SARS-CoV-2 vaccine composition

Abstract

The present disclosure provides compositions of an adjuvant SARS-CoV-2 vaccine and their use in the prevention and control of Covid-19 infection, including the excessive inflammatory response of a host to the infection, including the long-term symptoms associated with Covid infection, as well as the use of the composition in the prevention and control of Covid-19 infection, as well as the use of the composition in the prevention and control of Covid-19 infection, as well as the use of the composition in the prevention and control of Covid-19 infection.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0000.1] This application claims priority to U.S. Provisional Application Serial No. 63 / 329,850 filed on April 11, 2022, U.S. Provisional Application Serial No. 63 / 329,851 filed on April 11, 2022, and U.S. Provisional Application Serial No. 63 / 380,499 filed on October 21, 2022, the entire disclosure of each application is incorporated herein by reference.

[0001] FIELD OF THE INVENTION The present invention relates to compositions of adjuvanted SARS-CoV-2 vaccines and their use in preventing and controlling Covid-19 infection, including the host's hyperinflammatory response to infection, including long-term symptoms associated with Covid infection. Background Art

[0002] The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has resulted in more than five million deaths worldwide and has wreaked havoc on the world economy. While the development of safe and effective vaccines would be of great assistance in controlling the pandemic, there remains a need for vaccines that provide better protection at dose-sparing levels of antigen, that exhibit cross-reactivity against emerging variants of concern (VOCs), and that have superior overall durability of immune responses, particularly in susceptible populations, including immunocompromised and elderly individuals, who remain at particularly high risk for hospitalization and death from Covid-19 infection. One potential solution to these issues is the use of adjuvants in combination with multiple virus-associated protein antigen preparations, including, for example, the so-called acetylcholine (ACE-2) receptor binding domain (RBD) of the viral spike protein or the full-length stabilized trimeric spike protein (S-protein).

[0003] Currently, several different adjuvants are used in vaccines approved by the U.S. FDA. Aluminum-based adjuvants in the form of one or more of amorphous aluminum hydroxyphosphate sulfate (AAHS), aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate (Alum) are used in a variety of vaccines, including Anthrax, DT, DTaP (Daptacel), DTaP (Infanrix), DTaP-IPV (Kinrix), DTaP-IPV (Quadracel), DTaP-HepB-IPV (Pediarix), DTaP–IPV / Hib (Pentacel), Hep A (Havrix), Hep A (Vaqta), Hep B (Engerix-B), Hep B (Recombivax), HepA / Hep B (Twinrix), HIB (Pedvax HIB), HPV (Gardasil 9), Japanese encephalitis (Ixiaro), MenB (Bexsero, Trumenba), Pneumococcal (Prevnar 13), Td (Tenivac), Td (Mass Biologics), Tdap (Adacel) and Tdap (Boostrix).

[0004] MF59 is an oil-in-water emulsion adjuvant containing squalene, which is included in Fluad, a trivalent and / or quadrivalent inactivated vaccine against seasonal influenza licensed for use in adults aged 65 years and older. AS01 is a liposome-based adjuvant for the herpes zoster vaccine Shingrix, which contains two immunostimulants: 3-O-deacylated-4'-monophosphoryl lipid A (MPL), a non-toxic derivative of lipopolysaccharide from Salmonella minnesota, and QS-21, a saponin fraction extracted from the soap tree (Quillaja saponaria Molina). AS03 is an adjuvant system consisting of alpha-tocopherol, squalene and polysorbate 80 in an oil-in-water emulsion, used in the influenza vaccines Pandemrix and Arepanrix, for the emerging 2009 pandemic influenza A / H1N1 strain. CpG 1018 is a Toll-like receptor 9 (TLR9) agonist used in the Heplisav-B vaccine. It consists of a CpG motif, a synthetic form of DNA that mimics the genetic material of bacteria and viruses and stimulates pro-inflammatory cytokines and Th1-driven immune responses.

[0005] Adjuvants are commonly used to promote enhanced antibody and cellular immune responses against antigens that are not sufficiently immunogenic on their own. Adjuvants induce biased Th1 or Th2-type immune responses in different ways. However, there are limitations with respect to these currently available vaccine adjuvants, particularly with respect to promoting durable and cross-reactive immunity expressed in at-risk populations (especially immunocompromised populations, including elderly individuals); currently approved Covid vaccines based on mRNA and adenoviral vector DNA vaccine technologies also exhibit similar limitations. However, despite their previous widespread use, the exact molecular mechanisms by which available adjuvants actually work in humans are not well understood and vary greatly from antigen to antigen between different adjuvants, requiring an empirical trial-and-error approach to formulation selection and vaccine development strategies. Summary of the invention

[0006] The present disclosure provides an adjuvanted SARS-CoV-2 vaccine. The SARS-CoV-2 protein antigen is formulated as a water-in-oil nanosphere emulsion vaccine composition, which comprises an aqueous phase and an oil phase. By weight, the buffered aqueous phase accounts for 25% to 35% of the emulsion, in the form of aqueous nanospheres with a median diameter of about 0.3 μm to about 1 μm, and contains one or more SARS-CoV-2 antigens. The oil phase accounts for 65% to 75% of the emulsion by weight, and the oil phase contains 85% to 90% squalene and squalane, 9% to 12% mannitol monooleate (mannide monooleate) and 0.5% to 0.7% polyoxyl-40-hydrogenated castor oil (polyoxyl-40-hydrogenated castor oil), each by weight of the oil phase.

[0007] In some embodiments, the SARS-CoV-2 antigen is a protein antigen. In some embodiments, the nanoemulsion vaccine composition comprises more than one SARS-CoV-2 antigen. In some embodiments, the SARS-CoV2 antigen comprises components derived from more than one SARS-CoV-2 strain. In some embodiments, the SARS-CoV2 antigen comprises SARS-CoV-2 antigens derived from more than one SARS-CoV-2 strain.

[0008] In some embodiments, squalene comprises about 40% to about 60% by weight of the oil phase. In some embodiments, squalane comprises about 40% to about 60% by weight of the oil phase. In some embodiments, the weight ratio of squalene to squalane is about 1:1.

[0009] In some embodiments, the median globule size is about 300 nm. In some embodiments, the composition has a viscosity of 100 cP or less. In some embodiments, the aqueous phase further comprises a protein solubilizing agent. In some embodiments, the protein solubilizing agent is urea or DMSO.

[0010] The present disclosure also provides a method for SARS-CoV-2 prevention, comprising administering a vaccine composition as described herein to a patient in need. The present disclosure also provides a method for attenuating SARS-CoV-2 infection, comprising administering a vaccine composition as described herein to a patient in need. The present disclosure also provides a method for generating a protective immune response against SARS-CoV-2 infection, comprising administering a vaccine composition as described herein to a patient in need.

[0011] The present disclosure also provides a method for long-term SARS-CoV-2 prevention, comprising administering a vaccine composition as described herein to a patient in need. The present disclosure also provides an immunotherapy method for attenuating one or more symptoms of long-term SARS-CoV-2 infection, comprising administering a therapeutic vaccine composition as described herein to a patient in need. The present disclosure also provides a method for producing a protective immune response against long-term SARS-CoV-2 infection, comprising administering a vaccine composition as described herein to a patient in need. The present disclosure also provides a method for producing a protective immune response against long-term SARS-CoV-2 infection, comprising administering a vaccine composition as described herein to a patient in need, wherein the vaccine composition simultaneously stimulates a durable antibody response to viral fragments and a balanced anti-inflammatory response to the patient's long-term hyperinflammation. The present disclosure also provides a method for increasing the efficacy and / or persistence of an immune response to a SARS-CoV-2 antigen, comprising administering an antigen in an oil-in-water nanoemulsion vaccine composition as described herein.

[0012] The present disclosure also provides a kit for point-of-use administration of a water-in-oil nanoemulsion vaccine against a SARS-CoV-2 infectious agent, comprising: a vial of adjuvant oil comprising mannitol monooleate, squalene and squalane, a vial of aqueous PBS comprising a SARS-CoV-2 antigen, at least one syringe, an anti-lipid three-way stopcock, at least one needle, and a vial for storing the formulated water-in-oil nanoemulsion vaccine. In some embodiments, the kit is used for point-of-use administration of a nanoparticle water-in-oil emulsion vaccine against the SARS-CoV-2 infectious agent, the vaccine comprising: a vial of adjuvant oil comprising mannitol monooleate, squalene and squalane, a vial of aqueous PBS for combination with the antigen, two syringes, an anti-lipid three-way stopcock, two needles and a vial for storing the formulated water-in-oil emulsion vaccine. In some embodiments, the nanoparticle water-in-oil emulsion vaccine produced by combining the kit components is a composition as described herein. The present disclosure also provides a water-in-oil nanoemulsion vaccine composition produced by the components of the above kit.

[0013] Vaccine emulsions can be prepared in batches by batch homogenization or pulsed continuous flow-through homogenization procedures, or in single-dose or repeat-dose vials at the point of use (POU) by handheld procedures. POU preparation of vaccine emulsions conserves antigen and oil phase supplies and facilitates adjustment of vaccine formulations in the event of a pandemic, as viral mutations generate new VOCs that may evade immunity conferred by existing vaccines or generated by prior infection.

[0014] The vaccine can be administered as a single or multiple initial doses to immunologically naive individuals. Alternatively, the vaccine can be administered as a booster dose to individuals who have previously received a heterologous vaccine (e.g., mRNA-based or DNA-based vaccine). In addition, the vaccine can be administered as an immunotherapy to individuals who exhibit long-term symptoms (long-term Covid), thereby suppressing hyperinflammatory symptoms and achieving a balanced state of Th1 / Th1 humoral and cellular immune profiles. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A Depicted are the mean minimum log endpoint titers for Group 3 (10 μg alum-adsorbed CoV-2RBD), Group 7 (10 μg CoV-2RBD in MAS-1), and Group 9 (10 μg CoV-1RBD in MAS-1) over 168 days post-inoculation (d0, d28). Figure 2AIndividual minimum log endpoint titers of 10 μg alum-adsorbed CoV-2RBD positive control in Group 3 over 168 days post-inoculation (d0, d28) are depicted. Figure 2B Depicted are individual lowest log endpoint titers of CoV-2RBD in 10 μg MAS-1 in Group 7 up to 168 days post-vaccination (d0, d28). Figure 2C Individual lowest log endpoint titers of CoV-1 RBD in 10 μg MAS-1 in group 9 within 168 days after vaccination (d0, d28) are described. Figure 3A Group 3 alum-adsorbed CoV-2RBD depicted: comparison of mean lowest log titers against CoV-2RBD and cross-reactive titers against CoV-1RBD Figure 3B Describes group 7 MAS-1 adjuvanted CoV-2RBD: comparison of mean lowest log titers against CoV-2RBD and cross-reactive titers against CoV-1RBD Figure 3C Depicted are group 9 MAS-1 adjuvanted CoV-1RBD: comparison of mean lowest log titers against CoV-1RBD and cross-reactive titers against CoV-2RBD. Figure 4A An in vitro virus neutralization assay of a wild-type (WT-DG614G) CoV-2 strain is described. Figure 4B An in vitro virus neutralization assay of the βVOC (B.1.351) CoV-2 strain is described. Figure 5A Group 3 alum-adsorbed CoV-2RBD is described: comparison of mean log endpoint titers against CoV-2RBD and IgG isotypes IgG1, IgG2a, IgG2b, and IgG 3. Figure 5B Group 7 MAS-1 adjuvanted CoV-2RBD: comparison of mean log endpoint titers and IgG isotypes IgG1, IgG2a, IgG2b, and IgG3 against CoV-2RBD is described. Figure 5C Group 9 MAS-1 adjuvanted CoV-1RBD: comparison of mean log endpoint titers and IgG isotypes IgG1, IgG2a, IgG2b, and IgG3 against CoV-2RBD are described. DETAILED DESCRIPTION

[0015] The vaccine composition of the present disclosure comprises a water-in-oil nanoemulsion, wherein the vaccine antigen is contained within the buffered aqueous phase of the emulsion. The term "water-in-oil nanoemulsion" refers to an emulsion in which the aqueous phase is dispersed in a continuous oil phase in the form of aqueous nanospheres having a median diameter of about 0.3 μm to about 1 μm.

[0016] The term "RBD" refers to the SARS-CoV-2 spike protein receptor binding domain, which binds to the ACE-2 receptor on the surface of human and other mammalian cells to facilitate the entry of the virus into mammalian cells after membrane fusion.

[0017] The term spike protein refers to the complete Covid viral protein that carries the RBD.

[0018] The vaccines of the present invention can be produced either "point of use" or as a "bulk fill" final drug product.

[0019] Vaccine emulsions can be aseptically prepared using sterile filtered ingredients by repeated batch homogenization or pulsed continuous flow homogenization procedures and then aseptically filled into single or reusable vials and can be stored at refrigerated (2-8°C) temperatures for three years. As a "point-of-use" product, the vaccine is formulated at room temperature as a nanoparticle emulsion, which is prepared by a simple but robust and reproducible manual mixing procedure. This is illustrated by the results of the 50% distribution of emulsion particle size diameters (D(v, 0.5) determined by laser diffraction between multiple operators and their stability at room temperature. At T zero, the "point of use" emulsion particle size distributions prepared by 4 operators at the lower and upper mixing limits of the method were N=29; mean D(v, 0.5)±SEM=0.44±0.03μm and N=26; mean D(v, 0.5)±SEM=0.66±0.03μm, respectively. Particle size measurements at 2, 18-24 and 48 hours after preparation showed that this parameter did not change significantly over a two-day storage period. To maintain sterile integrity, the vaccine emulsion can be placed at room temperature for up to 4 hours before injection, allowing repeated administration of up to 0.3mL of dose volume per vial of POU vaccine emulsion to multiple subjects.

[0020] The components of the oil adjuvant vehicle suitable for the present invention include a first sugar ester emulsifier such as mannitol monooleate (MMO) or sorbitan monooleate, a second emulsifier such as hydrogenated castor oil, for example, polyoxyethylene-40-hydrogenated castor oil (POCO) and naturally occurring and metabolizable oils, preferably squalene and squalane. By weight, metabolizable oils generally account for about 85% to about 90% of the oil, the first sugar ester emulsifier accounts for about 6% to 15% of the oil, i.e., about 9% to about 12% of the oil by weight, or about 10% or 11%, and the second emulsifier accounts for about 0.1% to 1.1% of the oil by weight, i.e., 0.2% to about 1%, 0.4 to about 0.8%, 0.5% to about 0.7%, or about 0.6%. The metabolizable oil component can be about 10% to about 90% squalene and about 10% to about 90% squalane by weight. In one embodiment, the weight ratio of squalene to squalane is 1:1.

[0021] The components of the oil vehicle, including their starting materials, which may be derived from animal or plant sources or a combination thereof, are commercially available from a variety of sources. Preferably, the components of the oil vehicle, including their starting materials, are derived from plant sources to avoid the risk of transmissible spongiform encephalitis (TSE) contamination. MAS-1 is available from Mercia Pharma, Scarsdale, New York (www.merciapharma.com).

[0022] In addition to MMO, sugar esters suitable as the first emulsifier include polysorbates, in particular sorbitan monooleate. In addition to POCO, sorbitan esters (such as sorbitan monopalmitate), polysorbates (such as Tweens family emulsifiers) and Hypermers B239 and B246 can also be used as the second emulsifier.

[0023] The nanoparticle vaccine emulsion of the present invention generally contains about 65% to about 75% by weight of an adjuvant oil vehicle and about 25% to about 35% by weight of an aqueous phase containing a protein antigen. In certain embodiments of the present invention, the aqueous phase accounts for about 27% to about 33% of the vaccine emulsion by weight.

[0024] The water-in-oil vaccine emulsion used in the present invention should be formulated such that the median diameter of the aqueous spheres carrying the antigen in the emulsion is less than 1 micron, wherein the median diameter ranges from about 100 nanometers to about 1 micron, and the average diameter is generally about 300 nanometers. The oil component of the adjuvant is preferably a metabolizable naturally occurring biological oil, which is different from the mineral oil comprising the oil phase of the well-known Freund's adjuvant (incomplete and complete preparations).

[0025] The vaccine emulsions of the present invention should tolerate high concentrations of antigens, such as 0.1 mg / mL to 20 mg / mL, and should be compatible with commonly used protein solubilizers (e.g., 4 M urea, 30% DMSO). Unlike IFA emulsions, they should be compatible with aqueous phases having a wider pH range, i.e., about 4-9, preferably 6-8, and should not be affected by a wider range of salt concentrations. Unlike high-viscosity IFA emulsions (>1,500 cP), the vaccine emulsions of the present invention should have low viscosity (<100 cP), providing a free-flowing emulsion to allow high-precision low-volume (0.05 mL) administration.

[0026] In some preferred embodiments, the vaccines of the present disclosure employ the Mercia Pharma MAS-1 adjuvant / delivery system. The MAS-1 adjuvant sterile-filtered oil vehicle has a shelf life of at least 5 years when stored at room temperature, making it suitable for stockpiling for pandemic preparedness and convenient for distribution without worrying about cold chain issues. The mass-produced MAS-1 adjuvanted vaccine emulsion has a shelf life of up to 3 years when stored refrigerated at 2-8°C. The MAS-1 adjuvant oil vehicle can also be formulated with an aqueous solution containing an antigen by a rapid (90-120 seconds), robust and repeatable, validated point-of-use (POU) manual method.

[0027] MAS-1 adjuvant emulsion consists of aqueous spheres (300 nm in diameter) containing antigen dispersed in a continuous oil phase. The emulsion is free-flowing (viscosity less than 100 cP) and can be accurately formulated into low-volume doses of 0.05 to 0.5 mL, preferably 0.05 to 0.3 mL.

[0028] Thus, in certain embodiments, the water-in-oil nanoemulsion vaccine composition of the present disclosure comprises an aqueous phase and an oily phase, wherein: The aqueous phase contains 25% to 35% by weight of the emulsion, and in the form of aqueous nanospheres having a median diameter of about 0.3 μm to about 1 μm; and One or more SARS-CoV-2 antigens; and Oil phase contains 65% to 75% by weight of an emulsion wherein the oil phase comprises 85% to 90% squalene and squalane, 9% to 12% mannitol monooleate, and 0.5% to 0.7% polyoxyethylene-40-hydrogenated castor oil, each by weight of said oil phase; For example, MAS-1 adjuvanted vaccine.

[0029] In certain embodiments, the water-in-oil nanoemulsion vaccine composition of the present disclosure comprises an oil phase comprising 65% to 75% by weight of an emulsion wherein the oil phase comprises 85% to 90% squalene and squalane, 9% to 12% mannitol monooleate, and 0.5% to 0.7% polyoxyethylene-40-hydrogenated castor oil, each by weight of said oil phase;

[0030] For example, MAS-1 adjuvant oil vehicle. The present invention is further described by the following examples. These examples are merely illustrative and do not in any way limit the scope of the invention described and claimed.

[0031] Example

[0032] Overall goal Evaluating the potential of MAS-1 adjuvanted Covid vaccines to stimulate robust, cross-reactive, durable and balanced Th1 / Th2 immune responses against SARS CoV-2 virus

[0033] Limitations of current Covid-19 vaccines:

[0034] Although SARS CoV-1 causes severe disease in a high proportion of infected individuals and up to 10% of infected individuals die, CoV-1 is not highly contagious. However, although SARS CoV-2 is a less lethal virus than SARS CoV-1, it is a highly contagious virus and has caused severe disease and death in many infected individuals worldwide. SARS CoV-2 is particularly troublesome in immunocompromised and elderly populations. Moreover, SARS CoV-2 is highly mutagenic, and in the short time since it was first identified, many variants have emerged and continue to emerge. Each mutant has the potential to be more or less contagious, more or less pathogenic, and, to varying degrees, to evade the host's immune system in individuals who have been primed through prior natural exposure to CoV-2 or after immunization with a CoV-2 vaccine derived from an existing strain. Furthermore, the durability of immune responses following either natural exposure to coronaviruses, including CoV-2, or following vaccination with currently available CoV-2 vaccines is limited and requires repeated booster vaccinations 4-6 months later with mRNA vaccines, while adenovirus vaccines typically require repeat vaccinations 2 months after the initial dose. Furthermore, SARS CoV-2 infection, which results in severe Covid-19 infection, is associated with a hyperimmune Th1-type inflammatory response, characterized by the host initiation of a so-called inflammatory “cytokine storm” that leads to severe disease and death in infected individuals. Therefore, vaccination strategies that promote a more balanced Th1 / Th2 immune response are more likely to elicit a more regulated protective immune response from the host that reduces the likelihood of a life-threatening host Th1-type hyperimmune response. A further complication associated with SARS CoV-2 infection is that a large proportion of individuals, including those who initially display mild to moderate symptoms following infection, develop complex and varied long-term symptoms, so-called “Long Haul” Covid. The underlying causes of prolonged symptoms are not fully understood, but to the extent that the syndrome is caused or propagated by the host immune response to Covid-19 infection or residual viral particles and / or antigens, immune intervention using prophylactic or therapeutic vaccines to stimulate a more balanced Th1 / Th2 immune response may play a beneficial role by modulating the excessive inflammatory Th1 response toward a more balanced Th1 / Th2 state. In addition, prophylactic or therapeutic vaccines that stimulate a persistent humoral response may also eliminate residual viral fragments that may also contribute to the prolonged stimulation of the host inflammatory response.

[0035] Therefore, the development of new and improved Covid-19 vaccines should ideally better address the challenges that exist with current Covid-19 strains and respond to the emergence of new variants by demonstrating broader cross-reactive protection, responses that are more durable and therefore require less frequent booster doses, and responses that are effective in high-risk groups such as immunocompromised individuals such as those with cancer and in elderly populations where immune recovery is compromised by immunoscenescence. In addition, improved Covid-19 vaccines should stimulate a balanced Th1 / Th2 type immune response to offset the potential for breakthrough infection, thereby promoting the excessive inflammatory "cytokine storm" associated with severe Covid-19 infection and Covid-19 death. In addition, vaccines that stimulate a balanced Th1 / Th2 type immune response at both the humoral and cellular levels may rebalance the host inflammatory profile and may therefore play a role in preventing the emergence and / or spread of "long-term" COVID symptoms and / or therapeutic reversal.

[0036] Overall strategy

[0037] To address these questions, we evaluated the ability of MAS-1 adjuvant to enhance immunity against SARS CoV-1 RBD and SARS CoV-2 RBD antigens for at least 6 months after vaccination in a mouse model to assess the potency and durability of the immune response.

[0038] To assess the potential for improved cross-reactivity against future variants, we evaluated the ability of antisera raised against MAS-1 / CoV-2RBD to cross-react with SARS CoV-1RBD and vice versa, the ability of antisera raised against MAS-1 / CoV-1RBD to cross-react with SARS CoV-2RBD. The amino acid sequence of SARS CoV-1 differs from that of SARS CoV-2 by up to 30% of its sequence, significantly higher than the differences between currently emerging SARS CoV-2 variants of concern (VOCs), including α, β, δ, and ο (Omicron), and therefore represents an extremely challenging model for assessing the potential of MAS-1 to induce cross-reactive protection against yet-to-emerge Covid-19 mutants.

[0039] To assess the potential of the vaccine to elicit a balanced Th1 / Th2 immune response to prevent the potential for an excessively inflammatory “cytokine storm,” and / or to prevent a host inflammatory response that could lead to and / or propagate “long” Covid, the symptoms, IgG isotype responses, and cytokine profiles stimulated by the MAS-1 adjuvanted Covid vaccine were evaluated.

[0040] Study Summary: On days 0 and 28, a total of 9 groups of female BALB / cJ mice were immunized with the test articles as shown in Table 1. Each test article was administered intramuscularly in 2×0.05 mL doses into each posterior thigh muscle. Serum was collected on days 0 (pre-dose 1), 14, 28 (pre-dose 2), 42, 56, 84, and 169. On each of the 42 and 84 days, two animals in each group were euthanized by cardiac puncture to harvest spleens for preparation of splenocytes. Serum from these heart bleeds was used to provide serum controls for all ELISA assays. On day 169, the remaining 5 animals in each group were euthanized by cardiac puncture to collect 6-month serum to evaluate durability, harvest spleens to evaluate cellular immune responses, and collect various tissues stored in formalin and frozen for future toxicological evaluations to evaluate product safety. In addition, the animals' general health was monitored and body weight was measured weekly throughout the study to assess the overall safety of the MAS-1 adjuvanted Covid vaccine formulation.

[0041] Use the following test groups: Group 1: Tris-buffered saline (TBS) negative control Group 2: MAS-1 / TBS placebo control Group 3: CoV-2RBD positive control adsorbed to alum 10 μg RBD / 0.1 mL dose Group 4: CoV-2RBD on alum in MAS-1 5 μg RBD / 0.1 mL dose Group 5: CoV-2RBD on alum in MAS-1 1.5 μg RBD / 0.1 mL dose Group 6: CoV-2RBD on alum in MAS-1 0.5 μg RBD / 0.1 mL dose Group 7: CoV-2RBD in MAS-1 10μg RBD / 0.1mL dose Group 8: CoV-1RBD on alum in MAS-1 5μg RBD / 0.1mL dose Group 9: CoV-1RBD in MAS-1 10μg RBD / 0.1mL dose

[0042] For logistical reasons, the nine groups were divided into three cohorts of three groups each to facilitate evaluation: Cohort 1: groups 2, 3, and 4; Cohort 2: groups 5, 6, and 7; and Cohort 3: groups 1, 8, and 9.

[0043] ELISA assay Preliminary estimates of titers were performed on serially diluted sera from euthanized mice on day 42 to establish the optimal initial dilution, allowing for evaluation of antibody titers for the various test sera. For Groups 3, 4, and 8, the initial "optimal" dilution was determined to be 20-fold. For Groups 7 and 9, the initial "optimal" dilution was determined to be 5,000-fold. Endpoint titer estimates were performed for each animal and for all 9 animals.

[0044] In each case, the baseline "threshold" was based on the mean baseline OD450 + 3 x SD (in this case, the "baseline" was the OD450 value obtained from serum samples of unimmunized or MAS-1 placebo-immunized mice, which were assayed in the same ELISA assay format as the test samples). The "highest" log titer was the dilution that first exceeded the baseline "threshold"; the "middle" log titer was the dilution that was 2-fold lower than the dilution that first exceeded the baseline "threshold". The "lowest" log endpoint titer reported herein was estimated as the serum dilution that was 3-fold lower than the dilution that first exceeded the baseline "threshold" OD450 value, in order to conservatively estimate the lowest titer elicited by each test article.

[0045] Pseudovirus assay for determination of neutralizing antibodies

[0046] One day before the experiment, HEK-293T-hACE2 cells were plated at 1.5 × 10 4 Cells were inoculated in 100 μL DMEM medium. Pseudovirus was incubated with serial dilutions of serum samples at 37°C for 1 hour. Mouse serum samples were first diluted 50-fold, followed by 3-fold serial dilutions for a total of seven dilutions. Rat serum samples were first diluted 25-fold, followed by 3-fold serial dilutions for a total of seven dilutions.

[0047] 100 μL serum-pseudoviruses were added to 293T-hACE2 cells in 96-well poly-D-lysine coated culture plates. After incubation at 37 ° C for 48 hours in a 5% CO2 environment, the cells were lysed at RT for 15 minutes with 100 μL Promega Glo lysis buffer. Finally, 50 μL lysates were added to 50 μL luciferase substrates (Promega luciferase assay system). The amount of luciferase was quantified by luminescence (relative luminescence unit (RLU)) using a luminometer (BioSynergy H4).

[0048] Conditions were tested in duplicate wells on each plate, and virus controls (VC = no serum) and cell controls (CC = no pseudovirus) were included on each plate to determine the values ​​of 0% and 100% neutralization, respectively. The percentage inhibition of infection for each dilution of the sample was calculated based on the RLU value as follows: % inhibition = [1-(average RLU of the sample - average RLU of CC) / (average RLU of VC - average RLU of CC)] × 100%. The 50% inhibition dilution (EC50) is further defined as the serum dilution at which the relative luminescence units (RLU) are reduced by 50% compared to the virus control wells (virus + cells) after subtracting the background RLU in the control group containing only cells. The EC50 of each sample was calculated by the Reed–Muench method. ( https: / / www.nature.com / articles / s41596- 020-0394-5(4) ).

[0049] Human convalescent plasma was included as a positive control.

[0050] Study Results

[0051] Example 1: Efficacy and durability of MAS-1 induced mean log minimum endpoint titers

[0052] Figure 1A The mean log nadir endpoint titers for Group 3 (10 μg alum-adsorbed CoV-2RBD; positive control), Group 7 (10 μg CoV-2RBD in MAS-1), and Group 9 (10 μg CoV-1RBD in MAS-1) over 168 days post-inoculation (d0, d28) are shown. Compared to the positive control Group 3 (10 μg alum-adsorbed CoV-2RBD), the mean log nadir endpoint titers for Group 7 (10 μg CoV-2RBD in MAS-1) were at least 2 logs higher at all time points over the 168 days ( Figure 1A The mean minimum log endpoint titer in Group 9 (CoV-1RBD in 10 μg MAS-1) was also approximately 2 logs higher than the Group 3 positive control by Day 42 and remained the same at all time points up to 168 days post-vaccination, with no evidence of titer reduction ( Figure 1A ).

[0053] Example 2: Robustness of mean log lowest endpoint low

[0054] To assess the robustness of the immune response induced by the MAS-1 adjuvanted Covid vaccine, the following groups present the log minimum endpoint titers of individual animals in each group: Figure 2A , Group 3: 10 μg alum-adsorbed CoV-2RBD positive control; Figure 2B , Group 7: 10 μg CoV-2RBD in MAS-1; Figure 2C , Group 9: 10 μg CoV-1RBD in MAS-1.

[0055] To maintain inter-assay robustness, positive control reference sera from mice euthanized on day 42 from groups 7 and 9 were run on each ELISA plate, respectively. Figure 1A and Figure 2A , 2B As shown in Figure 2C. Likewise, in these studies, MAS-1 placebo serum on the 42nd day was run on each ELISA plate as a negative control. In addition, for each individual animal, the serum on the 42nd day was repeated as a control with the serum on the 84th day, and the titer on the 84th day was compared and standardized for the titer on the 42nd day repeated. Likewise, for each animal, the serum on the 84th day was re-determined as a control with the serum on the 168th day, and the titer on the 168th day was compared and standardized for the titer on the 84th day repeated. In all ELISA plates and between the different days of determination during the entire study until the 168th day, the respective lowest logarithmic endpoint titers assigned to the baseline thresholds of these positive and negative controls were highly consistent; The robustness of the research design itself was confirmed.

[0056] Group 3 alum-adsorbed positive control animals ( Figure 2A ) showed high variability between individual animals, and positive titers were often only observed after the second dose was administered on Day 28, indicating a lack of robustness in the immune response to the alum-adsorbed CoV-2RBD positive control. However, in all 9 individual animals, even after only a single dose of CoV-2RBD in MAS-1 achieved at least a 4-log minimum titer by Day 14 and before Dose 2 on Day 28, the lowest log endpoint titer of CoV-2RBD in MAS-1 in Group 7 ( Figure 2B ) were also robust, demonstrating the potential of a robust immunization regimen with only a single dose of CoV-2RBD in MAS-1 to provide effective protection against Covid-19. Similarly, although to a lesser extent than in Group 7, the lowest log endpoint titers of CoV-1RBD in MAS-1 in Group 9 also showed a robust response, with all animals expressing titers up to Day 14 after a single dose of CoV-1RBD in MAS-1 and up to Day 28 before Dose 2 ( Figure 2C Animals in Groups 7 and 9 showed further increases in nadir log endpoint titers after the second dose of MAS-1-adjuvanted CoV-2 and CoV-1RBD by day 42, with no evidence of reduction through day 168, whereas differences between individual animals in Group 3 remained evident for alum-adsorbed CoV-2RBD until day 168.

[0057] The data from Groups 7 and 9 further suggest that even a single dose regimen of MAS-1-adjuvanted CoV-2RBD (and CoV-1RBD), even as an initial dose, may be effective in a clinical setting. In addition, these data suggest that MAS-1-adjuvanted CoV-2 or CoV-1RBD should be highly effective as a single dose vaccine that is repeated annually in the setting of Covid-19, as is currently done with seasonal influenza vaccines.

[0058] Animals in Group 4 were immunized with alum-adsorbed CoV-2 formulated in MAS-1 at a 5 μg RBD dose, and the lowest log titers at 84 days were comparable but slightly lower than the titers of alum-adsorbed CoV-2 RBD at a 10 μg dose in Group 3. The lowest log endpoint titers of alum-adsorbed CoV-1 RBD at a 5 μg dose in Group 8 were comparable but only slightly higher than the titers of the 10 μg alum-adsorbed CoV-2 RBD positive control in Group 3 (data not shown). Sera from Groups 4 and 8, as well as Groups 5 and 6, were not further analyzed due to relatively poor responses compared with those of Groups 7 and 9.

[0059] Reproducibility between analyses across time points was assessed using common positive and negative controls included in each microtiter plate. The positive control reference serum used on each ELISA plate was from serum samples from euthanized mice on day 42 from CoV-2RBD in group 7 MAS-1 and CoV-1RBD in group 9 MAS-1, and the negative control used on each ELISA plate was from the MAS-1 placebo group on day 42, and the assigned titers of the positive controls were highly consistent with the mean baseline OD ± 3 × SD “threshold” of the MAS-1 placebo negative control in all analyses up to day 168 ( Figure 1A , 2A , 2B and 2C).

[0060] Summary: The data in Examples 1 and 2 demonstrate that alum-adsorbed CoV-2RBD Group 3 sera displayed a greater degree of variability between individual mice than either of the MAS-1 adjuvanted Groups 7 and 9. Similarly, at the same 10 μg CoV-2RBD, for the remaining 5 animals in each group, the mean lowest log endpoint titer for MAS-1 adjuvanted Group 7 was nearly 2 logs higher than that for alum-adjuvanted Group 3, with values ​​of 5.60 vs. 3.78, respectively, on Day 168, and similar responses were observed for MAS-1 adjuvanted CoV-1RBD. Therefore, MAS-1 promotes robust and durable humoral immune responses against either CoV-2RBD or CoV-1RBD that did not wane until Day 168.

[0061] The data suggest that the MAS-1 adjuvanted vaccines of the present disclosure have the potential to provide improved durability of immune responses against Covid infection. Notably, currently approved SARS-CoV-2 vaccines show waning titers over several months and require CDC-recommended booster doses 4 to 6 months after two doses of mRNA-based Covid-19 vaccines and as few as 2 months after a single dose of adenoviral vector-based Covid-19 vaccines (CDC, January 4, 2022). Recently, a fourth dose of mRNA vaccine has been recommended for individuals aged 50 years and over to offset the further weakening of immunity of boosted individuals to VOCs, especially to οVOCs (Watson, C (Feb 3, 2022) Nature 602: pp 17-18 "Three, four or more boosters: what's the magic number for booster shots"; Levine-Tiefenbrun, M (Feb 2022) MedRxiv https: / / doi.org / 10.1101 / 2021.12.27.21268424). Therefore, there is an urgent need for improved Covid-19 vaccines with more durable responses, especially to VOCs.

[0062] Example 3: Potential of MAS-1 to enhance cross-reactive immunity against emerging CoV-2 variants of concern (VOC)

[0063] Covid-19 viral evolution poses a major challenge to ongoing vaccine development. Since the initial outbreak of the Covid-19 pandemic, the SARS CoV-2 virus has evolved to produce many so-called variants of concern (VOCs). The wild-type WT-D614[G] strain, on which the first generation of anti-Covid-19 vaccines are based, and the subsequent α variant (strain B.1.1.7), were the dominant strains circulating in the United States, the United Kingdom, and elsewhere at the start of the pandemic in early 2020. In the fall of 2020 and early 2021, the β VOC (strain B.1.351) became dominant in South Africa. Both of these strains were replaced by VOCs that emerged later in 2021, first the δ VOC (strain B.1.617.2) and then the ο VOC (strain B.1.1.529BA.1 and more recently BA.2), which also first emerged in South Africa and are now the dominant VOCs worldwide. It is reported that the titers of sera isolated from individuals infected with B.1.1.7 and B.1.351 variants or vaccinated with Pfizer-BNT mRNA vaccine against B.1.351β variants are significantly lower than those against B.1.1.7 strains (Planas, D., Bruel, T., et al., March 26, 2021 Nature Medicine 27:917-924 "Sensitivity of Infectious SARS-CoV-2 B.1.1.7 and B.1.351 variants toneutralizing antibodies"). Although these sera have comparable efficacy against D614[G] and B.1.1.7 variants, the neutralizing titers of convalescent sera against B.1.351 VOC are reduced by at least 6 times, and the neutralizing titers of individuals vaccinated with Pfizer mRNA vaccine are reduced by 14 times. Compared with the WT D614[G] strain, the β variant has 5 significant mutations in the receptor binding domain (RBD) and 3 significant mutations in the N-terminal domain (NTD) of the spike protein, which are related to antibody escape (BL Sievers et al., Jan 13, 2022 Sci. Transl. Med. 10.1126 / scitranslmed.abn7842). The δ (B.1.617.2) variant has a single important mutation (L452R) related to antibody escape relative to the D614G WT virus, while the ο B.1.1.529 variant has 11 mutations in the NTD and 15 mutations in the RBD relative to the WT D614G strain. As a result, it is predicted that the ο variant, like the β variant, exhibits antibody escape relative to the WT D614G strain.Sievers et al. reported that convalescent sera from individuals infected with WT D614 were equally reactive to the δ strain as to the WT D614[G] strain, but significantly less reactive to both β and ο. Similarly, in subjects vaccinated with Pfizer or Moderna mRNA vaccines, both based on the WT D614[G] spike protein, antibody responses to the β and ο variants were reduced compared with antibody responses to the WT D614[G] and δ variants. However, despite the reduced relative titers to β and ο, these vaccines, especially after the third dose (and even the fourth booster dose), are effective in preventing severe infection and death caused by the β and ο variants. However, as discussed by Sievers et al. (BL Sievers et al., Jan. 13. 2022 Sci. Transl. Med. 10.1126 / scitranslmed.abn7842) and Watson (Watson, C (Feb 3, 2022) Nature 602: pp. 17-18), future protection requires that Covid vaccines need to have broader and more durable immune responses to fight future variants.

[0064] The spike protein of coronaviruses binds to the ACE2 receptor, initiating viral infection of human cells. The spike protein from the original SARSCoV-1 and the RBD from the current SARS CoV-2 that causes Covid-19 infection represent relatively extreme examples of the range of potential structural differences between functional coronavirus spike proteins. Therefore, the assessment of the cross-reactivity of CoV-2RBD-specific antisera against CoV-1RBD, and vice versa, provides a standard approach to assess the potential of a specific Covid-19 vaccine to induce broadly cross-reactive protection against emerging CoV-2 VOCs.

[0065] Sera from groups 3 and 7 were evaluated by ELISA for reactivity to their vaccine-matched CoV-2 RBD target antigen and cross-reactivity to CoV-1 RBD heterologous antigens. Similarly, group 9 sera were evaluated for cross-reactivity to matched CoV-1 RBD and to CoV-2 RBD.

[0066] The mean lowest log endpoint titers of cross-reactivity of group 3 alum-adjuvanted CoV-2RBD positive control sera to matched CoV-2RBD and to CoV-1RBD are shown in Figure 3.A. The mean lowest log endpoint titers of cross-reactivity of group 7 MAS-1 adjuvanted CoV-2RBD to matched CoV-2RBD and to CoV-1RBD are shown in Figure 3.B. The mean lowest log endpoint titers of cross-reactivity of group 9 MAS-1 adjuvanted CoV-1RBD sera to matched CoV-1RBD and to CoV-2RBD are shown in Figure 3.C.

[0067] The average minimum log endpoint titers of Group 3 sera on days 42, 56, and 84 showed that alum-adsorbed CoV-2 10 μg RBD induced cross-reactive titers against CoV-1 RBD that were approximately two logs lower (approximately log 1.9) than titers expressed against the “vaccine-matched” CoV-2 RBD target antigen by day 85 (approximately log 3.9) (Figure 3.A).

[0068] The average minimum logarithmic endpoint titers of group 7 sera on days 42, 56, and 84 showed that the high cross-reactive titers against CoV-1 RBD induced by MAS-1-adjuvanted CoV-2 10 μg RBD on day 85 (approximately log 5.1) were almost equivalent to the titers expressed against the “vaccine-matched” CoV-2 RBD target antigen by day 85 (approximately log 5.5) (Figure 3.B).

[0069] The mean minimum log endpoint titers of group 9 sera on days 42, 56, and 84 showed that the cross-reactive titers induced by MAS-1-adjuvanted CoV-1 10 μg RBD against CoV-2 RBD were approximately 1 log lower (approximately log 4.4) than the titers expressed against the “vaccine-matched” CoV-1 RBD target antigen by day 85 (approximately log 5.4) ( Figure 3.C ), but they were still higher than the titers induced by alum-adjuvanted CoV-2 RBD against the matched CoV-2 RBD on day 85 (approximately log 3.9) ( Figure 3A ), showing that MAS-1 promoted a meaningful degree of cross-reactive immunity when formulated with CoV-1 and CoV-2 RBD antigens, thereby showing great promise for developing Covid vaccines that protect against emerging VOCs.

[0070] The day 42 reference standard sera from groups 7 and 9 run on their respective microtiter plates were highly reproducible, Figure 3A , 3BThe titer assessments shown in Figures 3C and 3C provide confidence. To the extent that MAS-1-adjuvanted CoV-2 and CoV-1RBD induce neutralizing titers against live coronaviruses, these cross-reactive ELISA data support that MAS-1-adjuvanted CoV-2RBD will have the potential to provide robust and durable responses against emerging genetic variants of the SARS CoV-2 virus. These data further suggest that MAS-1-adjuvanted vaccines containing a combination of CoV-1 and CoV-2RBD (or their trimeric spike proteins) may also have the potential to provide robust, broad-spectrum and durable protective immunity.

[0071] Summary: MAS-1 induced antibody responses to CoV-2 and CoV-1 RBD, which showed robust and broad cross-reactivity to their respective counterpart CoV RBDs. In terms of neutralization titers induced by MAS-1-adjuvanted CoV-2 and CoV-1 RBD against live coronaviruses, these cross-reactivity data support that MAS-1-adjuvanted CoV-2 RBD shows great potential to provide robust and durable responses to emerging genetic variations of the virus due to mutations to reduce the possibility of immune escape, thereby extending the effective life of the vaccine.

[0072] Example 4: Evaluation of MAS-1-induced in vitro pseudovirus neutralization activity and cross-neutralization activity against Covid-19 VOCs A virus neutralization assay (VNA) was performed at the School of Tropical Medicine, Baylor College of Medicine to evaluate the potential of the pooled sera of groups 3, 7, 9 and 1:1 combination of groups 7+9 at days 42 and 84 to neutralize virus-infected human cells in vitro. Procedure: To this end, serial dilutions of antisera from each MAS-1 adjuvanted test group were assayed in vitro for their ability to neutralize infection of human cells by pseudoviruses carrying the spike protein of the original WT-D614G (wild-type CoV-2 virus) and the βVOC originally reported in South Africa (CoV-2 strain B.1.351), in order to assess the potential of the MAS-1 adjuvant to enhance long-lasting and cross-reactive antibody responses to VOCs. Results: The data shown in Figure 4.A show neutralization titers against the WT-D614G strain, which was the dominant strain circulating in the United States, the United Kingdom, and elsewhere at the beginning of the Covid-19 pandemic in 2020. The data shown in Figure 4.B show neutralization titers against the β(B.1.351) VOC strain that was dominant in South Africa in the fall of 2020. Titers of convalescent sera were included as positive controls in both tests. The neutralization titers are summarized as follows: Neutralization titer against CoV-2WT-D614G strain: Convalescent plasma (control) = 3.69E+03 Group 3 CoV-2RBD-Alum (positive control) = 1.0E+01 CoV-2 RBD in group 7 MAS-1 = 4.62E+03 CoV-1RBD in group 9 MAS-1 = <1.0E+01 Neutralization titers against CoV-2SA B.1.351 strain: Convalescent plasma (control) = 1.47E+03 Group 3 CoV-2RBD-Alum (positive control) = <1.0E+01 CoV-2 RBD in group 7 MAS-1 = 5.13E+03 CoV-1RBD in group 9 MAS-1 = <1.0E+01

[0073] The CoV-2RBD used in this study is derived from the original WT D614G strain, just as the spikes and RBDs used in all currently approved Covid vaccines. Compared with convalescent serum, the CoV-2RBD (Group 7) serum in MAS-1 showed excellent neutralizing activity for both WT D614G and β (SA B.1.351) strains. Sievers et al. (BL Sievers et al., Jan 13, 2022 Sci. Transl. Med. 10.1126 / scitranslmed.abn7842) reported that the β variant (strain B.1.351) was significantly less reactive to antibodies produced by currently approved Covid-19 vaccines, all of which were based on WT D614 [G] spike protein or its RBD. Consistent with the observations reported by Sievers et al., βB.1.351 VOC was significantly less sensitive to the above-mentioned convalescent control serum compared to the sensitivity to the WT D614G strain, 3.69E+03 vs. 1.47E+03, respectively. In contrast, βVOC was as sensitive to the group 7 MAS-1 adjuvanted CoV-2RBD serum as the WT D614G strain, with neutralization titers of 5.13E+03 and 4.62E+03, respectively. Figure 4A and Figure 4B These in vitro cross-neutralization test data against WTDG614G and βB.1.351 VOCs are consistent with the high cross-reactivity ELISA data shown by the Group 7 MAS-1 adjuvanted CoV-2RBD sera against the heterologous CoV-1RBD, indicating that the MAS-1-induced Covid vaccine exhibits the potential to be highly cross-reactive with Covid VOCs.

[0074] Cross-reactivity and persistence of neutralizing IgG responses

[0075] Table 1 below shows the log IC50 neutralizing antibody titer values ​​of convalescent plasma induced on day 168 after the initial vaccination of mice inoculated with SARS CoV-2RBD / MAS-1 and original SARS CoV-2 strains against original SARS CoV-2, ο, δ, β and SARS CoV-1: Table 1 Neutralizing antibody levels (IC50 values) in mouse sera collected on day 168 after primary vaccination These data show that MAS-1 induces potent, durable, cross-reactive IgG responses against CoV-2 RBD antigens in BALB / c mice, with no decrease in neutralization titers up to 6 months post-vaccination. Furthermore, neutralization titers cross-reactive to variants, including βVOC, were comparable or superior to those induced by human convalescent sera from the original SARS CoV-2 strain.

[0076] Summary: The data in Examples 3 and 4 demonstrate that MAS-1 induces a more robust, durable, and broadly protective immune response than that provided by natural exposure (convalescent sera) or by the immune responses reported by Sievers et al. or by contemporary approved Covid-19 vaccines based on the WT D614 strain. These characteristics suggest that, when combined with appropriate Covid-19 RBD or spike antigens, MAS-1 has the potential to provide broad and durable protection against existing VOCs and, therefore, potentially also against VOCs that have yet to emerge, for the purposes of pandemic planning.

[0077] Although it is difficult to predict the outcome of any specific antigen combined with any specific adjuvant, MAS-1-adjuvanted seasonal inactivated influenza virus (IIV) antigens have demonstrated improved robustness and durability of immune protection, including against both vaccine and non-vaccine virus strains, in clinical studies in the general adult population (Phase 1A; 18-49 years) and high-risk elderly individuals (Phase 1B / 1B extension; 65 years and older), who are also at particular risk for severe Covid-19 infection. See U.S. Patent Application Publication No. 2012 / 0219605, which is incorporated herein by reference in its entirety; and Gorse et al., 2022 Vaccine 40:1271-1281 (A phase 1 dose-sparing, randomized clinical trial of seasonal trivalent inactivated influenza vaccine combined with MAS-1, a novel water-in-oil adjuvant / delivery system) and Gorse et al., 2022 Vaccine 40; 1472-1482 (MAS-1, a novel water-in-oil adjuvant / delivery system, with reduced seasonal influenza vaccine hemagglutination dose may enhance potency, durability and cross-reactivity of antibody responses in the Therefore, the data presented here for CoV-2 and CoV-1 RBD in MAS-1 suggest that MAS-1 has the potential to improve the durability and cross-reactivity of protection against Covid-19 in human individuals, including elderly individuals who are most susceptible to severe infection from both Covid-19 and influenza virus infections.

[0078] Example 5: Understanding the Th1 / Th2 specificity of immune responses and potential consequences of Covid-19 infection through IgG isotype analysis

[0079] Background: Despite being associated with various other infections including influenza, severe Covid-19 infection often results in death, especially in vulnerable high-risk populations such as the elderly infected with SARS CoV-2, illustrating the critical role of an effective host immune response and the severe consequences of host immune dysregulation and the so-called "cytokine storm" on short- and long-term effects. (Tan et al., Frontiers Immunol. (30 Sept 2021) "Hyperinflammatory Immune Response and Covid-19: A Double Edged Sword". Early activation of type 1 interferon (IFN)-mediated innate immunity after infection is a key event for protection, and viral interference with this process can enhance viral replication and lead to excessive host inflammation and cytokine storm (Tan 18). Subsequent adaptive immunity includes activation of antibody-producing B cells, CD4+ and CD8+ T cells, each of which is stimulated to control pathogen infection. Covid-19 severity is associated with a Th1 / Th17-biased cytokine storm, including IL-1β, IL-2R, IL-6, IL-17 and TNF-α, which is associated with dysregulated immune response and disease severity (Tan 43, 45). The extent to which long-Covid (or long-term) symptoms can be attributed to dysfunction of the host's immune response to Covid-19 infection is unclear, but the wide range of symptoms associated with long-Covid suggests that a common link similar to autoimmune disorders mediated by the host immune response may be involved, as well as possibly contributing to blood-brain barrier damage and microglial activation, leading to neurological symptoms (Tan 81, 95, 96). Various attempts have been made to block the effects of these cytokines in an attempt to mitigate the effects of excessive inflammation and associated cytokine storms, but there is a limited window of opportunity to implement these interventions when excessive inflammation begins. In addition, it is theoretically possible that excessive immunity, particularly CD8 excessive inflammation and excessive inflammatory microglia, may be involved in triggering long Covid. See Mesa, N., "Multiple Possible Causes of Long COVID Come into Focus;" The Scientist, 10 / 28 / 22. These observations suggest that Covid-19 vaccines that promote a more balanced immune response could prime the host immune response to respond to infection with a more balanced humoral and cellular response, thereby mitigating the risk of host-mediated excessive inflammation and subsequent sequelae.To the extent that host inflammatory responses are associated with long Covid, vaccination to elicit a balanced immune response to reduce the risk of Th1 / Th17-mediated excessive inflammation may reduce the risk of developing long Covid after a "breakthrough" infection, and may even have immunotherapeutic benefits in subjects who already exhibit symptoms of long Covid. Currently approved vaccines are reported to have at best a modest effect on the incidence of long Covid, or none at all (Ledford, H "How Vaccination Effects the Risk of Long Covid" Nov 23, 2021 Nature 599 pp 546-547).

[0080] Adjuvants are often used to promote immune responses to antigens that are poorly immunogenic. Alum adjuvant is the most commonly used adjuvant in clinical practice, mainly producing Th2 responses. Both water-in-oil adjuvants MF59 and AS03 have been approved for use in influenza vaccines Fluad (ElSahly H. MF59 and AS03, respectively). TMas a vaccine adjuvant: a review of safety and immunogenicity. Expert Rev Vaccines 2010;9:1135-41. Doi.org / 10.1586 / erv.10.111) and Pandemrix (McElhaney JE, Beran J, Devaster JM, Esen M, Launay O, Leroux-RoelsG et al. AS03-adjuvanted versus non-adjuvanted inactivated trivalent influenza vaccine against seasonal influenza in elderly people: a phase 3 randomised trial. Lancet Infect Dis 2013;13:485-96) induce proinflammatory cytokines and chemokines (Wilkins, AL et al., Frontiers in Immunol Dec 13, 2017 | AS03-and MF59-Adjuvanted Influenza Vaccines in Children). Other adjuvants, such as Toll-like receptor (TLR) agonists, including CpG, can change the immune balance by, for example, stimulating type 1 interferon (IFN) expression to promote Th1 responses. Type 1 IFN responses are induced by mRNA vaccines, thereby promoting Th1-biased responses (Cagigi, A and Lore, K Jan 18, 2021 Vaccines 9: 61-75 Immune Responses Induced by mRNA Vaccination in Mice, Monkeys and Humans).

[0081] This article compares the immune response promoted by MAS-1 together with CoV-2 RBD and spike protein with the immune response promoted by alum-adjuvanted CoV-2 RBD as a control.

[0082] Results: Specificity of immune response analyzed by IgG isotype

[0083] IgG isotype responses (Th2-type IgG1 and IgG2b; Th1-type IgG2a and IgG3) were measured on days 14, 28 (before dose 2), 42, 56 and 84 in groups 3, 7 and 9 to assess the Th2 / Th1 balance of the humoral immune response. IgG isotype analysis was performed on pooled sera from groups 3, 7 and 9 over 85 days. The log-minimum endpoint titers of IgG isotypes against CoV-2RBD in group 3 sera are shown in Figure 5.A. Alum-adsorbed CoV-2RBD induced a Th2-dominated IgG response, with IgG1 reaching a log titer of 4.5 on day 42 (+14 days after dose 2). Titers of IgG2a, IgG2b and IgG3 were comparable, reaching log titers of approximately 2.0 by day 42. So far, all isotype titers remained constant over 85 days, while the overall mean log endpoint titer continued to rise until day 85, reaching a mean log titer of 3.7. On day 85, based on relative IgG1 / IgG2a titers, the Th2 / Th1 balance was 4.5:2.0=2.27.

[0084] Figure 5.B shows the lowest mean log endpoint titers of IgG isotypes against CoV-2RBD for group 7 sera. MAS-1 adjuvanted CoV-2RBD induced a Th2-biased IgG response, with IgG1 reaching a log titer of approximately 6.0 on day 42 (+14 days after dose 2) and rising to 6.6 by day 85. IgG2a and IgG2b reached log titers of approximately 4.0 on day 42 and remained constant over 85 days. IgG3 titers were slightly lower and reached log titers between 2.3 and 3.0 between days 42 and 85. All isotype titers remained constant over 85 days to date, while total IgG titers peaked on day 56 and remained constant thereafter, reaching an average log titer of 5.49 by day 85. The Th2 / Th1 balance based on relative IgG1 / IgG2a titers on day 85 was 6.0:3.95=1.67, indicating a more balanced Th2 / Th1 response than that observed in response to Group 3 alum-adsorbed CoV-2RBD controls.

[0085] The lowest mean logarithmic endpoint titers of IgG isotypes for group 9 sera against CoV-1RBD are shown in Figure 5.C. MAS-1 adjuvanted CoV-1RBD induced a Th2-biased IgG response, with IgG1 reaching a logarithmic titer of approximately 5.40 on day 42 (+14 days after dose 2), rising to 5.65 on day 85. IgG2a and IgG2b reached a logarithmic titer of approximately 4.0 on day 42 and remained constant over 85 days. IgG3 titers were slightly lower and reached logarithmic titers between 2.74 and 3.35 between days 42 and 85. So far, all isotype titers have remained constant over 85 days, while total IgG titers have continued to rise until day 85, reaching an average logarithmic titer of 5.50. The Th2 / Th1 balance based on IgG1 / IgG2a on day 85 was 5.65:4.20=1.35.

[0086] Summary: IgG isotype analysis showed that alum-adsorbed CoV-2RBD induced a Th2-dominated response with a log Th2 / Th1 ratio of 2.27, while MAS-1-adjuvanted CoV-2 and CoV-1RBD induced a more balanced isotype profile with log Th2 / Th1 ratios of 1.67 and 1.35, respectively. The lowest log Th1 IgG2a endpoint titer induced by MAS-1-adjuvanted CoV-2RBD was 3.94 on day 42, remained stable until day 85, and exceeded the total log titer induced by alum-adsorbed CoV-2RBD from day 42 to day 85, which increased from 2.63 to 3.70, respectively. Similarly, MAS-1-adjuvanted CoV-1RBD induced Th1 IgG2a log titers of 3.95 and 4.20 between days 42 and 85, which were comparable to the Th1 IgG2a log titer of 3.95 induced by MAS-1-adjuvanted CoV-2RBD.

[0087] Example 6 Comparative adjuvant study: Comparison of MAS-1 adjuvanted CoV-2 RBD (3 μg dose) and spike (1 μg dose) with AS03 and MF59 adjuvants. In a confirmatory follow-up study, MAS-1 adjuvant with a) CoV-2RBD antigen (original SARSCoV-2 strain) and b) full-length trimeric spike ("Spike") antigen (original SARS CoV-2 strain) was compared to ASO3-like and MF59 (Addavax) in a collaborative study conducted in a different mouse strain (C57BL / 6J) in an NIH-sponsored adjuvant comparator evaluation at the University of Montana. Endpoint titers measured against the target RBD were assessed for the different treatment groups. Response specificity was assessed by IgG isotype analysis of Th2 IgG1 and Th1 IgG2c titers. Adjuvant-stimulated cellular responses were assessed by measuring the cytokine profile expressed by isolated splenocytes and adjacent lymph nodes.

[0088] Procedure: Mice were injected twice (100 μl intramuscularly and 50 μl into each calf muscle) with an interval of 21 days between each injection, with 3 μg RBD or 1 μg Spike per injection. ELISA for RBD-specific or Spike-specific serum antibodies was performed 21 days after the second injection. Spleen and draining LN cells were collected 21 days after the second injection and cultured with RBD or Spike for 72 hours. Cytokines in culture supernatants were detected using a multiplex ELISA kit (Mesoscale Discovery).

[0089] Results: The results of the body fluid response spectrum are shown in Table 2: Table 2 Humoral response spectrum

[0090] *The AS03 adjuvant used in this study is not GMP grade. Its composition is the same as AS03 but it is not necessarily manufactured according to GMP standards.

[0091] **The MF59 used in this study is the animal-use version called Addavax

[0092] ***Non-adjuvanted antigen control only.

[0093] The results showed that MAS-1 induced potent IgG titers against CoV-2 RBD and spike antigens that were superior to AS03 or MF59 water-in-oil adjuvants in C57BL / 6J mice. This was more pronounced in the RBD than in the spike antigen, which is more immunogenic in nature.

[0094] MAS-1 adjuvanted CoV-2RBD produced the highest levels of anti-RBD IgG and anti-spike, outperforming ASO3 and MF59 (Addavax). As shown in Table 2, non-adjuvanted RBD and spike induced Th2-biased IgG responses, while both AS03 and MF59 promoted Th2-biased IgG responses. In contrast, the data confirmed that based on the isotype spectrum, MAS-1 induced a relatively balanced Th2-type IgG1 to Th1-type IgG2c humoral response compared to AS03 or MF59, which were dominated by Th2-type IgG1 responses.

[0095] The cell response spectrum results are shown in Table 3: Table 3 Cellular response profile

[0096]

[0097] The data evaluated the cellular immune response based on the expression of cytokines IFNγIL-5IL-10, IL-17, TNF-a IL-2. These data suggest that MAS-1 induces a Th1 type cellular response, inducing more IFNγ compared to AS03 or MF59 (with RBD and spike). In addition, MAS-1 does not stimulate IL-17, which is associated with an excessive inflammatory cytokine storm. MAS-1 also induces a modest IL-10 response, while MAS-1 does not induce IL-5, whereas both AS03 and MF59 induce IL-5, which is associated with eosinophil activation.

[0098] Cytokine profiles showed that MAS-1 increased IFNγ-producing cells, indicative of Th1 cell development, and that MAS-1 did not induce Th17 cell development associated with a Th1 / Th17 hyperinflammatory response and cytokine storm. MAS-1-adjuvanted RBD did stimulate a modest increase in IL-10, which, along with IFNγ expression, is consistent with stimulation of balanced immunity rather than a response dominated by proinflammatory Th1 or Th17 cytokines.

[0099] Summary: These data show clear IgG isotype and cytokine analysis, confirming the data presented in Examples 1, 2, and 5 above in BALB / cJ mice, demonstrating that MAS-1 promotes a balanced Th1 / Th2 immune response that should both prevent infection and protect the host from immunopathology, and further supporting that MAS-1 adjuvanted Covid vaccines may have the potential for therapeutic intervention against Covid and long Covid.

[0100] In addition, MAS-1 exhibits significant dose-saving, enhanced persistence and cross-reactivity protection against Covid strains, and MAS-1 can inhibit excessive inflammatory responses by restoring the balance of the immune system, showing immunotherapeutic potential for certain autoimmune and inflammatory disorders (e.g., long-term Covid).

[0101] Example 7: Point of Use Formulation

[0102] Preparation of MAS-1 adjuvanted vaccines at the point of use (POU) rather than in batches allows for universal formulation of vaccines with any candidate SARS-CoV-2 viral antigen, without the need to put precious candidate antigens into large-scale formulations that may not produce "protective" immunity. Typically, a vial of sterile MAS-1 adjuvant vehicle is combined with 0.5 mL of sterile antigen aqueous solution to produce 1.9 mL of vaccine emulsion. The dose volume for elderly subjects is a maximum of 0.3 mL, and the dose volume for young adults is a maximum of 0.2 mL or possibly 0.1 mL, so each vial can provide at least 4 0.3 mL doses, each vial can provide at least 6 to 7 0.2 mL doses, or can provide 10 to 12 repeated doses of 0.1 mL dose volume. Based on the formulation validation studies filed with the FDA, although the physicochemical integrity of the emulsion remains stable at room temperature for at least 48 hours, in order to minimize the risk of microbial contamination, the dose obtained from a single vial of POU-prepared vaccine emulsion should be administered within 4 hours of emulsification.

[0103] Although various embodiments of the present invention have been shown and described herein, it should be emphasized that these embodiments are provided by way of example only. Many changes, modifications and substitutions may be made in its various embodiments without departing from the present invention. Specifically, when any range is described herein, unless otherwise expressly stated, the range includes all values ​​therein and all subranges therein.

[0104] In addition, more generally, according to the disclosure, discussion, examples and embodiments herein, conventional molecular biology, cell biology, microbiology and recombinant DNA techniques within the technical scope of the art can be adopted. The resources incorporated herein by reference are for their corresponding contents and the teachings found therein. Such incorporation is at least for the specific teachings and / or other purposes that may be noted when citing the references herein. If no specific teachings and / or other purposes are noted, the published resources are specifically incorporated for the teachings indicated by one or more of the titles, abstracts and / or overviews of the references. If the teachings and / or other purposes of the specific identifications are not noted and may be so relevant, the published resources are incorporated to more fully describe the prior art involved in the present invention, and / or provide teachings that may be applicable that are generally known to those skilled in the art. However, it is particularly noted that the references to the disclosed resources herein should not be interpreted as admitting that they are prior art of the present invention. In addition, in the case where one or more of the incorporated published resources are different from the present application or contradict the present application, including but not limited to the defined terms, term usage, described technology, etc., the present application is a preferred embodiment, and any contradiction can be regarded as an alternative embodiment.

[0105] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided as examples only. Without departing from the present invention, those skilled in the art will appreciate that many variations, changes and substitutions may be employed in implementing the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed.

Claims

1. A water-in-oil nanoemulsion vaccine composition, comprising an aqueous phase and an oil phase, wherein: The aqueous phase contains 25% to 35% by weight of the emulsion, and in the form of aqueous nanospheres having a median diameter of about 0.3 μm to about 1 μm; and One or more SARS-CoV-2 antigens; and Oil phase contains 65% to 75% by weight of the emulsion, wherein the oil phase comprises 85% to 90% squalene and squalane, 9% to 12% mannitol monooleate and 0.5% to 0.7% polyoxyl-40-hydrogenated castor oil, Each is based on the weight of the oil phase.

2. The nanoemulsion vaccine composition of claim 1, wherein the SARS-CoV-2 antigen is a protein antigen.

3. The nanoemulsion vaccine composition of claim 1 or claim 2, wherein the composition comprises more than one SARS-CoV-2 antigen.

4. The nanoemulsion vaccine composition of any of the preceding claims, wherein the SARS-CoV2 antigen comprises components derived from more than one SARS-CoV-2 strain.

5. The nanoemulsion vaccine composition of any of the preceding claims, wherein the SARS-CoV-2 antigen comprises SARS-CoV-2 antigens derived from more than one SARS-CoV-2 strain.

6. The nanoemulsion vaccine composition of any one of the preceding claims, wherein squalene comprises from about 40% to about 60% by weight of the oil phase.

7. The nanoemulsion vaccine composition of any of the preceding claims, wherein squalane comprises from about 40% to about 60% by weight of the oil phase.

8. The nanoemulsion vaccine composition of any one of the preceding claims, wherein the weight ratio of squalene to squalane is about 1:

1.

9. The nanoemulsion vaccine composition of any one of the preceding claims, wherein the median particle size is about 300 nm.

10. The nanoemulsion vaccine composition of any one of the preceding claims, wherein the composition has a viscosity of 100 cP or less.

11. The nanoemulsion vaccine composition of any one of the preceding claims, wherein the aqueous phase further comprises a protein solubilizer.

12. The nanoemulsion vaccine composition of claim 11, wherein the protein solubilizing agent is urea or DMSO.

13. A method for SARS-CoV-2 prevention, comprising administering a vaccine composition according to any one of claims 1-12 to a patient in need thereof.

14. A method for attenuating SARS-CoV-2 infection, comprising administering to a patient in need thereof a vaccine composition according to any one of claims 1-12.

15. A method of generating a protective immune response against SARS-CoV-2 infection, comprising administering to a patient in need thereof a vaccine composition according to any one of claims 1-12.

16. A method for long-haul SARS-CoV-2 prevention, comprising administering to a patient in need thereof a vaccine composition according to any one of claims 1-12.

17. A method for alleviating one or more symptoms of long-term SARS-CoV-2 infection, comprising administering to a patient in need thereof a vaccine composition according to any one of claims 1-12.

18. A method of generating a protective immune response against long-term SARS-CoV-2 infection, comprising administering to a patient in need thereof a vaccine composition according to any one of claims 1-12.

19. A method for increasing the potency and / or durability of an immune response to a SARS-CoV-2 antigen, comprising administering the antigen in a water-in-oil nanoemulsion vaccine composition comprising an aqueous phase and an oil phase, wherein: The aqueous phase contains 25% to 35% by weight of the emulsion, and in the form of aqueous nanospheres having a median diameter of about 0.3 μm to about 1 μm; and SARS-CoV-2 antigens; and Oil phase contains 65% to 75% by weight of the emulsion, wherein the oil phase comprises 85% to 90% squalene and squalane, 9% to 12% mannitol monooleate and 0.5% to 0.7% polyoxyethylene-40-hydrogenated castor oil, each by weight of the oil phase.

20. A kit for point-of-use administration of a water-in-oil nanoemulsion vaccine against a SARS-CoV-2 infectious agent, comprising: a vial of adjuvant oil comprising mannitol monooleate, squalene, and squalane, a vial of aqueous PBS comprising a SARS-CoV-2 antigen, at least one syringe, a lipid-resistant three-way stopcock, at least one needle, and a vial for storing the formulated water-in-oil nanoemulsion vaccine.

21. The kit of claim 20, wherein the kit is for point-of-use administration of a nanoparticle water-in-oil emulsion vaccine against the SARS-CoV-2 infectious agent, the kit comprising: a vial of adjuvant oil comprising mannitol monooleate, squalene and squalane, a vial of aqueous PBS for combination with an antigen, two syringes, a lipid-resistant three-way stopcock, two needles and a vial for storing the formulated water-in-oil emulsion vaccine.

22. The kit of claim 20, wherein the nanoparticle water-in-oil emulsion vaccine produced by combining the kit components is the composition of any one of claims 1-12.

23. A water-in-oil nanoemulsion vaccine composition produced from the components of the kit of any one of claims 20-22.

Images