 4.4-5.9) log 10 RNA copy mL -1 In animals immunized with 2.5 μg of HA / strain, median 5.5 (range 3.6~6.0) log 10 RNA copy mL -1 The levels were high. Viral RNA was hardly or not detected at all in BAL samples obtained from animals immunized with CoV2373 (1 μg or 5 μg) or a combination of qNIV / CoV2373 (Figure 13E).

[0375] Lung tissue was collected at autopsy and viral load was evaluated. Lung homogenates from animals administered placebo and animals administered qNIV had the highest viral load: placebo median 7.1 (range 6.4~8.9) log 10 RNA copygram -1 , 10 μg HA / strain 7.3 (range 6.7~7.8) log 10 RNA copygram -1 , and 2.5 μg of HA / strain 6.7 (range 6.0~7.7) log 10 RNA copygram -1 Lung homogenates derived from animals immunized with CoV2373 or a combination of qNIV / CoV2373 contained little to no detectable virus (Figure 13F).

[0376] Macroscopic and microscopic observations: All animals survived until the scheduled necropsy (day 42 of the study). Lungs were collected and weighed. Lung weight was significantly higher in animals immunized with placebo or qNIV compared to animals immunized with CoV2373 or a combination of qNIV / CoV2373 (p ≤ 0.003) (Figure 13G). Microscopic findings in the airways of placebo-administered and qNIV-immunized animals were identical, consisting of bronchioloalveolar hyperplasia, mixed alveolar inflammation, perivasculitis with edema of surrounding tissues, and rare syncytial cells. No significant findings were observed in the lungs of animals immunized with CoV2373 or qNIV / CoV2373 vaccine (Figures 14A-J). These results were consistent with high viral load and pneumonia in animals administered placebo and qNIV, whereas the lungs of animals vaccinated with CoV2373 and qNIV / CoV2373 were normal and virus-free.

[0377] An additional feature necessary for the success of respiratory combination vaccines is to address and cover the inevitable evolution of the virus. RNA respiratory viruses are particularly prone to rapid evolution, often under immune pressure from the host and antigenic changes from zoonotic sources. The evolution of the SARS-CoV-2 virus under immune pressure in South Africa has led to a clear epidemic in a population where some degree of herd immunity had been established. In light of seasonal influenza, viral evolution is a major challenge for effective immunization. Recently, there have been several severe A(H3N2)-predominant influenza epidemics, in the face of recurring reports of inadequate field vaccine efficacy. This is likely driven by a mismatch in antigenicity from both egg-based vaccines and the virus itself, due to the rapid rate of antigenic evolution.

[0378] In this study, the inventors demonstrate that the qNIV / CoV2373 vaccine combination induced a competitive polyclonal antibody response not only against US-WA but also against the B.1.351 South African variant of the SARS-CoV-2 spike protein RBD neutralizing epitope. This study also showed the potential for neutralizing the epitope common to both US-WA and B.1.352 RBD, which is induced by the CoV2373 vaccine.

[0379] Notable events in 2019-2020 included the near absence of seasonal influenza, along with the emergence and surge of COVID-19 cases. Currently, it has been suggested that COVID-19 control measures may be contributing to the decrease in seasonal influenza cases, while others propose ecological mechanisms. In any case, past centuries have shown that influenza can recirculate and cause disease, and that highly infectious and clinically significant SARS-CoV-2 and newly emerging variants can continue to evolve globally. Co-infection with influenza and SAR-CoV-2 has been reported. Common clinical symptoms of COVID-19, such as fever, chills, cough, and dyspnea, make diagnosis of influenza virus infection difficult. A retrospective study of hospitalized SARS-CoV-2 positive patients with severe illness found that 12% (64 out of 544) were co-infected with influenza A (84%, 54 out of 64) and influenza B (16%, 10 out of 64). However, the clinical impact of co-infection with influenza and SARS-CoV-2 remains unclear.

[0380] The future need for seasonal influenza vaccines and the ongoing need for SARS-CoV-2 vaccines, due to the logistics of immunization with two vaccines each year, strongly demand the possibility of a combined vaccine. In this report, we describe a co-formulated influenza and SARS-CoV-2 nanoparticle vaccine that offers broad protection from simultaneously spreading seasonal influenza and COVID-19 viruses and has the potential for a broad protective response to address the challenges of newly emerging influenza strains and SARS-CoV-2 variants.

[0381] Materials and Methods: Virus stocks and receptors: SARS-CoV-2 (strain 2019-nCoV / USA-WA1 / 2020) isolates were obtained from the Centers for Disease Control and Prevention, and prepared by passage of the stock viruses in Vero E6 cells. Virus stocks A / Kansas / 14 / 17, A / Brisbane / 02 / 016, B / Maryland / 15 / 16, and B / Phuket / 3073 / 13 were provided by Novavax, Inc. (Gaithersburg, MD, USA). Histidine-tagged human ACE2 receptors were purchased from Sino Biologics (Beijing, CHN). Monoclonal antibody CR3022

[23] was obtained from Creative BioLabs (Shirley, NY, USA, catalog number MRO-1214LC). The SARS-CoV-2 US-WA recombinant 6-histidine-tagged receptor-binding domain (RBD) was provided by Novavax, Inc. (Gaithersburg, MD, USA). The histidine-tagged B.1.351 spike RBD was obtained from Sino Biologics (catalog number 40592-V08H85, Beijing, CHN).

[0382] The NVX-CoV2373 spike (S) protein and recombinant hemagglutinin vaccine: The CoV2373 vaccine was constructed from the full-length wild-type SARS-CoV-2 S glycoprotein based on the GenBank gene sequence MN908947, nucleotides 21563-25384. The native full-length S protein was modified by mutating a putative furin cleavage site (682-RRAR-685~682-QQAQ-685) located within the S1 / S2 cleavage domain to confer protease resistance. Two proline amino acid substitutions were inserted at positions K986P and V987P(2P) within the 7-base repeat 1 (HR1) domain to stabilize SARS-CoV-2S in the pre-fusion structure.

[0383] The synthetic transgene was codon-optimized and engineered into a baculovirus vector for expression in Spodoptera frugiperda (Sf9) insect cells (GenScript, Piscataway, NJ, USA). The spike trimer (named CoV2373) was extracted from the plasma membrane using Tris buffer containing TERGITOL NP-9 surfactant and clarified by centrifugation. The S trimer was purified using TMAE anion exchange and lentil lectin affinity chromatography. The purified CoV2373 was formulated in 25 mM sodium phosphate (pH 7.2), 300 mM NaCl, and 0.02% (v / v) polysorbate.

[0384] Cloning and Expression of Hemagglutinin (HA) Nanoparticles: The HA genes of influenza viruses A / Kansas / 14 / 17, A / Brisbane / 02 / 016, B / Maryland / 15 / 16, and B / Phuket / 3073 / 13 were codon-optimized for expression in Sf9 insect cells. The synthetic codon-optimized HA genes were cloned into the pBac1 baculovirus transfer vector (Millipore Sigma, Billerica, MA, USA). The pBac1 plasmid was transfected into Sf9 cells using Flash-bacGOLD bacmid (Oxford Expression Technology, Oxford UK) containing the Autographa californica polydedrosis viral genome. Sf9 cell cultures were infected with recombinant baculovirus expressing the HA gene. Recombinant HA was purified as described above.

[0385] Immunogenicity in ferrets. Ferrets (n=30, 15 males, 15 females) were randomized into 5 groups. Animals were immunized with 15 μg or 60 μg of HA / strain, together with 5 μg of CoV2373, without, or in combination with, IM. The control group was immunized with 5 μg of CoV2373. All vaccines were adjuvanted with 50 μg of saponin adjuvant. All groups were immunized with prime / boost regimens at 21-day intervals. Serum for analysis was collected 21 days after priming and 14 days after booster immunization.

[0386] Immunogenicity and SARS-CoV-2 antigen administration in hamsters. Hamsters 6-9 weeks old and weighing approximately 100 grams (n=54, 27 males, 27 females) were randomized into 10 groups (n=5-6 / group). Animals were immunized by intramuscular (IM) injection with a combination of 10 μg HA / strain and 5 μg or 1 μg CoV2373, a combination of 2.5 μg HA / strain and 5 μg or 1 μg CoV2373, 10 μg HA / strain, 2.5 μg HA / strain, 5 μg CoV2373, or 1 μg CoV2373. On the day of inj...

Claims

1. An immunogenic composition, (a) Coronavirus S (CoV S) glycoprotein in the form of surfactant core nanoparticles, wherein the surfactant is a nonionic surfactant, the glycoprotein, (b) at least three hemagglutinin (HA) glycoproteins, each of which is derived from a different influenza strain, and (c) The immunogenic composition comprising a pharmaceutically acceptable buffer.

2. The above-mentioned at least three types of HA glycoproteins, (a) Surfactant core nanoparticles containing hemagglutinin (HA), (b) HaSMaN (hemagglutinin saponin matrix nanoparticles), (c) Inactivated whole influenza virus, (d) A hemagglutinin composition extracted from influenza virus, optionally an influenza split virion composition or a subunit influenza composition. The immunogenic composition according to claim 1, which is a form selected from the group consisting of and any combination thereof.

3. The immunogenic composition according to claim 2, wherein at least one HA glycoprotein is in the form of a surfactant core nanoparticle containing HA, and at least one HA glycoprotein is in the form of HaSMaN.

4. The immunogenic composition according to claim 3, wherein the hemagglutinin glycoprotein of the surfactant core nanoparticles is derived from influenza B strain.

5. The immunogenic composition according to claim 3, wherein the hemagglutinin glycoprotein of the surfactant core nanoparticles is derived from an influenza A strain.

6. The immunogenic composition according to claim 3, wherein the surfactant core nanoparticles are trypsin-resistant nanoparticles.

7. The immunogenic composition according to claim 3, wherein the HaSMaN is a trypsin-resistant nanoparticle.

8. The immunogenic composition according to any one of claims 1 to 7, wherein each HA glycoprotein is derived from a different influenza strain.

9. An immunogenic composition according to any one of claims 1 to 8, comprising up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10 types of HA glycoproteins.

10. The immunogenic composition according to any one of claims 1 to 9, wherein each HA glycoprotein is in the form of nanoparticles.

11. The immunogenic composition according to claim 10, wherein each nanoparticle contains an HA glycoprotein derived from a single influenza strain.

12. The immunogenic composition according to claim 11, wherein each nanoparticle is a surfactant core nanoparticle or HaSMaN.

13. An immunogenic composition according to any one of claims 1 to 12, comprising an adjuvant.

14. The immunogenic composition according to claim 13, wherein the adjuvant is a saponin adjuvant.

15. The saponin adjuvant comprises at least two ISCOM particles, The first Iskom particle contains fraction A of Quillaja Saponaria Molina, but does not contain fraction C of Quillaja Saponaria Molina. The immunogenic composition according to claim 14, wherein the second iscom particle comprises fraction C of Quillaja Saponaria Molina and does not contain fraction A of Quillaja Saponaria Molina.

16. The immunogenic composition according to claim 15, wherein, with respect to the total weight of fraction A and fraction C of Quillaja Saponaria Molina in the adjuvant, fraction A of Quillaja Saponaria Molina accounts for 50 to 96% by weight, and fraction C of Quillaja Saponaria Molina accounts for the remainder.

17. The immunogenic composition according to claim 15, wherein, with respect to the total weight of fraction A and fraction C of Quillaja Saponaria Molina in the adjuvant, fraction A of Quillaja Saponaria Molina accounts for at least 70% by weight, and fraction C of Quillaja Saponaria Molina accounts for the remainder.

18. The immunogenic composition according to claim 15, wherein, with respect to the total weight of fraction A and fraction C of Quillaja Saponaria Molina in the adjuvant, fraction A of Quillaja Saponaria Molina accounts for at least 85% by weight, and fraction C of Quillaja Saponaria Molina accounts for the remainder.

19. The immunogenic composition according to claim 15, wherein fraction A and fraction C of Quillaja Saponaria Molina in the adjuvant account for approximately 85% by weight and approximately 15% by weight, respectively, of the total weight of fraction A and fraction C of Quillaja Saponaria Molina.

20. The immunogenic composition according to claim 15, wherein fraction A and fraction C of Quillaja Saponaria Molina in the adjuvant account for approximately 92% by weight and approximately 8% by weight, respectively, of the total weight of fraction A and fraction C of Quillaja Saponaria Molina.

21. An immunogenic composition according to any one of claims 13 to 20, comprising approximately 50 μg or approximately 75 μg of a saponin adjuvant.

22. The immunogenic composition according to any one of claims 1 to 21, wherein the surfactant is PS80.

23. The immunogenic composition according to any one of claims 1 to 22, wherein the influenza strain is a subtype selected from the group consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and H18.

24. The immunogenic composition according to any one of claims 1 to 23, wherein the pharmaceutically acceptable buffer comprises (i) about 25 mM sodium phosphate, (ii) about 150 mM sodium chloride, (iii) about 100 mM arginine hydrochloride, and (iv) about 5% trehalose, and the pH of the composition is about 7.

5.

25. The aforementioned CoV S glycoprotein, (i) An S1 subunit having an inactivated furin cleavage site, comprising an N-terminal domain (NTD), a receptor-binding domain (RBD), and subdomains 1 and 2 (SD1 / 2), wherein the inactivated furin cleavage site has the amino acid sequence QQAQ (SEQ ID NO: 7), The aforementioned NTD may optionally, (a) Deletion of one or more amino acids selected from the group consisting of amino acids 56, 57, 131, 132, 144, 145, 228, 229, 230, 231, 234, 235, 236, 237, 238, 239, 240 and combinations thereof, (b) Insertion of 1, 2, 3, or 4 amino acids after amino acid 132, and (c) comprising one or more modifications selected from the group consisting of mutations of one or more amino acids selected from the group consisting of amino acids 5, 6, 7, 13, 51, 53, 56, 57, 62, 63, 67, 82, 125, 129, 131, 132, 133, 139, 143, 144, 145, 177, 200, 201, 202, 209, 229, 233, 240, 245 and combinations thereof, The RBD optionally includes mutations in one or more amino acids selected from the group consisting of amino acids 333, 404, 419, 426, 439, 440, 464, 465, 471, 477, 481, 488 and combinations thereof. The SD1 / 2 domain optionally includes mutations in one or more amino acids selected from the group consisting of 557, 600, 601, 642, 664, 668 and combinations thereof, as well as the S1 subunit, and (ii) An S2 subunit in which amino acids 973 and 974 are proline, Optionally, (a) Deletion of one or more amino acids from 676-685, 676-702, 702-711, 775-793, 806-815 and combinations thereof, (b) Mutations of one or more amino acids selected from the group consisting of 688, 703, 846, 875, 937, 969, 1014, 1058, 1105, and 1163 and combinations thereof, and (c) The S2 subunit comprising one or more modifications selected from the group consisting of the deletion of one or more amino acids from the TMCT, The immunogenic composition according to any one of claims 1 to 24, wherein the amino acids of the CoV S glycoprotein are numbered relative to the polypeptide having the sequence of SEQ ID NO:

2.

26. The immunogenic composition according to claim 25, wherein the CoV S glycoprotein contains or comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NOs: 86-89, 105, 106, 112-115, 174, 175, 176, 181-184, 186, 188, 190, 195, 217-228, 233-236, 243, 255-264, 273-280, and 298-302.

27. The immunogenic composition according to claim 26, wherein the CoV S glycoprotein comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

87.

28. An immunogenic composition according to any one of claims 1 to 27, comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 CoV S glycoproteins.

29. An immunogenic composition according to any one of claims 1 to 28, comprising approximately 1 μg to approximately 50 μg of CoV S glycoprotein and approximately 5 μg to approximately 60 μg of hemagglutinin per strain.

30. The immunogenic composition according to claim 29, comprising approximately 20 μg to approximately 50 μg of CoV S glycoprotein and approximately 24 μg to approximately 40 μg of hemagglutinin per strain.

31. An immunogenic composition according to any one of claims 1 to 29, comprising approximately 2.5 μg, approximately 3 μg, approximately 5 μg, approximately 7.5 μg, approximately 22.5 μg, approximately 25 μg, or approximately 30 μg of CoV S glycoprotein.

32. An immunogenic composition according to any one of claims 1 to 29, comprising approximately 5 μg, approximately 10 μg, approximately 24 μg, approximately 25 μg, approximately 26 μg, approximately 27 μg, approximately 28 μg, approximately 29 μg, approximately 30 μg, approximately 31 μg, approximately 32 μg, approximately 33 μg, approximately 34 μg, approximately 35 μg, approximately 36 μg, approximately 37 μg, approximately 38 μg, approximately 39 μg, approximately 40 μg, or approximately 60 μg of hemagglutinin per strain.

33. The immunogenic composition according to any one of claims 1 to 32, wherein the ratio of the amount of hemagglutinin to the amount of CoV S glycoprotein per strain in the immunogenic composition is about 1:1 to about 5:

1.

34. The immunogenic composition according to claim 33, wherein the ratio of the amount of hemagglutinin to the amount of CoV S glycoprotein for each strain in the immunogenic composition is about 1:1 to about 3:

1.

35. The immunogenic composition according to claim 33, wherein the ratio of the amount of hemagglutinin to the amount of CoV S glycoprotein for each strain in the immunogenic composition is about 1.2:1 to about 3:1 or about 1.3:

1.

36. In the immunogenic composition, the ratio of the amount of hemagglutinin to the amount of CoV S glycoprotein for each strain is approximately 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3.0:1, 3.1:1, and The immunogenic composition according to claim 33, wherein the ratio is approximately 3.2:1, approximately 3.3:1, approximately 3.4:1, approximately 3.5:1, approximately 3.6:1, approximately 3.7:1, approximately 3.8:1, approximately 3.9:1, approximately 4.0:1, approximately 4.1:1, approximately 4.2:1, approximately 4.3:1, approximately 4.4:1, approximately 4.5:1, approximately 4.6:1, approximately 4.7:1, approximately 4.8:1, approximately 4.9:1, or approximately 5:

1.

37. An immunogenic composition according to any one of claims 1 to 31 and 33 to 36, comprising approximately 30 μg to approximately 54 μg of hemagglutinin per strain.

38. An immunogenic composition according to any one of claims 1 to 31 and 33 to 36, comprising approximately 30 μg, approximately 31 μg, approximately 32 μg, approximately 33 μg, approximately 34 μg, approximately 35 μg, approximately 36 μg, approximately 37 μg, approximately 38 μg, approximately 39 μg, approximately 40 μg, approximately 41 μg, approximately 42 μg, approximately 43 μg, approximately 44 μg, approximately 45 μg, approximately 46 μg, approximately 47 μg, approximately 48 μg, approximately 49 μg, approximately 50 μg, approximately 51 μg, approximately 52 μg, approximately 53 μg, approximately 54 μg, approximately 55 μg, approximately 56 μg, approximately 57 μg, approximately 58 μg, approximately 59 μg, or approximately 60 μg of hemagglutinin per strain.

39. An immunogenic composition according to any one of claims 1 to 29 and 32 to 38, comprising approximately 13 μg to approximately 25 μg of CoV S glycoprotein.

40. An immunogenic composition according to any one of claims 1 to 30 and 32 to 39, comprising approximately 25 μg to approximately 35 μg of CoV S glycoprotein.

41. An immunogenic composition according to any one of claims 1 to 37 and 39 to 40, comprising approximately 30 μg of hemagglutinin and approximately 25 μg of CoV S glycoprotein per strain.

42. An immunogenic composition according to any one of claims 1 to 29, 32 to 36, and 38 to 41, comprising approximately 60 μg of hemagglutinin and approximately 35 μg of coronavirus S glycoprotein per strain.

43. An immunogenic composition according to any one of claims 1 to 30, 32 to 38, and 40 to 41, comprising approximately 13 μg, approximately 14 μg, approximately 15 μg, approximately 16 μg, approximately 17 μg, approximately 18 μg, approximately 19 μg, approximately 20 μg, approximately 21 μg, approximately 22 μg, approximately 23 μg, approximately 24 μg, or approximately 25 μg of CoV S glycoprotein.

44. The immunogenic composition according to any one of claims 1 to 40 and 43, comprising approximately 33 μg of hemagglutinin and approximately 25 μg of CoV S glycoprotein per strain.

45. An immunogenic composition according to any one of claims 1 to 30, 33 to 37, 39, and 43, comprising approximately 30 μg to approximately 40 μg of hemagglutinin and approximately 20 μg of CoV S glycoprotein per strain.

46. An immunogenic composition according to any one of claims 1 to 29, 31, 33 to 40, and 43, comprising approximately 54 μg of hemagglutinin and approximately 25 μg of CoV S glycoprotein per strain.

47. An immunogenic composition according to any one of claims 1 to 29, 33 to 37, and 39, comprising approximately 33 μg to approximately 39 μg of hemagglutinin and approximately 14 μg to approximately 15 μg of CoV S glycoprotein per strain.

48. An immunogenic composition according to any one of claims 1 to 29, 33 to 37, and 39, comprising approximately 39 μg of hemagglutinin and approximately 13 μg of CoV S glycoprotein per strain.

49. An immunogenic composition according to any one of claims 1 to 29, 32 to 36, 38, 40, and 42, comprising approximately 60 μg of hemagglutinin, 75 μg of saponin adjuvant, and approximately 35 μg of CoV S glycoprotein per strain.

50. The immunogenic composition according to any one of claims 33 to 49, wherein the ratio of the amount of hemagglutinin to the amount of coronavirus S glycoprotein for each strain in the immunogenic composition is about 1.2:1, 1.3:1, about 1.5:1, about 2:1, about 2.2:1, about 2.4:1, about 2.8:1, about 2.6:1, or about 3:

1.

51. An immunogenic composition, (a) At least three hemagglutinin (HA) glycoproteins, each HA glycoprotein derived from a different influenza strain, with 30 to 60 μg of HA present in the composition for each strain, and (b) The immunogenic composition comprising a pharmaceutically acceptable buffer.

52. Furthermore, the immunogenic composition according to claim 51, comprising coronavirus S (CoV S) glycoprotein in the form of surfactant core nanoparticles, wherein the surfactant is a nonionic surfactant.

53. The immunogenic composition according to any one of claims 51 to 52, wherein approximately 30 μg, approximately 45 μg, or approximately 60 μg of HA is present in the composition per strain.

54. The immunogenic composition according to any one of claims 51 to 53, further comprising an adjuvant.

55. The immunogenic composition according to any one of claims 51 to 54, wherein the adjuvant is a saponin adjuvant.

56. The saponin adjuvant comprises at least two ISCOM particles, The first Iskom particle contains fraction A of Quillaja Saponaria Molina, but does not contain fraction C of Quillaja Saponaria Molina. The immunogenic composition according to claim 55, wherein the second iscom particle comprises fraction C of Quillaja Saponaria Molina and does not contain fraction A of Quillaja Saponaria Molina.

57. The immunogenic composition according to claim 56, wherein, with respect to the total weight of fraction A and fraction C of Quillaja Saponaria Molina in the adjuvant, fraction A of Quillaja Saponaria Molina accounts for 50 to 96% by weight, and fraction C of Quillaja Saponaria Molina accounts for the remainder.

58. The immunogenic composition according to claim 56, wherein, with respect to the total weight of fraction A and fraction C of Quillaja Saponaria Molina in the adjuvant, fraction A of Quillaja Saponaria Molina accounts for at least 70% by weight, and fraction C of Quillaja Saponaria Molina accounts for the remainder.

59. The immunogenic composition according to claim 56, wherein, with respect to the total weight of fraction A and fraction C of Quillaja Saponaria Molina in the adjuvant, fraction A of Quillaja Saponaria Molina accounts for at least 85% by weight, and fraction C of Quillaja Saponaria Molina accounts for the remainder.

60. The immunogenic composition according to claim 56, wherein fraction A and fraction C of Quillaja Saponaria Molina in the adjuvant account for approximately 85% by weight and approximately 15% by weight, respectively, of the total weight of fraction A and fraction C of Quillaja Saponaria Molina.

61. The immunogenic composition according to claim 56, wherein fraction A and fraction C of Quillaja Saponaria Molina in the adjuvant account for approximately 92% by weight and approximately 8% by weight, respectively, of the total weight of fraction A and fraction C of Quillaja Saponaria Molina.

62. An immunogenic composition according to any one of claims 54 to 61, comprising approximately 50 μg or approximately 75 μg of an adjuvant.

63. A method for stimulating an immune response to SARS-CoV-2, a heterologous SARS-CoV-2 strain, an influenza virus, or a combination thereof in a subject, comprising administering an immunogenic composition according to any one of claims 1 to 62.

64. The method according to claim 63, wherein the subject is administered a first dose on day 0 and a boost dose on day 56.

65. The method according to claim 63 or 64, wherein the immunogenic composition is administered intramuscularly.

66. The method according to any one of claims 63 or 65, wherein a single dose of the immunogenic composition is administered.

67. The method according to any one of claims 63 to 66, wherein the heterologous SARS-CoV-2 strain is selected from the group consisting of Cal. 20C SARS-CoV-2 strain, P. 1 SARS-CoV-2 strain, B. 1.351 SARS-CoV-2 strain, B. 1.1.7 SARS-CoV-2 strain, SARS-CoV-2 B. 1.617.2 strain, B. 1.525 strain, B. 1.526 strain, B. 1.617.1 strain, C. 37 strain, B. 1.621 strain, or SARS-CoV-2 Omicron strain.

68. The efficacy of the immunogenic composition for the prevention of COVID-19 is determined to last for up to approximately 2 months, 2.5 months, 3 months, 3.5 months, 4 months, 4.5 months, 5 months, 5.5 months, 6 months, 6.5 months, 7 months, 7.5 months, 8 months, 8.5 months, 9 months, 9.5 months, 10 months, 10.5 months, 11 months, 11.5 months, or up to The method according to any one of claims 63 to 67, wherein over a period of approximately 12 months, the percentage is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or about 100%.

69. The efficacy of the immunogenic composition for the prevention of COVID-19 is observed for up to approximately 2 months, 2.5 months, 3 months, 3.5 months, 4 months, 4.5 months, 5 months, 5.5 months, 6 months, 6.5 months, 7 months, 7.5 months, 8 months, 8.5 months, 9 months, 9.5 months, 10 months, 10.5 months, 11 months, 11.5 months, or 12 months from the administration of the immunogenic composition. The method according to any one of claims 63 to 67, wherein the percentages range from approximately 50% to approximately 99%, approximately 50% to approximately 95%, approximately 50% to approximately 90%, approximately 50% to approximately 85%, approximately 50% to approximately 80%, approximately 60% to approximately 99%, approximately 60% to approximately 95%, approximately 60% to approximately 90%, approximately 60% to approximately 85%, approximately 60% to approximately 80%, approximately 40% to approximately 99%, approximately 40% to approximately 95%, approximately 40% to approximately 90%, approximately 40% to approximately 85%, approximately 40% to approximately 80%, approximately 40% to approximately 75%, approximately 40% to approximately 70%, approximately 40% to approximately 65%, approximately 40% to approximately 55%, or approximately 40% to approximately 50%.

70. A pre-filled syringe comprising the immunogenic composition according to any one of claims 1 to 62.

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