Compositions for Coronavirus and Influenza and Methods for Using Them

Description

Compositions for Coronavirus and Influenza and Methods for Using Them REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0001] This application claims priority over U.S. Provisional Application No. 63 / 508,798, filed June 16, 2023, and 63 / 493,884, filed April 3, 2023, the contents of which are incorporated herein by reference.

[0002] This request contains a Sequence Listing, which was submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy, created on April 3, 2024, is named 1450_099WO1_Sequence_Listing_04_03_2024 and is 768,523 bytes in size. FIELD OF DISSEMINATION

[0003] This disclosure relates to compositions and methods for inducing immune responses against influenza and coronavirus. FUNDAMENTALS OF THE INVENTION

[0004] Influenza and coronavirus disease 2019 (COVID-19) are life-threatening illnesses caused by the influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respectively. The mortality rate for patients diagnosed with influenza is approximately 0.1%, and the mortality rate for patients diagnosed with COVID-19 ranges from 0.2% to 7.7%.

[0005] The development of vaccines that prevent or reduce the severity of life-threatening infectious diseases is desirable. However, the development of human vaccines remains a challenge due to the highly sophisticated evasion mechanisms of pathogens and the difficulties in stabilizing vaccines. Ideally, a vaccine should induce antibodies that block or neutralize infectious agents and remain stable in various environments, including environments that do not allow refrigeration. The combination of two antigens from two pathogens in a single composition of Petition 870250108300, dated 11 / 26 / 2025, p. 9 / 284 The 2 / 210 vaccine is particularly challenging because the antigens can interact with each other, preventing a sufficient immune response to either of the two pathogens. SUMMARY OF THE INVENTION

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

[0007] This document provides an immunogenic composition comprising: (a) a coronavirus S glycoprotein (CoV S) in the form of a detergent-core nanoparticle, wherein the detergent is a nonionic detergent; (b) at least three hemagglutinin (HA) glycoproteins, wherein each HA glycoprotein is from a different influenza strain; and (c) a pharmaceutically acceptable buffer. The ratio of the amount of hemagglutinin per strain to the amount of coronavirus S glycoprotein in the immunogenic composition 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 document provides an immunogenic composition comprising (a) at least three hemagglutinin (HA) glycoproteins, wherein each HA glycoprotein is from a different strain of influenza, wherein 30 to 60 pg of HA per strain are present in the composition; and (b) a pharmaceutically acceptable buffer.

[0009] In embodiments, the immunogenic compositions comprise an adjuvant. In embodiments, the adjuvant comprises at least two iscom particles, wherein: the first iscom particle comprises fraction A of Quillaja Saponaria Molina and not fraction C of Quillaja Saponaria Molina; and the second iscom particle comprises fraction C of Quillaja Saponaria Molina and not fraction A of Quillaja Saponaria Molina.

[0010] In embodiments, methods are provided in this document for stimulating an immune response against SARS-CoV-2, a heterogeneous strain of SARS-CoV-2, an influenza virus or a combination thereof comprising the administration of an immunogenic composition described in this document. Petition 870250108300, dated 11 / 26 / 2025, p. 10 / 284 3 / 210 BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Fig. 1 shows a schematic of a wild-type amino acid sequence of the SARS-CoV-2 Spike (S) protein (SEQ ID NO: 1). The RRAR of the furin cleavage site (SEQ ID NO: 6) is highlighted in bold and the signal peptide is underlined.

[0012] Fig. 2 shows the purification of the CoV S polypeptides BV2364, BV2365, BV2366, BV2367, BV2368, BV2369, BV2373, BV2374, and BV2375. The data reveal that BV2365 (SEQ ID NO: 4) and BV2373 (SEQ ID NO: 87), which has an inactive furin cleavage site with an amino acid sequence of QQAQ (SEQ ID NO: 7), is expressed as a single chain (S0). In contrast, the 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] Fig. 3 shows the primary structure of the BV2373 CoV S polypeptide and modifications at the furin cleavage site, K986P and V987P.

[0014] Fig. 4 shows the purification of wild-type CoV S polypeptide and CoV S polypeptides BV2365 and BV2373.

[0015] Fig. 5 shows a diagram of the Spike (S) protein of the coronavirus (SEQ ID NO: 86) (BV2373). The furin cleavage site QQAQ (SEQ ID NO: 7) is underlined once and the K986P and V987P mutations are underlined twice.

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

[0017] Fig. 7 shows examples of transmission electron microscopy (TEM) images of influenza HA detergent core nanoparticles alone (left), saponin adjuvant alone (middle) (i.e., 85% Fraction A ISCOM matrix and 15% Fraction C ISCOM matrix) Petition 870250108300, dated 11 / 26 / 2025, p. 11 / 284 4 / 210 and the combination of HA nanoparticles and saponin adjuvant, which forms Hemagglutinin Saponin Matrix Nanoparticles (HaSMaNs) (right).

[0018] Figs. 8A-F show the immune response to SARS-CoV-2 spike glycoprotein and influenza hemagglutinin in ferrets immunized with qNIV / CoV2373 combined vaccine. (Fig. 8A) Male and female ferret groups (n = 6 / group) were immunized with a combination of 15 μg or 60 μg of hemagglutinin (HA) per strain with 5 μg of CoV2373 with 50 μg of saponin adjuvant. Comparator groups were immunized with 15 μg or 60 μg of HA per strain or with 5 μg of CoV2373 with 50 μg of saponin adjuvant. All groups were immunized with two doses spaced 21 days apart. Red triangles indicate serum collection days. (Fig. 8B) Human angiotensin-converting enzyme-2 receptor (hACE2) blocking antibody titer. Hemagglutinin inhibitor (HAI) antibody titers 21 days after 1 dose and 14 days after booster immunization (study day 35). (Fig. 8C) A / Kansas / 14 / 2017. (Fig. 8D) A / Brisbane / 02 / 2018. (Fig. 8E) B / Phuket / 3070 / 2013. (Fig. 8F) B / Maryland / 15 / 2016. The bars indicate the geometric mean oil (GMT) and the error bars indicate the 95% confidence interval.

[0019] Figs. 9A-E show the immune response to SARS-CoV-2 spike glycoprotein and receptor-blocking antibodies in hamsters immunized with qNIV / CoV2373 combined vaccine. (Fig. 9A) Groups of male and female hamsters (n = 5-6 per group) were immunized with a combination of 2.5 or 10 μg hemagglutinin (HA) / strain with 1 or 5 μg CoV2373 with 15 μg saponin adjuvant. Comparator groups were immunized with 2.5 or 10 μg HA / strain or 1 or 5 μg CoV2373 with 15 μg saponin adjuvant. Animals were immunized intramuscularly (IM) with 2 doses spaced 14 days apart. The placebo group received formulation buffer. Sera were collected for analysis as indicated by the red triangles. Immunized and placebo animals were infected intranasally (IN) with 2.0 x 10⁴ pfu of SARS-CoV-2 21 days after the second immunization. Petition 870250108300, dated 11 / 26 / 2025, page 12 / 284 5 / 210 Oral swabs were collected 2, 4, and 7 days post-infection (dpi, blue triangles). Alveolar white blood lavage and lung samples were collected 7 dpi (black triangle). (Fig. 9B, Fig. 9C) Anti-spike IgG titer 14 days after 1 dose and 14 days after booster immunization (study day 28), respectively. (Fig. 9D, Fig. 9E) Human ACE-2 receptor blocking antibody (hACE2) titer 14 days after 1 dose and 14 days after booster immunization, respectively. Bars indicate the geometric mean titer (GMT) of the group, and error bars indicate the 95% confidence interval (95% CI). Individual animal values ​​are indicated by colored symbols. The horizontal dashed black line indicates the limit of detection (LOD).

[0020] Figs. 10A-H show the influenza HA immune response in hamsters immunized with the qNIV / CoV2373 combined vaccine. Hamster groups were immunized with the qNIV / CoV2373 combination vaccine or with the component vaccines, as indicated in Fig. 8A. Serum was analyzed for hemagglutinin inhibitor (HAI) antibody titers 14 days after a dose and 14 days after booster immunization (study day 28). (Fig. 10A, Fig. 10B) A / Kansas / 14 / 2017. (Fig. 10C, Fig. 10D) A / Brisbane / 02 / 2018. (Fig. 10E, Fig. 10F) B / Phuket / 3070 / 2013. (Fig. 10G, Fig. 10H) B / Maryland / 15 / 2016. The bars indicate the geometric mean title, and the error bars indicate the 95% confidence interval.

[0021] Figs. 11A-H show influenza virus neutralizing antibodies in hamsters immunized with qNIV / CoV2373 combination vaccine. Hamster groups were immunized with the qNIV / CoV2373 combination or component vaccines, as shown in Fig. 8A. Virus neutralization titers were determined 14 days after 1 dose and 14 days after booster immunization (study day 28) in response to A / Kansas / 14 / 2017 (Fig. 11A, Fig. 11B), A / Brisbane / 02 / 2018 (Fig. 11C, Fig. 11D), B / Phuket / 3073 / 2013 (Fig. 11E, Fig. 11F) and B / Maryland / 15 / 2016 (Fig. 11G, Fig. 11H). Petition 870250108300, dated 11 / 26 / 2025, p. 13 / 284 6 / 210 limit of detection (LOD).

[0022] Figs. 12A-E show that the qNIV / CoV2373 combination vaccine induces antibodies that bind to highly conserved cryptic epitopes in the receptor-binding domain (RBD) of SARS-CoV-2 as determined by biolayer interferometry (BLI). The specificity of antibodies induced by the qNIV / CoV2373 combination or monovalent CoV2373 was determined by competitive antibody binding of immune serum with receptor-site-specific neutralizing monoclonal antibodies by BLI. Horizontal bars indicate the geometric mean of the group and error bars indicate the 95% confidence interval. Colored symbols indicate individual animal values. Figs. 12A-C show antibodies against SARS-CoV-2 US-WA RBD. Figs. 12D-E show antibodies against the RBD of SARS-CoV-2 B.1.351 from South Africa.

[0023] Figs. 13A-G show the change in weight and protection against upper and lower respiratory infection with SARS-CoV-2 in hamsters immunized with the combined qNIV / CoV2373 vaccine. Groups of male and female hamsters (n = 5-6 / group) were immunized with a qNIV / CoV2373 starter / booster regimen or the component vaccines spaced 14 days apart. Three weeks after the second immunization (study day 35), the animals were exposed intranasally to 2.0 x 10⁴ pfu. Figs. 13A-B show the change in body weight (percent change following SARS-CoV-2 exposure) for up to 7 days post-infection (dpi). Data are mean ± SEM for the vaccinated, placebo, and sham groups. Figures 13C-D show that subgenomic RNA (sg) in buccal swabs was collected 2, 4, and 7 days post-infection (dpi). sgRNA was analyzed by qRT-PCR. Figure 13E shows the viral load of sgRNA in alveolar white blood cell wash fluids collected 7 days post-infection (dpi).Figure 13F shows the sgRNA viral load in lung homogenates collected 7 dpi. In the box plots, the median is indicated by the horizontal line, the top and bottom of the box indicate the interquartile range, and the whiskers indicate minimums and maximums. Petition 870250108300, dated 11 / 26 / 2025, page 14 / 284 7 / 210 each experimental group (n = 5-6 / group). Individual animal values ​​are indicated by colored symbols. The dashed line indicates the assay's limit of detection (LOD). Student's t-test (paired, two-tailed) was used to determine significant differences in viral sgRNA levels between placebo-treated animals and immunized animals at 2, 4, and 7 dpi. Not significant (ns), ***p < 0.001, ****p < 0.0001. Fig. 13G shows the weight of lungs collected at 7 dpi from vaccinated, placebo-treated, and untreated sham animals. Bars indicate the mean and error bars indicate the ± standard deviation (SD). Individual animal values ​​are indicated by colored symbols. Student's t-test (paired, two-tailed) was used to determine significant differences in lung weight between the indicated paired groups.

[0024] Figs. 14A-J show histopathological changes in the lungs of hamsters immunized with the qNIV / CoV2373 combined vaccine and exposed to SARS-CoV-2. Male and female hamster groups were immunized with the qNIV / CoV2373 combination with 2.5 μg or 10 μg of hemagglutinin (HA) per strain combined with 1 μg or 5 μg of CoV2373 adjuvanted with saponin adjuvant. Comparator groups were immunized with 2.5 μg or 10 μg of HA / strain or with 1 μg or 5 μg of recombinant CoV2373 peak adjuvanted with 15 μg of saponin adjuvant. All groups were immunized with 2 doses administered 14 days apart. The placebo group received formulation buffer. Three weeks after the second immunization (study day 35), all animals were exposed intranasally to 2.0 x 10⁴ pfu of SARS-CoV-2 and lungs were collected 7 dpi. Histological images were stained with hematoxylin and eosin (H&E). Fig. 14A is a sham control image showing a normal lung.Figure 14B shows microscopic findings in the lungs of animals treated with placebo that showed that the airways were consolidated by bronchioloalveolar hyperplasia (arrows) with mixed alveolar inflammation. Mononuclear inflammatory cells surround the blood vessels (arrowhead) with edema expanding the surrounding tissues (arrowhead). Figures 14C-D show microscopic findings in the lungs. Petition 870250108300, dated 11 / 26 / 2025, page 15 / 284 8 / 210 of animals immunized with qNIV show histological changes similar to the placebo group, including extensive bronchioloalveolar hyperplasia (arrows) with mixed alveolar inflammation with perivascular inflammation and expanded vessel walls (arrowheads). Figs. 14E-F show images of lungs from animals immunized with monovalent CoV2373 that show no significant microscopic findings. Figs. 14G-J are images of lungs from animals immunized with combined qNIV / CoV2373 vaccine that show no significant microscopic findings. 10X magnification.

[0025] Fig. 15 shows the anti-Spike IgG antibody titer as a function of hemagglutinin and CoV S polypeptide dose from day 0 to day 28 after immunization according to Example 5.

[0026] Fig. 16 shows the geometric mean titers of HAI against A / Brisbane H1N1 as a function of hemagglutinin and CoV S polypeptide dose from day 0 to day 28 after immunization according to Example 5.

[0027] Fig. 17 shows the geometric mean titers of HAI against A / Kansas H3N2 as a function of hemagglutinin and CoV S polypeptide dose from day 0 to day 28 after immunization according to Example 5.

[0028] Fig. 18 shows the geometric mean HAI titers against B / Maryland (Vic) as a function of hemagglutinin and CoV S polypeptide dose from day 0 to day 28 after immunization according to Example 5.

[0029] Fig. 19 shows the geometric mean titers of HAI against B / Phuket (Yam) as a function of hemagglutinin and CoV S polypeptide dose from day 0 to day 28 after immunization according to Example 5.

[0030] Fig. 20 shows the five second-order models from Example 6.

[0031] Fig. 21 shows the geometric mean HAI titers against A / Brisbane H1N1 as a function of hemagglutinin and CoV S polypeptide dose from day 0 to day 182 after immunization according to Example 5.

[0032] Fig. 22 shows the titer of anti-Spike IgG antibodies (in geometric mean ELISA units) as a function of the dose of Petition 870250108300, dated 11 / 26 / 2025, p. 16 / 284 9 / 210 hemagglutinin and CoV S polypeptide from day 0 to day 182 after immunization according to Example 5.

[0033] Fig. 23 shows the number of CD4+ T cells producing dual cytokines as a function of hemagglutinin and CoV S polypeptide dose from day 0 to day 7 after immunization according to Example 5. (parental SARS-CoV-2 strain (homologous))

[0034] Fig. 24 shows the geometric mean titers of HAI against A / Hong Kong H3N2 (heterologous) as a function of hemagglutinin and CoV S polypeptide dose from day 0 to day 28 after immunization according to Example 5.

[0035] Fig. 25 shows the IgG antibody titer against SARS-CoV-2 Omicron BA. 1 (heterologous strain) (in geometric mean ELISA units) as a function of hemagglutinin and CoV S polypeptide dose from day 0 to day 28 after immunization according to Example 5.

[0036] Fig. 26 shows the amount of CD4+ T cells producing dual cytokines as a function of hemagglutinin and CoV S polypeptide dose from day 0 to day 7. (A / Cambodia (H3N2 heterologous).

[0037] Fig. 27 shows that serum from patients administered with the compositions described in Example 8, which contain hemagglutinin, CoV S polypeptide, and saponin adjuvant, contains anti-Spike IgG antibodies and antibodies that neutralize SARS-CoV-2. M50 refers to a saponin adjuvant dose of 50 pg. M75 refers to a saponin adjuvant dose of 75 pg. HA30 refers to a strain-specific hemagglutinin dose of 30 pg. HA45 refers to a strain-specific hemagglutinin dose of 45 pg. HA60 refers to a strain-specific hemagglutinin dose of 60 pg. rS15 refers to a CoV S polypeptide dose of 15 pg. rS25 refers to a CoV S polypeptide dose of 25 pg. rS35 refers to a dose of 35 pg of CoV S polypeptide.

[0038] Figs. 28A-B show the geometric mean HAI titers against influenza strains, A / Wisconsin (H1N1) (Fig. 28A), A / Darwin (H3N2) (Fig. 28B), B / Victoria (Fig. 28A) and B / Yamagata (Fig. 28B), for patients who Petition 870250108300, dated 11 / 26 / 2025, p. 17 / 284 10 / 210 received the compositions from Example 8. M50 refers to an adjuvant saponin dose of 50 μg. M75 refers to an adjuvant saponin dose of 75 μg. HA30 refers to a hemagglutinin dose per strain of 30 μg. HA45 refers to a hemagglutinin dose per strain of 45 μg. HA60 refers to a hemagglutinin dose per strain of 60 μg. rS15 refers to a CoV S polypeptide dose of 15 μg. rS25 refers to

[0039] Fig. 29 shows the anti-Spike IgG and neutralizing antibody titer (in geometric mean ELISA units) of compositions F and K from Example 8. Composition F contained 30 μg per strain of HA, 25 μg of CoV S polypeptide, and 75 μg of saponin adjuvant. Composition K contained 60 μg per strain of HA, 35 μg of CoV S polypeptide, and 75 μg of saponin adjuvant. These compositions induce titers comparable to a composition containing a CoV S polypeptide of SEQ ID NO: 87 (NVX-CoV2373) and without hemagglutinin protein.

[0040] Fig. 30 shows 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 who received Compositions F and K of Example 8 compared with those in the serum of patients who received commercial influenza vaccines (FLUZONE® HD or FLUAD®). Composition F contained 30 μg per strain of HA, 25 μg of CoV S polypeptide, and 75 μg of saponin adjuvant. Composition K contained 60 μg per strain of HA, 35 μg of CoV S polypeptide, and 75 pg of saponin adjuvant. These compositions induce titers comparable to a composition containing a CoV S polypeptide with SEQ ID NO: 87 (NVX-CoV2373) and without hemagglutinin.

[0041] Fig. 31 shows the HAI titers induced by commercially available influenza vaccines, FLUZONE® HD (Composition S of Example 8) and FLUAD® (Composition T of Example 8) and by compositions L, M and N of Example 8, which contained HA glycoprotein and saponin adjuvant, but no CoV S polypeptide. DETAILED DESCRIPTION OF THE INVENTION Petition 870250108300, dated 11 / 26 / 2025, p. 18 / 284 11 / 210 Definitions

[0042] As used in this document and the accompanying claims, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a protein may refer to a protein or to mixtures of such a protein, and reference to the method includes reference to equivalent steps and / or methods known to those skilled in the art, and so forth.

[0043] As used in this document, the term adjuvant refers to a compound that, when used in combination with an immunogen, enhances or otherwise alters or modifies the immune response induced against the immunogen. Modification of the immune response may include enhancing or broadening the specificity of one or both of the antibodies and cellular immune responses.

[0044] As used in this document, the term about or approximately, when preceding a numerical value, indicates the value plus or minus a range of 10%. For example, about 100 covers 90 and 110.

[0045] As used in this document, the terms immunogen, antigen, and epitope refer to substances such as proteins, including glycoproteins, and peptides that are capable of inducing an immune response.

[0046] As used in this document, an immunogenic composition is a composition comprising an antigen wherein administration of the composition to a subject results in the development in the subject of a humoral and / or cellular immune response to the antigen.

[0047] As used in this document, a subunit composition, for example, a vaccine, which includes one or more selected antigens, but not all antigens of a pathogen. Such a composition is substantially free of intact virus or lysate of such cells or particles and is typically prepared from at least partially purified immunogenic polypeptides, often substantially purified from the pathogen. The antigens in the subunit composition disclosed in this Petition 870250108300, dated 11 / 26 / 2025, page 19 / 284 12 / 210 documents are typically prepared in a recombinant manner, often using a baculovirus system.

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

[0049] The terms treat, treatment and treating, as used in this document, refer to an approach to obtain beneficial or desired results, for example, clinical results. For the purposes of this disclosure, beneficial or desired results may include inhibiting or suppressing the initiation or progression of an infection or disease; improving or reducing the development of symptoms of an infection or disease; or a combination thereof.

[0050] Prevention, as used in this document, is used interchangeably with prophylaxis and may mean complete prevention of an infection or disease, or prevention of the development of symptoms of that infection or disease; a delay in the onset of an infection or disease or its symptoms; or a decrease in the severity of a subsequently developed infection or disease or its symptoms.

[0051] As used in this document, an effective dose or effective amount refers to a quantity of an immunogen sufficient to induce an immune response that reduces at least one symptom of pathogen infection. An effective dose or effective amount can be determined, for example, by measuring amounts of neutralizing secretion antibodies and / or serum, for example, by plaque neutralization, complement fixation, Petition 870250108300, dated 11 / 26 / 2025, p. 20 / 284 13 / 210 enzyme-linked immunosorbent assay (ELISA) or microneutralization assay.

[0052] As used in this document, the term vaccine refers to an immunogenic composition, such as an immunogen derived from a pathogen, that is used to induce an immune response against the pathogen. The protective immune response may include antibody formation and / or a cell-mediated response. Depending on the context, the term vaccine may also refer to a suspension or solution of an immunogen that is administered to a subject to produce an immune response. Preferably, vaccines induce an immune response that is effective in preventing infection by SARS-CoV-2, a variant of SARS-CoV-2, influenza, or a combination thereof.

[0053] As used in this document, the term subject includes humans and other animals. Typically, the subject is a human. For example, the subject may be an adult, an adolescent, a child (2 to 14 years old), an infant (from birth to 2 years old), or a newborn (up to 2 months old). In particular aspects, the subject is up to 4 months old or up to 6 months old. In aspects, adults are elderly, about 65 years or older, or about 60 years or older. In aspects, the subject is a pregnant woman or a woman intending to become pregnant. In other aspects, the subject is not a human; for example, a non-human primate; for example, a baboon, a chimpanzee, a gorilla, or a monkey. In certain aspects, the subject may be a pet, such as a dog or a cat.

[0054] In aspects, the subject is immunocompromised. In modalities, the immunocompromised subject receives a medication that causes immunosuppression. Non-limiting examples of medications that cause immunosuppression include corticosteroids (e.g., prednisone), alkylating agents (e.g., cyclophosphamide), antimetabolites (e.g., azathioprine or 6-mercaptopurine), transplant-related immunosuppressive drugs (e.g., cyclosporine, tacrolimus, sirolimus, or mycophenolate mofetil), mitoxantrone, chemotherapeutic agents, methotrexate, agents Petition 870250108300, dated 11 / 26 / 2025, page 21 / 284 14 / 210 tumor necrosis factor (TNF) blockers (e.g., etanercept, adalimumab, infliximab). In some modalities, the immunocompromised subject is infected with a virus (e.g., human immunodeficiency virus or Epstein-Barr virus). In some modalities, the virus is a respiratory virus, such as respiratory syncytial virus, influenza, parainfluenza, adenovirus, or a picornavirus. In some modalities, the immunocompromised subject has acquired immunodeficiency syndrome (AIDS). In some modalities, the immunocompromised subject is a person living with human immunodeficiency virus (HIV). In some modalities, the immunocompromised subject is immunocompromised due to a treatment regimen designed to prevent inflammation or prevent transplant rejection. In some modalities, the immunocompromised subject is a subject who has received a transplant. In some modalities, the immunocompromised subject has undergone radiotherapy or splenectomy. In some modalities,The immunocompromised subject has been diagnosed with cancer, an autoimmune disease, tuberculosis, a substance use disorder (e.g., an alcohol, opioid, or cocaine use disorder), stroke or cerebrovascular disease, a solid organ or blood stem cell transplant, sickle cell disease, thalassemia, autoimmune lymphoproliferative syndrome (ALPS), autoimmune polyglandular syndrome type 1 (APS-1), B-cell expansion with NF-κB and T-cell anergy disease (BENTA), caspase-eight deficiency state (CEDS), chronic granulomatous disease (CGD), common variable immunodeficiency (CVID), congenital neutropenia syndromes, a deficiency in cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), a DOCK8 deficiency, a GATA2 deficiency, a glycosylation disorder with immunodeficiency, a hyperimmunoglobulin E syndrome (HIES), hyperimmunoglobulin M syndrome, diabetes, type 2 diabetes 1, type 2 diabetes,Interferon gamma deficiency, interleukin 12 deficiency, interleukin 23 deficiency, leukocyte adhesion deficiency, lipopolysaccharide-responsive beige-type anchor (LRBA) deficiency, PI3 kinase disease, PLCG2-associated antibody and immune dysregulation deficiency (PLAID), immunodeficiency. Petition 870250108300, dated 11 / 26 / 2025, page 22 / 284 Severe combined immune disease (SCID), STAT3 dominant-negative disease, STAT3 gain-of-function disease, warts, hypogammaglobulinemia, infections and myelocathexis syndrome (WHIM), Wiskott-Aldrich syndrome (WAS), X-linked agammaglobulinemia (XLA), X-linked lymphoproliferative disease (XLP), uremia, malnutrition, or XMEN disease. In some cases, the immunocompromised subject is currently or was previously a cigarette smoker. In others, the immunocompromised subject has a B-cell defect, T-cell defect, macrophage defect, cytokine defect, phagocyte deficiency, phagocyte dysfunction, complement deficiency, or a combination thereof.

[0055] In modalities, the subject is overweight or obese. In modalities, an overweight subject has a body mass index (BMI) > 25 kg / m2 and < 30 kg / m2. In modalities, an obese subject has a BMI that is > 30 kg / m2. In modalities, the subject has a mental health condition. In modalities, the mental health condition is depression, schizophrenia, or anxiety.

[0056] As used in this document, the term pharmaceutically acceptable means approved by a Federal or State Government regulatory agency or listed in the U.S. Pharmacopoeia, European Pharmacopoeia, or other generally recognized pharmacopoeia for use in mammals and, more particularly, in humans. These compositions may be useful as a vaccine and / or antigenic compositions to induce a protective immune response in a vertebrate.

[0057] As used in this document, the term about means approximately 10% of the indicated numerical value.

[0058] As used in this document, the term NVX-CoV2373 refers to a vaccine composition comprising the Spike glycoprotein BV2373 (SEQ ID NO: 87) and the Fraction A and Fraction C iscom matrix (e.g., MATRIX-MTM).

[0059] As used in this document, Quad-NIV, QuadNIV or Petition 870250108300, dated 11 / 26 / 2025, page 23 / 284 16 / 210 quadrivalent influenza nanoparticle vaccine or qNIV refers to influenza vaccine formulations containing antigens from four strains of influenza virus.

[0060] As used in this document, the term modification, as it refers to a CoV S polypeptide, refers to the mutation, deletion, or addition of one or more amino acids to the CoV S polypeptide. The location of a modification within a CoV S polypeptide can be determined by aligning the polypeptide sequence to SEQ ID NO: 1 (CoV S polypeptide containing a signal peptide) or SEQ ID NO: 2 (mature CoV S polypeptide without a signal peptide).

[0061] The term SARS-CoV-2 variant, used interchangeably in this document with a heterogeneous strain of SARS-CoV-2, refers to a SARS-CoV-2 virus comprising a CoV S polypeptide with one or more modifications compared to a SARS-CoV S polypeptide with 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 a CoV S polypeptide with 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 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to Petition 870250108300, dated 11 / 26 / 2025, p. 24 / 284 17 / 210 18, up to 19, up to 20, up to 21, up to 22, up to 23, up to 24, up to 25, up to 26, up to 27, up to 28, up to 29, up to 30, up to 31, up to 32, up to 33, up to 34, up to 35 modifications, up to 40 modifications, up to 45 modifications, up to 50 modifications, up to 55 modifications, up to 60 modifications, up to 65 modifications, up to 70 modifications, up to 75 modifications, up to 80 modifications, up to 85 modifications, up to 90 modifications, up to 95 modifications or up to 100 modifications compared to a CoV S polypeptide with the amino acid sequence SEQ ID NO: 2.In terms of aspects, a SARS-CoV-2 variant can have between approximately 2 and approximately 35 modifications, between approximately 5 and approximately 10 modifications, between approximately 5 and approximately 20 modifications, between approximately 10 and approximately 20 modifications, between approximately 15 and approximately 25 modifications, between approximately 20 and 30 modifications, between approximately 20 and approximately 40 modifications, between approximately 25 and approximately 45 modifications, between approximately 25 and approximately 100 modifications, between approximately 25 and approximately 45 modifications, between approximately 35 and approximately 100 modifications, compared to a CoV S polypeptide with the amino acid sequence SEQ ID NO: 2.

[0062] In embodiments, the heterogeneous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide with 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 a CoV S polypeptide having the amino acid sequence SEQ ID NO: 2. In embodiments, the heterogeneous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide with between about 70% and about 99.9% identity with a CoV S polypeptide having the amino acid sequence SEQ ID NO: 2. In embodiments, the heterogeneous SARS-CoV-2 strain is a SARS-CoV-2 virus. comprising a CoV S polypeptide with between approximately 70% and approximately 99.5% identity to a CoV S polypeptide with the amino acid sequence of SEQ ID NO: 2. In embodiments, the heterogeneous SARS-CoV-2 strain is a SARS-CoV-2 virus. Petition 870250108300, dated 11 / 26 / 2025, p. 25 / 284 18 / 210 comprising a CoV S polypeptide with between about 90% and about 99.9% identity to a CoV S polypeptide with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In embodiments, the heterogeneous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide with between about 90% and about 99.8% identity to a CoV S polypeptide with the amino acid sequence of SEQ ID NO: 2. In embodiments, the heterogeneous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide with between about 95% and about 99.9% identity to a CoV S polypeptide with the amino acid sequence of SEQ ID NO: 2. In embodiments, the heterogeneous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide with between about 95% and approximately 99.8% identity with a CoV S polypeptide with the amino acid sequence SEQ ID NO: 2.In embodiments, the heterogeneous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide with between about 95% and about 99% identity to a CoV S polypeptide with the amino acid sequence SEQ ID NO: 2. In embodiments, the heterogeneous SARS-CoV-2 strain has a World Health Organization Label of alpha, beta, gamma, delta, epsilon, eta, iota, kappa, zeta, mu, or omicron. In embodiments, the heterogeneous 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 document describes the designation of the Pango lineage and is incorporated by reference into this document in its entirety: O'Toole et al. BMC Genomics, 23, 121 (2022).

[0063] In embodiments, the heterogeneous SARS-CoV-2 strain has a World Health Organization label of omicron. In embodiments, the heterogeneous SARS-CoV-2 strain with a World Health Organization label of omicron has at least 35 modifications compared to the wild-type SARS-CoV-2 S polypeptide of SEQ ID NO: 2. In embodiments, the strain Petition 870250108300, dated 11 / 26 / 2025, page 26 / 284 19 / 210 heterogeneous SARS-CoV-2 with a World Health Organization label of omicron has 35 to 55, 35 to 65, 35 to 75, 35 to 85, 35 to 95 or 35 to 105 modifications compared to the wild-type SARS-CoV-2 S polypeptide of SEQ ID NO: 2.In terms of modifications, the models are selected from the group consisting of T6I, T6R, A14S, A54V, V70A, T82I, G129D, H133Q, K134E, W139R, E143G, F144L, Q170E, I197V, L199I, V200E, V200G, G239V, G244S, G326D, G326H, R333T, L355I, S358F, S358L, S360P, S362F, T363A, D392N, R395S, K404N, N427K, K431T, V432P, G433S, L439R, L439Q, N447K, S464N, T465K, E471A, F473V, F473S, F477S, Q480R, G483S, Q485R, N488Y, Y492H, T534K, T591I, D601G, G626V, H642Y, N645S, N666K, P668H, S691L, N751K, D783Y, N843K, Q941H, N956K, L968F, D1186N, amino acid deletion 11, amino acid deletion 12, amino acid deletion 13, amino acid deletion 56, amino acid deletion 57, amino acid deletion 130, amino acid deletion 131, amino acid deletion 132, amino acid deletion 144, amino acid deletion 145, deletion of amino acid 198 and insertion of a tripeptide with the EPE amino acid sequence between amino acids 214 and 215 and combinations thereof.

[0064] In embodiments, the CoV S polypeptide of the variant comprises a combination of selected modifications from the group consisting of: (i) A54V, T82I, G129D, L199I, G326D, S358L, S360P, S362F, K404N, N427K, G433S, S464N, T465K, E471A, Q480R, G483S, Q485R, N488Y, Y492H, T534K, D601G, H642Y, N666K, P668H, N751K, D783Y, N843K, Q941H, N956K, L968F, amino acid 56 deletion, amino acid 57 deletion, amino acid 130 deletion, amino acid 131 deletion, amino acid 132 deletion, amino acid 198 deletion and insertion of a tripeptide with the EPE amino acid sequence 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, amino acid 11 deletion, amino acid 12 deletion and amino acid 11 deletion Petition 870250108300, dated 11 / 26 / 2025, p. 27 / 284 20 / 210 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 with the amino acid sequence of 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, amino acid 11 deletion, amino acid 12 deletion and amino acid 13 deletion; (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, amino acid 11 deletion, amino acid 12 deletion and amino acid 13 deletion; (vi) T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, D601G, H642Y, N645S, N666K, N751K, D783Y, Q941H, N956K, amino acid 11 deletion, amino acid 12 deletion and amino acid 13 deletion, amino acid 56 deletion and amino acid 57 deletion; (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, amino acid 11 deletion, amino acid 12 deletion, amino acid 13 deletion, amino acid 56 deletion, and amino acid 57 deletion; (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, amino acid 11 deletion, amino acid 12 deletion, amino acid 13 deletion, amino acid 56 deletion, and amino acid 57 deletion; Petition 870250108300, dated 11 / 26 / 2025, p. 28 / 284 21 / 210 (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, amino acid 11 deletion, amino acid 12 deletion, 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, amino acid 11 deletion, amino acid 12 deletion and amino acid 13 deletion; (xi) T6I, A14S, G129D, K134E, W139R, F144L, I197V, V200G, G244S, G326H, R333T, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, G433S, L439R, N447K, S464N, T465K, E471A, F473S, Q485R, N488Y, Y492H, T591I, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, D1186N, amino acid 11 deletion, amino acid 12 deletion, and amino acid 13 deletion; (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, amino acid 11 deletion, amino acid 12 deletion, amino acid 13 deletion, amino acid 56 deletion and amino acid 57 deletion; (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, amino acid 11 deletion, amino acid 12 deletion, amino acid 13 deletion, amino acid 56 deletion, and amino acid 57 deletion; (xiv) T6I, A14S, V70A, G129D, H133Q, Q170E, V200E, G239V, G326H, R333T, L355I, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, V432P, G433S, N447K, S464N, T465K, E471A, F473S, F477S, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, Petition 870250108300, dated 11 / 26 / 2025, p. 29 / 284 22 / 210 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, S362F, T363A, D392N, R395S, K404N, N427K, V432P, G433S, N447K, S464N, T465K, E471A, F473S, F477S, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, amino acid 11 deletion, amino acid 12 deletion, amino acid 13 deletion, 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, amino acid 11 deletion, amino acid 12 deletion, amino acid 13 deletion, amino acid 56 deletion and amino acid 57 deletion; (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, amino acid 11 deletion, amino acid 12 deletion, amino acid 13 deletion, amino acid 56 deletion and amino acid 57 deletion; 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, amino acid 11 deletion, amino acid 12 deletion, amino acid 13 deletion, amino acid 56 deletion, amino acid 57 deletion, and amino acid 131 deletion; (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; Petition 870250108300, dated 11 / 26 / 2025, p. 30 / 284 23 / 210 (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) amino acid 144 deletion, amino acid 145 deletion, T6R, G129D, E143G, W245I, K404N, N426K, L439R, T465K, E471K, N488Y, D601G, P668R and D937N; (xxx) amino acid 144 deletion, amino acid 145 deletion, 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) Q39R, A54V, E471K; D601G, Q664H, F875L and deletion of 1, 2, 3 or 4 of amino acids 56, 57, 131, 132; (xxxiii) T82I, D240G, E471K, D601G and A688V; (xxxiv) L439R, E471Q, D601G, P668R and Q1058H; (xxxv) G62V, T63I, R233N, L439Q, F477S, D601G, T846N and deletion of 1, 2, 3, 4, 5 or 6 of amino acids 234-240; (xxxvi) T82I, Y131S, Y132N, R333K, E471K, N488Y, D601G, P668H and D937N; and (xxxvii) G129D, G326D, S360P, S362F, K404N, N427K, T465K, E471A Petition 870250108300, dated 11 / 26 / 2025, p. 31 / 284 24 / 210 or E471K, Q480K or Q480R, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H and N953K; where the amino acids of the CoV S glycoprotein are numbered relative to a polypeptide with the sequence SEQ ID NO: 2.

[0065] A subject who is positive for SARS-CoV-2 or a variant thereof has a positive serological or PCR test for SARS-CoV-2 or a variant thereof. A positive PCR test detects genetic material from SARS-CoV-2 or a variant thereof. A positive serological test shows the presence of antibodies against a SARS-CoV-2 protein, typically the SARS-CoV-2 nucleocapsid protein or a variant thereof.

[0066] The term asymptomatic refers to a subject who is positive for SARS-CoV-2 or a variant of SARS-CoV-2 thereof, but does not experience any symptoms of COVID-19.

[0067] The term mild, as it relates to COVID-19, refers to a subject who has a positive serological or PCR test for SARS-CoV-2 or a variant thereof and has one or more of the following symptoms: (i) fever; (ii) recent onset cough; (iii) or two additional COVID-19 symptoms selected from recent onset or worsening shortness of breath or difficulty breathing; fatigue; generalized muscle or body aches; headache; loss of taste or smell; sore throat, congestion or runny nose; or nausea, vomiting or diarrhea.

[0068] The term moderate, as it relates to COVID-19, refers to a subject who has a positive serological or PCR test for SARS-CoV-2 or a variant thereof and one or more of the following symptoms: (i) a high fever of > 38.4°C for three or more days; (ii) any evidence of significant lower respiratory tract infection (LRTI), wherein the evidence is selected from: (a) shortness of breath with or without exertion; (b) tachypnea (24 to 29 breaths per minute at rest); (c) SpO2 of 94% to 95%; (d) an abnormal chest X-ray or computed tomography (CT) scan consistent with pneumonia or LRTI; or (e) adventitious sounds on lung auscultation (e.g., crackles / rattles, wheezes, rhonchi, pleural flush, stridor). Petition 870250108300, dated 11 / 26 / 2025, p. 32 / 284 25 / 210

[0069] The term severe, as it relates to COVID-19, refers to a subject who has a positive serological or PCR test for SARS-CoV-2 or a variant thereof and one or more of the following symptoms: (i) tachypnea of ​​> 30 breaths per minute at rest; (ii) resting heart rate of > 125 beats per minute; (iii) SpO2 of < 93% or PaO2 / FiO2 < 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 bilevel positive airway pressure (BiPAP)); (v) need for mechanical ventilation or extracorporeal membrane oxygenation (ECMO); (vi) one or more major organ system dysfunctions or failures selected from (a) acute respiratory failure, including acute respiratory distress syndrome (ARDS); (b) acute renal failure; (c) acute liver failure;(d) acute right or left heart failure; (e) septic or cardiogenic 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, such as acute myocardial infarction (AMI), deep vein thrombosis (DVT), or pulmonary embolism (PE); (h) a requirement for vasopressors, systemic corticosteroids, or hemodialysis; (vii) admission to an intensive care unit; or (viii) death.

[0070] As used in this document, the terms bedside mixture, bedside formulations, bedside vaccine compositions, bedside vials, bedside vial formulations refer to vaccine formulations that are prepared immediately before administration. Such vaccine formulations contain viral antigens and adjuvants that are stored separately in different containers and are administered to a subject (e.g., by administering two consecutive injections or by combining the antigens and adjuvants into one injection before administration). Petition 870250108300, dated 11 / 26 / 2025, page 33 / 284 26 / 210

[0071] As used in this document, the terms co-formulation mixture, co-formulation, co-formulation vaccine compositions, pre-filled syringes, premix refer to vaccine formulations that are prepared for short- to long-term storage prior to the time of administration to a subject. Such vaccine formulations contain a combination of antigens and adjuvant in the same container and are prepared prior to administration. In embodiments, the formulations containing hemagglutinin and adjuvant (e.g., saponin adjuvant) form HaSMaNs (Hemagglutinin Saponin Matrix Nanoparticles).

[0072] The term efficacy of an immunogenic composition or vaccine composition described in this document refers to the percentage reduction in disease (e.g., COVID-19) in a group that received an immunogenic composition compared with a group that did not receive the immunogenic composition. In embodiments, efficacy (E) is calculated using the following equation: E (%) = (1 - RR) χ 100, where RR = relative risk of incidence rates between the group that received the immunogenic composition and the group that did not receive the immunogenic composition.In embodiments, the immunogenic compositions described in this document have an efficacy against a SARS-CoV-2 virus or heterogeneous SARS-CoV-2 strain that 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%, at least about 99%, between about 50% and about 99%, between about 50% and about 98%, between about 60% and about 99%, between approximately 60% and approximately 98%, between approximately 70% and approximately 98%, between approximately 70% and approximately 95%, between approximately 70% and approximately 99%, between approximately 80% and approximately 99%, between approximately 80% and approximately 98%, between approximately 80% and approximately 95%, between approximately 85% and approximately 99%, in between. Petition 870250108300, dated 11 / 26 / 2025, p. 34 / 284 27 / 210 approximately 85% and approximately 98%, between approximately 85% and approximately 95%, between approximately 90% and approximately 95%, between approximately 90% and 98%, or between approximately 90% and approximately 99%.

[0073] As used in this document, the term split virion refers to a virus (e.g., an influenza virus or a SARS-CoV-2 virus) that has a viral membrane that has been disrupted by a surfactant. Examples of surfactants are described throughout this disclosure. Split virions do not undergo further purification, so they typically contain multiple viral proteins.

[0074] As used in this document, the term recombinant refers to a protein (e.g., hemagglutinin) that is produced in a cell by transcription and translation of a nucleic acid that is introduced into a cell. The nucleic acid may be introduced by a vector or a virus that encodes the nucleic acid.

[0075] As used in this document, the term whole influenza virus refers to a virus comprising its entire envelope, viral membrane, nucleocapsid, and genetic material. In embodiments, the whole influenza virus is inactivated.

[0076] As used in this document, the term inactivated virus refers to a virus that has been treated to substantially reduce or eliminate its virulence compared to the wild-type virus. Immunogenic Compositions Immunogenic compositions against Influenza Viruses

[0077] Immunogenic compositions and vaccine compositions containing (i) at least three hemagglutinin (HA) glycoproteins, wherein the three hemagglutinin (HA) glycoproteins are from different influenza strains and (ii) a pharmaceutically acceptable buffer, are provided in this document. In embodiments, the at least three HA glycoproteins are in a form selected from the group consisting of (a) detergent core nanoparticles comprising hemagglutinin (HA); (b) HaSMaNs (Hemagglutinin Matrix Nanoparticles) Petition 870250108300, dated 11 / 26 / 2025, page 35 / 284 28 / 210 (a) Hemagglutinin saponin); (b) an inactivated whole influenza virus; (c) a hemagglutinin composition extracted from an influenza virus; optionally, an influenza split virion composition or an influenza subunit composition; and any combination thereof. In embodiments, methods are provided in this document for using the immunogenic compositions and vaccine compositions mentioned above to stimulate an immune response against an influenza virus. In embodiments, the compositions comprise four HA glycoproteins, wherein each HA glycoprotein is from a different strain.

[0078] In embodiments, the compositions comprise from about 25 pg to about 75 pg of hemagglutinin per strain. In embodiments, the compositions comprise from about 30 pg to about 60 pg of hemagglutinin per strain.In embodiments, the compositions comprise about 30 μg, about 31 μg, about 32 μg, about 33 μg, about 34 μg, about 35 μg, about 36 μg, about 37 μg, about 38 μg, about 39 μg, about 40 μg, about 41 μg, about 42 μg, about 43 μg, about 44 μg, about 45 μg, about 46 μg, about 47 μg, about 48 μg, about 49 μg, about 50 μg, about 51 μg, about 52 μg, about 53 μg, about 54 μg, about 55 μg, about 56 μg, about 57 μg, about 58 μg, about 59 μg, about 60 μg, about 61 μg, about 62 μg, about 63 μg, about 64 μg, about 65 μg, 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, or about 75 μg of hemagglutin per strain, including all values ​​and subranges in between. In some modalities, the compositions comprise approximately 30 pg of hemagglutinin per strain.In embodiments, the compositions comprise from approximately 45 pg of hemagglutinin per strain. In embodiments, the compositions comprise from approximately 60 pg of hemagglutinin per strain.

[0079] In embodiments, the compositions also include any pharmaceutically acceptable buffer described in this document. In Petition 870250108300, dated 11 / 26 / 2025, p. 36 / 284 In embodiments, the compositions also include any adjuvant described in this document. In embodiments, the compositions include a saponin adjuvant. In embodiments, the saponin adjuvant comprises at least two iscom particles, wherein: the first iscom particle comprises fraction A of Quillaja Saponaria Molina and not fraction C of Quillaja Saponaria Molina; and the second iscom particle comprises fraction C of Quillaja Saponaria Molina and not fraction A of Quillaja Saponaria Molina. In embodiments, fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina represent approximately 92% by weight and approximately 8% by weight, respectively, of the sum of the weights of fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina in the adjuvant.In embodiments, fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina represent approximately 85% by weight and approximately 15% by weight, respectively, of the sum of the weights of fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina in the adjuvant. In embodiments, fraction A of Quillaja Saponaria Molina represents at least 85% by weight of the adjuvant and fraction C of Quillaja Saponaria represents the remainder by weight of the adjuvant. In embodiments, fraction A of Quillaja Saponaria Molina represents at least 70% by weight of the adjuvant and fraction C of Quillaja Saponaria represents the remainder by weight of the adjuvant. In embodiments, the adjuvant is present in the composition in an amount of 30 pg to approximately 100 pg.In terms of modalities, the compositions comprise approximately 30 pg, approximately 31 pg, approximately 32 pg, approximately 33 pg, approximately 34 pg, approximately 35 pg, approximately 36 pg, approximately 37 pg, approximately 38 pg, approximately 39 pg, approximately 40 pg, approximately 41 pg, approximately 42 pg, approximately 43 pg, approximately 44 pg, approximately 45 pg, approximately 46 pg, approximately 47 pg, approximately 48 pg, approximately 49 pg, approximately 50 pg, approximately 51 pg, approximately 52 pg, approximately 53 pg, approximately 54 pg, approximately 55 pg, approximately 56 pg, approximately 57 pg, approximately 58 pg, approximately 59 pg, about 60 pg, about 61 pg, about 62 pg, about 63 pg, about 64 pg, about 65 pg, about 66 pg, about 67 pg, about 68 pg, about 69 pg, about 70 pg, about 71 pg, about 72 pg,. Petition 870250108300, dated 11 / 26 / 2025, page 37 / 284 30 / 210 about 73 pg, about 74 pg, about 75 pg, about 76 pg, about 77 pg, about 78 pg, about 79 pg, about 80 pg, about 81 pg, about 82 pg, about 83 pg, about 84 pg, about 85 pg, about 86 pg, about 87 pg, about 88 pg, about 89 pg, about 90 pg, about 91 pg, about 92 pg, about 93 pg, about 94 pg, about 95 pg, about 96 pg, about 97 pg, about 98 pg, about 99 pg or about 100 pg of adjuvant. In some formulations, approximately 50 pg or 75 pg of adjuvant are present in the composition. Immunogenic compounds against Coronavirus and Influenza Virus

[0080] Immunogenic compositions and vaccine compositions containing (i) at least three hemagglutinin (HA) glycoproteins, wherein the three hemagglutinin (HA) glycoproteins are from different influenza strains; (ii) a CoV S polypeptide in the form of a detergent core nanoparticle, wherein the detergent is a non-ionic detergent; 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) detergent core nanoparticles comprising hemagglutinin (HA); (b) HaSMaNs (Hemagglutinin Saponin Matrix Nanoparticles); (c) an inactivated whole influenza virus; (d) a hemagglutinin composition extracted from an influenza virus; optionally, an influenza split virion composition or an influenza subunit composition; and any combination thereof.In embodiments, methods are provided in this document for using the immunogenic compositions and vaccine compositions mentioned above to stimulate an immune response against a coronavirus, an influenza virus, or a combination thereof.

[0081] This document also provides methods for manufacturing the aforementioned nanoparticles and immunogenic compositions. Advantageously, the methods provide nanoparticles that are substantially free from contamination by other proteins, such as proteins associated with recombinant protein expression in insect cells. In Petition 870250108300, dated 11 / 26 / 2025, page 38 / 284 In 31 / 210 modalities, the expression occurs in baculovirus / Sf9 systems. (i) non-naturally occurring CoV S nanoparticles or polypeptides

[0082] The immunogenic compositions of the disclosure contain unnaturally occurring CoV S nanoparticles or polypeptides comprising the same. The CoV S polypeptides may be derived from coronaviruses, including, but not limited to, SARS-CoV-2, for example, from SARS-CoV2, MERS CoV and SARS CoV. In embodiments, the immunogenic compositions of the disclosure contain from about 1 to about 15, from about 2 to about 15, from about 3 to about 15, from about 3 to about 12, from about 4 to about 6, from about 3 to about 7, from about 4 to about 12, from about 5 to about 8, or from about 6 to about 9 unnaturally occurring CoV S polypeptides.

[0083] In embodiments, the CoV S polypeptide is from a heterogeneous strain of SARS-CoV-2. In embodiments, the heterogeneous strain of SARS-CoV-2 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 heterogeneous strain of SARS-CoV-2 has a World Health Organization label of alpha, beta, gamma, delta, epsilon, eta, iota, kappa, zeta, mu or omicron.

[0084] In embodiments, the SARS-CoV-2 virus has a CoV S polypeptide with the amino acid sequence of SEQ ID NO: 1 and the SARS-CoV-2 variant comprises a CoV S polypeptide with 1-100 modifications compared to the CoV S polypeptide of SEQ ID NO: 1. In embodiments, the SARS-CoV-2 virus has a CoV S polypeptide with the amino acid sequence of SEQ ID NO: 1 and the SARS-CoV-2 variant comprises a CoV S polypeptide with 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, Petition 870250108300, dated 11 / 26 / 2025, p. 39 / 284 32 / 210 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, 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, at least about 50, at least about 51, at least about 52, at least about 53, at least about 54,at least about 55, at least about 56, at least about 57, at least about 58, at least about 59, at least about 60, at least about 61, at least about 62, at least about 63, at least about 64, at least about 65, at least about 66, at least about 67, at least about 68, at least about 69, at least about 70, at least about 71, at least about 72, at least about 73, at least about 74, at least about 75, at least about 76, 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 approximately 95, at least approximately 96, at least approximately 97, at least approximately 98, at least approximately 99, or at least approximately 100 modifications compared to SEQ ID NO: 1. In, Petition 870250108300, dated 11 / 26 / 2025, p. 40 / 284 33 / 210 forms, the SARS-CoV-2 virus has a CoV S polypeptide with the amino acid sequence SEQ ID NO: 1 and the SARS-CoV-2 variant comprises a CoV S polypeptide with 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, up to about 22, up to about 23, up to about 24, up to about 25, up to about 26, up to about 27, up to about 28, until about 29, until about 30, until about 31, until about 32, until about 33, until about 34, until about 35, until about 36, until about 37, until about 38, until about 39, until about 40, until about 41, until about 42, until about 43, until about 44, until about 45, until about 46, until about 47, until about 48,until about 49, until about 50, until about 51, until about 52, until about 53, until about 54, until about 55, until about 56, until about 57, until about 58, until about 59, until about 60, until about 61, until about 62, until about 63, until about 64, until about 65, until about 66, until about 67, until about 68, until about 69, until about 70, until about 71, until about 72, until about 73, until about 74, until about 75, until about 76, until about 77, until about 78, until about 79, until about 80, until about 81, until about 82, until about 83, until 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 compared to SEQ ID NO: 1.,

[0085] In contrast to the SARS-CoV S protein, the SARS-CoV-2 S protein has a four-amino acid insertion 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) that is proteolytically cleaved at the furin cleavage site in Petition 870250108300, dated 11 / 26 / 2025, p. 41 / 284 34 / 210 S1 and S2 subunits that remain non-covalently linked to form prefusion trimers. The S2 domain of the SARS-CoV-2 S protein comprises a fusion peptide (FP), two heptad repeats (HR1 and HR2), a transmembrane domain (TM), and a cytoplasmic tail (CT). The S1 domain of the SARS-CoV-2 S protein folds into four distinct domains: the N-terminal domain (NTD) and the C-terminal domain, which contains the receptor-binding domain (RBD) and two subdomains SD1 and SD2. The prefusion trimers of the SARS-CoV-2 S protein undergo a structural rearrangement from a pre-fusion to a post-fusion conformation upon binding and cleavage of the S protein receptor.

[0086] In embodiments, the CoV S polypeptides are glycoproteins, due to post-translational glycosylation. The glycoproteins comprise one or more of a signal peptide, an S1 subunit, an S2 subunit, an NTD, an RBD, two subdomains (SD1 and SD2, labeled SD1 / 2 in Figs. 6A-B and referred to as SD1 / 2 in this document), an intact or modified fusion peptide, an HR1 domain, an HR2 domain, a TM, and a CD. In embodiments, the amino acids for each domain are given in Fig. 6A (shown according to SEQ ID NO: 1) and Fig. 6B (shown according to SEQ 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 the sequences for each domain as in SEQ ID NO: 1 or SEQ ID NO: 2. Each domain may have a deletion, insertion, or mutation of 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 20, up to about 30, up to about 35, up to about 40, up to about 45, or up to about 50 amino acids compared with those shown in SEQ ID NO: 1 or SEQ ID NO: 2. Each domain can have a deletion, insertion, or mutation between approximately 1 and approximately 5 amino acids, between approximately 3 and approximately 10 amino acids, between approximately 5 and 10 amino acids, or between approximately 8 and 12 amino acids. Petition 870250108300, dated 11 / 26 / 2025, p. 42 / 284 35 / 210 amino acids, between about 10 and 15 amino acids, between about 12 and 17 amino acids, between about 15 and 20 amino acids, between about 18 and 23 amino acids, between about 20 and 25 amino acids, between about 22 and about 27 amino acids, or between about 25 and 30 amino acids compared to those shown in SEQ ID NO: 1 or SEQ ID NO: 2. Note that Figs. 2 and 3 illustrate the 13-amino acid N-terminal signal peptide that is absent from the mature peptide. CoV S polypeptides can be used to stimulate immune responses against the native CoV spike (S) polypeptide.

[0088] In embodiments, the native CoV Spike (S) polypeptide (SEQ ID NO: 2) is modified resulting in non-naturally occurring CoV Spike (S) polypeptides

[0089] In embodiments, the native CoV Spike (S) polypeptide (SEQ ID NO: 2) is modified resulting in unnaturally occurring CoV Spike (S) polypeptides (Fig. 1). In embodiments, the CoV Spike (S) glycoproteins comprise an S1 subunit and an S2 subunit, wherein the S1 subunit comprises an NTD, an RBD, an SD1 / 2 and an inactive furin cleavage site (amino acids 669-672) and wherein the S2 subunit comprises amino acid mutations 973 and 974; where NTD (amino acids 1-318) optionally comprises one or more selected modifications of 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) mutation 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; wherein the RBD optionally comprises the mutation of one or more amino acids selected from the group consisting of amino acids 333, 404, Petition 870250108300, dated 11 / 26 / 2025, p. 43 / 284 36 / 210 419, 426, 439, 440, 464, 465, 471, 477, 481, 488 and combinations thereof; wherein the SD1 / 2 domain optionally comprises a mutation of one or more amino acids selected from the group consisting of 557, 600, 601, 642, 664, 668 and combinations thereof; and wherein the S2 subunit optionally comprises one or more modifications selected from the group consisting of: (a) deletion of one or more amino acids from 676-685, 676-702, 702-711, 775-793, 806-815 and combinations thereof; (b) mutation 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 domain (TMCT) (amino acids 1201-1260), wherein the amino acids of the CoV S glycoprotein are numbered relative to SEQ ID NO: 2.

[0090] In embodiments, the CoV S polypeptides described in this document exist in a pre-fusion conformation. In embodiments, the CoV S polypeptides described in this document comprise a flexible HR2 domain. Unless otherwise stated, the flexibility of a domain is determined by transition electron microscopy (TEM) and 2D class averaging. A reduction in electron density corresponds to a flexible domain. Modifications to the S1 subunit

[0091] In embodiments, the CoV S polypeptides contain one or more modifications in the S1 subunit with an amino acid sequence of SEQ ID NO: 121.

[0092] The amino acid sequence of the S1 subunit (SEQ ID NO: 121) is shown below. QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSN VTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQ SLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTF Petition 870250108300, dated 11 / 26 / 2025, page 44 / 284 37 / 210 EYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSA LEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFL LKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFP NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVS PTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWN SNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYF PLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFN FNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVS VITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAG CLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRAR

[0093] In embodiments, the CoV S polypeptides described in this document comprise an S1 subunit with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the S1 subunit of SEQ ID NO: 1 or SEQ ID NO: 2. The S1 subunit may have a deletion, insertion, or mutation of 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 acids 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 a deletion, insertion, or mutation between approximately 1 and approximately 5 amino acids, between approximately 3 and approximately 10 amino acids, between approximately 5 and 10 amino acids, between approximately 8 and 12 amino acids, between approximately 10 and 15 amino acids, between approximately 12 and 17 amino acids, between approximately 15 and 20 amino acids, between approximately 18 and 23 amino acids, between approximately 20 and 25 amino acids, between approximately 22 and approximately 27 amino acids, or between approximately 25 and 30 amino acids compared to the S1 subunit at SEQ ID NO: 1 or SEQ ID NO: 2.

[0094] In embodiments, the S1 subunit may contain any combination of modifications shown in Table 1A. Table 1A Petition 870250108300, dated 11 / 26 / 2025, p. 45 / 284 38 / 210 Modifications in S1 (SEQ ID NO: 121) * amino acids 14-685 of SEQ ID NO: 1 and amino acids 1-672 of SEQ ID NO: 2 Position in SEQ ID NO: 1 Position in SEQ ID NO: 2 Position in SEQ ID NO: 121 Potential Modifications 14-305 1-292 1-292 • deletion of up to approximately 1, 2, 3, 4, 5, 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 18 5 5 • mutation to phenylalanine • mutation to tyrosine • mutation to tryptophan 19 6 6 • mutation to arginine • mutation to lysine • mutation to histidine • mutation to isoleucine 20 7 7 • mutation to asparagine • mutation to glutamine • mutation to isoleucine • mutation to valine 24 11 11 • mutation to serine • mutation to threonine • deletion 25 12 12 • insertion of proline-proline-alanine (PPA) amino acids after amino acid 25 • deletion 26 13 13 • mutation to serine • mutation to threonine • deletion 27 14 14 • mutation to serine • mutation to threonine 52 39 39 • mutation to arginine • mutation to lysine, Petition 870250108300, dated 11 / 26 / 2025, page 46 / 284 39 / 210 Modifications in S1 (SEQ ID NO: 121) * amino acids 14-685 of SEQ ID NO: 1 and amino acids 1-672 of SEQ ID NO: 2 Position in SEQ ID NO: 1 Position in SEQ ID NO: 2 Position in SEQ ID NO: 121 Potential Modifications • mutation to histidine 64 51 51 • mutation to histidine • mutation to lysine • mutation to arginine 66 53 53 • mutation to tryptophan • mutation to tyrosine • mutation to phenylalanine 67 54 54 • mutation to valine • mutation to isoleucine • mutation to leucine 69 56 56 • Amino acid deletion 70 57 57 • Amino acid deletion • Mutation to phenylalanine • Mutation to tyrosine • Mutation to tryptophan 75 62 62 • Mutation to valine • Mutation • Mutation for leucine • Mutation for isoleucine 76 63 63 • Mutation for isoleucine • Mutation for valine • Mutation for leucine 80 67 67 • Mutation for alanine • Mutation for glycine 83 70 70 • Mutation for alanine 95 82 82 • Mutation for branched beta amino acid • Mutation for isoleucine • Mutation for valine 138 125 125 • Mutation forTyrosine mutation • mutation to phenylalanine • mutation to tryptophan Petition 870250108300, dated 11 / 26 / 2025, page 47 / 284 40 / 210 Modifications in S1 (SEQ ID NO: 121) * amino acids 14-685 of SEQ ID NO: 1 and amino acids 1-672 of SEQ ID NO: 2 Position in SEQ ID NO: 1 Position in SEQ ID NO: 2 Position in SEQ ID NO: 121 Potential Modifications 142 129 129 • mutation to aspartic acid • mutation to glutamic acid 143 130 130 • amino acid deletion 144 131 131 • Amino acid deletion • Mutation to serine 145 132 132 • Amino acid deletion • Mutation to histidine • Mutation to asparagine • Mutation to glutamine 146 133 133 • mutation to aromatic amino acid • mutation to tyrosine • mutation to phenylalanine • mutation to tryptophan • mutation to glutamine 147 134 134 • mutation for glutamic acid • mutation for aspartic acid 152 139 139 • mutation for cysteine ​​• mutation for methionine • mutation for serine • mutation for threonine • mutation for arginine • mutation for lysine 156 143 143 • mutation for glycine • mutation for alanine 157 144 144 • amino acid deletion • mutation for leucine • mutation forisoleucine • mutation to valine • mutation to beta-branched amino acid 158 145 145 • amino acid deletion Petition 870250108300, dated 11 / 26 / 2025, page 48 / 284 41 / 210 Modifications in S1 (SEQ ID NO: 121) * amino acids 14-685 of SEQ ID NO: 1 and amino acids 1-672 of SEQ ID NO: 2 Position in SEQ ID NO: 1 Position in SEQ ID NO: 2 Position in SEQ ID NO: 121 Potential Modifications 180 167 167 • mutation to beta-branched amino acid • mutation to valine • mutation to isoleucine • mutation to leucine 183 170 170 • mutation to glutamic acid • mutation to aspartic acid 190 177 177 • mutation to serine • mutation to threonine • mutation to cysteine ​​210 197 197 • mutation to valine • mutation to isoleucine • mutation to leucine • mutation to beta-branched amino acid 211 198 198 • mutation to isoleucine • mutation to valine • mutation to leucine • mutation for branched beta amino acid • deletion of amino acid 212 199 199 • mutation for isoleucine • mutation for valine • mutation for leucine • mutation for branched beta amino acid • deletion of amino acid 213 200 200 • mutation for valine • mutation for leucine • mutation for isoleucine •mutation for beta amino acid Petition 870250108300, dated 11 / 26 / 2025, page 49 / 284 42 / 210 Modifications in S1 (SEQ ID NO: 121) * amino acids 14-685 of SEQ ID NO: 1 and amino acids 1-672 of SEQ ID NO: 2 Position in SEQ ID NO: 1 Position in SEQ ID NO: 2 Position in SEQ ID NO: 121 Potential Modifications branched • mutation to proline • mutation to glycine • mutation to glutamic acid • mutation to aspartic acid 214 201 201 • mutation to arginine • mutation to lysine • mutation to histidine • mutation to aspartic acid • mutation to glutamic acid • insertion of glutamic acid-proline-glutamic acid (EPE) amino acids after 214 215 202 202 • mutation to glycine • mutation to alanine • insertion of glutamic acid-proline-glutamic acid (EPE) amino acids after 215 222 209 209 • mutation to valine • mutation to leucine • mutation to isoleucine 241-244 228-231 228-231 • deletion of 1,23 or 4 amino acids 242 229 229 • mutation to histidine • mutation to lysine • mutation to arginine 246 233 233 • mutation to branched-chain beta amino acid • mutation to isoleucine • mutation to valine • mutation to threonine • mutation to asparagine, Petition 870250108300, dated 11 / 26 / 2025, page 50 / 284 43 / 210 Modifications in S1 (SEQ ID NO: 121) * amino acids 14-685 from SEQ ID NO: 1 and amino acids 1-672 from SEQ ID NO: 2 Position in SEQ ID NO: 1 Position in SEQ ID NO: 2 Position in SEQ ID NO: 121 Potential Modifications 247 234 234 • amino acid deletion 248 235 235 • amino acid deletion 249 236 236 • amino acid deletion 250 237 237 • amino acid deletion 251 238 238 • amino acid deletion 252 239 239 • amino acid deletion • mutation to valine • mutation to leucine • mutation to isoleucine • mutation to branched beta amino acid 253 240 240 • mutation to glycine • amino acid deletion 257 244 244 • mutation to serine • mutation to threonine • mutation to asparagine • mutation to glutamine 258 245 245 • mutation to isoleucine • mutation to valine • mutation to leucine • mutation to branched beta amino acid 339 326 326 • mutation to aspartic acid • mutation to glutamic acid • mutation to histidine 346 333 333 • mutation to lysine • mutation to arginine • mutation tohistidine • mutation to threonine • mutation to serine 368 355 355 • mutation to isoleucine Petition 870250108300, dated 11 / 26 / 2025, page 51 / 284 44 / 210 Modifications in S1 (SEQ ID NO: 121) * amino acids 14-685 of SEQ ID NO: 1 and amino acids 1-672 of SEQ ID NO: 2 Position in SEQ ID NO: 1 Position in SEQ ID NO: 2 Position in SEQ ID NO: 121 Potential Modifications • mutation to leucine • mutation to valine • mutation to beta-branched amino acid 371 358 358 • mutation to leucine • mutation to isoleucine • mutation to valine • mutation to phenylalanine 373 360 360 • mutation to proline 375 362 362 • mutation to phenylalanine • mutation to tyrosine • mutation to tryptophan 376 363 363 • mutation to alanine • mutation to glycine 405 392 392 • mutation to asparagine • mutation to glutamine 408 395 395 • mutation to serine • mutation to threonine 417 404 404 • mutation to asparagine • mutation to threonine • mutation to isoleucine • mutation to valine • mutation to serine • mutation to glutamine • mutation to beta-branched amino acid 432 419 419 • mutation to lysine • mutation to arginine • mutation to histidine 439 426426 • mutation for lysine • mutation for arginine Petition 870250108300, dated 11 / 26 / 2025, page 52 / 284 45 / 210 Modifications in S1 (SEQ ID NO: 121) * amino acids 14-685 of SEQ ID NO: 1 and amino acids 1-672 of SEQ ID NO: 2 Position in SEQ ID NO: 1 Position in SEQ ID NO: 2 Position in SEQ ID NO: 121 Potential Modifications • mutation to histidine 440 427 427 • mutation to lysine • mutation to arginine • mutation to histidine 444 431 431 • mutation to threonine • mutation to serine 445 432 432 • mutation to proline 446 433 433 • mutation to serine • mutation to threonine • mutation to asparagine • mutation to glutamine 452 439 439 • mutation to arginine • mutation to lysine • mutation to histidine • mutation to glutamine • mutation to asparagine 453 440 440 • mutation to phenylalanine • mutation to tryptophan 460 447 447 • mutation to lysine • mutation to arginine 477 464 464 • mutation to asparagine • mutation to glutamine 478 465 465 • mutation to lysine • mutation to arginine • mutation to histidine 484 471 471 • mutation to alanine • mutation to lysine • mutation toarginine • mutation to histidine • mutation to glutamine • mutation to asparagine Petition 870250108300, dated 11 / 26 / 2025, page 53 / 284 46 / 210 Modifications in S1 (SEQ ID NO: 121) * amino acids 14-685 of SEQ ID NO: 1 and amino acids 1-672 of SEQ ID NO: 2 Position in SEQ ID NO: 1 Position in SEQ ID NO: 2 Position in SEQ ID NO: 121 Potential Modifications 486 473 473 • mutation to valine • mutation to leucine • mutation to isoleucine • mutation to serine • mutation to threonine • mutation to proline 490 477 477 • mutation to serine • mutation to threonine 493 480 480 • mutation to lysine • mutation to arginine • mutation to histidine 494 481 481 • mutation to proline 496 483 483 • mutation to serine • mutation to threonine 498 485 485 • mutation to lysine • mutation to arginine • Histidine mutation 501 488 488 • Tyrosine mutation • Phenylalanine mutation • Tryptophan mutation 505 492 492 • Histidine mutation 521 508 508 • Serine mutation • Threonine mutation 547 534 534 • Lysine mutation • Arginine mutation • Histidine mutation 570 557 557 • Aspartic acid mutationfor glutamic acid 604 591 591 • mutation for isoleucine • mutation for leucine • mutation for valine Petition 870250108300, dated 11 / 26 / 2025, page 54 / 284 47 / 210 Modifications in S1 (SEQ ID NO: 121) * amino acids 14-685 of SEQ ID NO: 1 and amino acids 1-672 of SEQ ID NO: 2 Position in SEQ ID NO: 1 Position in SEQ ID NO: 2 Position in SEQ ID NO: 121 Potential Modifications • mutation for branched beta amino acid 613 600 600 • mutation for histidine • mutation for lysine • mutation for arginine 614 601 601 • mutation for glycine • mutation for alanine 639 626 626 • mutation for valine • mutation for leucine • mutation for isoleucine 655 642 642 • mutation for tyrosine • mutation for phenylalanine • mutation for tryptophan 658 645 645 • mutation for serine • mutation for threonine 677 664 664 • mutation for histidine 679 666 666 • mutation to lysine • mutation to arginine • mutation to histidine 681 668 668 • mutation to histidine • mutation to lysine • mutation to arginine 682-685 669-672 669-672 • inactive furin cleavage site (see Table 1E) Modifications to the S1-NTD subunit

[0095] In embodiments, the CoV S polypeptides contain one or more modifications in NTD. In embodiments, NTD has an 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 an NTD (SEQ ID NO: 118) is Petition 870250108300, dated 11 / 26 / 2025, p. 55 / 284 48 / 210 shown below. QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSN VTWFHAIHySGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQ SLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTF EYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSA LEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFL LKYNENGTITDAVDCALDPLSETKCTLKS

[0097] The underlined regions of SEQ ID NO: 118 represent amino acids within the NTD that can be modified.

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

[0099] QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLP FFSNVTWFHAIHySGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLD SKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSAN NCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQ GFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQP RTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIV RFPN

[0100] In modalities, NTD and RBD overlap by up to about 1 amino acid, up to about 5 amino acids, up to about 10 amino acids, or up to about 20 amino acids.

[0101] In embodiments, an NTD, as provided in this document, may be extended at the C-terminus by up to 5, up to 10, up to 15, up to 20, up to 25, or up to 30 amino acids.

[0102] In the embodiments, the CoV S polypeptides described in this document comprise an NTD with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the NTD of SEQ ID NO: 1 or SEQ ID NO: 2. The NTD may have a Petition 870250108300, dated 11 / 26 / 2025, p. 56 / 284 49 / 210 deletion, an insertion or mutation of 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 acids compared to the amino acid sequence of the NTD from SEQ ID NO: 1 or SEQ ID NO: 2. The NTD may have a deletion, insertion or mutation between about 1 and about 5 amino acids, between about 3 and about 10 amino acids, between about 5 and 10 amino acids, between about 8 and 12 amino acids, between 12 and 17 amino acids, between 18 and 23 amino acids, between about 10 and 15, between about 15 and 20, between about 20 and 25 amino acids, between about 22 and about 27 amino acids, or between about 25 and 25. 30 amino acids compared to SEQ ID NO: 1 or SEQ ID NO: 2 NTD.

[0103] In embodiments, the CoV S polypeptides contain a 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 polypeptides contain a deletion of up to about 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 embodiments, the CoV S polypeptides contain a deletion of one or more amino acids from the NTD (corresponding to amino acids 1-318 of SEQ ID NO: 2). In embodiments, the CoV S polypeptides contain a deletion of amino acids 1-318 from the NTD of SEQ ID NO: 2. In embodiments, the NTD deletion enhances the expression of the CoV Spike (S) polypeptide protein. In embodiments, the CoV S polypeptides that have an NTD deletion have amino acid sequences represented by SEQ ID NO: 46, 48, 49, 51, 52, and 54. In embodiments, the CoV S polypeptides that have an NTD deletion are encoded by an isolated nucleic acid sequence selected from the group consisting of SEQ ID NO: 47, SEQ ID NO: 50, and SEQ ID NO: 53.

[0105] In modalities, the NTD may contain any combination of modifications shown in Table 1B. Modifications are shown in Petition 870250108300, dated 11 / 26 / 2025, p. 57 / 284 50 / 210 in relation to SEQ ID NO: 2, the mature S polypeptide sequence for reference. Table 1B Modifications of NTD (SEQ ID NO: 118) *amino acids 14-305 of SEQ ID NO: 1 and amino acids 1-292 of SEQ ID NO: 2 Position in SEQ ID NO: 1 SEQ ID NO: 2 residue SEQ ID NO: 118 or SEQ ID NO: 45 residue Modifications 14-305 1-292 1-292 • deletion of up to approximately 1, 2, 3, 4, 5, 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 18 5 5 • mutation for phenylalanine • mutation for tyrosine • mutation for tryptophan 19 6 6 • mutation for arginine • mutation for lysine • mutation for histidine • mutation for isoleucine 20 7 7 • mutation for asparagine • mutation for glutamine • mutation for isoleucine • mutation for valine 24 11 11 • mutation for serine • mutation for threonine • deletion 25 12 12 • insertion of proline-proline-alanine (PPA) amino acids after amino acid 25 • deletion 26 13 13 • mutation for serine • mutation for threonine Petition 870250108300, dated 11 / 26 / 2025, page 58 / 284 51 / 210 Modifications of NTD (SEQ ID NO: 118) *amino acids 14-305 of SEQ ID NO: 1 and amino acids 1-292 of SEQ ID NO: 2 Position in SEQ ID NO: 1 SEQ ID NO: 2 residue SEQ ID NO: 118 or SEQ ID NO: 45 residue Modifications • deletion 27 14 14 • mutation to serine • mutation to threonine 52 39 39 • mutation to arginine • mutation to lysine • mutation to histidine 64 51 51 • mutation to histidine • mutation to lysine • mutation to arginine 66 53 53 • mutation to tryptophan • mutation to tyrosine • mutation to phenylalanine 67 54 54 • mutation to valine • mutation to isoleucine • mutation to leucine 69 56 56 • amino acid deletion 70 57 57 • amino acid deletion • mutation for phenylalanine • mutation for tyrosine • mutation for tryptophan 75 62 62 • mutation for valine • mutation for leucine • mutation for isoleucine 76 63 63 • mutation for isoleucine • mutation for valine • mutation for leucine 80 67 67 • mutation for alanine • mutation for glycine 83 70 70 • mutation for alanine 95 82 82 •mutation for beta amino acid Petition 870250108300, dated 11 / 26 / 2025, page 59 / 284 52 / 210 Modifications of NTD (SEQ ID NO: 118) *amino acids 14-305 of SEQ ID NO: 1 and amino acids 1-292 of SEQ ID NO: 2 Position in SEQ ID NO: 1 SEQ ID NO: 2 residue SEQ ID NO: 118 or SEQ ID NO: 45 residue Modifications branched • mutation to isoleucine • mutation to valine 138 125 125 • mutation to tyrosine • mutation to phenylalanine • mutation to tryptophan 142 129 129 • mutation to aspartic acid • mutation to glutamic acid 143 130 130 • amino acid deletion 144 131 131 • amino acid deletion • mutation to serine 145 132 132 • amino acid deletion • mutation to histidine • mutation to asparagine • mutation to glutamine 146 133 133 • mutation to aromatic amino acid • mutation to tyrosine • mutation to phenylalanine • mutation to tryptophan • mutation to glutamine • mutation to asparagine 147 134 134 • mutation to glutamic acid • mutation to aspartic acid 152 139 139 • mutation to cysteine ​​• mutation to methionine • mutation to serine • mutation to threonine •mutation for arginine • mutation for lysine Petition 870250108300, dated 11 / 26 / 2025, page 60 / 284 53 / 210 Modifications of NTD (SEQ ID NO: 118) *amino acids 14-305 of SEQ ID NO: 1 and amino acids 1-292 of SEQ ID NO: 2 Position in SEQ ID NO: 1 SEQ ID NO: 2 residue SEQ ID NO: 118 or SEQ ID NO: 45 residue Modifications 156 143 143 • mutation to glycine • mutation to alanine 157 144 144 • amino acid deletion • mutation to leucine 158 145 145 • amino acid deletion 180 167 167 • mutation to isoleucine • mutation to valine • mutation to leucine • mutation to branched beta amino acid 183 170 170 • mutation to glutamic acid • mutation to aspartic acid 190 177 177 • mutation to serine • mutation to threonine • mutation to cysteine ​​210 197 197 • mutation to valine • mutation to isoleucine • mutation to leucine • mutation to branched beta amino acid 211 198 198 • mutation to isoleucine • mutation to valine • mutation to leucine • mutation to branched beta amino acid • amino acid deletion 212 199 199 • mutation to isoleucine • mutation to valine • mutation to leucine •mutation for beta amino acid Petition 870250108300, dated 11 / 26 / 2025, page 61 / 284 54 / 210 Modifications of NTD (SEQ ID NO: 118) *amino acids 14-305 of SEQ ID NO: 1 and amino acids 1-292 of SEQ ID NO: 2 Position in SEQ ID NO: 1 SEQ ID NO: 2 residue SEQ ID NO: 118 or SEQ ID NO: 45 residue Modifications branched • amino acid deletion 213 200 200 • mutation to valine • mutation to leucine • mutation to isoleucine • mutation to branched beta amino acid • mutation to proline • mutation to glycine • mutation to glutamic acid • mutation to aspartic acid 214 201 201 • mutation to arginine • mutation to lysine • mutation to histidine • mutation to aspartic acid • mutation to glutamic acid • insertion of glutamic acid-proline-glutamic acid (EPE) amino acids after 214 215 202 202 • mutation to glycine • mutation to alanine • insertion of glutamic acid-proline-glutamic acid (EPE) amino acids after 215 222 209 209 • mutation to valine • mutation to leucine • mutation to isoleucine 241-244 228-231 228-231 • deletion of 1,2,3 or 4 amino acids 242 229 229 • mutation to histidine, Petition 870250108300, dated 11 / 26 / 2025, page 62 / 284 55 / 210 Modifications of NTD (SEQ ID NO: 118) *amino acids 14-305 of SEQ ID NO: 1 and amino acids 1-292 of SEQ ID NO: 2 Position in SEQ ID NO: 1 SEQ ID NO: 2 residue SEQ ID NO: 118 or SEQ ID NO: 45 residue Modifications • mutation to lysine • mutation to arginine 246 233 233 • mutation to beta-branched amino acid • mutation to isoleucine • mutation to valine • mutation to threonine • mutation to asparagine 247 234 234 • amino acid deletion 248 235 235 • amino acid deletion 249 236 236 • amino acid deletion 250 237 237 • amino acid deletion 251 238 238 • amino acid deletion 252 239 239 • amino acid deletion • mutation to valine • mutation to isoleucine • mutation to leucine • mutation to branched beta amino acid 253 240 240 • mutation to glycine • amino acid deletion 257 244 244 • mutation to serine • mutation to threonine 258 245 245 • mutation to isoleucine • mutation to valine • mutation to leucine • mutation to branched beta amino acid Modifications to the S1-RBD subunit

[0106] In embodiments, CoV S polypeptides contain one or more Petition 870250108300, dated 11 / 26 / 2025, p. 63 / 284 56 / 210 modifications to RBD.

[0107] In modalities, the RBD has an 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: NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKC YGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGC VIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEG FNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGP

[0109] The underlined regions of SEQ ID NO: 126 represent amino acids within the RBD subunit that can be modified.

[0110] In modalities, the RBD has an 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 (SEQ ID NO: 116) is shown below. LCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGV SPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAW NSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCY FPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKS

[0112] In embodiments, an RBD, as provided in this document, may be extended at the N-terminus or at the C-terminus 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.

[0113] In embodiments, the CoV S polypeptides described in this document comprise an RBD with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the RBD of SEQ ID NO: 1 or SEQ ID NO: 2. The RBD may have a deletion, insertion, or mutation of up to about 1, up to about 2, up to about Petition 870250108300, dated 11 / 26 / 2025, p. 64 / 284 57 / 210 of 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 acids compared to the amino acid sequence of the RBD of SEQ ID NO: 1 or SEQ ID NO: 2. The RBD may have a deletion, insertion, or mutation between about 1 and about 5 amino acids, between about 3 and about 10 amino acids, between about 5 and 10 amino acids, between about 8 and 12 amino acids, between about 10 and 15 amino acids, between about 12 and 17 amino acids, between about 15 and 20 amino acids, between about 18 and 23 amino acids, between about 20 and 25 amino acids, between about 22 and about 27 amino acids, or between about 25 and 30 amino acids compared to the RBD of SEQ ID NO: 1 or SEQ ID NO: 2.

[0114] In embodiments, the CoV S polypeptide has at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least one, at least one, at least one, at least one, at least one, at least one, at least one, at least one, at least one, at least one, at least one, at least one, at least one, or at least twenty mutations in the RBD. In embodiments, the RBD may contain any combination of modifications as shown in Table 1C. Table 1C Modifications in RBD (SEQ ID NO: 126) * amino acids 331-527 of SEQ ID NO: 1 and amino acids 318-514 of SEQ ID NO: 2 Position in SEQ ID NO: 1 Position in SEQ ID NO: 2 Position in SEQ ID NO: 126 Potential Modifications 339 326 9 • mutation to aspartic acid • mutation to glutamic acid • mutation to histidine 346 333 16 • mutation to lysine • mutation to arginine • mutation to histidine • mutation to threonine • mutation to serine Petition 870250108300, dated 11 / 26 / 2025, p. 65 / 284 58 / 210 Modifications in RBD (SEQ ID NO: 126) * amino acids 331-527 of SEQ ID NO: 1 and amino acids 318-514 of SEQ ID NO: 2 Position in SEQ ID NO: 1 Position in SEQ ID NO: 2 Position in SEQ ID NO: 126 Potential Modifications 368 355 38 • mutation to isoleucine • mutation to leucine • mutation to valine • mutation to beta-branched amino acid 371 358 41 • mutation to leucine • mutation to isoleucine • mutation to valine • mutation to phenylalanine • mutation to tyrosine • mutation to tryptophan 373 360 43 • mutation to proline 375 362 45 • mutation to phenylalanine • mutation to tyrosine • mutation to tryptophan 376 363 46 • mutation to alanine • mutation to glycine 405 392 75 • mutation to asparagine • mutation to glutamine 408 395 78 • mutation to serine • mutation to threonine 417 404 87 • mutation to asparagine • mutation to threonine • mutation to isoleucine • mutation to valine • mutation to serine • mutation to glutamine • mutation to beta-branched amino acid 432419 102 • mutation for lysine • mutation for arginine Petition 870250108300, dated 11 / 26 / 2025, page 66 / 284 59 / 210 Modifications in RBD (SEQ ID NO: 126) * amino acids 331-527 of SEQ ID NO: 1 and amino acids 318-514 of SEQ ID NO: 2 Position in SEQ ID NO: 1 Position in SEQ ID NO: 2 Position in SEQ ID NO: 126 Potential Modifications • mutation to histidine 439 426 109 • mutation to lysine • mutation to arginine • mutation to histidine 440 427 110 • mutation to lysine • mutation to arginine • mutation to histidine 444 431 114 • mutation to threonine • mutation to serine 445 432 115 • mutation to proline 446 433 116 • mutation to serine • mutation to threonine 452 439 122 • mutation to arginine • mutation to lysine • mutation to histidine • Glutamine mutation • Asparagine mutation 453 440 123 • Phenylalanine mutation • Tryptophan mutation 460 447 130 • Lysine mutation • Arginine mutation 477 464 147 • Asparagine mutation • Glutamine mutation 478 465 148 • Lysine mutation • Arginine mutation • Histidine mutation 484 471 154 • Alanine mutationmutation for lysine • mutation for arginine • mutation for histidine • mutation for glutamine Petition 870250108300, dated 11 / 26 / 2025, page 67 / 284 60 / 210 Modifications in RBD (SEQ ID NO: 126) * amino acids 331-527 of SEQ ID NO: 1 and amino acids 318-514 of SEQ ID NO: 2 Position in SEQ ID NO: 1 Position in SEQ ID NO: 2 Position in SEQ ID NO: 126 Potential Modifications • mutation to asparagine 486 473 156 • mutation to valine • mutation to leucine • mutation to isoleucine • mutation to serine • mutation to threonine 490 477 160 • mutation to serine • mutation to threonine 493 480 163 • mutation to lysine • mutation to arginine • mutation to histidine 494 481 164 • mutation to proline 496 483 166 • mutation to serine • mutation to threonine 498 485 168 • mutation to lysine • mutation for arginine • mutation for histidine 501 488 171 • mutation for tyrosine • mutation for phenylalanine • mutation for tryptophan 505 492 175 • mutation for histidine 521 508 191 • mutation for serine • mutation for threonine Modifications for SD1 / 2

[0115] In embodiments, the CoV S polypeptides contain one or more SD1 / 2 modifications with an 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 (SEQ ID NO: 122) is shown below. Petition 870250108300, dated 11 / 26 / 2025, p. 68 / 284 61 / 210 NFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCS FGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVF QTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSP

[0117] In embodiments, the CoV S polypeptides described in this document comprise an SD1 / 2 with 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 a deletion, insertion, or mutation of 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 acids compared to the amino acid sequence of the SD1 / 2 of SEQ ID NO: 1 or SEQ ID NO: 2.SD1 / 2 may have a deletion, insertion, or mutation between approximately 1 and approximately 5 amino acids, between approximately 3 and approximately 10 amino acids, between approximately 5 and 10 amino acids, between approximately 8 and 12 amino acids, between approximately 10 and 15 amino acids, between approximately 12 and 17 amino acids, between approximately 15 and 20 amino acids, between approximately 18 and 23 amino acids, between approximately 20 and 25 amino acids, between approximately 22 and approximately 27 amino acids, or between approximately 25 and 30 amino acids compared to SD1 / 2 in SEQ ID NO: 1 or SEQ ID NO: 2.

[0118] In embodiments, the CoV S polypeptide has at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least one, at least one, at least one, at least one, at least one, at least one, at least one, at least one, at least one, at least one, at least one, at least one, or at least twenty mutations in SD1 / 2. In embodiments, SD1 / 2 may contain any combination of modifications as shown in Table 1D. Table 1D Petition 870250108300, dated 11 / 26 / 2025, page 69 / 284 62 / 210 Modifications to SD1 / 2 (SEQ ID NO: 122) * amino acids 542-681 of SEQ ID NO: 1 or amino acids 529-668 of SEQ ID NO: 2 Position in SEQ ID NO: 1 Position in SEQ ID NO: 2 Position in SEQ ID NO: 122 Potential Modifications 547 534 6 • mutation to lysine • mutation to arginine • mutation to histidine 570 557 29 • mutation to aspartic acid • mutation to glutamic acid 604 591 63 • mutation to isoleucine • mutation to leucine • mutation to valine • mutation to beta-branched amino acid 613 600 72 • mutation to histidine • mutation to lysine • mutation to arginine 614 601 73 • mutation to glycine • mutation to alanine 639 626 98 • mutation to valine • mutation to leucine • mutation to isoleucine 655 642 114 • mutation to tyrosine • mutation to phenylalanine • mutation to tryptophan 658 645 117 • mutation to serine 677 664 136 • mutation to histidine 679 666 138 • mutation to lysine • mutation to arginine • mutation to histidine 681 668 140 • mutation to histidine •mutation for lysine • mutation for arginine 547 534 6 • mutation for lysine • mutation for arginine Petition 870250108300, dated 11 / 26 / 2025, page 70 / 284 63 / 210 Modifications to SD1 / 2 (SEQ ID NO: 122) * amino acids 542-681 of SEQ ID NO: 1 or amino acids 529-668 of SEQ ID NO: 2 Position in SEQ ID NO: 1 Position in SEQ ID NO: 2 Position in SEQ ID NO: 122 Potential Modifications • mutation to histidine 570 557 29 • mutation to aspartic acid • mutation to glutamic acid 613 600 72 • mutation to histidine • mutation to lysine • mutation to arginine 614 601 73 • mutation to glycine • mutation to alanine 655 642 114 • mutation to tyrosine • mutation to phenylalanine • mutation to tryptophan 677 664 136 • mutation to histidine 679 666 138 • mutation to lysine • mutation to arginine • mutation to histidine Modifications to the Furin Cleavage Site

[0119] In embodiments, the CoV S polypeptides contain a furin cleavage site (RRAR), which corresponds 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 embodiments, the CoV S polypeptides described in this document that contain an inactivated furin cleavage site are expressed as a single chain.

[0120] In embodiments, one or more of the amino acids comprising the native furin cleavage site are mutated into any natural amino acid. In embodiments, the amino acids are L-amino acids. Non-limiting examples of amino acids include alanine, arginine, glycine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, serine, threonine, histidine, lysine, methionine, Petition 870250108300, dated 11 / 26 / 2025, p. 71 / 284 64 / 210 proline, valine, isoleucine, leucine, tyrosine, tryptophan and phenylalanine.

[0121] In embodiments, one or more of the amino acids comprising the native furin cleavage site are mutated to glutamine. In embodiments, 1, 2, 3, or 4 amino acids may be mutated to glutamine. In embodiments, one of the arginines comprising the native furin cleavage site is mutated to glutamine. In embodiments, two of the arginines comprising the native furin cleavage site are mutated to glutamine. In embodiments, three of the arginines comprising the native furin cleavage site are mutated to glutamine.

[0122] In embodiments, one or more of the amino acids comprising the native furin cleavage site are mutated into alanine. In embodiments, 1, 2, 3, or 4 amino acids may be mutated into alanine. In embodiments, one of the arginines comprising the native furin cleavage site is mutated into alanine. In embodiments, two of the arginines comprising the native furin cleavage site are mutated into alanine. In embodiments, three of the arginines comprising the native furin cleavage site are mutated into alanine.

[0123] In embodiments, one or more of the amino acids comprising the native furin cleavage site are mutated to glycine. In embodiments, 1, 2, 3, or 4 amino acids may be mutated to glycine. In embodiments, one of the arginines of the native furin cleavage site is mutated to glycine. In embodiments, two of the arginines comprising the native furin cleavage site are mutated to glycine. In embodiments, three of the arginines comprising the native furin cleavage site are mutated to glycine.

[0124] In embodiments, one or more of the amino acids comprising the native furin cleavage site are mutated into asparagine. For example, 1, 2, 3, or 4 amino acids may be mutated into asparagine. In embodiments, one of the arginines comprising the native furin cleavage site is mutated into asparagine. In embodiments, two of the arginines comprising the native furin cleavage site are mutated into asparagine. In embodiments, three of the Petition 870250108300, dated 11 / 26 / 2025, p. 72 / 284 65 / 210 arginines comprising the native furin cleavage site are mutated into asparagine.

[0125] Non-limiting examples of the amino acid sequences of the inactivated furin sites contained in CoV S polypeptides are found in Table 1E. Table 1E Furin Cleavage Site Amino Acid Sequence Furin Cleavage Site Active or Inactive RRAR (SEQ ID NO: 6) Active QQAQ (SEQ ID NO: 7) Inactive QRAR (SEQ ID NO: 8) Inactive RQAR (SEQ ID NO: 9) Inactive RRAQ (SEQ ID NO: 10) Inactive QQAR (SEQ ID NO: 11) Inactive RQAQ (SEQ ID NO: 12) Inactive QRAQ (SEQ ID NO: 13) Inactive NNAN (SEQ ID NO: 14) Inactive NRAR (SEQ ID NO: 15) Inactive RNAR (SEQ ID NO: 16) Inactive RRAN (SEQ ID NO: 17) Inactive NNAR (SEQ ID NO: 18) Inactive RNAN (SEQ ID NO: 19) Inactive NRAN (SEQ ID NO: 20) Inactive AAAA (SEQ ID NO: 21) Inactive ARAR (SEQ ID NO: 22) Inactive RAAR (SEQ ID NO: 23) Inactive RRAA (SEQ ID NO: 24) Inactive AAAR (SEQ ID NO: 25) Inactive RAAA (SEQ ID NO: 26) Inactive Petition 870250108300, dated 11 / 26 / 2025, page 73 / 284 66 / 210 Furin Cleavage Site Amino Acid Sequence Active or Inactive Furin Cleavage Site ARAA (SEQ ID NO: 27) Inactive GGAG (SEQ ID NO: 28) Inactive GRAR (SEQ ID NO: 29) Inactive RGAR (SEQ ID NO: 30) Inactive RRAG (SEQ ID NO: 31) Inactive GGAR (SEQ ID NO: 32) Inactive RGAG (SEQ ID NO: 33) Inactive GRAG (SEQ ID NO: 34) Inactive GSAS (SEQ ID NO: 97) Inactive GSGA (SEQ ID NO: 111) Inactive

[0126] In embodiments, instead of an active furin cleavage site (SEQ ID NO: 6), the CoV S polypeptides described in this document contain an inactivated furin cleavage site. In embodiments, the amino acid sequence of the inactivated furin cleavage site is represented by either SEQ ID NO: 7-34 or SEQ ID NO: 97. In embodiments, the amino acid sequence of the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7). In embodiments, the amino acid sequence of the inactivated furin cleavage site is GSAS (SEQ ID NO: 97). In embodiments, the amino acid sequence of the inactivated furin cleavage site is GSGA (SEQ ID NO: 111). In some forms, 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). Modifications to the S2 subunit

[0127] In embodiments, the CoV S polypeptides contain one or more modifications in the S2 subunit with an 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. Petition 870250108300, dated 11 / 26 / 2025, pp. 74 / 284 67 / 210

[0128] The amino acid sequence of the S2 subunit (SEQ ID NO: 120) is shown below. SVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSV DCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIY KTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDI AARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFA MQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVV NQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTY VTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHG VVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYE PQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDL GDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFI AGLIAIVMTIMLCCMTSCCSCLKGCCSCCKFDEDDSEPVLKGVKLHYT

[0129] In embodiments, the CoV S polypeptides described in this document comprise an S2 subunit with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the S2 subunit of SEQ ID NO: 1 or SEQ ID NO: 2. The S2 subunit may have a deletion, insertion, or mutation of 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 acids 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 a deletion, insertion, or mutation between approximately 1 and approximately 5 amino acids, between approximately 3 and approximately 10 amino acids, between approximately 5 and 10 amino acids, between approximately 8 and 12 amino acids, between approximately 10 and 15 amino acids, between approximately 12 and 17 amino acids, between approximately 15 and 20 amino acids, between approximately 18 and 23 amino acids, between approximately 20 and 25 amino acids, between approximately 22 and approximately 27 amino acids, or between approximately 25 and 30 amino acids compared to the S2 subunit at SEQ ID NO: 1 or SEQ ID NO: 2.

[0130] In modalities, the S2 subunit may contain any Petition 870250108300, dated 11 / 26 / 2025, p. 75 / 284 68 / 210 combination of modifications shown in Table 1F. Table 1F Modifications in S2 (SEQ ID NO: 120) * amino acids 686-1273 from SEQ ID NO: 1 and amino acids 673-1260 from SEQ ID NO: 2 Position in SEQ ID NO: 1 Position in SEQ ID NO: 2 Position in SEQ ID NO: 120 Possible Modifications 689-698 676-685 4-13 • deletion of 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 or up to about 10 amino acids 701 688 16 • mutation to beta-branched amino acid • mutation to valine • mutation to isoleucine • mutation to threonine 704 691 19 • mutation to leucine • mutation to isoleucine • mutation to valine 715-724 702-711 30-39 • deletion of 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 or up to about 10 amino acids 716 703 31 • mutation to beta-branched amino acid • mutation to valine • mutation to isoleucine 764 751 79 • mutation to lysine • mutation to arginine • mutation to histidine, Petition 870250108300, dated 11 / 26 / 2025, page 76 / 284 69 / 210 Modifications in S2 (SEQ ID NO: 120) * amino acids 686-1273 of SEQ ID NO: 1 and amino acids 673-1260 of SEQ ID NO: 2 Position in SEQ ID NO: 1 Position in SEQ ID NO: 2 Position in SEQ ID NO: 120 Possible Modifications 788-806 775-793 103-121 • deletion of 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 or up to about 19 amino acids 796 783 111 • mutation for tyrosine • mutation for phenylalanine • mutation for tryptophan 819-828 806-815 134-143 • deletion of 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 or up to about 10 amino acids 856 843 171 • mutation to lysine • mutation to arginine • mutation to histidine 859 846 174 • mutation to asparagine • mutation to glutamine 888 875 203 • mutation to leucine • mutation to isoleucine • mutation to valine, Petition 870250108300, dated 11 / 26 / 2025, page 77 / 284 70 / 210 Modifications in S2 (SEQ ID NO: 120) * amino acids 686-1273 of SEQ ID NO: 1 and amino acids 673-1260 of SEQ ID NO: 2 Position in SEQ ID NO: 1 Position in SEQ ID NO: 2 Position in SEQ ID NO: 120 Possible Modifications 950 937 265 • mutation to asparagine • mutation to glutamine 954 941 269 • mutation to histidine • mutation to lysine • mutation to arginine 969 956 284 • mutation to lysine • mutation to arginine • mutation to histidine 981 968 296 • mutation to phenylalanine • mutation to tyrosine • mutation to tryptophan 982 969 297 • mutation to alanine • mutation to glycine • mutation to threonine 986 973 301 • mutation for proline • mutation for glycine 987 974 302 • mutation for proline • mutation for glycine 1027 1014 342 • mutation for isoleucine • mutation for valine • mutation for serine 1071 1058 386 • mutation for histidine • mutation for arginine • mutation for lysine 1118 1105 433 • mutation for histidine • mutation for lysine • mutation for arginine • mutation forasparagine Petition 870250108300, dated 11 / 26 / 2025, page 78 / 284 71 / 210 Modifications in S2 (SEQ ID NO: 120) * amino acids 686-1273 of SEQ ID NO: 1 and amino acids 673-1260 of SEQ ID NO: 2 Position in SEQ ID NO: 1 Position in SEQ ID NO: 2 Position in SEQ ID NO: 120 Possible Modifications • mutation for glutamine 1176 1163 491 • mutation for phenylalanine • mutation for tyrosine • mutation for tryptophan 1199 1186 514 • mutation for asparagine • mutation for glutamine 1214-1237 1201-1224 1-24 • deletion of one or more amino acids from TM 1238-1273 1225-1260 1-36 • deletion of one or more amino acids from CD

[0131] In embodiments, the CoV S polypeptides contain a deletion, corresponding to one or more deletions within amino acids 676-685 of the native CoV Spike (S) polypeptide (SEQ ID NO: 2). In embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from amino acids 676-685 of the native CoV Spike (S) polypeptide (SEQ ID NO: 2) are deleted. In embodiments, the amino acid deletions within amino acids 676-685 are consecutive, for example, amino acids 676 and 677 are deleted, or amino acids 680 and 681 are deleted. In embodiments, the amino acid deletions within amino acids 676-685 are non-consecutive, for example, amino acids 676 and 680 are deleted, or amino acids 677 and 682 are deleted. In embodiments, CoV S polypeptides containing a deletion, corresponding to one or more deletions within amino acids 676-685, have a selected amino acid sequence from the group consisting of SEQ ID NO: 62 and SEQ ID NO: 63. Petition 870250108300, dated 11 / 26 / 2025, p. 79 / 284 72 / 210

[0132] In embodiments, the CoV S polypeptides contain a deletion, corresponding to one or more deletions within amino acids 702-711 of the native CoV Spike (S) polypeptide (SEQ ID NO: 2). In embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from amino acids 702-711 of the native SARS-CoV-2 Spike (S) polypeptide (SEQ ID NO: 2) are deleted. In embodiments, the one or more amino acid deletions within amino acids 702-711 are consecutive, for example, amino acids 702 and 703 are deleted or amino acids 708 and 709 are deleted. In embodiments, the amino acid deletions within amino acids 702-711 are non-consecutive, for example, amino acids 702 and 704 are deleted or amino acids 707 and 710 are deleted. In embodiments, CoV S polypeptides containing a deletion, corresponding to one or more deletions within amino acids 702-711, have a selected amino acid sequence from the group consisting of SEQ ID NO: 64 and SEQ ID NO: 65.

[0133] In embodiments, the CoV S polypeptides contain a deletion, corresponding to one or more deletions within amino acids 775-793 of the native CoV S polypeptide (SEQ ID NO: 2). In embodiments, 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 native SARS-CoV-2 Spike (S) polypeptide (SEQ ID NO: 2) are deleted. In embodiments, the one or more amino acid deletions within amino acids 775-793 are consecutive, for example, amino acids 776 and 777 are deleted, or amino acids 780 and 781 are deleted. In some modalities, the amino acid deletions within amino acids 775-793 are non-consecutive; for example, amino acids 775 and 790 are deleted, or amino acids 777 and 781 are deleted.

[0134] In embodiments, the CoV S polypeptides contain a deletion of the fusion peptide (SEQ ID NO: 104), which corresponds to amino acids 806815 of SEQ ID NO: 2. In embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the fusion peptide of the CoV Spike (S) polypeptide (SEQ ID NO: 2) are deleted. In embodiments, amino acid deletions within the peptide of Petition 870250108300, dated 11 / 26 / 2025, page 80 / 284 In some embodiments, the fusion peptide deletions are consecutive, for example, amino acids 806 and 807 are deleted, or amino acids 809 and 810 are deleted. In others, the amino acid deletions within the fusion peptide are non-consecutive, for example, amino acids 806 and 808 are deleted, or amino acids 810 and 813 are deleted. In others, the CoV S polypeptides containing a deletion, corresponding to one or more amino acids of the fusion peptide, have an amino acid sequence selected from the SEQ ID NOS: 66, 77, and 105-108.

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

[0136] In embodiments, the CoV S polypeptides contain a mutation in Val-974 of the native CoV Spike (S) polypeptide (SEQ ID NO: 2). In embodiments, Val-974 is mutated to any natural amino acid. In embodiments, Val-974 is mutated to proline. In embodiments, Val-974 is mutated to glycine. In embodiments, the CoV S polypeptides containing a mutation in amino acid 974 are selected from the group consisting of SEQ ID NO: 8489, 105-106 and 109-110.

[0137] In embodiments, the CoV S polypeptides contain a mutation in Lys-973 and Val-974 of the native CoV Spike (S) polypeptide (SEQ ID NO: 2). In embodiments, Lys-973 and Val-974 are mutated in any natural amino acid. In embodiments, Lys-973 and Val-974 are mutated in proline. In embodiments, the CoV S polypeptides containing a mutation in amino acids 973 and 974 are selected from SEQ ID NOS: 84-89, 105-106 and 109-110. Modifications to the S2 subunit - HR1 domain

[0138] In embodiments, CoV S polypeptides contain one or more modifications in the HR1 domain with an amino acid sequence of SEQ ID Petition 870250108300, dated 11 / 26 / 2025, pp. 81 / 284 74 / 210 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. MAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQD VVNQNAQALNTLVKQLSSNFGAISSVLNDILSRL

[0140] In embodiments, the CoV S polypeptides described in this document comprise an HR1 domain with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the HR1 domain of SEQ ID NO: 1 or SEQ ID NO: 2. The HR1 domain may have a deletion, insertion, or mutation of 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 acids compared to the amino acid sequence of the HR1 domain of SEQ ID NO: 1 or SEQ ID NO: 2.The HR1 domain can have a deletion, insertion, or mutation between approximately 1 and approximately 5 amino acids, between approximately 3 and approximately 10 amino acids, between approximately 5 and 10 amino acids, between approximately 8 and 12 amino acids, between approximately 10 and 15 amino acids, between approximately 12 and 17 amino acids, between approximately 15 and 20 amino acids, between approximately 18 and 23 amino acids, between approximately 20 and 25 amino acids, between approximately 22 and approximately 27 amino acids, or between approximately 25 and 30 amino acids compared to the HR1 domain of SEQ ID NO: 1 or SEQ ID NO: 2.

[0141] In modalities, the HR1 domain can contain any combination of modifications as shown in Table 1G. Table 1G Modifications in HR1 (SEQ ID NO: 119) * amino acids 912-984 of SEQ ID NO: 1 and amino acids 889-971 of SEQ ID NO: 2) Position in SEQ ID NO: 1 Position in SEQ ID NO: 2 Position in SEQ ID NO: 119 Possible Modifications 950 937 49 • mutation to asparagine Petition 870250108300, dated 11 / 26 / 2025, page 82 / 284 75 / 210 • Glutamine mutation 954 941 53 • Histidine mutation 969 956 68 • Lysine mutation • Arginine mutation • Histidine mutation 981 968 80 • Phenylalanine mutation • Tyrosine mutation • Tryptophan mutation 982 969 81 • Alanine mutation • Glycine mutation • Threonine mutation Modifications to the S2 subunit - HR2 domain

[0142] In embodiments, the CoV S polypeptides contain one or more modifications in the HR2 domain with an 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. DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKW P

[0144] In embodiments, the CoV S polypeptides described in this document comprise an HR2 domain with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the HR2 domain of SEQ ID NO: 1 or SEQ ID NO: 2. The HR2 domain may have a deletion, insertion, or mutation of 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 acids 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 a deletion, insertion, or mutation between about 1 and about 5 amino acids, between about 3 and about 10 amino acids, between about 5 to 10 amino acids, between approximately 8 and 12 Petition 870250108300, dated 11 / 26 / 2025, pp. 83 / 284 76 / 210 amino acids, between about 10 and 15 amino acids, between about 12 and 17 amino acids, between about 15 and 20 amino acids, between about 18 and 23 amino acids, between about 20 and 25 amino acids, between about 22 and about 27 amino acids, or between about 25 and 30 amino acids compared to the HR2 domain of SEQ ID NO: 1 or SEQ ID NO: 2. Modifications to the TM domain

[0145] In embodiments, the CoV S polypeptides contain one or more modifications in the TM domain with an 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 polypeptides described in this document comprise a TM domain with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the TM domain of SEQ ID NO: 1 or SEQ ID NO: 2. The TM domain may have a deletion, insertion, or mutation of 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 acids 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 a deletion, insertion, or mutation between approximately 1 and approximately 5 amino acids, between approximately 3 and approximately 10 amino acids, between approximately 5 and 10 amino acids, between approximately 8 and 12 amino acids, between approximately 10 and 15 amino acids, between approximately 12 and 17 amino acids, between approximately 15 and 20 amino acids, between approximately 18 and 23 amino acids, between approximately 20 and 25 amino acids, between approximately 22 and approximately 27 amino acids, or between approximately 25 and 30 amino acids compared to the TM domain of SEQ ID NO: 1 or SEQ ID NO: 2.

[0148] In embodiments, the CoV S polypeptides described in this Petition 870250108300, dated 11 / 26 / 2025, pp. 84 / 284 77 / 210 documents do not have the entire TM domain. In other words, the CoV S polypeptides comprise the TM domain. Modifications to the CT

[0149] In embodiments, the CoV S polypeptides contain one or more CT modifications with an 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 polypeptides described in this document comprise a CT with 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 a deletion, insertion, or mutation of 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 acids compared to the amino acid sequence of the CT of SEQ ID NO: 1 or SEQ ID NO: 2.A CT may have a deletion, insertion, or mutation between approximately 1 and approximately 5 amino acids, between approximately 3 and approximately 10 amino acids, between approximately 5 and 10 amino acids, between approximately 8 and 12 amino acids, between approximately 10 and 15 amino acids, between approximately 12 and 17 amino acids, between approximately 15 and 20 amino acids, between approximately 18 and 23 amino acids, between approximately 20 and 25 amino acids, between approximately 22 and approximately 27 amino acids, or between approximately 25 and 30 amino acids compared to SEQ ID NO: 1 or SEQ ID NO: 2 CT.

[0152] In embodiments, the CoV S polypeptides described in this document do not have a CT. In embodiments, the CoV S polypeptides comprise CT.

[0153] In embodiments, CoV S polypeptides comprise a TM and Petition 870250108300, dated 11 / 26 / 2025, pp. 85 / 284 78 / 210 a CT. In embodiments, the Spike (S) polypeptides of CoV contain a deletion of one or more amino acids from the transmembrane cytoplasmic tail (TMCT) (corresponding to amino acids 1201-1260). The amino acid sequence of the TMCT is represented by SEQ ID NO: 39. In embodiments, the CoV S polypeptides that have a deletion of one or more TMCT residues have enhanced protein expression. In embodiments, CoV Spike (S) polypeptides that have one or more TMCT deletions have an amino acid sequence selected from the group consisting of SEQ ID NO: 40, 41, 42, 52, 54, 59, 61, 88, and 89. In embodiments, CoV S polypeptides that have one or more TM-CD deletions are encoded by an isolated nucleic acid sequence selected from the group consisting of SEQ ID NO: 39, 43, 53, and 60. Nanoparticles or polypeptides of non-naturally occurring CoV S, for example.

[0154] In embodiments, the CoV S polypeptides contain a deletion of amino acids 56 and 57 of the native CoV Spike (S) polypeptide (SEQ ID NO: 2).

[0155] In embodiments, the CoV S polypeptides contain deletions of amino acids 131 and 132 of the native CoV Spike (S) polypeptide (SEQ ID NO: 2).

[0156] In embodiments, the CoV S polypeptides contain a deletion of amino acids 56 and 131 from the native CoV Spike (S) polypeptide (SEQ ID NO: 2). In embodiments, the CoV S polypeptides contain a deletion of amino acids 57 and 131 from the native CoV Spike (S) polypeptide (SEQ ID NO: 2).

[0157] In embodiments, the CoV S polypeptides contain a deletion of amino acids 56, 57 and 131 of the native CoV Spike (S) polypeptide (SEQ ID NO: 2).

[0158] In embodiments, the CoV S polypeptides contain a deletion of amino acids 56 and 132 from the native CoV Spike (S) polypeptide (SEQ ID NO: 2).

[0159] In embodiments, the CoV S polypeptides contain a deletion of amino acids 57 and 132 from the native CoV Spike (S) polypeptide (SEQ ID NO: 2).

[0160] In some forms, the CoV S polypeptides contain a deletion of Petition 870250108300, dated 11 / 26 / 2025, page 86 / 284 79 / 210 amino acids 56, 57 and 132 of the native CoV Spike (S) polypeptide (SEQ ID NO: 2).

[0161] In embodiments, the CoV S polypeptides contain a deletion of amino acids 56, 57, 131 and 132 of the native CoV Spike (S) polypeptide (SEQ ID NO: 2).

[0162] In embodiments, CoV S polypeptides contain mutations that stabilize the pre-fusion conformation of the CoV S polypeptide. In embodiments, CoV S polypeptides contain proline or glycine substitutions that stabilize the pre-fusion conformation. This strategy was used to develop a pre-fusion stabilized MERS-CoV S protein, as described in the following documents, each of which is incorporated by reference herein in its entirety: Proc Natl Acad Sci USA. August 29, 2017;114(35): E7348-E7357; Sci Rep. October 24, 2018;8(1):15701; US ​​Publication 2020 / 0061185; and PCT Application No. PCT / US2017 / 058370.

[0163] In embodiments, the CoV S polypeptides contain a mutation in Lys-973 and Val-974 and an inactivated furin cleavage site. In embodiments, the CoV S polypeptides contain mutations of Lys-973 and Val-974 to proline and an inactivated furin cleavage site, with the amino acid sequence of QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96). An exemplary CoV S polypeptide containing a mutation in Lys-973 and Val-974 and an inactivated furin cleavage site is shown in Fig. 3. In embodiments, CoV S polypeptides containing mutations of Lys-973 and Val-974 to proline and an inactivated furin cleavage site have an amino acid sequence of SEQ ID NOS: 86 or 87 and a nucleic acid sequence of SEQ ID NO: 96.

[0164] In embodiments, the CoV S polypeptides contain a mutation in Lys-973 and Val-974, an inactivated furin cleavage site, and a deletion of one or more amino acids from the fusion peptide. In embodiments, the CoV S polypeptides contain mutations of Lys-973 and Val-974 to proline, an inactivated furin cleavage site with the amino acid sequence QQAQ. Petition 870250108300, dated 11 / 26 / 2025, p. 87 / 284 80 / 210 (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96) and deletion of one or more amino acids from the fusion peptide. In embodiments, the CoV S polypeptides contain Lys-973 and Val-974 mutations to proline, an inactivated furin cleavage site, and a deletion of one or more amino acids from the fusion peptide with an amino acid sequence of SEQ ID NO: 105 or 106. In embodiments, the CoV S polypeptide contains a Leu-5 mutation to phenylalanine, a Thr-7 mutation to asparagine, a Pro-13 mutation to serine, an Asp-125 mutation to tyrosine, an Arg-177 mutation to serine, a Lys-404 mutation to threonine, a Glu-471 mutation to lysine, an Asn-488 mutation to tyrosine, a His-642 mutation to tyrosine, a Thr-1014 mutation to isoleucine, Lys-973 and Val-974 mutations to proline, and an inactivated furin cleavage site with the sequence amino acid ratios of QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96) relative to the native CoV Spike (S) polypeptide (SEQ ID NO: 2).

[0165] In embodiments, the CoV S polypeptide contains a Trp-139 mutation for cysteine, a Leu-439 mutation for arginine, Lys-973 and Val-974 mutations for proline, and an inactivated furin cleavage site with the amino acid sequence of QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96) relative to the native CoV Spike (S) polypeptide (SEQ ID NO: 2). In embodiments, the CoV S polypeptide contains a Trp-152 mutation to cysteine, a Leu-452 mutation to arginine, a Ser-13 mutation to isoleucine, Lys-986 and Val-987 mutations to proline, and an inactivated furin cleavage site with the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96) relative to the native CoV Spike (S) polypeptide (SEQ ID NO: 1).

[0166] In embodiments, the CoV S polypeptide contains a Lys-404 mutation to threonine or asparagine, a Glu-471 mutation to lysine, an Asn-488 mutation to tyrosine, a Leu-5 mutation to phenylalanine, an Asp67 mutation to alanine, an Asp-202 mutation to glycine, a deletion of one or more amino acids 229-231, an Arg-233 ​​mutation to isoleucine, Lys973 and Val-974 mutations to proline, and a furin cleavage site inactivated with the Petition 870250108300, dated 11 / 26 / 2025, pp. 88 / 284 81 / 210 amino acid sequence of QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96) in relation to the native CoV Spike (S) polypeptide (SEQ ID NO: 2).

[0167] In embodiments, the CoV S polypeptide contains an Asn-488 mutation for tyrosine, Lys-973 and Val-974 mutations for proline, and an inactivated furin cleavage site with the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96) relative to the native CoV Spike (S) polypeptide (SEQ ID NO: 2). In embodiments, the CoV S polypeptide with an Asn-488 mutation for tyrosine, Lys-973 and Val-974 mutations for proline, and an inactivated furin cleavage site with the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96) comprises an amino acid sequence of SEQ ID NO: 112.

[0168] In embodiments, the CoV S polypeptide contains an Asp-601 mutation for glycine, an Asn-488 mutation for tyrosine, Lys973 and Val-974 mutations for proline, and an inactivated furin cleavage site with the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96) relative to the native CoV Spike (S) polypeptide (SEQ ID NO: 2). In embodiments, the CoV S polypeptide with an Asn-488 mutation for tyrosine, Lys-973 and Val-974 mutations for proline, and an inactivated furin cleavage site with the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96) comprises an amino acid sequence of SEQ ID NO: 113.

[0169] In embodiments, the CoV S polypeptide contains a deletion of amino acids 56, 57, and 131, a mutation of Asn-488 to tyrosine, a mutation of Ala-557 to aspartate, 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 with the amino acid sequence QQAQ (SEQ ID NO: 7), GSAS (SEQ ID NO: 96), or GG relative to the native CoV Spike (S) polypeptide (SEQ ID NO: 2). In embodiments, the CoV S polypeptide with a deletion of amino acids 56, 57, and 131, Petition 870250108300, dated 11 / 26 / 2025, pp. 89 / 284 82 / 210 mutation of Asn-488 to tyrosine, a mutation of Ala-557 to aspartate, 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 Asp1105 to histidine, mutations of Lys-973 and Val-974 to proline, and an inactivated furin cleavage site with the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96) comprises an amino acid sequence of SEQ ID NO: 114.In embodiments, the CoV S polypeptide with deletion of amino acids 56, 57 and 131, mutation of Asn-488 to tyrosine, a mutation of Ala-557 to aspartate, mutation of Asp-601 to glycine, mutation of Pro-668 to histidine, mutation of Thr-703 to isoleucine, mutation of Ser969 to alanine, mutation of Asp-1105 to histidine, mutations of Lys-973 and Val-974 to proline and an inactivated furin cleavage site with the amino acid sequence of QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96) or GG comprises an amino acid sequence of SEQ ID NO: 136.In embodiments, the CoV S polypeptide with deletions of amino acids 56, 57, and 131, a mutation of Asn-488 to tyrosine, a mutation of Ala-557 to aspartate, 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 Asp1105 to histidine, mutations of Lys-973 and Val-974 to proline, and an inactivated furin cleavage site with the amino acid sequence GG comprises an amino acid sequence with SEQ ID NO: 137 or SEQ ID NO: 138. In some embodiments, the CoV S polypeptide with an amino acid sequence with SEQ ID NO: 114 or SEQ ID NO: 136 is encoded by a nucleic acid with a nucleic acid sequence with SEQ ID NO: 135. In some In these modalities, the CoV S polypeptide with an amino acid sequence of SEQ ID NO: 137 or SEQ ID NO: 138 is encoded by a nucleic acid with a sequence of SEQ ID NO: 139.

[0170] In embodiments, the CoV S polypeptide contains deletions of amino acids 56, 57 and 132, a mutation of Asn-488 to tyrosine, a mutation of Ala-557 to aspartate, a mutation of Asp-601 to glycine, a mutation of Pro-668 Petition 870250108300, dated 11 / 26 / 2025, pp. 90 / 284 83 / 210 for histidine, Thr-703 mutation for isoleucine, Ser-969 mutation for alanine, Asp-1105 mutation for histidine, Lys-973 and Val-974 mutations for proline, and an inactivated furin cleavage site with the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96 relative to the native CoV Spike (S) polypeptide (SEQ ID NO: 2)). In embodiments, the CoV S polypeptide with a deletion of amino acids 56, 57 and 132, mutation of Asn-488 to tyrosine, a mutation of Ala-557 to aspartate, mutation of Asp-601 to glycine, mutation of Pro-668 to histidine, mutation of Thr-703 to isoleucine, mutation of Ser-969 to alanine, mutation of Asp-1105 to histidine, mutations of Lys-973 and Val-974 to proline and an inactivated furin cleavage site with the amino acid sequence of QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96) comprises an amino acid sequence of SEQ ID NO: 114.

[0171] In embodiments, the CoV S polypeptide contains Asn488 mutation to tyrosine, Asp-67 mutation to alanine, Leu-229 mutation to histidine, Asp-202 mutation to glycine, Lys-404 mutation to asparagine, Glu-471 mutation to lysine, Ala-688 mutation to valine, Asp-601 mutation to glycine, Lys-973 and Val-974 mutations to proline, and an inactivated furin cleavage site with the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96) relative to the native CoV Spike (S) polypeptide (SEQ ID NO: 2).In embodiments, the CoV S polypeptide with an Asn-488 mutation to tyrosine, an Asp-67 mutation to alanine, a Leu-229 mutation to histidine, an Asp-202 mutation to glycine, a Lys-404 mutation to asparagine, a Glu-471 mutation to lysine, an Ala688 mutation to valine, an Asp-601 mutation to glycine, Lys-973 and Val974 mutations to proline, and an inactivated furin cleavage site with the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96) comprises an amino acid sequence of SEQ ID NO: 115.

[0172] In embodiments, the CoV S polypeptide contains one or more modifications selected from: K973P, V974P, a furin cleavage site Petition 870250108300, dated 11 / 26 / 2025, pp. 91 / 284 84 / 210 inactivated, amino acid deletions 56, amino acid deletion 57, amino acid deletion 131, N488Y, A557D, D601G, P668H, T703I, S969A and D1105H, where the amino acids are numbered relative to a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 2. In embodiments, the inactivated furin cleavage site has the amino acid sequence QQAQ (SEQ ID NO: 7). In embodiments, the inactivated furin cleavage site has the amino acid sequence GG.

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

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

[0175] In embodiments, the CoV S polypeptide contains one or more modifications selected from: K973P, V974P, an inactivated furin cleavage site with the amino acid sequence QQAQ (SEQ ID NO: 7), amino acid deletions 229-231, L5F, D67A, D202G, K404N, E471K, N488Y, D601G, and A688V, wherein the amino acids are numbered relative to a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 2. In embodiments, the CoV S polypeptide with one or more modifications selected from K973P, V974P, an inactivated furin cleavage site with the amino acid sequence QQAQ (SEQ ID NO: 7), amino acid deletions 229-231, L5F, D67A, D202G, K404N, E471K, N488Y, D601G and A688V, in which the amino acids Petition 870250108300, dated 11 / 26 / 2025, pp. 92 / 284 85 / 210 are numbered relative to a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 2 comprising the amino acid sequence of SEQ ID NO: 144. In embodiments, the CoV S polypeptide with the amino acid sequence of SEQ ID NO: 144 is encoded by a nucleic acid with a sequence of SEQ ID NO: 145.

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

[0177] In embodiments, the CoV S polypeptide contains 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 a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 2. In embodiments, the CoV S polypeptide containing 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 a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 2, has an amino acid sequence of SEQ ID NO: 151. In embodiments, the polypeptide Petition 870250108300, dated 11 / 26 / 2025, pp. 93 / 284 86 / 210 CoV S with an amino acid sequence of SEQ ID NO: 151 is encoded by a nucleic acid with a sequence of SEQ ID NO: 150.

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

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

[0180] In embodiments, the CoV S polypeptide contains one or more modifications selected from: K973P, V974P, an inactivated furin cleavage site, K404N, E471K, and N488K, wherein the amino acids are numbered relative to a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 2. In embodiments, the CoV S polypeptide contains one or more modifications selected from: K973P, V974P, an inactivated furin cleavage site, K404N, E471K, and N488Y. In embodiments, the CoV S polypeptide is the RBD of the CoV S polypeptide with one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, K404N, E471K, and N488K, wherein the amino acids are numbered relative to a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 2.In embodiments, the CoV S polypeptide is the RBD of the CoV S polypeptide with one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, K404N, E471K, and N488Y, wherein the amino acids are numbered in. Petition 870250108300, dated 11 / 26 / 2025, pp. 94 / 284 87 / 210 in relation to a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 2.

[0181] In embodiments, the CoV S polypeptide contains one or more modifications selected from: K973P, V974P, an inactivated furin cleavage site with the amino acid sequence GG, D601G, E404N, E471K, and N488Y. In embodiments, the CoV S polypeptide contains one or more modifications selected from: K973P, V974P, an inactivated furin cleavage site with the amino acid sequence GG, and a D601G mutation, wherein the amino acids are numbered relative to a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 2. In embodiments, the CoV S polypeptide containing modifications selected from: K973P, V974P, an inactivated furin cleavage site with the amino acid sequence GG, and a D601G mutation has an amino acid sequence of SEQ ID NO: 133.

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

[0183] In embodiments, the CoV S polypeptide contains one or more Petition 870250108300, dated 11 / 26 / 2025, pp. 95 / 284 88 / 210 selected modifications from among: K973P, V974P, an inactivated furin cleavage site, W139C, and L439R, wherein the amino acids are numbered relative to a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 2. In embodiments, the CoV S polypeptide comprising K973P, V974P, an inactivated furin cleavage site, modifications with W139C and L439R is expressed with a signal peptide with an amino acid sequence of SEQ ID NO: 117 or SEQ ID NO: 5. In embodiments, the CoV S polypeptide comprises one or more selected modifications from among: K973P, V974P, an inactivated furin cleavage site, D601G, W139C, and L439R, wherein the amino acids are numbered relative to a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 2.In embodiments, the CoV S polypeptide comprises K973P, V974P, an inactivated furin cleavage site, modifications with D601G, W139C and L439R and is expressed with a signal peptide with an amino acid sequence of SEQ ID NO: 117 or SEQ ID NO: 5.

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

[0185] In embodiments, the CoV S polypeptide contains one or more modifications selected from: K973P, V974P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), W139C, S481P, D601G and L439R, wherein the amino acids are numbered relative to a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 2. In embodiments, the CoV S polypeptide contains one or more modifications selected from: K973P, V974P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), W139C, D601G and L439R, wherein the amino acids are numbered relative to a CoV S polypeptide with a Petition 870250108300, dated 11 / 26 / 2025, pp. 96 / 284 89 / 210 amino acid sequence of SEQ ID NO: 2. In embodiments, the CoV S polypeptide contains one or more modifications selected from: K973P, V974P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), W139C, S481P, and D601G, wherein the amino acids are numbered relative to a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 2. In embodiments, the CoV S polypeptide containing one or more modifications selected from: K973P, V974P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), W139C, S481P, D601G, and L439R has the amino acid sequence of SEQ ID NO: 153. In embodiments, the CoV S polypeptide with amino acid sequence SEQ ID NO: 153 comprises a signal peptide with amino acid sequence SEQ ID NO: 117.In embodiments, the CoV S polypeptide with amino acid sequence SEQ ID NO: 153 comprises a signal peptide with an amino acid sequence SEQ ID NO: 5.

[0186] In embodiments, the CoV S polypeptide contains one or more modifications selected from: K973P, V974P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), T82I, D240G, E471K, D601G and A688V, wherein the amino acids are numbered relative to a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 2. In embodiments, the CoV S polypeptide containing one or more modifications selected from: K973P, V974P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), T82I, D240G, E471K, D601G and A688V, wherein the Amino acids are numbered in relation to a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 2, which has an amino acid sequence of SEQ ID NO: 156.In embodiments, the CoV S polypeptide containing one or more modifications selected from: K973P, V974P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), T82I, D240G, E471K, D601G and. Petition 870250108300, dated 11 / 26 / 2025, p. 97 / 284 90 / 210 A688V, wherein the amino acids are numbered relative to a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 2, comprises a signal peptide with an amino acid sequence of SEQ ID NO: 154 or SEQ ID NO: 5.

[0187] In embodiments, the CoV S polypeptide contains one or more modifications selected from: K973P, V974P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), T82I, D240G, S464N, D601G and A688V, wherein the amino acids are numbered relative to a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 2. In embodiments, the CoV S polypeptide containing one or more modifications selected from: K973P, V974P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), T82I, D240G, S464N, D601G and A688V, wherein the Amino acids are numbered in relation to a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 2, which has an amino acid sequence of SEQ ID NO: 158.In embodiments, the CoV S polypeptide containing one or more modifications selected from: K973P, V974P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), T82I, D240G, S464N, D601G and A688V, wherein the amino acids are numbered relative to a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 2, comprises a signal peptide of SEQ ID NO: 154.

[0188] In embodiments, the CoV S polypeptide contains one or more modifications selected from: K973P, V974P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), amino acid 56 deletion, amino acid 57 deletion, amino acid 131 deletion, an N488Y mutation, an A557D mutation, a D601G mutation, a P668H mutation, a T703I mutation, an S969A mutation, and a D1105H mutation, wherein the CoV S polypeptide is numbered relative to the wild-type SARS-CoV-2 S polypeptide with the sequence of Petition 870250108300, dated 11 / 26 / 2025, pp. 98 / 284 91 / 210 amino acids of SEQ ID NO: 2. In embodiments, the CoV S polypeptide contains one or more modifications selected from: K973P, V974P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), amino acid 56 deletion, amino acid 57 deletion, amino acid 132 deletion, an N488Y mutation, an A557D mutation, a D601G mutation, a P668H mutation, a T703I mutation, an S969A mutation, and a D1105H mutation, wherein the CoV S polypeptide is numbered relative to the wild-type SARS-CoV-2 S polypeptide with the amino acid sequence of SEQ ID NO: 2.

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

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

[0191] In embodiments, the CoV S polypeptide contains one or more modifications selected from: K986P, V987P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), an S13I mutation, a W152C mutation, and a mutation Petition 870250108300, dated 11 / 26 / 2025, page 99 / 284 92 / 210 L452R, wherein the CoV S polypeptide is numbered relative to the wild-type SARS-CoV-2 S polypeptide with the amino acid sequence of SEQ ID NO: 1. In embodiments, the CoV S polypeptide contains one or more modifications selected from: K986P, V987P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), an S13I mutation, a W152C mutation, and an L452R mutation, wherein the CoV S polypeptide is numbered relative to the wild-type SARS-CoV-2 S polypeptide with the amino acid sequence of SEQ ID NO: 1 and does not have an N-terminal signal peptide.

[0192] In embodiments, the Spike (S) polypeptides of CoV comprise a polypeptide linker. In embodiments, the polypeptide linker contains 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 embodiments, the polypeptide linker has a repeat of (SGGG)n (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 some embodiments, the polypeptide linker has an amino acid sequence corresponding to SEQ ID NO: 90.

[0194] In embodiments, the polypeptide linker has a repeat of (GGGGS)n (SEQ ID NO: 93), 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 modalities, the polypeptide linker has a repeat of (GGGS)n (SEQ ID NO: 92), 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). Petition 870250108300, dated 11 / 26 / 2025, pp. 100 / 284 93 / 210

[0196] In some embodiments, the polypeptide linker is a poly-(Gly)n linker, wherein 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 quadripeptides. In other embodiments, the linker is a dipeptide selected from the group consisting of alanine-serine (AS), leucineglutamic acid (LE), and serine-arginine (SR).

[0197] In embodiments, the polypeptide linker comprises between 1 and 100 contiguous amino acids of a naturally occurring CoV S polypeptide or of a CoV S polypeptide disclosed in this document. In embodiments, the polypeptide linker has an amino acid sequence corresponding to SEQ ID NO: 94.

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

[0199] In embodiments, the disclosure provides CoV S polypeptides comprising a fragment of a coronavirus S protein and nanoparticles and vaccines comprising the same. In embodiments, the coronavirus S protein fragment is between about 10 and about 1500 amino acids in length (e.g., 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, Petition 870250108300, dated 11 / 26 / 2025, pp. 101 / 284 94 / 210 approximately 650, approximately 700, approximately 750, approximately 800, approximately 850, approximately 900, approximately 950, approximately 1000, approximately 1050, approximately 1100, approximately 1150, approximately 1200, approximately 1250, approximately 1300, approximately 1350, approximately 1400, approximately 1450 or approximately 1500 amino acids in length). In embodiments, the coronavirus S protein fragment is selected from the group consisting of the receptor-binding domain (RBD), subdomain 1, subdomain 2, top helix, fusion peptide, connecting region, heptad repeat 1, core helix, heptad repeat 2, NTD and TMCT.

[0200] In embodiments, the CoV S polypeptide comprises an RBD and a subdomain 1. In embodiments, the CoV S polypeptide comprising an RBD and a subdomain 1 are amino acids 319 to 591 of SEQ ID NO: 1.

[0201] In embodiments, the CoV S polypeptide contains a fragment of a coronavirus S protein, wherein the coronavirus S protein fragment is the 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 embodiments, the CoV S polypeptide contains two or more RBDs, which are connected by a polypeptide linker. In embodiments, the polypeptide linker has an amino acid sequence of SEQ ID NO: 90 or SEQ ID NO: 94.

[0203] In embodiments, 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 contains two or more SARS-CoV-2 RBDs, which are connected by a polypeptide linker. In embodiments, the antigen containing two or more SARS-CoV-2 RBDs has an amino acid sequence corresponding to one of the SEQ IDs NOS: 72-75.

[0205] In embodiments, the CoV S polypeptide contains a SARS-CoV-2 RBD and a SARS RBD. In embodiments, the CoV S polypeptide comprises a SARS-CoV-2 RBD and a SARS RBD, wherein each RBD is separated Petition 870250108300, dated 11 / 26 / 2025, pp. 102 / 284 95 / 210 by a polypeptide linker. In embodiments, the CoV S polypeptide comprising a SARS-CoV-2 RBD and a SARS RBD has an amino acid sequence selected from the group consisting of SEQ ID NOS: 76-79.

[0206] In embodiments, the CoV S polypeptide contains a SARS-CoV-2 RBD and a MERS RBD. In embodiments, the CoV S polypeptide comprises a SARS-CoV-2 RBD and a MERS RBD, wherein each RBD is separated by a polypeptide linker.

[0207] In embodiments, the CoV S polypeptide comprises a SARS RBD and a MERS RBD. In embodiments, the CoV S polypeptide comprises a SARS RBD and a MERS RBD, wherein each RBD is separated by a polypeptide linker.

[0208] In embodiments, the CoV S polypeptide contains a SARS-CoV-2 RBD, a SARS RBD, and a MERS RBD. In embodiments, the CoV S polypeptide contains a SARS-CoV-2 RBD, a SARS RBD, and a MERS RBD, wherein each RBD is separated by a polypeptide linker. In embodiments, the CoV S polypeptide comprising a SARS-CoV-2 RBD, a SARS RBD, and a MERS RBD has an amino acid sequence selected from the group consisting of SEQ ID NOS: 80-83.

[0209] In embodiments, the CoV S polypeptides described in this document are expressed with an N-terminal signal peptide. In embodiments, the N-terminal signal peptide has an amino acid sequence of SEQ ID NO: 5 (MFVFLVLLPLVSS). In embodiments, the N-terminal signal peptide has an amino acid sequence of SEQ ID NO: 117 (MFVFLVLLPLVSI). In embodiments, the N-terminal signal peptide has an amino acid sequence of SEQ ID NO: 154 (MFVFFVLLPLVSS). In embodiments, the signal peptide can be replaced by any signal peptide that allows the expression of the CoV S protein. In embodiments, one or more of the amino acids of the CoV S protein signal peptide can be deleted or mutated. An initiation methionine residue is retained to initiate expression. In embodiments, the polypeptides Petition 870250108300, dated 11 / 26 / 2025, pp. 103 / 284 96 / 210 CoV S are encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 95, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 96, SEQ ID NO: 60, SEQ ID NO: 131, SEQ ID NO: 135, SEQ ID NO: 142, SEQ ID NO: 145, SEQ ID NO: 148, and SEQ ID NO: 150. In embodiments, the N-terminal signal peptide of the CoV S polypeptide contains a mutation in Ser-13 relative to the native CoV Spike (S) signal polypeptide (SEQ ID NO: 5). In embodiments, Ser-13 is mutated to any natural amino acid. In some modalities, Ser-13 is mutated to alanine, methionine, isoleucine, leucine, threonine, or valine. In others, Ser-13 is mutated to isoleucine.

[0210] Following the expression of the CoV S protein in a host cell, the N-terminal signal peptide is cleaved to provide the mature CoV protein sequence (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 some forms, the signal peptide is cleaved by proteases from the host cell. In others, the full-length protein can be isolated from the host cell and the signal peptide subsequently cleaved.

[0211] Following cleavage of the signal peptide of the CoV Spike (S) polypeptide with an amino acid sequence corresponding to 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, 173, 177-180, 185, 189, 191, 194, During expression and purification, a mature polypeptide with an amino acid sequence selected from the group consisting 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, was obtained. 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, 283, Petition 870250108300, dated 11 / 26 / 2025, pp. 104 / 284 97 / 210 284, 287, 288, 291, 292 and 298-302 are obtained and used to produce a CoV S nanoparticle vaccine or CoV S nanoparticles.

[0212] Advantageously, the disclosed CoV S polypeptides may have enhanced protein expression and stability compared to the native CoV Spike (S) protein.

[0213] In embodiments, the CoV S polypeptides described in this document contain additional modifications of the native coronavirus S protein (SEQ ID NO: 2). In embodiments, the coronavirus S proteins described in this document exhibit 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 the native coronavirus S protein. One skilled in the art would use known techniques to calculate the percentage identity of the recombinant coronavirus S protein with the native protein or with any of the CoV S polypeptides described in this document. For example, the percentage identity can be calculated using the CLUSTALW2 tool, which is available online.The following standard parameters can be used for CLUSTALW2 pair alignment: Protein Weight Matrix = Gonnet; Gap Open = 10; Gap Extension = 0.1.

[0214] In embodiments, the amino acid sequence of the CoV S polypeptides described in this document 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% identical or 100% identical to the CoV S polypeptide with an amino acid sequence of any of the following: SEQ ID NO: 87, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 181-184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 195; SEQ ID NOS: 217-228, SEQ ID NOS: 233-236, SEQ ID NO: 243, SEQ ID NOS: 255-302. A CoV S polypeptide may have a deletion, insertion, or mutation of up to approximately 1, up to approximately 2, up to approximately 3, up to approximately 4, up to Petition 870250108300, dated 11 / 26 / 2025, pp. 105 / 284 98 / 210 approximately 5, up to approximately 10, up to approximately 15, up to approximately 20, up to approximately 25, up to approximately 30, up to approximately 35, up to approximately 40, up to approximately 45 or up to approximately 50 amino acids compared to the amino acid sequence of the CoV S polypeptide with an amino acid sequence of any of the following: SEQ ID NO: 87, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 181-184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 195; SEQ ID NOS: 217-228, SEQ ID NOS: 233-236, SEQ ID NO: 243, SEQ ID NOS: 255-302.A CoV S polypeptide may have a deletion, insertion, or mutation between approximately 1 and approximately 5 amino acids, between approximately 3 and approximately 10 amino acids, between approximately 5 and 10 amino acids, between approximately 8 and 12 amino acids, between approximately 10 and 15 amino acids, between approximately 12 and 17 amino acids, between approximately 15 and 20 amino acids, between approximately 18 and 23 amino acids, between approximately 20 and 25 amino acids, between approximately 22 and approximately 27 amino acids, between approximately 25 and 30 amino acids, between approximately 30 and 35 amino acids, between approximately 35 and 40 amino acids, between approximately 40 and 45 amino acids, or between approximately 45 and 50 amino acids, compared to the CoV S polypeptide with an amino acid sequence of any of the following: SEQ ID NO: 87, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NOS: 181-184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 195; SEQ ID NOS: 217-228, SEQ ID NOS: 233-236, SEQ ID NO: 243, SEQ ID NOS: 255-302.In embodiments, the CoV S polypeptides described in this document comprise approximately 1, approximately 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 7, approximately 8, approximately 9, approximately 10, approximately 11, approximately 12, approximately 13, approximately 14, approximately 15, approximately 16, approximately 17, approximately 18, approximately 19, approximately 20, approximately 21, approximately 22, approximately 23, approximately 24, or approximately 25 substitutions compared with the CoV S polypeptide of any of the SEQ ID NO: 87, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 181-184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 195; SEQ ID NOS: 217-228, SEQ ID NOS: 233236, SEQ ID NO: 243, SEQ ID NOS: 255-302. Petition 870250108300, dated 11 / 26 / 2025, pp. 106 / 284 99 / 210

[0215] In embodiments, the CoV S polypeptides described in this document are 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% identical, or 100% identical to the CoV S polypeptide with an amino acid sequence selected from any of the following 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 embodiments, the coronavirus S polypeptide is extended at the N-terminus, the C-terminus, or both the N-terminus and C-terminus. In aspects, the extension is a useful tag for a function, such as purification or detection. In aspects, the tag contains an epitope. For example, the tag may be a polyglutamate tag, a FLAG tag, an HA tag, a polyHis tag (approximately 5-10 histidines) (SEQ ID NO: 101), a hexahistidine tag (SEQ ID NO: 100), an 8X-His tag (with eight histidines) (SEQ ID NO: 102), a Myc tag, a Glutathione-S-transferase tag, a green fluorescent protein tag, a Maltose-binding protein tag, a Thioredoxin tag, or an Fc tag. In other aspects, the extension may be an N-terminal signal peptide fused to the protein to enhance expression. Although such signal peptides are often cleaved during expression in the cell, some nanoparticles may contain the antigen with an intact signal peptide.Thus, when a nanoparticle comprises an antigen, the antigen may contain an extension and thus may be a fusion protein when incorporated into nanoparticles. For identity calculation purposes for the sequence, extensions are not included. In 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 embodiments, the protease cleavage site is a... Petition 870250108300, dated 11 / 26 / 2025, page 107 / 284 100 / 210 HRV3C protease cleavage site. In embodiments, the protease cleavage site comprises an amino acid sequence with SEQ ID NO: 98.

[0217] In embodiments, the CoV S glycoprotein comprises a fusion protein. In embodiments, the CoV S glycoprotein comprises an N-terminal fusion protein. In embodiments, the CoV S glycoprotein comprises a C-terminal fusion protein. In embodiments, the fusion protein encompasses a tag useful for protein expression, purification, or detection. In embodiments, the tag is a polyHis tag (with approximately 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 some embodiments, the tag is an Fc tag. In embodiments, the Fc tag is monomeric, dimeric, or trimeric. In embodiments, the tag is a hexahistidine tag, for example, a polyHis tag containing six histidines (SEQ ID NO: 100). In embodiments, the tag is a Twin-Strep tag with an amino acid sequence of SEQ ID NO: 99.

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

[0219] In some respects, the CoV S protein can be truncated. For example, the N-terminus can be truncated by about 10 amino acids, about 30 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, or about 200 amino acids. The C-terminus can be truncated instead of or in addition to the N-terminus. For example, the C-terminus can be truncated by about 10 amino acids, about 30 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, or about 200 amino acids. For the purposes of calculating the identity of the truncated protein, the identity is measured over the remaining portion of the protein. Detergent core nanoparticles containing CoV Spike (S) polypeptides Petition 870250108300, dated 11 / 26 / 2025, page 108 / 284 101 / 210

[0220] In embodiments, the compositions disclosed in this document comprise detergent core nanoparticles comprising a CoV S polypeptide, which is associated with a detergent core.

[0221] In embodiments, the nanoparticles of the present disclosure comprise a mature CoV S polypeptide. In embodiments, the nanoparticles of the present disclosure comprise CoV S polypeptides associated with a detergent core. The presence of the detergent facilitates the formation of the nanoparticles forming a core that organizes and presents the antigens. In embodiments, the nanoparticles may contain the CoV S polypeptides assembled into multioligomeric glycoprotein detergent nanoparticles (e.g., PS80) with the head regions projecting outwards and hydrophobic regions and PS80 detergent forming a central core surrounded by the glycoprotein. In embodiments, the CoV S polypeptide inherently contains or is adapted to contain a transmembrane domain to promote the association of the protein into a detergent core. In embodiments, the CoV S polypeptide contains a head domain.Primarily, the transmembrane domains of a CoV S polypeptide trimer associate with detergent; however, other portions of the polypeptide can also interact. Advantageously, the nanoparticles improved resistance to environmental stresses, thus providing enhanced stability and / or improved presentation to the immune system due to the organization of multiple protein copies around the detergent.

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

[0223] In embodiments, the CoV S polypeptide forms a trimer. In embodiments, the CoV S polypeptide nanoparticles are composed of Petition 870250108300, dated 11 / 26 / 2025, pp. 109 / 284 102 / 210 multiple polypeptide trimers around a nonionic detergent core. In embodiments, the nanoparticles contain at least about 1 trimer or more. In embodiments, the nanoparticles contain at least about 5 trimers to about 30 trimers of the Spike protein. In 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 in between. The compositions disclosed in this document may contain nanoparticles with different numbers of trimers. For example, a composition may contain nanoparticles in which the number of trimers ranges from 2-9; in embodiments, the nanoparticles in a composition may contain from 2-6 trimers. In embodiments, the compositions contain a heterogeneous population of nanoparticles with 2 to 6 trimers per nanoparticle, or 2 to 9 trimers per nanoparticle. In embodiments, the compositions may contain a substantially homogeneous population of nanoparticles.For example, the population may contain approximately 95% nanoparticles with 5 trimers.

[0224] The nanoparticles disclosed in this document vary in particle size. In embodiments, the nanoparticles disclosed in this document vary in particle size from a Z-bird size of about 20 nm to about 60 nm, about 20 nm to about 50 nm, about 20 nm to about 45 nm, about 20 nm to about 35 nm, about 20 nm to about 30 nm, about 25 nm to about 35 nm, or about 25 nm to about 45 nm. The particle size (Z-bird) is measured by dynamic light scattering (DLS) using a Zetasizer NanoZS (Malvern, UK), unless otherwise specified.

[0225] In embodiments, the CoV S polypeptide nanoparticles disclosed in this document have a reduced particle size compared to nanoparticles comprising a wild-type CoV S polypeptide. In embodiments, the CoV S polypeptides are at least about 40% smaller in particle size, for example, at least about 40%, at least about 45%, at least about 50%, at least Petition 870250108300, dated 11 / 26 / 2025, p. 110 / 284 103 / 210 less 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 in particle size.

[0226] The CoV S polypeptide nanoparticles disclosed in this document are more homogeneous in size, shape, and mass than nanoparticles comprising a wild-type CoV S polypeptide. The polydispersity index (PDI), which is a measure of heterogeneity, is measured by dynamic light scattering using a Malvern Setasizer, unless otherwise specified. In embodiments, the particles measured in this document 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 in this document have a PDI that is at least about 25% lower than the PDI of nanoparticles comprising 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] CoV S polypeptides and nanoparticles comprising them have enhanced thermal stability compared to wild-type CoV S polypeptide or a nanoparticle thereof. The thermal stability of CoV S polypeptides is measured using differential scanning calorimetry (DSC), unless otherwise specified. The enthalpy of transition (ΔHcal) is the energy required to unfold a CoV S polypeptide. In embodiments, CoV S polypeptides have an increased ΔHcal compared to wild-type CoV S polypeptide. In embodiments, the ΔHcal of a CoV S polypeptide is approximately 2 times, approximately 3 times, approximately 4 times, approximately 5 times, approximately 6 times, approximately 7 times, approximately 8 times, approximately 9 times, or approximately 10 times greater than the ΔHcal of a wild-type CoV S polypeptide. Petition 870250108300, dated 11 / 26 / 2025, p. 111 / 284 104 / 210

[0228] Several types of nanoparticles can be included in the vaccine compositions disclosed in this document. In some aspects, the nanoparticle type is in the form of an anisotropic rod, which can be a dimer or a monomer. In other aspects, the nanoparticle type is a spherical oligomer. In still other aspects, the nanoparticle can be described as an intermediate nanoparticle, with sedimentation properties intermediate between the first two types. The formation of nanoparticle types can be regulated by controlling the detergent and protein concentration during the production process. The type of nanoparticle can be determined by measuring the sedimentation coefficient. (ii) at least three HA glycoproteins

[0229] In embodiments, an immunogenic composition of the disclosure comprises at least three HA glycoproteins or at least four HA glycoproteins. In embodiments, an immunogenic composition of the disclosure comprises three or four HA glycoproteins. In embodiments, each of the HA glycoproteins is from a different influenza strain. In embodiments, three HA glycoproteins are from a Type A influenza strain and one HA glycoprotein is from a Type B influenza strain. In embodiments, two HA glycoproteins are from a Type A influenza strain and two HA glycoproteins are from a Type B influenza strain. In embodiments, two HA glycoproteins are from a Type A influenza strain and one HA glycoprotein is from a Type B influenza strain.

[0230] In embodiments, each of at least three HA glycoproteins is isolated separately using an egg-based manufacturing process. In embodiments, an egg-based manufacturing process comprises (a) propagating an influenza virus in an egg and (b) harvesting the influenza virus.

[0231] In embodiments, the influenza virus is a live virus. In embodiments, the influenza virus is a weakened or attenuated virus. In embodiments, the influenza virus is optimized to grow in an egg. In embodiments, the optimized influenza virus does not possess the cleavage site. Petition 870250108300, dated 11 / 26 / 2025, p. 112 / 284 105 / 210 polybasic hemagglutinin. The following article describes the development of an optimized influenza virus (referred to as a vaccine virus candidate) in detail and is incorporated by reference in this document in its entirety: Belser et al. Virologia. 2017 Nov; 511: 135-141.

[0232] In some embodiments, the egg is a chicken egg. In some embodiments, the chicken egg is an embryonated chicken egg. In some embodiments, the egg is a pathogen-free egg. In embodiments, an influenza virus is propagated by inoculating the virus into the allantoic cavity of an embryonated chicken egg. The following article describes an exemplary inoculation method and is incorporated by reference into this document in its entirety: Brauer et al. J Vis Exp. 2015; (97): 52421.

[0233] In embodiments, the egg-based manufacturing process comprises the purification of the influenza virus. In embodiments, an influenza virus is purified using any of the following techniques: centrifugation, chromatography, precipitation, or nanofiltration. In embodiments, the centrifugation technique is ultracentrifugation. In embodiments, the virus is purified using zone centrifugation. In some embodiments, the zone centrifugation is continuous-flow zone centrifugation.

[0234] In embodiments, the egg-based manufacturing process comprises the inactivation (also referred to in this document as killing) of an influenza virus. In embodiments, an influenza virus is inactivated using low pH (e.g., a pH of about 3.5-5.5), heat, ethanol, ultraviolet light, exposure to a detergent (e.g., octylphenol), or exposure to a chemical (e.g., 2-propanol, ethanol, iodopovidone). In embodiments, the purified virus is inactivated with ultraviolet light, betapropiolactone, sodium deoxycholate, formaldehyde, or any combination thereof.

[0235] In embodiments, the egg-based manufacturing process comprises exposing the influenza virus to a surfactant. The surfactant may be sodium taurodeoxycholate, octylphenol ethoxylate (Triton®-X 100) or cetyltrimethylammonium bromide. Exposure of an influenza virus to a surfactant Petition 870250108300, dated 11 / 26 / 2025, pp. 113 / 284 106 / 210 results in the formation of an influenza split virion. In embodiments, at least three HA glycoproteins are in the form of an influenza split virion.

[0236] In embodiments, the egg-based manufacturing process comprises the purification of an influenza antigen (e.g., hemagglutinin) from the virus.

[0237] The following influenza vaccines approved by the Food and Drug Administration (FDA) are produced using an egg-based manufacturing process: AFLURIA® QUADRIVALENT, FLUARIX® QUADRIVALENT, FLULAVAL QUADRIVALENT, FLUZONE® QUADRIVALENT, FLUZONE® HIGH-DOSE QUADRIVALENT, FLUMIST® QUADRIVALENT, FLUAD®, and FLUAD® QUADRIVALENT.

[0238] In embodiments, each of at least three HA glycoproteins is isolated using a cell culture-based process. In embodiments, a cell culture-based process comprises (i) growing an influenza virus in a cell and (ii) harvesting the virus from the cell. In embodiments, a cell culture-based process comprises (i) transfecting a cell with a vector comprising a hemagglutinin and (ii) harvesting the hemagglutinin from the cell. In embodiments, a cell culture-based process comprises (i) transducing a cell with a virus encoding a hemagglutinin and (ii) harvesting the hemagglutinin from the cell. In embodiments, the harvested hemagglutinin is recombinant hemagglutinin.

[0239] In embodiments, the cell is an animal cell, a bacterial cell, an insect cell, or a fungal cell. In embodiments, the animal is a human, a bird (e.g., a chicken), a dog, a reptile, a goat, a pig, a mouse, a rabbit, or a rat.

[0240] In modalities, the virus that encodes a hemagglutinin is a baculovirus, a lentivirus, or an adeno-associated virus.

[0241] In embodiments, an influenza virus produced using a cell culture-based process is purified. Any of the purification techniques described for purifying influenza virus produced using a Petition 870250108300, dated 11 / 26 / 2025, pp. 114 / 284 107 / 210 egg-based manufacturing process can be used to purify influenza virus produced using a cell culture-based process.

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

[0243] The Food and Drug Administration (FDA) approved FLUCELVAX® QUADRIVALENT influenza vaccine is produced using a cell culture-based process.

[0244] In embodiments, each of at least three HA glycoproteins is a recombinant hemagglutinin. The FLUBLOK® QUADRIVALENT influenza vaccine approved by the Food and Drug Administration (FDA) is a recombinant hemagglutinin.

[0245] In embodiments, at least three hemagglutinins are in the form of recombinant hemagglutinin. Recombinant hemagglutinin is isolated from a cell that produces hemagglutinin. In embodiments, a cell that produces hemagglutinin has been transfected with a vector that encodes hemagglutinin. In embodiments, a cell that produces hemagglutinin has been transduced with a virus that encodes hemagglutinin. (iia) at least three HA glycoprotein detergent core nanoparticles comprising hemagglutinin

[0246] In embodiments, the compositions disclosed in this document comprise detergent core nanoparticles comprising hemagglutinin from an influenza virus. The aforementioned detergent 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] Detergent core nanoparticles comprise a hemagglutinin from an influenza virus, which is associated with a detergent core. In some 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. Petition 870250108300, dated 11 / 26 / 2025, pp. 115 / 284 108 / 210 In embodiments, the nanoparticle contains between about 2 to about 9, about 2 to about 6, or about 5 hemagglutinin trimers. The hemagglutinin is associated with the non-ionic detergent-containing core of the nanoparticle. In embodiments, the detergent is selected from polysorbate-20 (PS20), polysorbate-40 (PS40), polysorbate-60 (PS60), polysorbate-65 (PS65), and polysorbate-80 (PS80). The presence of the detergent facilitates the formation of the nanoparticles, forming a core that organizes and presents the antigens. In embodiments, the nanoparticles may contain antigens assembled in multioligomeric glycoprotein-PS80 detergent nanoparticles with head regions projecting outwards and hydrophobic regions, and PS80 detergent forming a central core surrounded by the antigens.

[0248] The nanoparticles disclosed in this document range in Z-ave size from about 20 nm to about 60 nm, about 20 nm to about 50 nm, about 20 nm to about 45 nm, or about 25 nm to about 45 nm.

[0249] In embodiments, a detergent-core nanoparticle is produced in insect cells expressing HA proteins using a baculovirus expression system and extracting the HA protein with a detergent. During purification, the first detergent is replaced by a second detergent, typically a non-ionic detergent, resulting in nanoparticles with a non-ionic detergent core to which the transmembrane domain of the HA protein, in trimer form, is incorporated. Fig. 7 (left panel) illustrates these structures as observed under an electron microscope.

[0250] The hemagglutinin contained in the detergent-core nanoparticles or HaSMaNs described in this document may be from any strain of influenza virus. Human influenza viruses Type A and Type B cause seasonal disease outbreaks nearly every winter in the United States.

[0251] The HA protein can be selected from the subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and H18. Phylogenetically, influenza is divided into groups. For HA, Group 1 contains Petition 870250108300, dated 11 / 26 / 2025, p. 116 / 284 109 / 210 Group 1 contains H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17 and H18, and group 2 contains H3, H4, H7, H10, H14 and H15.

[0252] In embodiments, detergent core nanoparticles or HaSMaNs are trypsin-resistant nanoparticles produced using neutral pH purification. Trypsin resistance is achieved by the neutral pH range of above 6.9 to 8.5 during the purification and formulation of the HA nanoparticles. Trypsin-resistant influenza glycoproteins and trypsin-resistant influenza nanoparticles; and methods of production thereof are described in detail in U.S. Patent No. 10,426,829.

[0253] In embodiments, the hemagglutinin of detergent core nanoparticles or HaSMaNs described in this document comprises the full-length wild-type hemagglutinin amino acid sequence. In embodiments, hemagglutinin is a variant of hemagglutinin. 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 with the wild-type hemagglutinin protein. The percent identity can be calculated using the ClustalW2 alignment program, available at www.ebi.ac.uk / Tools / msa / clustalw2 / . The following standard parameters can be used for pairwise alignment: Protein Weight Matrix = Gonnet; Gap Open = 10; Gap Extension = 0.1.

[0254] HA is a homotrimer with each monomer consisting of ~550 amino acid residues. Each HA monomer has been conceptually divided into three domains: the ectodomain of ~515 residues constitutes the extraviral portion of the molecule; a single 27-residue stretch defines the transmembrane (TM) domain; and ~10 residues constitute the cytoplasmic tail (CT). Although some changes can be made to hemagglutinin, the formation of both detergent core nanoparticles and HaSMaNs requires an intact transmembrane (TM) domain. Thus, in particular examples, a modified HA protein sequence comprises 100% identity with the wild-type TM and CT domains with some flexibility in the ectodomain portion. Petition 870250108300, dated 11 / 26 / 2025, page 117 / 284 110 / 210 remaining, where identity can be at least 90% or at least 95%. Domains can be identified by homology with the amino acid sequences of the TM and CT domains of Japan / 305 / 57 HA shown in Figure 1 of Melikyan et al. (Mol Biol Cell. 1999 Jun; 10(6): 1821-1836), although it should be noted that the boundaries between the ectodomain, TM, and CT domains can vary from HA protein to HA protein by up to three amino acids. (iib) influenza vaccine - HaSMaNs (Hemagglutinin Saponin Matrix Nanoparticles)

[0255] In embodiments, the immunogenic compositions and vaccine compositions described in this document comprise a HaSMaN (Hemagglutinin Saponin Matrix Nanoparticle). Fig. 7 (right panel) illustrates the HaSMaN structures as observed under an electron microscope. HA glycoproteins decorate the Matrix cage-like structures. HaSMaN structures are formed by preparing detergent core nanoparticles comprising hemagglutinin from an influenza virus and then incubating them with ISCOM matrix adjuvant particles for a period of time. The ISCOM matrix particles are shown in the center panel of Fig. 7. Notably, HaSMaNs readily form with HA proteins from influenza Type A, but not with HA proteins from influenza Type B.

[0256] The HaSMaNs disclosed in this document are produced by incubating detergent core nanoparticles with an adjuvant of ISCOM matrix comprising a saponin fraction, cholesterol, and a phospholipid. In embodiments, a HaSMaN is formed by incubating a detergent core nanoparticle with an ISCOM matrix adjuvant for between approximately 24 hours and approximately 48 hours. For example, the detergent core nanoparticle can be incubated for approximately 24 hours, approximately 25 hours, approximately 28 hours, approximately 29 hours, approximately 32 hours, approximately 33 hours, and an ISCOM matrix adjuvant for approximately 26 hours, approximately 27 hours, approximately 30 hours, approximately 31 hours, approximately 34 hours, approximately 35 hours. Petition 870250108300, dated 11 / 26 / 2025, pp. 118 / 284 111 / 210 approximately 36 hours, approximately 37 hours, approximately 40 hours, approximately 41 hours, approximately 44 hours, approximately 45 hours, approximately 38 hours, approximately 39 hours, approximately 42 hours, approximately 43 hours, approximately 46 hours, approximately 47 hours, approximately 48 hours or more.In some embodiments, a HaSMaN is formed by incubating a detergent core nanoparticle 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 embodiments, a HaSMaN is formed by incubating a detergent core nanoparticle with an ISCOM matrix adjuvant at a temperature of about 4 °C to about 25 °C. For example, HaSMaN can be formed by incubating a detergent core nanoparticle with an ISCOM matrix adjuvant at a temperature of about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C or higher.In embodiments, a HaSMaN is formed by incubating a detergent core nanoparticle with an ISCOM matrix adjuvant at a temperature 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. Petition 870250108300, dated 11 / 26 / 2025, p. 119 / 284 112 / 210 °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, at least about 25 °C. Typically, about 24 to 48 hours at 4 °C or 25 °C of incubation are required for formation. HaSMaN formation is promoted by higher temperatures. In embodiments, HaSMaN formation occurs by incubating detergent core nanoparticles with an ISCOM Matrix adjuvant for at least 24 hours at about 25 °C. Mixing detergent-core nanoparticles with Matrix ISCOM adjuvant shortly before administration to a subject—i.e., bedside mixing—does not produce HaSMaNs. Longer incubation periods do not negatively impact HaSMaN formation. Production of detergent core nanoparticles

[0257] The nanoparticles of this disclosure are products of non-natural occurrence, whose components do not occur together in nature. In embodiments, the methods disclosed in this document use a detergent exchange approach, whereby a first detergent is used to isolate a protein and then this first detergent is exchanged for a second detergent to form the nanoparticles.

[0258] The antigens contained in the nanoparticles are typically produced by recombinant expression in host cells. Standard recombinant techniques can be used. In embodiments, the CoV S polypeptides are expressed in insect host cells using a baculovirus system. In some embodiments, the baculovirus is a cathepsin-L knockout baculovirus, a chitinase knockout baculovirus. Optionally, the baculovirus is a double knockout for cathepsin-L and chitinase. High-level expression can be achieved in insect cell expression systems. Non-limiting examples of insect cells are Spodoptera frugiperda (Sf) cells, for example, Sf9, Sf21 cells, Trichoplusiani, for example, Petition 870250108300, dated 11 / 26 / 2025, pp. 120 / 284 113 / 210 High Five cells and Drosophila S2 cells. In embodiments, the CoV S polypeptide or hemagglutinin described in this document is produced in any suitable host cell. In embodiments, the host cell is an insect cell. In embodiments, the insect cell is an Sf9 cell.

[0259] Typical methods of transfection and cell growth can be used to culture the cells. Vectors, for example, vectors comprising 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 culturing host cells include, among others, batch, fed-batch, continuous, and perfusion cell culture techniques. Cell culture means the growth and propagation of cells in a bioreactor (a fermentation chamber) where the cells propagate and express protein (e.g., recombinant proteins) for purification and isolation. Typically, cell culture is performed under sterile, temperature- and atmosphere-controlled conditions in a bioreactor. A bioreactor is a chamber used to culture cells in which 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 pre-sterilized plastic bag (e.g., Cellbag®, Wave Biotech, Bridgewater, NJ). In yet another embodiment, the pre-sterilized plastic bags are bags of approximately 50 L to 3500 L. Extraction and Purification of Nanoparticles

[0261] After the host cells have grown, the protein or nanoparticle can be harvested from the host cells using detergents and purification protocols. Once the host cells have grown by Petition 870250108300, dated 11 / 26 / 2025, pp. 121 / 284 For 114 / 210 to 96 hours, the cells are isolated from the medium and a detergent-containing solution is added to solubilize the cell membrane, releasing the protein into a detergent extract. Triton X-100 and TERGITOL® nonylphenol ethoxylate, also known as NP-9, are preferred detergents for extraction. The detergent can be added at a final concentration of about 0.1% to about 1.0%. For example, the concentration can 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 can be from about 0.1% to about 0.3%. In embodiments, the concentration is about 0.5%.

[0262] In terms of modalities, different first detergents can be used to isolate the protein from the host cell. For example, the first detergent Bis(polyethylene glycol bis[imidazoylcarbonyl]), nonoxynol-9, Bis(polyethylene glycol bis[imidazoyl carbonyl]), 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 mono-oleyl ether 92V, BRIJ® Polyoxyethylene (10) oleyl ether 97, BRIJ® Polyethylene glycol hexadecyl ether 58P, CREMOPHOR® EL Macrogolglycerol ricinoleate, Decaethylene glycol monododecyl ether, N-Decanoyl-N-methylglucamine, n-Decyl alpha-D-glucopyranoside, Decyl beta-Dmaltopyranoside, n-Dodecanoyl-N-methylglucamide, nDodecyl 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 mono-hexadecyl ether, Hexaethylene glycol mono-octadecyl ether, Hexaethylene glycol monotetradecyl ether, Igepal CA-630, Igepal CA-630, Methyl-6-O-(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 mono-hexadecyl ether, Octaethylene glycol mono-octadecyl ether, Octaethylene glycol monotetradecyl ether, Octyl-beta-D-glucopyranoside, Pentaethylene glycol monodecyl ether Petition 870250108300, dated 11 / 26 / 2025, page. 122 / 284 115 / 210 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(imidazolyl carbonyl), Polyoxyethylene 25 propylene glycol stearate, Quillaja bark saponin, 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 ethoxylate alcohol Type 15-S-12, TERGITOL® secondary ethoxylate alcohol Type 15-S-30, TERGITOL® secondary ethoxylate alcohol Type 15-S-5, TERGITOL® secondary ethoxylate alcohol Type 15-S-7,TERGITOL® secondary ethoxylate alcohol 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, TERGITOL® branched secondary ethoxylate alcohol Type TMN-10, TERGITOL® branched secondary ethoxylate alcohol Type TMN-6, TRITON™ X100 Polyethylene glycol tert-octylphenyl ether or combinations thereof.

[0263] Nanoparticles can then be isolated from cellular debris using centrifugation. In embodiments, gradient centrifugation, such as using cesium chloride, sucrose, and iodixanol, can be used. Other techniques can be used as alternatives or in addition, such as standard purification techniques including, for example, ion exchange, affinity, and gel filtration chromatography.

[0264] For example, the first column could be an ion-exchange chromatography resin, such as FRACTOGEL® EMD TMAE methacrylate-based polymer beads (EMD Millipore), the second column could be a lentil (Lens culinaris) lectin affinity resin, and the third column could be a cation-exchange column, such as a polymer bead resin. Petition 870250108300, dated 11 / 26 / 2025, pp. 123 / 284 116 / 210 methacrylate-based FRACTOGEL® EMD SO3 (EMD Millipore). In other respects, the cation exchange column may be an MMC column or a Nuvia C Prime column (Bio-Rad Laboratories, Inc.). Preferably, the methods disclosed in this document do not use a detergent extraction column; for example, a hydrophobic interaction column. This column is frequently used to remove detergents during purification, but it may negatively impact the methods disclosed herein.

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

[0266] In embodiments, detergent exchange is performed using affinity chromatography to link glycoproteins through their carbohydrate moiety. For example, affinity chromatography can use a legume lectin column. Legume 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; Liener, “The Lectins: Properties, Functions, and Applications in Biology and Medicine,” Elsevier, 2012. Suitable lectins include concanavalin A (con A), pea lectin, sanfena lectin, and lentil lectin. Lentil lectin is a preferred column for detergent exchange due to its binding properties.Lectin columns are commercially available; for example, Capto Lentil Lectin is available from GE Healthcare. In certain aspects, the lentil lectin column may use a recombinant lectin. At the molecular level, it is believed that carbohydrate fractions bind to the lentil lectin, releasing amino acids from the protein. Petition 870250108300, dated 11 / 26 / 2025, pp. 124 / 284 117 / 210 to agglutinate around the detergent, resulting in the formation of a detergent core that provides nanoparticles with multiple copies of the antigen, for example, glycoprotein oligomers that can be dimers, trimers, or tetramers anchored in the detergent. In embodiments, the CoV S polypeptides and / or hemagglutinins form trimers. In embodiments, the trimers of CoV S polypeptide and / or hemagglutinins are anchored in detergent. In embodiments, each nanoparticle contains at least one trimer associated with a non-ionic core.

[0267] The detergent, when incubated with the protein to form nanoparticles during detergent changeover, can be present at up to about 0.1% (w / v) during the initial purification steps and this amount is reduced to achieve the final nanoparticles with ideal stability. For example, the non-ionic detergent (e.g., PS80) can be from about 0.005% (v / v) to about 0.1% (v / v), for example, about 0.005% (v / v), about 0.006% (v / v), about 0.007% (v / v), about 0.008% (v / v), about 0.009% (v / v), about 0.01% (v / v), about 0.015% (v / v), about 0.02% (v / v), about 0.025% (v / v), about 0.03% (v / v), about 0.035% (v / v), about 0.04% (v / v), about 0.045% (v / v), about 0.05% (v / v), about 0.055% (v / v), about 0.06% (v / v), about 0.065% (v / v), about 0.07% (v / v), about 0.075% (v / v), about 0.08% (v / v), about 0.085% (v / v), about 0.09% (v / v), about 0.095% (v / v), or about 0.1% (v / v) PS80.In embodiments, the nanoparticle contains approximately 0.03% to approximately 0.05% PS80. In embodiments, the nanoparticle contains approximately 0.01% (v / v) PS80.

[0268] In embodiments, purified CoV S polypeptides and / or hemagglutinin are dialyzed. In embodiments, dialysis occurs after purification. In embodiments, CoV S polypeptides and / or hemagglutinin are dialyzed in a solution comprising sodium phosphate, NaCl, and PS80. In embodiments, the dialysis solution comprising sodium phosphate contains between 5 mM and 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 Petition 870250108300, dated 11 / 26 / 2025, pp. 125 / 284 118 / 210 mM, approximately 40 mM, approximately 45 mM, approximately 50 mM, approximately 55 mM, approximately 60 mM, approximately 65 mM, approximately 70 mM, approximately 75 mM, approximately 80 mM, approximately 85 mM, approximately 90 mM, approximately 95 mM, or approximately 100 mM of sodium phosphate. In embodiments, the pH of the solution comprising sodium phosphate is approximately 6.5, approximately 6.6, approximately 6.7, approximately 6.8, approximately 6.9, approximately 7.0, approximately 7.1, approximately 7.2, approximately 7.3, approximately 7.4, or approximately 7.5.In embodiments, the dialysis solution comprising sodium chloride comprises from 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, about 260 mM, about 270 mM, about 280 mM, about 290 mM, about 300 mM, about 310mM, about 320mM, about 330mM, about 340mM, about 350mM, about 360mM, about 370mM, about 380mM, about 390mM, about 400mM, about 410mM, about 420mM, about 430mM, about 440mM, about 450mM mM, about 460 mM, about 470 mM, about 480 mM, about 490 mM or about 500 mM NaCl.In various forms, the dialysis solution comprising PS80 comprises 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), 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) of PS80. In embodiments, the dialysis solution comprises approximately 25 mM sodium phosphate (pH 7.2), approximately 300 mM NaCl and approximately 0.01% (v / v) of PS80.

[0269] Detergent replacement can be performed with purified proteins Petition 870250108300, dated 11 / 26 / 2025, pp. 126 / 284 119 / 210 as discussed above and purified, frozen for storage and then thawed for detergent change.

[0270] The stability of the compositions disclosed in this document can be measured in several ways. In one approach, a peptide map can be prepared to determine the integrity of the antigen protein after various treatments designed to stress the nanoparticles mimicking adverse storage conditions. Thus, a measure of stability is the relative abundance of antigen peptides in a stressed sample compared to a control sample. For example, the stability of nanoparticles containing CoV S polypeptides can be evaluated by exposing the nanoparticles to various pHs, proteases, salt, oxidizing agents, including but not limited to hydrogen peroxide, various temperatures, freeze / thaw cycles, and agitation. (iic) at least three hemagglutinins: a complete inactivated influenza virus

[0271] In embodiments, the at least three hemagglutinins are in the form of a complete influenza virus. A complete influenza virus comprises its entire envelope, viral membrane, nucleocapsid, and genetic material. In embodiments, the complete influenza virus is inactivated as described throughout this disclosure. (iid) at least three hemagglutinins: hemagglutinin composition extracted from an influenza virus

[0272] In embodiments, the at least three hemagglutinins are in the form of a hemagglutinin composition extracted from an influenza virus. In embodiments, the hemagglutinin composition extracted from an influenza virus is either an influenza split virion composition or an influenza subunit composition.

[0273] A split influenza virion is an influenza virus that has an influenza virus membrane that has been disrupted by a surfactant. In embodiments, surfactant is any surfactant described in this document. Petition 870250108300, dated 11 / 26 / 2025, pp. 127 / 284 In embodiments 120 / 210, the surfactant is sodium taurodeoxycholate, octylphenol ethoxylate (Triton®-X 100), or cetyltrimethylammonium bromide. In embodiments, the split influenza virion is produced using an egg-based or cell culture-based manufacturing method. In embodiments, a split influenza virion is produced by propagating an influenza virus in Madin Darby Canine Kidney (MDCK) cells, inactivating the virus with β-propiolactone, exposing the virus to cetyltrimethylammonium bromide detergent, and purifying the virion.

[0274] Like an influenza split virion, an influenza subunit composition is generated by disrupting the influenza virus membrane with a surfactant. However, unlike an influenza split virion, the influenza subunit composition undergoes further purification. In embodiments, the influenza subunit composition is purified by different sedimentation to remove the internal subviral core. In embodiments, the influenza subunit composition is purified by centrifugation, chromatography, precipitation, or nanofiltration. The surfactant can be any surfactant described in this document. In embodiments, the surfactant is sodium taurodeoxycholate, octylphenol ethoxylate (Triton®-X 100), or cetyltrimethylammonium bromide. In embodiments, the influenza subunit vaccine is produced using an egg-based or cell culture-based manufacturing method.

[0275] In embodiments, a hemagglutinin composition extracted from a virus comprises hemagglutinin and excludes viral ribonucleoprotein (vRNP), M1 matrix protein, and the viral envelope.

[0276] In embodiments, a hemagglutinin composition extracted from a virus is obtained by (i) producing a whole influenza virus using an egg-based manufacturing method; (ii) harvesting 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 zonal centrifugation; and (v) centrifuging the inactivated influenza virus in the presence of cetyltrimethylammonium bromide. Petition 870250108300, dated 11 / 26 / 2025, pp. 128 / 284 121 / 210 Immunogenic Composition Formulations

[0277] The disclosure provides immunogenic compositions containing (i) at least three hemagglutinin (HA) glycoproteins, wherein the three HA glycoproteins are from different influenza strains; (ii) a CoV S polypeptide in the form of a detergent core nanoparticle, wherein the detergent is a non-ionic detergent; 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) detergent core nanoparticles comprising hemagglutinin (HA); (b) HaSMaNs (Hemagglutinin Saponin Matrix Nanoparticles); (c) a split influenza virion; (d) a complete influenza virus; (e) recombinant hemagglutinin; and (f) a hemagglutinin composition extracted from a virus. In embodiments, the immunogenic composition may contain nanoparticles with antigens from more than one viral strain of the same virus species.

[0278] In embodiments, the immunogenic compositions comprise (i) a CoV S polypeptide in the form of a detergent core nanoparticle, wherein the detergent is a nonionic detergent, (ii) at least three hemagglutinin (HA) glycoproteins, wherein the three HA glycoproteins are from different influenza strains, wherein the at least three HA glycoproteins are in the form of detergent core nanoparticles comprising HA glycoproteins and HaSMaNs. In embodiments, the immunogenic compositions comprise (i) at least three hemagglutinin (HA) glycoproteins, wherein the three HA glycoproteins are from different influenza strains, wherein the at least three HA glycoproteins are in the form of an influenza split virion and (ii) a CoV S polypeptide in the form of a detergent core nanoparticle, wherein the detergent is a nonionic detergent.In embodiments, the immunogenic compositions comprise (i) at least three hemagglutinin (HA) glycoproteins, wherein the three HA glycoproteins are from different influenza strains, wherein the at least three HA glycoproteins are in the form of a hemagglutinin composition extracted from a virus and. Petition 870250108300, dated 11 / 26 / 2025, pp. 129 / 284 122 / 210 (ii) a CoV S polypeptide in the form of a detergent core nanoparticle, wherein the detergent is a non-ionic detergent.

[0279] In embodiments, the compositions comprise a total of between about 1 and about 10 detergent core nanoparticles comprising hemagglutinin and HaSMaNs, for example, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9 or about 10 nanoparticles in total. In embodiments, each detergent core nanoparticle and HaSMaN contains a hemagglutinin from a different influenza strain. In embodiments, each of the HaSMaNs contains hemagglutinin from a strain of influenza Type A. In embodiments, the hemagglutinin of each of the detergent core nanoparticles is from a strain of influenza Type B. In embodiments, each of the HaSMaNs contains hemagglutinin from a strain of influenza Type B. In embodiments, the hemagglutinin of each of the detergent core nanoparticles is from a strain of influenza Type A.In embodiments, the hemagglutinin of each detergent core nanoparticle is from a Type B influenza strain and the hemagglutinin of each HaSMaN is from a Type A influenza strain. In embodiments, the compositions comprise a total of four detergent core nanoparticles comprising hemagglutinin and HaSMaNs. In embodiments, the compositions comprise a total of three detergent core nanoparticles comprising hemagglutinin and HaSMaNs.

[0280] In another embodiment, the disclosures provide a pharmaceutical package or kit comprising one or more containers filled with one or more of the components of the immunogenic compositions.

[0281] The compositions disclosed in this document may be used prophylactically or therapeutically, but will typically be prophylactic. Therefore, the disclosure includes methods for treating or preventing infection. The methods involve administering to the subject a therapeutic or prophylactic amount of the immunogenic compositions of the disclosure. In embodiments, the pharmaceutical composition is a vaccine composition that provides an effect Petition 870250108300, dated 11 / 26 / 2025, pp. 130 / 284 123 / 210 protective. In embodiments, the protective effect includes the improvement of a symptom associated with infection in a percentage of the exposed population. For example, the composition may prevent or reduce one or more selected viral disease symptoms from among: fever-related fatigue, muscle pain, headache, sore throat, vomiting, diarrhea, rash, symptoms of renal and hepatic insufficiency, internal bleeding, and external bleeding, compared to an untreated subject.

[0282] Nanoparticles can be formulated for administration as vaccines in the presence of various excipients, buffers, and the like. For example, vaccine compositions 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 particular cases, about 22 mM of 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, when present, may be about 150 mM. In certain compositions, sodium chloride may be present in higher concentrations, for example, from about 200 mM to about 500 mM.In embodiments, sodium chloride is present in a high concentration, including, but not limited to, approximately 200 mM, approximately 250 mM, approximately 300 mM, approximately 350 mM, approximately 400 mM, approximately 450 mM, or approximately 500 mM.

[0283] In embodiments, the nanoparticles described in this document have improved stability at certain pH levels. In embodiments, the nanoparticles are stable at slightly acidic pH levels. For example, nanoparticles that are stable at a slightly acidic pH, for example, from pH 5.8 to pH 7.0. In embodiments, nanoparticles and compositions containing nanoparticles can be stable at pHs ranging from about pH 5.8 to about pH 7.0, including about pH 5.9 to about pH 6.8, about pH 6.0 to about pH 6.5, about pH 6.1 to about pH 6.4, about pH 6.1 to about pH 6.3, or about pH 6.2. In embodiments, the nanoparticles and compositions described Petition 870250108300, dated 11 / 26 / 2025, pp. 131 / 284 The nanoparticles and compositions described in this document are stable at neutral pHs, including from about pH 7.0 to about pH 7.4. In embodiments, the nanoparticles and compositions described in this document are stable at slightly alkaline pHs, for example, from about pH 7.0 to about pH 8.5, from about pH 7.0 to about pH 8.0, or from about pH 7.0 to about pH 7.5, including all values ​​and ranges in between. Adjuvants

[0284] In certain embodiments, the compositions disclosed in this document may be combined with one or more adjuvants to enhance an immune response. In embodiments, the compositions are prepared without adjuvants and are thus available for administration as adjuvant-free compositions. Advantageously, the adjuvant-free compositions disclosed in this document may provide protective immune responses when administered as a single dose. Alum-free compositions that induce robust immune responses are especially useful in adults aged 60 years or older. In embodiments, the adjuvant is an oil-in-water emulsion composed of squalene as the oil phase, stabilized with the surfactants polysorbate 80 and sorbitan trioleate, in citrate buffer. Aluminum-based adjuvants

[0285] In embodiments, the adjuvant may be alum (e.g., AlPO4 or Al(OH)3). Typically, the nanoparticle is substantially bound to the alum. For example, the nanoparticle may be at least 80% bound, at least 85% bound, at least 90% bound, or at least 95% bound to the alum. Frequently, the nanoparticle is 92% to 97% bound to the alum in a composition. The amount of alum present per dose is typically in a range between about 400 pg to about 1250 pg. For example, alum may be present in a quantity per dose of approximately 300 pg to approximately 900 pg, approximately 400 pg to approximately 800 pg, approximately 500 pg to approximately 700 pg, approximately 400 pg to approximately 600 pg, or approximately 400 pg to approximately 500 pg. Typically, alum is present in approximately 400 pg to Petition 870250108300, dated 11 / 26 / 2025, pp. 132 / 284 125 / 210 a dose of 120 pg of the protein nanoparticle. Saponin Adjuvants

[0286] Adjuvants containing saponins may also be combined with the immunogens disclosed in this document. Saponins are glycosides derived from the bark of the Quillaja saponaria Molina tree. Typically, saponin is prepared using a multi-step purification process, resulting in multiple fractions. As used in this document, the term a saponin fraction of Quillaja saponaria Molina is used generically to describe a semi-purified or defined saponin fraction of Quillaja saponaria or a substantially pure fraction thereof. Saponin Fractions

[0287] Several approaches to the production of saponin fractions are suitable. Fractions A, B, and C are described in U.S. Pat. No. 6,352,697 and can be prepared as follows. A lipophilic fraction of 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 semi-preparative HPLC with elution using a gradient of 25% to 60% acetonitrile in acidic water. The fraction referred to in this document as Fraction A or QH-A is, or corresponds to, the fraction that is eluted to approximately 39% acetonitrile. The fraction referred to in this document as Fraction B or QH-B is, or corresponds to, the fraction that is eluted to approximately 47% acetonitrile. The fraction referred to in this document as Fraction C or QH-C is, or corresponds to, the fraction that is eluted at approximately 49% acetonitrile. Additional information on fraction purification can be found in Pat.US Patent No. 5,057,540. When prepared as described in this document, Fractions A, B, and C of Quillaja saponaria Molina each represent groups or families of chemically related molecules with definable properties. The chromatographic conditions under which they are obtained are such that batch-to-batch reproducibility in terms of elution profile and biological activity is highly high. Petition 870250108300, dated 11 / 26 / 2025, pp. 133 / 284 126 / 210 consistent.

[0288] Other saponin fractions have been described. Fractions B3, B4 and B4b are described in EP 0436620. Fractions QA1-QA22 are described in EP03632279. B2, Q-VAC (Nor-Feed, AS Denmark), Quillaja saponaria Molina Spikoside (Isconova AB, Ultunaallén 2B, 756 51 Uppsala, Sweden). Fractions QA1, 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 EP 0 3632 279 B2, especially QA-7, QA-17, QA-18 and QA-21 can be used. They are obtained as described in document EP 0 3632 279 B2, especially on page 6 and in Example 1 on pages 8 and 9.

[0289] The saponin fractions described in this document and used to form adjuvants are frequently substantially pure fractions; that is, the fractions are substantially free from the presence of contamination from other materials. In particular aspects, a substantially pure saponin fraction 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. ISCOM Structures

[0290] Saponin fractions can be administered in the form of a cage-like particle referred to as an ISCOM (Immune Stimulating COM Complex). ISCOMs can be prepared as described in EP0109942B1, EP0242380B1 and EP0180546 B1. In particular embodiments, a carrier and / or passenger antigen may be used, as described in EP 9600647-3 (PCT / SE97 / 00289). Matrix Adjuvants

[0291] In some embodiments, ISCOM is an ISCOM matrix complex. An ISCOM matrix complex comprises at least one saponin moiety and a lipid. The lipid is at least one sterol, such as cholesterol. In specific aspects, the ISCOM matrix complex also contains a Petition 870250108300, dated 11 / 26 / 2025, pp. 134 / 284 127 / 210 phospholipid. ISCOM matrix complexes may also contain one or more other immunomodulatory substances (active adjuvants), not necessarily a glycoside, and may be produced as described in EP0436620B1, which is incorporated herein by reference in its entirety.

[0292] In other respects, ISCOM is an ISCOM complex. An ISCOM complex contains at least one saponin, at least one lipid, and at least one type of antigen or epitope. The ISCOM complex contains antigen associated by detergent treatment so that a portion of the antigen integrates into the particle. In contrast, the ISCOM matrix is ​​formulated as a mixture with antigen, and the association between the ISCOM matrix particles and the antigen is mediated by electrostatic and / or hydrophobic interactions.

[0293] According to one embodiment, the saponin fraction integrated into an ISCOM matrix complex or an ISCOM complex, or at least one additional adjuvant, which is also integrated into the ISCOM matrix complex or ISCOM or mixed with them, 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 any purified sub-fraction, for example, QA 1-21.

[0294] In particular aspects, each ISCOM particle may contain at least two saponin fractions. Any combinations of % by weight of different saponin fractions may be used. Any combination of % by weight of any two fractions may be used. For example, the particle may contain any % by weight of fraction A and any % by weight of another saponin fraction, such as a crude saponin fraction or fraction C, respectively. Therefore, in particular aspects, each ISCOM matrix particle or each ISCOM complex particle may contain from 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 of a fraction Petition 870250108300, dated 11 / 26 / 2025, pp. 135 / 284 128 / 210 of saponin, for example, fraction A, and the remainder up to 100% in each case of another saponin, for example, any crude fraction or any other fraction, for example, fraction C. 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. Published Application No. 2013 / 0129770, which is incorporated herein by reference in its entirety.

[0295] In particular embodiments, the ISCOM matrix or ISCOM complex comprises 5-99% by weight of one fraction, for example, fraction A, and the remainder up to 100% by weight of another fraction, for example, a crude saponin fraction or fraction C. The weight is calculated as the total weight of the saponin fractions.

[0296] In another embodiment, the ISCOM matrix or ISCOM complex comprises from 40% to 99% by weight of one fraction, for example, fraction A, and from 1% to 60% by weight of another fraction, for example, a crude saponin fraction or fraction C. The weight is calculated as the total weight of the saponin fractions.

[0297] In yet another embodiment, the ISCOM matrix or ISCOM complex comprises 70% to 95% by weight of one fraction, for example, fraction A, and 30% to 5% by weight of another fraction, for example, a crude saponin fraction or fraction C. The weight is calculated as the total weight of the saponin fractions. In other embodiments, the saponin fraction of Quillaja saponaria Molina is selected from any one of QA 1-21.

[0298] In addition to particles containing mixtures of saponin fractions, ISCOM matrix particles and ISCOM complex particles may be formed using only one saponin fraction. The compositions disclosed in this document may contain multiple particles, wherein each particle contains only one saponin fraction. That is, certain compositions may contain one or more different types of ISCOM matrix complex particles and / or one or more different types of ISCOM complex particles, wherein each individual particle contains a saponin fraction of Petition 870250108300, dated 11 / 26 / 2025, pp. 136 / 284 129 / 210 Quillaja saponaria Molina, in which the saponin fraction in one complex is different from the saponin fraction in the other complex particles.

[0299] In particular aspects, one type of saponin fraction or a crude saponin fraction may be integrated into an ISCOM matrix complex or particle and another type of substantially pure saponin fraction, or a crude saponin fraction, may be integrated into another ISCOM matrix complex or particle. A composition or vaccine may comprise at least two types of complexes or particles, each type with a type of saponins integrated into physically different particles.

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

[0301] The ISCOM matrix or ISCOM complex particles, each with a saponin fraction, may be present in the composition in any combination of % by weight. In particular aspects, a composition may contain 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, of an ISCOM complex or matrix containing a first saponin fraction with the remaining portion consisting of an ISCOM complex or matrix containing a different saponin fraction. In some aspects, the remaining portion is one or more ISCOM complexes or matrices, where each complex or matrix particle contains only one saponin fraction. In other aspects, ISCOM complex or matrix particles may contain more than one saponin fraction.

[0302] In particular compositions, the only saponin fraction in an ISCOM matrix particle or ISCOM complex is Fraction A and the only Petition 870250108300, dated 11 / 26 / 2025, pp. 137 / 284 The 130 / 210 saponin fraction in a second ISCOM matrix or ISCOM complex particle is Fraction C.

[0303] Preferred compositions comprise a first ISCOM matrix containing Fraction A and a second ISCOM matrix containing Fraction C, wherein the ISCOM matrix of Fraction A constitutes about 70% by weight of the total saponin adjuvant and the ISCOM matrix of Fraction C constitutes about 30% by weight of the total saponin adjuvant. In another preferred composition, the ISCOM matrix of Fraction A constitutes about 85% by weight of the total saponin adjuvant and the ISCOM matrix of Fraction C constitutes about 15% by weight of the total saponin adjuvant. Thus, in certain compositions, the ISCOM matrix of Fraction A is present in a range of about 70% to about 85% and the ISCOM matrix of Fraction C is present in a range of about 15% to about 30% of the total weight amount of saponin adjuvant in the composition.In embodiments, the ISCOM matrix of Fraction A represents 50-96% by weight and the ISCOM matrix of Fraction C represents the remainder, respectively, of the sums of the weights of the ISCOM matrix of Fraction A and ISCOM matrix of Fraction C in the adjuvant. In a particularly preferred composition, referred to in this document as MATRIX-Mtm, the ISCOM matrix of Fraction A is present in about 85% and the ISCOM matrix of Fraction C is present in about 15% of the total weight of saponin adjuvant in the composition. MATRIX-MTM may be referred to interchangeably as Matrix-M1.

[0304] Fractions QS-7 and QS-21 exemplars, their production and use are described in U.S. Pat. Nos. 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 by reference herein.

[0305] In various applications, other adjuvants may be used additionally or as an alternative. The inclusion of any adjuvant described in Vogel et al., A Compendium of Vaccine Adjuvants and Excipients (2nd Edition), incorporated herein by reference in its entirety. Petition 870250108300, dated 11 / 26 / 2025, pp. 138 / 284 131 / 210 for all purposes, is provided for in the scope of this disclosure. Other adjuvants include Freund's complete adjuvant (a non-specific immune response stimulator containing killed Mycobacterium tuberculosis), Freund's incomplete adjuvants, and aluminum hydroxide adjuvant. Other adjuvants comprise GMCSP, BCG, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A and monophosphoryl lipid A (MPL), MF-59, RIBI, which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS) in a 2% squalene / TWEEN® polysorbate 80 emulsion. In embodiments, the adjuvant may be a paucilamellar lipid vesicle; for example, NOVASOMES®. NOVASOMES® are paucilamellar nonphospholipid vesicles ranging in size from about 100 nm to about 500 nm. They comprise BRIJ® 72 alcohol ethoxylate, cholesterol, oleic acid, and squalene. NOVASOMES® has been shown to be an effective adjuvant (see U.S. Pat. No.° 5,629,021,6,387,373 and 4,911,928. Administration and Dosage

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

[0307] In embodiments, the compositions described in this document are administered with an adjuvant. In embodiments, the compositions described in this document are administered without an adjuvant. In aspects, the adjuvant may be linked to the nanoparticle, such as by a non-interaction. Petition 870250108300, dated 11 / 26 / 2025, pp. 139 / 284 132 / 210 covalent. In other respects, the adjuvant is co-administered with the nanoparticle, but the adjuvant and the nanoparticle do not interact substantially.

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

[0309] In embodiments, the compositions described in this document have an efficacy against a SARS-CoV-2 virus or a heterogeneous SARS-CoV-2 strain that is between about 50% and about 99%, between about 80% and about 99%, between about 75% and about 99%, between about 80% and about 95%, between about 90% and about 98%, between about 75% and 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 CoV S nanoparticles or polypeptides described in this document have efficacy against a Cal strain.20C of SARSCoV-2 which is between approximately 50% and approximately 99%, between approximately 80% and approximately 99%, between approximately 75% and approximately 99%, between approximately 80% and approximately 95%, between approximately 90% and approximately 98%, between approximately 75% and approximately 95%. Petition 870250108300, dated 11 / 26 / 2025, pp. 140 / 284 133 / 210 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 CoV S nanoparticles or polypeptides described in this document have an efficacy against a P strain.1 of SARS-CoV-2 that is between about 50% and about 99%, between about 80% and about 99%, between about 75% and about 99%, between about 80% and about 95%, between about 90% and about 98%, between about 75% and 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 approximately 98% or at least approximately 99%. In embodiments, the CoV S nanoparticles or polypeptides described in this document have efficacy against a B.1 strain.351 of SARS-CoV-2 that is between about 50% and about 99%, between about 80% and about 99%, between about 75% and about 99%, between about 80% and about 95%, between about 90% and about 98%, between about 75% and 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 approximately 98% or at least approximately 99%. In embodiments, the CoV S nanoparticles or polypeptides described in this document have an efficacy against a B.1.1.7 strain of SARS-CoV-2 which is... Petition 870250108300, dated 11 / 26 / 2025, pp. 141 / 284 134 / 210 between about 50% and about 99%, between about 80% and about 99%, between about 75% and about 99%, between about 80% and about 95%, between about 90% and about 98%, between about 75% and 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 around 99%.

[0310] In embodiments, the immunogenicity of a composition described in this document against an influenza virus is determined using an HAI assay or by measuring neutralizing antibodies. The method for performing an HAI assay is described in the following article, which is incorporated by reference herein in its entirety: Cowling et al. Clin Infect Dis. 2019; 68(10): 1713-1717. In embodiments, the immunogenicity of nanoparticle influenza vaccines can be compared to a commercially available influenza vaccine composition. As used in this document, a commercially available influenza vaccine composition can be any influenza vaccine composition that is available for medical use. For example, a commercially available influenza vaccine composition may be formulated for a trivalent or quadrivalent injection. In aspects of the embodiments, the formulation for an injection may comprise the inactivated form of the virus.In embodiments, the commercially available influenza vaccine composition may be formulated for a nasal spray. In embodiments, the nasal spray formulation may comprise attenuated or weakened forms of the virus. In embodiments, the compositions disclosed in this document induce neutralizing antibodies that bind to an influenza strain that has deviated (i.e., undergone a slight mutation) from the sequence used in the virus within the same influenza subtype. In embodiments, one, two, three, four, or all of the strains used in... Petition 870250108300, dated 11 / 26 / 2025, pp. 142 / 284 135 / 210 compositions induce neutralizing antibodies against one deflected strain, against two deflected strains, against three deflected strains, against four deflected strains, or against five deflected strains.

[0311] The compositions disclosed in this document may be administered systemically, via mucosal routes, transdermally, or directly into a specific tissue. As used in this document, the term systemic administration includes parenteral routes of administration. In particular, parenteral administration includes subcutaneous, intraperitoneal, intravenous, intra-arterial, intramuscular, or intrasternal injection techniques, intravenous or renal dialysis infusion. Typically, systemic parenteral administration is intramuscular injection. As used in this document, the term mucosal administration includes oral, intranasal, intravaginal, intrarectal, intratracheal, intestinal, and ophthalmic administration. Preferably, administration is intramuscular.

[0312] The compositions can be administered on a single-dose schedule or a multiple-dose schedule. Multiple doses can be used in a primary immunization schedule or in a booster immunization schedule. In a multiple-dose schedule, the various doses can be administered by the same or different routes, for example, a parenteral initiation and mucosal booster, a mucosal initiation and parenteral booster, etc. In some modalities, a follow-up booster dose is administered approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 5 weeks, or approximately 6 weeks after the previous dose. In others, the follow-up booster dose is administered 3 weeks after the administration of the previous dose. In others, the first dose is administered on day 0 and the booster dose is administered on day 21. In others, the first dose is administered on day 0 and the booster dose is administered on day 28.

[0313] In embodiments, the dose, measured in pg, may be the total weight of the dose including the solute, or the weight of the nanoparticles, or the weight of a protein. Petition 870250108300, dated 11 / 26 / 2025, pp. 143 / 284 136 / 210 in a nanoparticle (e.g., hemagglutinin weight or a CoV S polypeptide). The dose is measured using the A280 protein concentration assay or ELISA.

[0314] The dose of the CoV S polypeptide, including for pediatric administration, can range from about 1 pg to about 25 pg, about 3 pg to about 25 pg, about 5 pg to about 25 pg, about 5 pg to about 50 pg, about 1 pg to about 300 pg, about from 90 pg to about 270 pg, about 100 pg to about 160 pg, about 110 pg to about 150 pg, about 120 pg to about 140 pg, or about 140 pg to about 160 pg. In some cases, a dose of about 120 pg, administered with aluminum. In aspects, the dose varies from about 1 pg to about 90 pg. In modalities, the dose of the Spike (S) polypeptide of CoV is about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 11 pg, about 12 pg, about 13 pg, about 14 pg, about 15 pg, about 16 pg, about 17 pg, about 18 pg, about 19 pg, about 20 pg, about 21, about 22, about 23, about 24,about 25 pg, about 26 pg, about 27 pg, about 28 pg, about 29 pg, about 30 pg, about 40 pg, about 50, about 60, about 70, about 80, about 90 about 100 pg, about 110 pg, about 120 pg, about 130 pg, about 140 pg, about 150 pg, about 160 pg, about 170 pg, about 180 pg, about 190 pg, about 200 pg, about 210 pg, about 220 pg, about 230 pg, about 240 pg, about 250 pg, about 260 pg, about 270 pg, approximately 280 pg, approximately 290 pg, or approximately 300 pg, including all values ​​and ranges in between. In embodiments, the dose of the CoV S polypeptide is approximately 3 pg. In embodiments, the dose of the CoV S polypeptide is approximately 5 pg. In embodiments, the dose of the CoV S polypeptide is approximately 25 pg. In embodiments, the dose of the CoV S polypeptide is approximately 20 pg. In embodiments, the dose of the CoV S polypeptide is approximately 35 pg.

[0315] The total amount of hemagglutinin in immunogenic compositions can vary from about 25 pg to about 200 pg, about 30 pg Petition 870250108300, dated 11 / 26 / 2025, pp. 144 / 284 137 / 210 to about 150 pg, about 50 pg to about 100 pg, about 45 pg to about 180 pg, about 60 pg to about 190 pg, or about 100 pg to about 200 pg. In certain embodiments, the amount of influenza HA protein in the immunogenic composition may range from about 5 pg per strain to about 80 pg per strain, about 10 pg per strain to about 75 pg per strain, about 15 pg per strain to about 70 pg per strain, about 20 pg per strain to about 65 pg per strain, about 25 pg per strain to about 60 pg per strain, about 30 pg per strain to about 55 pg per strain, about 35 pg per strain to about 50 pg per strain, about 15 pg per strain to about 60 pg per strain. A dose per strain, for example, 10 pg per strain, refers to a dose of a hemagglutinin from a particular influenza strain. In certain modalities, a subject is administered an immunogenic composition comprising between approximately 5 pg and approximately 100 pg of hemagglutinin per strain. For example, in certain modalities,The compositions comprise approximately 5 pages, approximately 6 pages, approximately 7 pages, approximately 7.5 pages, approximately 8 pages, approximately 9 pages, approximately 10 pages, approximately 11 pages, approximately 12 pages, approximately 13 pages, approximately 14 pages, approximately 15 pages, approximately 16 pages, approximately 17 pages, approximately 18 pages, approximately 19 pages, approximately 20 pages, approximately 21 pages, approximately 22 pages, approximately 23 pages, approximately 24 pages, approximately 25 pages, approximately 26 pages, approximately 27 pages, approximately 28 pages, approximately 29 pages, approximately 30 pages, approximately 31 pages, approximately 32 pages, approximately 33 pages, approximately 34 pages pg, about 35 pg, about 36 pg, about 37 pg, about 38 pg, about 39 pg, about 40 pg, about 41 pg, about 42 pg, about 43 pg, about 44 pg, about 45 pg, about 46 pg, about 47 pg, about 48 pg, about 49 pg, about 50 pg, about 51 pg, about 52 pg, about 53 pg, about 54 pg, about 55 pg, about 56 pg, about 57 pg, about 58 pg, about 59 pg, about 60 pg, about 61 pg, about 62 pg, about 63 pg, about 64 pg,approximately 65 pages, approximately 66 pages, approximately 67 pages, approximately 68 pages, approximately 69 pages, approximately 70 pages, approximately 71 pages, approximately 72 pages, approximately 73 pages, approximately 74 pages, approximately 75 pages, approximately 76 pages, approximately 77 pages, approximately 78 pages, approximately 79 pages, approximately 80 pages, approximately 81 pages, approximately 82 pages, approximately 83 pages, approximately 84 pages, approximately 85 pages, approximately 86 pages, approximately 87 pages Petition 870250108300, dated 11 / 26 / 2025, page. 145 / 284 138 / 210 about 88 pg, about 89 pg, about 90 pg, about 91 pg, about 92 pg, about 93 pg, about 94 pg, about 95 pg, about 96 pg, about 97 pg, about 98 pg, about 99 pg or about 100 pg of hemagglutinin per strain. In embodiments, the composition comprises from about 24 pg to about 40 pg of hemagglutinin per strain. In embodiments, the composition comprises from about 24 pg to 75 pg of hemagglutinin per strain. In embodiments, the composition comprises about 30 pg of hemagglutinin per strain. In embodiments, the composition comprises about 60 pg of hemagglutinin per strain.

[0316] In embodiments, a patient receives an immunogenic composition containing approximately 5 to 60 pg of hemagglutinin per strain and approximately 2.5 to 22.5 pg of CoV S polypeptide. In embodiments, the composition comprises approximately 24 pg to 40 pg of hemagglutinin per strain and 5 pg to approximately 25 pg of CoV S polypeptide. In embodiments, the immunogenic composition contains 5, 10, 35, or 60 pg of hemagglutinin per strain. In embodiments, the immunogenic composition contains 2.5, 7.5, or 22.5 pg of CoV S polypeptide. In embodiments, the immunogenic composition contains 30 pg to approximately 54 pg of hemagglutinin per strain. In some embodiments, the immunogenic composition contains from 13 pg to approximately 25 pg of CoV S polypeptide. In some embodiments, the immunogenic composition contains hemagglutinin from three or four influenza strains. In some embodiments, the immunogenic composition contains approximately 40 pg of a saponin adjuvant, for example, MATRIX-Mtm.In embodiments, the immunogenic composition contains approximately 50 pg or 75 pg of a saponin adjuvant, for example, MATRIX-MTM. In embodiments, the hemagglutinin dose per strain and the CoV S polypeptide dose are found in Table 1H. In embodiments, a patient receives 30 pg of hemagglutinin per strain and 25 pg of CoV S polypeptide. In embodiments, a patient receives 60 pg of hemagglutinin per strain and 35 pg of CoV S polypeptide.

[0317] In modalities, the ratio between the amount of hemagglutinin per strain and the amount of CoV S polypeptide in the immunogenic composition is Petition 870250108300, dated 11 / 26 / 2025, pp. 146 / 284 139 / 210 approximately 1:1 to approximately 5:1. In embodiments, the ratio between the amount of hemagglutinin per strain and the amount of CoV S polypeptide in the immunogenic composition is approximately 1:1 to approximately 3:1. In embodiments, the ratio between the amount of hemagglutinin per strain and the amount of CoV S polypeptide in the immunogenic composition is approximately 1.3:1 to approximately 3:1. In embodiments, the ratio between the amount of hemagglutinin per strain and the amount of CoV S polypeptide in the immunogenic composition is approximately 1.3:1, approximately 1.4:1, about 1.5:1, about 1.6:1, 1.9:1, about 2.0:1, about 2.1:1, 2.4:1, about 2.5:1, about 2.6:1, 2.9:1, about 3.0:1, about 3.1:1, about 1.7:1, about 1.8:1, about about 2.2:1, about 2.3:1, about about 2.7:1, about 2.8:1, about about 3.2:1, about 3.3:1, about 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.

[0318] In embodiments, a patient is administered a booster dose of an immunogenic composition 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61 days after the initial administration of the immunogenic composition. In embodiments, a patient is administered a booster dose of the immunogenic composition 56 days after the initial administration of the immunogenic composition. In embodiments, a patient is administered a booster dose of the immunogenic composition 56 days (+ 5 days) after the initial administration of the immunogenic composition. In embodiments, the booster dose contains the same amount of hemagglutinin per strain and CoV S polypeptide as the initial dose. In embodiments, the booster dose contains a different amount of hemagglutinin per strain, CoV S polypeptide, or a combination thereof than the initial dose. In each modality, the amount of hemagglutinin per strain and the amount of CoV S polypeptide in the booster dose of the immunogenic composition are selected from Table 1H. Table 1H Petition 870250108300, dated 11 / 26 / 2025, pp. 147 / 284 140 / 210 Option Hemagglutinin per strain (pg) CoV S Polypeptide (pg) A 60 22.5 B 10 2.5 C 60 22.5 D 10 7.5 E 10 22.5 F 35 7.5 G 5 22.5 H 60 2.5 I 5 7.5 J 5 2.5 K 35 22.5 L 35 2.5 M 60 7.5 N 60 2.5 O 24 25 P 25 25 Q 26 25 R 27 25 S 28 25 T 29 25 U 30 25 V 31 25 W 32 25 X 33 25 Y 34 25 Z 35 25 AA 36 25 Petition 870250108300, dated 11 / 26 / 2025, pp. 148 / 284 141 / 210 Option Hemagglutinin per strain (pg) CoV S Polypeptide (pg) AB 37 25 AC 38 25 AD 39 25 AE 40 25 AF 33 25 AG 30-40 20 AH 54 25 AI 33-39 14-15 AJ 39 13 AK 30 20 AL 31 20 AM 32 20 AN 33 20 AO 34 20 AP 35 20 AQ 39 20 AR 37 20 AS 38 20 AT 39 20 AU 40 20 AV 33 14 AW 34 14 AX 35 14 AY 36 14 AZ 37 14 BA 38 14 BB 39 14 Petition 870250108300, dated 11 / 26 / 2025, pp. 149 / 284 142 / 210 Option Hemagglutinin by strain (pg) CoV S Polypeptide (pg) BC 33 15 BD 34 15 BE 35 15 BF 36 15 BG 37 15 BH 38 15 BI 39 15 BJ 30 15 BK 30 15 BL 45 15 BM 45 15 BN 30 25 BO 30 25 BP 45 25 BQ 45 25 BR 60 25 BS 60 25 BT 60 35

[0319] In embodiments, a patient receives a first immunogenic composition containing about 5 to 60 μg of hemagglutinin per strain and a second immunogenic composition containing about 2.5 to about 22.5 μg of CoV S polypeptide. In embodiments, a patient receives 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 embodiments, a patient receives a first immunogenic composition containing 30 μg of hemagglutinin per strain and a second immunogenic composition containing 25 μg of CoV S polypeptide. In embodiments, a patient receives a first immunogenic composition. Petition 870250108300, dated 11 / 26 / 2025, pp. 150 / 284 143 / 210 containing 60 pg of hemagglutinin per strain and a second immunogenic composition containing 35 pg of CoV S polypeptide. In embodiments, the first immunogenic composition contains about 5, about 10, about 35, or about 60 pg of hemagglutinin per strain. In embodiments, the second immunogenic composition contains about 2.5 pg, about 7.5 pg, about 22.5 pg, or about 25 pg of CoV S polypeptide. In embodiments, the first immunogenic composition contains hemagglutinin from three or four influenza strains. In 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 provided in Table 1H. In some modalities, the first immunogenic composition, the second immunogenic composition, or both contain approximately 50 pg of a saponin adjuvant, for example, MATRIXMTM.In embodiments, a patient is administered a booster dose of the first immunogenic composition, second immunogenic composition, or both, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61 days after the initial administration of the first immunogenic composition, second immunogenic composition, or both. In embodiments, a patient receives a booster dose of the immunogenic composition 56 days after the initial administration of the first immunogenic composition, second immunogenic composition, or both. In embodiments, a patient receives a booster dose of the immunogenic composition 56 days (+ 5 days) after the initial administration of the first immunogenic composition, second immunogenic composition, or both. In embodiments, the booster dose contains the same amount of hemagglutinin per strain and CoV S polypeptide as the initial dose. In embodiments, the booster dose contains a different amount of hemagglutinin per strain, CoV S polypeptide, or a combination thereof than the initial dose.In certain modalities, the amount of hemagglutinin per strain and the amount of CoV S polypeptide in the booster dose of the first or second immunogenic composition are selected from Table 1H. Certain populations may be administered with or without adjuvants. In certain aspects, the... Petition 870250108300, dated 11 / 26 / 2025, pp. 151 / 284 144 / 210 compositions may be free of added adjuvant. Under such circumstances, the dose may be increased by approximately 10%.

[0320] In embodiments, a patient is administered approximately 24 pg to approximately 40 pg of hemagglutinin per strain and a dose of CoV S polypeptide greater than approximately 20 pg. In embodiments, a patient is administered 24-40 pg of hemagglutinin per strain and a dose of CoV S polypeptide of approximately 25 pg.

[0321] In modality, the dose of the adjuvant is from approximately 1 pg to approximately 100 pg, for example, approximately 1 pg, approximately 2 pg, approximately 3 pg, approximately 4 pg, approximately 5 pg, approximately 6 pg, approximately 7 pg, approximately 8 pg, approximately 9 pg, approximately 10 pg, approximately 11 pg, approximately 12 pg, approximately 13 pg, approximately 14 pg, approximately 15 pg, approximately 16 pg, approximately 17 pg, approximately 18 pg, approximately 19 pg, approximately 20 pg, approximately 21, approximately 22, approximately 23, approximately 24, approximately 25 pg, approximately 26 pg, approximately 27 pg, approximately 28 pg, about 29 pg, about 30 pg, about 31 pg, about 32 pg, about 33 pg, about 34 pg, about 35 pg, about 36 pg, about 37 pg, about 38 pg, about 39 pg, about 40 pg, about 41 pg, about 42 pg, about 43 pg, about 44 pg, about 45 pg, about 46 pg, about 47 pg, about 48 pg, about 49 pg, about 50 pg, about 51 pg, about 52 pg, about 53 pg, about 54 pg, about 55 pg, about 56 pg, about 57 pg, about 58 pg,approximately 59 pages, approximately 60 pages, approximately 61 pages, approximately 62 pages, approximately 63 pages, approximately 64 pages, approximately 65 pages, approximately 66 pages, approximately 67 pages, approximately 68 pages, approximately 69 pages, approximately 70 pages, approximately 71 pages, approximately 72 pages, approximately 73 pages, approximately 74 pages, approximately 75 pages, approximately 76 pages, approximately 77 pages, approximately 78 pages, approximately 79 pages, approximately 80 pages, approximately 81 pages, approximately 82 pages, approximately 83 pages, approximately 84 pages, approximately 85 pages, approximately 86 pages, approximately 87 pages, approximately 88 pages, approximately 89 pages, approximately 90 pg, about 91 pg, about 92 pg, about 93 pg, about 94 pg, about 95 pg, about 96 pg, about 97 pg, about 98 pg, about 99 pg, or cercade, 100 pg of adjuvant. In some cases, a dose of adjuvant is about 50 pg. In some ways, the adjuvante is a saponin adjuvant, for example, Petition 870250108300, dated 11 / 26 / 2025, page. 152 / 284 145 / 210 MATRIX-Mtm.

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

[0323] In embodiments, the dose may comprise a CoV S polypeptide or hemagglutinin concentration of about 1 pg / mL to about 50 pg / mL, 10 pg / mL to about 100 pg / mL, about 10 pg / mL to about 50 pg / mL, about 175 pg / mL to about 325 pg / mL, about 200 pg / mL to about 300 pg / mL, about 220 pg / mL to about 280 pg / mL, or about 240 pg / mL to about 260 pg / mL.

[0324] In embodiments, the immunogenic compositions described in this document are administered in combination with an additional immunogenic composition. In embodiments, the additional immunogenic composition induces an immune response against SARS-CoV-2.In various modalities, the additional immunogenic composition is administered within approximately 1 minute, approximately 5 minutes, approximately 10 minutes, approximately 20 minutes, approximately 30 minutes, approximately 40 minutes, approximately 50 minutes, approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately 1 day, approximately 2 days, approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days, approximately 7 days, approximately 8 days, approximately 9 days, approximately 10 days, approximately 11 days, approximately 12 days, approximately 13 days, approximately 14 days. Petition 870250108300, dated 11 / 26 / 2025, pp. 153 / 284 146 / 210 days, approximately 15 days, approximately 16 days, approximately 17 days, approximately 18 days, approximately 19 days, approximately 20 days, approximately 21 days, approximately 22 days, approximately 23 days, approximately 24 days, approximately 25 days, approximately 26 days, approximately 27 days, approximately 28 days, approximately 29 days, approximately 30 days, or approximately 31 days of the disclosed immunogenic compositions. In embodiments, the additional composition is administered with a first dose of a composition described in this document. In embodiments, the additional composition is administered with a booster dose of a composition comprising CoV S nanoparticles or polypeptides, detergent core nanoparticles, HaSMaNs, or combinations thereof.In embodiments, the additional composition is administered with a (a) first immunogenic composition comprising (i) a CoV S glycoprotein in the form of a detergent-core nanoparticle, wherein the detergent is a nonionic detergent and (ii) a pharmaceutically acceptable buffer and (b) a second immunogenic composition comprising (i) at least three HA glycoproteins, wherein each HA glycoprotein is from a different influenza strain and (ii) a pharmaceutically acceptable buffer. In embodiments, the additional composition is administered with an initial dose of a first immunogenic composition and / or second immunogenic composition. In embodiments, the additional composition is administered with a booster dose of a first immunogenic composition and / or second immunogenic composition.

[0325] In embodiments, a method is provided in this document for inducing an immune response against one or more coronaviruses and / or influenza viruses comprising administering an immunogenic composition described in this document. In embodiments, a method is provided in this document for inducing an immune response against one or more coronaviruses and / or influenza viruses comprising (a) administering a first immunogenic composition comprising (i) a CoV S glycoprotein in the form of a detergent-core nanoparticle, wherein the detergent is a nonionic detergent and (ii) a pharmaceutically acceptable buffer and (b) administering a second composition Petition 870250108300, dated 11 / 26 / 2025, pp. 154 / 284 147 / 210 immunogenic comprising (i) at least three HA glycoproteins, wherein each HA glycoprotein is from a different influenza strain and (ii) a pharmaceutically acceptable buffer. In these modalities, the first immunogenic composition is administered approximately 20 minutes, approximately 30 minutes, approximately 40 minutes, approximately 50 minutes, approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately 1 day before the second immunogenic composition.In various modalities, the first immunogenic composition is administered approximately 20 minutes, approximately 30 minutes, approximately 40 minutes, approximately 50 minutes, approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately 1 day after the second immunogenic composition. Typically, the first immunogenic composition and the second immunogenic composition are administered at the same time (i.e., within 15 minutes of each other). In some modalities, the first immunogenic composition is administered intramuscularly. In others, the second immunogenic composition is administered intramuscularly.In some modalities, the second immunogenic composition is administered intranasally. In some modalities, the first immunogenic composition is administered intramuscularly and the second immunogenic composition is administered intramuscularly. In some modalities, the first immunogenic composition is administered intramuscularly and the second immunogenic composition is administered intranasally. In some modalities, the first immunogenic composition and the second composition. Petition 870250108300, dated 11 / 26 / 2025, pages 155 / 284 148 / 210 immunogenic compounds are administered intramuscularly to the same arm. In other modalities, the first immunogenic compound and the second immunogenic compound are administered intramuscularly to different arms.

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

[0327] In embodiments, the additional immunogenic composition comprises mRNA encoding for a CoV S polypeptide. In embodiments, the mRNA encodes for a CoV S polypeptide comprising proline substitutions at positions 986 and 987 of SEQ ID NO: 1. In embodiments, the mRNA encodes for a CoV S polypeptide comprising an intact furin cleavage site. In embodiments, the mRNA encodes for a CoV S polypeptide comprising proline substitutions at positions 986 and 987 of SEQ ID NO: 1 and an intact furin cleavage site. In embodiments, the mRNA encodes for a CoV S polypeptide comprising proline substitutions at positions 986 and 987 of SEQ ID NO: 1 and an inactive furin cleavage site. In embodiments, the mRNA encodes a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 87. In embodiments, the mRNA encoding a CoV S polypeptide is encapsulated in a lipid nanoparticle.An exemplary immunogenic composition comprising mRNA encoding for a CoV S polypeptide is described in Jackson et al. N. Eng. J. Med. 2020. An mRNA vaccine against SARS-CoV-2 is a preliminary report, which is incorporated herein by reference in its entirety. In embodiments, the composition comprising mRNA encoding for a CoV S polypeptide is administered at a dose of 25 pg, 100 pg, or 250 pg.

[0328] In embodiments, the additional immunogenic composition comprises an adenovirus vector encoding a CoV S polypeptide. In embodiments, the AAV vector encodes a CoV S polypeptide of type Petition 870250108300, dated 11 / 26 / 2025, pages 156 / 284 149 / 210 wild type. In embodiments, the AAV vector encodes for a CoV S polypeptide comprising proline substitutions at positions 986 and 987 of SEQ ID NO: 1 and an intact furin cleavage site. In embodiments, the AAV vector encodes for a CoV S polypeptide comprising proline substitutions at positions 986 and 987 of SEQ ID NO: 1 and an inactive furin cleavage site. In embodiments, the AAV vector encodes for a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 87. The following publications describe immunogenic compositions comprising an adenovirus vector encoding a CoV S polypeptide, each of which is incorporated by reference in its entirety herein: 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 embodiments, the additional immunogenic composition comprises deoxyribonucleic acid (DNA). In embodiments, the additional immunogenic composition comprises plasmid DNA. In embodiments, the plasmid DNA encodes for a CoV S polypeptide. In embodiments, the DNA encodes for a CoV S polypeptide comprising proline substitutions at positions 986 and 987 of SEQ ID NO: 1 and an intact furin cleavage site. In embodiments, the DNA encodes for a CoV S polypeptide comprising proline substitutions at positions 986 and 987 of SEQ ID NO: 1 and an inactive furin cleavage site. In embodiments, the DNA encodes for a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 87.

[0330] In modalities, the additional immunogenic composition comprises an inactivated virus vaccine.

[0331] In embodiments, immunogenic compositions comprising CoV S nanoparticles or polypeptides, detergent core nanoparticles and HaSMaNs are administered to a patient who has or has previously had a confirmed infection caused by SARS-CoV-2 or a heterogeneous strain of Petition 870250108300, dated 11 / 26 / 2025, pp. 157 / 284 150 / 210 SARS-CoV-2. Infection with SARS-CoV-2 or a heterogeneous strain of SARS-CoV-2 can be confirmed by a nucleic acid amplification test (e.g., polymerase chain reaction) or serological test (e.g., antibody test against a SARS-CoV-2 viral antigen). In embodiments, the composition comprising CoV S nanoparticles or polypeptides, detergent core nanoparticles, HaSMaNs, or combinations thereof, is administered to a patient at least about 3 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks after a patient has been diagnosed with COVID-19.In embodiments, the composition comprising CoV S nanoparticles or polypeptides, detergent core nanoparticles, HaSMaNs, or combinations thereof, is administered to a patient between 1 week and 1 year after the patient's diagnosis with COVID-19, for example, approximately 1 week, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 5 weeks, approximately 6 weeks, approximately 1 month, approximately 2 months, approximately 3 months, approximately 4 months, approximately 5 months, approximately 6 months, approximately 7 months, approximately 8 months, approximately 9 months, approximately 10 months, approximately 11 months, or approximately 1 year.In embodiments, the composition comprising CoV S nanoparticles or polypeptides, detergent core nanoparticles, HaSMaNs, or combinations thereof, is administered to a patient between 1 week and 20 years after the patient's diagnosis with COVID-19, for example, approximately 1 week, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 5 weeks, approximately 6 weeks, approximately 1 month, approximately 2 months, approximately 3 months, approximately 4 months, approximately 5 months, approximately 6 months, approximately 7 months, approximately 8 months, approximately 9 months, approximately 10 months, approximately 11 months, approximately 1 year, approximately 2 years, approximately 3 years, approximately 4 years, approximately 5 years, approximately 6 years, approximately 7 years, approximately 8 years, approximately 9 years, approximately 10 years, approximately 11 years, approximately 12 years, approximately 13 years old, approximately 14 years old, approximately 15 years old, approximately 16 years old, approximately 17 years old, approximately 18 years old, approximately 19 years old, or approximately 20 years old. Petition 870250108300, dated 11 / 26 / 2025, pp. 158 / 284 151 / 210

[0332] In embodiments, the composition comprising CoV S nanoparticles or polypeptides, detergent core nanoparticles, HaSMaNs, or combinations thereof, is administered after the patient has received a first immunogenic composition. Non-limiting examples of first immunogenic compositions include a SARS-CoV-2 Spike glycoprotein, an mRNA encoding a SARS-CoV-2 Spike glycoprotein, a plasmid DNA encoding a SARS-CoV-2 Spike glycoprotein, a viral vector encoding a SARS-CoV-2 Spike glycoprotein, or an inactivated SARS-CoV-2 virus.In various embodiments, the CoV S polypeptides or nanoparticles comprising them are administered between approximately 1 week and approximately 1 year, between approximately 1 week and 1 month, between approximately 3 weeks and 4 weeks, between approximately 1 week and 5 years, between approximately 1 year and approximately 5 years, between approximately 1 year and approximately 3 years, between approximately 3 years and approximately 5 years, between approximately 5 years and approximately 10 years, between approximately 1 year and approximately 10 years, or between approximately 1 year and approximately 2 years after administration of the first immunogenic composition.In embodiments, the CoV S polypeptides or nanoparticles comprising the same are administered between approximately 1 week and approximately 1 year after administration of the first immunogenic composition, for example, approximately 1 week, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 5 weeks, approximately 6 weeks, approximately 7 weeks, approximately 8 weeks, approximately 9 weeks, approximately 10 weeks, approximately 1 month, approximately 2 months, approximately 3 months, approximately 4 months, approximately 5 months, approximately 6 months, approximately 7 months, approximately 8 months, approximately 9 months, approximately 10 months, approximately 11 months, or approximately 1 year after administration of the first immunogenic composition.

[0333] In embodiments, the composition comprising CoV S polypeptides or nanoparticles comprising CoV S polypeptides, detergent core nanoparticles, HaSMaNs or combinations thereof, is useful for preparing immunogenic compositions to stimulate an immune response that confers immunity or substantial immunity to one or more of MERS, SARS, SARS-CoV. Petition 870250108300, dated 11 / 26 / 2025, p. 159 / 284 152 / 210 and a heterogeneous strain of SARS-CoV-2. Mucosal and cellular immunity can contribute to immunity to infection and disease. Antibodies secreted locally in the upper respiratory tract are an important factor in resistance to natural infection. Secretory immunoglobulin A (sIgA) is involved in upper respiratory tract protection and serum IgG in lower respiratory tract protection. The immune response induced by an infection protects against reinfection with the same virus or an antigenically similar viral strain. Antibodies produced in a host after immunization with the nanoparticles disclosed in this document can also be administered to others, thus providing passive administration to the subject.

[0334] In embodiments, the composition comprising CoV S nanoparticles or polypeptides, detergent core nanoparticles, HaSMaNs or combinations thereof, induces cross-neutralizing antibodies against SARS-CoV-2 viruses containing S proteins with one or more modifications selected from: (a) deletion of one or more amino acids from the NTD, wherein 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; and (b) mutation of one or more amino acids of the 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) mutation of one or more amino acids of the 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) mutation for one or more amino acids of SD1 / 2, wherein the one or more amino acids are selected from the group consisting of 557, 600, 601, 642, 664, 668 and combinations thereof; Petition 870250108300, dated 11 / 26 / 2025, pp. 160 / 284 153 / 210 (e) an inactive furin cleavage site (corresponding to one or more mutations in amino acids 669-672); (f) deletion of one or more amino acids from 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) mutation of one or more amino acids from the S2 subunit, wherein the amino acids are 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 from the TMCT (amino acids 12011260), wherein the amino acids of the CoV S glycoprotein are numbered in relation to SEQ ID NO: 2.

[0335] In embodiments, the composition comprising CoV S polypeptides or nanoparticles comprising CoV S polypeptides, detergent core nanoparticles, HaSMaNs or combinations thereof, induces cross-neutralizing antibodies against SARS-CoV-2 virus containing S proteins with one or more modifications selected from: amino acid 56 deletions, amino acid 57 deletion, amino acid 131 deletion, N488Y, A557D, D601G, P668H, T703I, S969A, D1105H, N426K and Y440F, wherein the amino acids are numbered relative to a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 2.

[0336] In embodiments, the composition comprising CoV S polypeptides or nanoparticles comprising CoV S polypeptides, detergent core nanoparticles, HaSMaNs or combinations thereof, induces cross-neutralizing antibodies against SARS-CoV-2 virus containing S proteins with one or more modifications selected from: amino acid 56 deletions, amino acid 57 deletion, amino acid 131 deletion, N488Y, A557D, D601G, P668H, T703I, S969A and D1105H, wherein the amino acids are numbered relative to a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 2.

[0337] In embodiments, the composition comprising polypeptides Petition 870250108300, dated 11 / 26 / 2025, pp. 161 / 284 154 / 210 CoV S or nanoparticles comprising CoV S polypeptides, detergent core nanoparticles, HaSMaNs or combinations thereof, induce cross-neutralizing antibodies against SARS-CoV-2 viruses containing S proteins with one or more modifications selected from: D67A, D202G, L229H, K404N, E471K, N488Y, D601G and A688V, wherein the amino acids are numbered relative to a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 2.

[0338] In embodiments, the composition comprising CoV S polypeptides or nanoparticles comprising CoV S polypeptides, detergent core nanoparticles, HaSMaNs or combinations thereof, induces cross-neutralizing antibodies against SARS-CoV-2 virus containing S proteins with one or more modifications selected from: deletion of amino acids 229231, D67A, D202G, K404N, E471K, N488Y, D601G and A688V, wherein the amino acids are numbered relative to a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 2.

[0339] In embodiments, the composition comprising CoV S polypeptides or nanoparticles comprising CoV S polypeptides, detergent core nanoparticles, HaSMaNs or combinations thereof, induces cross-neutralizing antibodies against SARS-CoV-2 virus containing S proteins with one or more modifications selected from: 229-231, L5F, D67A, D202G, K404N, E471K, N488Y, D601G and A688V, wherein the amino acids are numbered relative to a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 2.

[0340] In embodiments, the composition comprising CoV S polypeptides or nanoparticles comprising CoV S polypeptides, detergent core nanoparticles, HaSMaNs or combinations thereof, induces cross-neutralizing antibodies against SARS-CoV-2 viruses containing S proteins with one or more modifications selected from: L5F, T7N, P13S, D125Y, R177S, K404T, E471K, N488Y, D601G, H642Y, T1014I and V1163F, where the amino acids are numbered relative to a CoV S polypeptide with a Petition 870250108300, dated 11 / 26 / 2025, pp. 162 / 284 155 / 210 amino acid sequence of SEQ ID NO: 2.

[0341] In embodiments, the composition comprising CoV S polypeptides or nanoparticles comprising CoV S polypeptides, detergent core nanoparticles, HaSMaNs, or combinations thereof, induces cross-neutralizing antibodies against SARS-CoV-2 virus with an S protein comprising one or more modifications selected from: W139C and L439R, wherein the amino acids are numbered relative to a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 2. In embodiments, the CoV S protein comprising modifications with W139C and L439R is expressed with a signal peptide with an amino acid sequence of SEQ ID NO: 117 or SEQ ID NO: 5. In embodiments, the CoV S protein or nanoparticle comprising a CoV S protein induces cross-neutralizing antibodies against SARS-CoV-2 virus with one or more modifications selected from: D601G, W139C, and L439R, wherein the amino acids are numbered with respect to a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 2.In embodiments, the CoV S protein or nanoparticle comprising D601G, W139C, and L439R modifications is expressed with a signal peptide with an amino acid sequence of SEQ ID NO: 117 or SEQ ID NO: 5.

[0342] In embodiments, the composition comprising CoV S polypeptides or nanoparticles comprising CoV S polypeptides, detergent-core nanoparticles, HaSMaNs, or combinations thereof, induces cross-neutralizing antibodies against SARS-CoV-2 virus with one or more modifications selected from: D601G, L5F, D67A, D202G, amino acid deletions 229-231, R233I, K404N, E471K, N488Y, and A688V, wherein the amino acids are numbered relative to a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 2. In embodiments, the composition comprising nanoparticles or CoV S polypeptides, detergent-core nanoparticles, HaSMaNs, or combinations thereof, induces cross-neutralizing antibodies against SARS-CoV-2 virus with one or more modifications Petition 870250108300, dated 11 / 26 / 2025, pp. 163 / 284 156 / 210 selected from: L5F, D67A, D202G, amino acid deletions 229-231, R233I, K404N, E471K, N488Y and A688V, wherein the amino acids are numbered relative to a CoV S polypeptide with an amino acid sequence of SEQ ID NO: 2.

[0343] In embodiments, the composition comprising CoV S polypeptides or nanoparticles comprising CoV S polypeptides, detergent core nanoparticles, HaSMaNs or combinations thereof, described in this document, has efficacy in preventing coronavirus disease 19 (COVID-19) caused by a SARS-CoV-2 virus or by a heterogeneous strain of SARS-CoV-2 (for example, a SARS-CoV-2 strain 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 or a strain of SARS-CoV-2 Cal. 20C) which is from about 50% to about 99%, from about 80% to about 99%, from about 75% to about 99%, from about 80% to about 95%, from about 90% to about 98%, from about 75% to about 95%, from about 80% to about 90%, from about 85% to about 95%, from about 80% 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% for up to about 1 month, up to about 2 months, up to about 2.5 months, up to about 3 months, up to about 3.5 months, up to about 4 months, up to about 4.5 months, up to about 5 months, up to about 5.5 months, up to about 6 months, up to about 6.5 months, up to about 7 months, up to about 7.5 months, up to about 8 months, up to about 8.5 months, up to about 9 months, up to about 9.5 months, up to about 10 months, up to about 10.5 months, up to about 11 months, Petition 870250108300, dated 11 / 26 / 2025, pages 164 / 284 157 / 210 until approximately 11.5 months, until approximately 12 months, until approximately 12.5 months, until approximately 13 months, until approximately 13.5 months, until approximately 14 months, until approximately 14.5 months, until approximately 15 months, until approximately 15.5 months, until approximately 16 months, until approximately 16.5 months, until approximately 17 months, until approximately 17.5 months, until approximately 18 months, until approximately 18.5 months, until approximately 19 months, until approximately 19.5 months, until approximately 20 months, until approximately 20.5 months, until approximately 21 months, until approximately 21.5 months, until approximately 22 months, until approximately 22.5 months, until approximately 23 months, until approximately 23.5 months, until approximately 24 months, until 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 2.9 years, up to approximately 3 years or more after administration of the last dose of CoV S nanoparticles or polypeptides described in this document.In certain modalities, COVID-19 is mild. In certain modalities, COVID-19 is moderate. In certain modalities, COVID-19 is severe. In certain modalities, COVID-19 is asymptomatic.

[0344] In embodiments, the present 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 the nanoparticles disclosed in this document are produced by administering an immunogenic composition comprising CoV S nanoparticles or polypeptides, detergent core nanoparticles, HaSMaNs, or combinations thereof to an animal, collecting the animal's serum and / or plasma, and purifying the antibody from the serum and / or plasma. In one embodiment, the animal is a human. In embodiments, the animal is a chicken, mouse, guinea pig, rat, rabbit, goat, human, horse, sheep, or cow. In one embodiment, the animal is bovine or equine. In another embodiment, the bovine or equine animal is transgenic. In yet another embodiment, the transgenic bovine or equine animal produces human antibodies. In embodiments, the animal produces monoclonal antibodies.In modalities, the. Petition 870250108300, dated 11 / 26 / 2025, pp. 165 / 284 158 / 210 animal produces polyclonal antibodies. In one embodiment, the method further comprises the administration of an adjuvant or immunostimulatory compound. In another embodiment, 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 MERS, SARS, SARS-CoV-2, and influenza viruses.

[0345] In some embodiments, the disclosure provides co-formulation strategies (i.e., pre-filled syringes or premixing) for immunogenic compositions comprising nanoparticles. Typical vaccine delivery strategies currently being used are bedside mixing formulations. That is, vaccine and adjuvant compositions are stored separately and are mixed before administration. Premixing, co-formulation, or pre-filled syringe strategies for the vaccine are less common due to concerns about the stability of the antigens (e.g., hemagglutinin and CoV S polypeptide) and their subsequent immunogenic capabilities. The present disclosure provides immunogenic compositions that can be premixed and stored in advance. The disclosed vaccination strategies and formulations may improve vaccination efficiency and may reduce the risks of bedside mixing errors while maintaining overall safety and immunogenicity.

[0346] A variety of containers can be used to store and transport premixed formulations, including syringes for single administrations and plastic ampoules. In some cases, plastic ampoules can be manufactured using the blow-fill-seal manufacturing technique or method. In general, the blow-fill-seal (BFS) manufacturing method involves extruding a plastic material (e.g., resin) to form a parison, which is then placed in a mold and cut to size. A filling needle or mandrel is then used to inflate the plastic, which in turn results in a hollow ampoule that conforms substantially to the shape of the mold. Once inflated, a volume Petition 870250108300, dated 11 / 26 / 2025, pp. 166 / 284 159 / 210 of the desired liquid can be injected into the ampoule, the needle or filling stylet can be removed, and the ampoule can be sealed. Therefore, BFS can be an automated process that can be performed in a sterile environment without direct human intervention.

[0347] In some cases, the ability to aseptically manufacture sterile ampoules containing a desired liquid can make ampoules manufactured with BFS particularly suitable for the pharmaceutical industry. However, BFS technology has not been compatible with all products, pharmaceutical liquids, etc. For example, some known BFS manufacturing methods involve dispensing the liquid or product into the ampoule while the plastic is still relatively warm, which can result in adverse effects for temperature-sensitive liquids and / or products such as vaccines, biological products, etc. Advances in cold BFS technology, however, have increased the range of suitable products, liquids, etc., allowing some temperature-sensitive vaccines, biological products, and / or other pharmaceuticals to be contained 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 a desired pharmaceutical liquid or dosage that the ampoule is configured to contain. For example, some known BFS ampoules may include a piercing top, a twist top, a top including a male or female luer, and / or the like. Some known BFS ampoules may have a size and / or shape based on the volume of liquid or dosage configured to be dispensed therein. Additionally, some known BFS ampoules may be manufactured in a strip of multiple temporarily connected ampoules, which may increase manufacturing, packaging, and / or storage efficiencies and / or the like.

[0349] In embodiments, the immunogenic compositions described in this document are supplied in pre-filled syringes. When the immunogenic composition is prepared in a pre-filled syringe, an antigen and a Petition 870250108300, dated 11 / 26 / 2025, pp. 167 / 284 160 / 210 adjuvants are combined before administration. In embodiments, the pre-filled syringe contains hemagglutinin and CoV S polypeptide. In embodiments, the pre-filled syringe contains hemagglutinin and does not contain CoV S polypeptide. In embodiments, the pre-filled syringe contains CoV S polypeptide and does not contain hemagglutinin.

[0350] In embodiments, a subject is administered an immunogenic composition from a pre-filled syringe. In embodiments, the subject is administered an immunogenic composition containing hemagglutinin and CoV S polypeptide in a single pre-filled syringe. In embodiments, the subject is administered an immunogenic composition from a pre-filled syringe that contains a CoV S polypeptide but does not contain hemagglutinin. In embodiments, the subject...

Claims

1. Immunogenic composition characterized in that it comprises: (a) a coronavirus (CoV S) glycoprotein in the form of a detergent-core nanoparticle, wherein the detergent is a nonionic detergent; (b) at least three hemagglutinin (HA) glycoproteins, wherein each HA glycoprotein is from a different influenza strain; and (c) a pharmaceutically acceptable buffer.

2. Immunogenic composition, according to claim 1, characterized in that the at least three HA glycoproteins are in a form selected from the group consisting of: (a) detergent core nanoparticles comprising hemagglutinin (HA); (b) HaSMaNs (Hemagglutinin Saponin Matrix Nanoparticles); (c) an inactivated whole influenza virus; (d) a hemagglutinin composition extracted from an influenza virus; optionally, an influenza split virion composition or an influenza subunit composition; and any combination thereof.

3. Immunogenic composition, according to claim 2, characterized in that at least one HA glycoprotein is in the form of a detergent core nanoparticle comprising HA and at least one HA glycoprotein is in the form of a HaSMaN.

4. Immunogenic composition, according to claim 3, characterized in that the hemagglutinin glycoprotein of the detergent core nanoparticle is from an influenza Type B strain.

5. Immunogenic composition, according to claim 3, characterized in that the hemagglutinin glycoprotein of the detergent core nanoparticle is from an influenza Type A strain. Petition 870250089963, dated 10 / 02 / 2025, page 21 / 35 2 / 13 6. Immunogenic composition, according to claim 3, characterized in that the detergent core nanoparticle is a trypsin-resistant nanoparticle.

7. Immunogenic composition, according to claim 3, characterized in that HaSMaN is a trypsin-resistant nanoparticle.

8. Immunogenic composition, according to any one of claims 1 to 7, characterized in that each HA glycoprotein is from a different influenza strain.

9. Immunogenic composition, according to any one of claims 1 to 8, characterized in that it comprises up to 4, up to 5, up to 6, up to 7, up to 8, up to 9 or up to 10 HA glycoproteins.

10. Immunogenic composition, according to any one of claims 1-9, characterized in that each HA glycoprotein is in the form of a nanoparticle.

11. Immunogenic composition, according to claim 10, characterized in that each nanoparticle comprises an HA glycoprotein from a single influenza strain.

12. Immunogenic composition, according to claim 11, characterized in that each nanoparticle is a detergent core nanoparticle or a HaSMaN.

13. Immunogenic composition, according to any one of claims 1-12, characterized in that it comprises an adjuvant.

14. Immunogenic composition, according to claim 13, characterized in that the adjuvant is a saponin adjuvant.

15. Immunogenic composition, according to claim 14, characterized in that the adjuvant comprises at least two iscom particles, wherein: the first iscom particle comprises fraction A of Quillaja Saponaria Molina and not fraction C of Quillaja Saponaria Molina; and Petition 870250089963, dated 02 / 10 / 2025, page 22 / 35 3 / 13 the second iscom particle comprises fraction C of Quillaja Saponaria Molina and not fraction A of Quillaja Saponaria Molina.

16. Immunogenic composition, according to claim 15, characterized in that fraction A of Quillaja Saponaria Molina represents 50-96% by weight and fraction C of Quillaja Saponaria Molina represents the remainder, respectively, of the sum of the weights of fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina in the adjuvant.

17. Immunogenic composition, according to claim 15, characterized in that fraction A of Quillaja Saponaria Molina represents at least 70% by weight and fraction C of Quillaja Saponaria Molina represents the remainder, respectively, of the sum of the weights of fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina in the adjuvant.

18. Immunogenic composition, according to claim 15, characterized in that fraction A of Quillaja Saponaria Molina represents at least 85% by weight and fraction C of Quillaja Saponaria Molina represents the remainder, respectively, of the sum of the weights of fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina in the adjuvant.

19. Immunogenic composition, according to claim 15, characterized in that fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina represent about 85% by weight and about 15% by weight, respectively, of the sum of the weights of fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina in the adjuvant.

20. Immunogenic composition, according to claim 15, characterized in that fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina represent approximately 92% by weight and approximately 8% by weight, respectively, of the sum of the weights of fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina in the adjuvant. Petition 870250089963, dated 02 / 10 / 2025, page 23 / 35 4 / 13 21. Immunogenic composition, according to any one of claims 13-20, characterized in that it comprises about 50 pg or about 75 pg of saponin adjuvant.

22. Immunogenic composition, according to any one of claims 1-21, characterized in that the detergent is PS80.

23. Immunogenic composition, according to any one of claims 1-22, characterized in that the influenza strain is of 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. Immunogenic composition, according to any one of claims 1-23, characterized in that the pharmaceutically acceptable buffer comprises (i) sodium phosphate at about 25 mM; (ii) sodium chloride at about 150 mM; (iii) arginine hydrochloride at about 100 mM; and (iv) trehalose at about 5%; wherein the pH of the composition is about 7.

5.

25. Immunogenic composition, according to any one of claims 1-24, characterized in that the CoV S glycoprotein comprises: (i) an S1 subunit with an inactivated furin cleavage site, wherein the S1 subunit comprises an N-terminal domain (NTD), a receptor-binding domain (RBD), subdomains 1 and 2 (SD1 / 2), wherein the inactivated furin cleavage site has an amino acid sequence of QQAQ (SEQ ID NO: 7); wherein NTD optionally comprises one or more selected modifications of the group consisting of: (a) deletion of one or more selected amino acids 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) mutation 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, Petition 870250089963, dated 02 / 10 / 2025, p. 24 / 35 5 / 13 131, 132, 133, 139, 143, 144, 145, 177, 200, 201, 202, 209, 229, 233, 240, 245 and combinations thereof; wherein the RBD optionally comprises the mutation of 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; wherein the SD1 / 2 domain optionally comprises the mutation of one or more amino acids selected from the group consisting of 557, 600, 601, 642, 664, 668 and combinations thereof; and (ii) an S2 subunit, wherein amino acids 973 and 974 are proline, wherein the S2 subunit optionally comprises one or more selected modifications of the group consisting of: (a) deletion of one or more amino acids from 676-685, 676-702, 702-711, 775-793, 806-815 and combinations thereof;(b) mutation 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) deletion of one or more amino acids from TMCT; wherein the amino acids of the CoV S glycoprotein are numbered relative to a polypeptide with the sequence SEQ ID NO: 2.; 26. Immunogenic composition, according to claim 25, characterized in that the CoV S glycoprotein comprises or consists of an amino acid sequence with 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 of the following SEQ IDs: 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. Immunogenic composition, according to claim 26, characterized in that 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. Immunogenic composition, according to any one of claims 1-27, characterized in that it comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 CoV S glycoproteins.

29. Immunogenic composition, according to any one of claims 1-28, characterized in that it comprises from about 1 pg to about 50 pg of CoV S glycoprotein and from about 5 pg to about 60 pg of hemagglutinin per strain.

30. Immunogenic composition, according to claim 29, characterized in that it comprises from about 20 pg to about 50 pg of CoV S glycoprotein and from about 24 pg to about 40 pg of hemagglutinin per strain.

31. Immunogenic composition, according to any one of claims 1 to 29, characterized in that it comprises about 2.5 pg, about 3 pg, about 5 pg, about 7.5 pg, about 22.5 pg, about 25 pg or about 30 pg of CoV S glycoprotein.

32. Immunogenic composition, according to any one of claims 1-29, characterized in that it comprises about 5 pg, about 10 pg, about 24 pg, about 25 pg, about 26 pg, about 27 pg, about 28 pg, about 29 pg, about 30 pg, about 31 pg, about 32 pg, about 33 pg, about 34 pg, about 35 pg, about 36 pg, about 37 pg, about 38 pg, about 39 pg, about 40 pg or about 60 pg of hemagglutinin per strain.

33. Immunogenic composition, according to any one of claims 1-32, characterized in that the ratio between the amount of hemagglutinin per strain and the amount of CoV S glycoprotein in the immunogenic composition is from about 1:1 to about 5:

1.

34. Immunogenic composition, according to claim 33, characterized in that the ratio between the amount of hemagglutinin per strain and the amount of CoV S glycoprotein in the immunogenic composition is from about 1:1 to about 3:

1.

35. Immunogenic composition, according to claim 33, characterized in that the ratio between the amount of hemagglutinin per strain and the amount of CoV S glycoprotein in the immunogenic composition is from about 1.2:1 to about 3:1 or about 1.3:

1.

36. Immunogenic composition, according to claim 33, characterized in that the ratio between the amount of hemagglutinin per strain and the amount of CoV S glycoprotein in the immunogenic composition is about 1.2:1, 1.3:1, about 1.4:1, about 1.5:1, about 1.6:1, about 1.7:1, about 1.8:1, about 1.9:1, about 2.0:1, about 2.1:1, about 2.2:1, about 2.3:1, about 2.4:1, about 2.5:1, about 2.6:1, about 2.7:1, about 2.8:1, about 2.9:1, about 3.0:1, about 3.1:1, about 3.2:1, about 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. Immunogenic composition, according to any one of claims 1-31 and 33-36, characterized in that it comprises from about 30 pg to about 54 pg of hemagglutinin per strain.

38. Immunogenic composition, according to any one of claims 1-31 and 33-36, characterized in that it comprises approximately 30 pg, approximately 31 pg, approximately 32 pg, approximately 33 pg, approximately 34 pg, approximately 35 pg, approximately 36 pg, approximately 37 pg, approximately 38 pg, approximately 39 pg, approximately 40 pg, approximately 41 pg, approximately 42 pg, approximately 43 pg, approximately 44 pg, approximately 45 pg, approximately 46 pg, approximately 47 pg, approximately 48 pg, approximately 49 pg, approximately 50 pg, approximately 51 pg, approximately 52 pg, approximately 53 pg, approximately 54 pg, approximately 55 pg, approximately 56 pg, about 57 pg, about 58 pg, about 59 pg, or about 60 pg of hemagglutinin per strain.

39. Immunogenic composition, according to any of the Petition 870250089963, dated 10 / 02 / 2025, page 27 / 35 8 / 13 claims 1-29 and 32-38, characterized in that it comprises from about 13 μg to about 25 μg of CoV S glycoprotein.

40. Immunogenic composition, according to any one of claims 1-30 and 32-39, characterized in that it comprises from about 25 μg to about 35 μg of CoV S glycoprotein.

41. Immunogenic composition, according to any one of claims 1-37 and 39-40, characterized in that it comprises about 30 μg of hemagglutinin per strain and about 25 μg of CoV S glycoprotein.

42. Immunogenic composition, according to any one of claims 1-29, 32-36 and 38-41, characterized in that it comprises about 60 μg of hemagglutinin per strain and about 35 μg of coronavirus S glycoprotein.

43. Immunogenic composition, according to any one of claims 1-30, 32-38 and 40-41, characterized in that it comprises about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg or about 25 μg of CoV S glycoprotein.

44. Immunogenic composition, according to any one of claims 1-40 and 43, characterized in that it comprises about 33 μg of hemagglutinin per strain and about 25 μg of CoV S glycoprotein.

45. Immunogenic composition, according to any one of claims 1-30, 33-37, 39 and 43, characterized in that it comprises from about 30 μg to about 40 μg of hemagglutinin per strain and about 20 μg of CoV S glycoprotein.

46. ​​Immunogenic composition, according to any one of claims 1-29, 31, 33-40 and 43, characterized in that it comprises approximately 54 μg of hemagglutinin per strain and approximately 25 μg of CoV S glycoprotein.

47. Immunogenic composition, according to any of claims 1-29, 33-37 and 39, characterized in that it comprises from Petition 870250089963, dated 02 / 10 / 2025, page 28 / 35 9 / 13 about 33 pg to about 39 pg of hemagglutinin per strain and from about 14 pg to about 15 pg of CoV S glycoprotein.

48. Immunogenic composition, according to any one of claims 1-29, 33-37 and 39, characterized in that it comprises about 39 pg of hemagglutinin per strain and about 13 pg of CoV S glycoprotein.

49. Immunogenic composition, according to any one of claims 1-29, 32-36, 38, 40 and 42, characterized in that it comprises about 60 pg of hemagglutinin per strain and 75 ug of saponin adjuvant and about 35 pg of CoV S glycoprotein.

50. Immunogenic composition, according to any one of claims 33-49, characterized in that the ratio between the amount of hemagglutinin per strain and the amount of coronavirus S glycoprotein 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. Immunogenic composition characterized in that it comprises: (a) at least three hemagglutinin (HA) glycoproteins, wherein each HA glycoprotein is from a different influenza strain, wherein 30 to 60 pg of HA per strain are present in the composition; and (b) a pharmaceutically acceptable buffer.

52. Immunogenic composition, according to claim 51, characterized in that it further comprises a coronavirus (CoV S) glycoprotein in the form of a detergent core nanoparticle, wherein the detergent is a nonionic detergent.

53. Immunogenic composition, according to any one of claims 51-52, characterized in that about 30 pg, about 45 pg or about 60 pg of HA per strain are present in the composition.

54. Immunogenic composition, according to any of claims 51-53, characterized in that it further comprises an adjuvant.

55. Immunogenic composition, according to any one of claims 51-54, characterized in that the adjuvant is a saponin adjuvant.

56. Immunogenic composition, according to claim 55, characterized in that the adjuvant comprises at least two iscom particles, wherein: the first iscom particle comprises fraction A of Quillaja Saponaria Molina and not fraction C of Quillaja Saponaria Molina; and the second iscom particle comprises fraction C of Quillaja Saponaria Molina and not fraction A of Quillaja Saponaria Molina.

57. Immunogenic composition, according to claim 56, characterized in that fraction A of Quillaja Saponaria Molina represents 50-96% by weight and fraction C of Quillaja Saponaria Molina represents the remainder, respectively, of the sum of the weights of fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina in the adjuvant.

58. Immunogenic composition, according to claim 56, characterized in that fraction A of Quillaja Saponaria Molina represents at least 70% by weight and fraction C of Quillaja Saponaria Molina represents the remainder, respectively, of the sum of the weights of fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina in the adjuvant.

59. Immunogenic composition, according to claim 56, characterized in that fraction A of Quillaja Saponaria Molina represents at least 85% by weight and fraction C of Quillaja Saponaria Molina represents the remainder, respectively, of the sum of the weights of fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina in the adjuvant.

60. Immunogenic composition, according to claim 56, Petition 870250089963, dated 10 / 02 / 2025, page 30 / 35 11 / 13 characterized in that fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina represent about 85% by weight and about 15% by weight, respectively, of the sum of the weights of fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina in the adjuvant.

61. Immunogenic composition, according to claim 56, characterized in that fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina represent about 92% by weight and about 8% by weight, respectively, of the sum of the weights of fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina in the adjuvant.

62. Immunogenic composition, according to any one of claims 54-61, characterized in that it comprises about 50 pg or about 75 pg of adjuvant.

63. A method for stimulating an immune response against SARS-CoV-2, a heterogeneous strain of SARS-CoV-2, an influenza virus, or a combination thereof in a subject, characterized in that it comprises administering the immunogenic composition as defined in any of claims 1 and 62.

64. Method according to claim 63, characterized in that the subject receives a first dose on day 0 and a booster dose on day 56.

65. Method according to claim 63 or 64, characterized in that the immunogenic composition is administered intramuscularly.

66. A method according to either of claims 63 or 65, characterized in that a single dose of the immunogenic composition is administered.

67. Method, according to any one of claims 63-66, characterized in that the heterogeneous strain of SARS-CoV-2 is selected from the group consisting of SARS-CoV-2 strain 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 B.1.617.2, strain B.1.525, strain B.1.526, strain B.1.617.1, strain C.37, strain B.1.621 or SARS-CoV-2 omicron strain.

68. Method, according to any one of claims 63-67, characterized in that the effectiveness of the immunogenic composition in preventing coronavirus disease-19 (COVID-19) is at least 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% for up to about 2 months, up to about 2.5 months, up to about 3 months, up to about from 3.5 months, up to about 4 months, up to about 4.5 months, up to about 5 months, up to about 5.5 months, up to about 6 months, up to about 6.5 months, up to about 7 months, up to about 7.5 months, up to about 8 months, up to about 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, or up to approximately 12 months after administration of the immunogenic composition.

69. Method, according to any one of claims 63-67, characterized in that the effectiveness of the immunogenic composition in preventing coronavirus disease-19 (COVID-19) is from about 50% to about 99%, from about 50% to about 95%, from about 50% to about 90%, from about 50% to about 85%, from about 50% to about 80%, from about 60% to about 99%, from about 60% to about 95%, from about 60% to about 90%, from about 60% to about 85%, from about 60% to about 80%, from about 40% to about 99%, from about 40% to about 95%, from about 40% to about 90%, from about 40% to approximately 85%, from approximately 40% to approximately 80%, from approximately 40% to approximately 75%, from approximately 40% to approximately 70%, from approximately 40% to approximately 65%, from approximately 40% to approximately 55%, or from approximately 40% to approximately 50% for up to approximately 2 months, up to approximately 2.5 months, up to Petition 870250089963, dated 02 / 10 / 2025, page.32 / 35 13 / 13 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, or up to approximately 12 months after administration of the immunogenic composition.

70. Pre-filled syringe characterized in that it comprises the immunogenic composition as defined in any one of claims 1-62.

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