SARS-CoV-2 Vaccine Composition

Abstract

The present disclosure relates to compositions of adjuvanted SARS-CoV-2 vaccines and their uses for preventing and managing Covid-19 infection, including the host's hyperinflammatory response to the infection, including long-term symptoms associated with Covid infection.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. provisional patent applications No. 63 / 329,850, filed April 11, 2022, No. 63 / 329,851, filed April 11, 2022, and No. 63 / 380,499, filed October 21, 2022, which are incorporated by reference in their entireties. The field relates to compositions of adjuvanted SARS-CoV-2 vaccines and their uses for preventing and managing Covid-19 infection, including the host's hyperinflammatory response to infection, including long-term symptoms associated with Covid infection. [Background technology]

[0002] The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused over 5 million deaths worldwide and significant disruption to the global economy. Although the development of safe and effective vaccines has contributed greatly to controlling the pandemic, there remains a need for vaccines that provide improved protection with dose-sparing levels of antigen, cross-reactivity against variants of concern (VOCs), and better durability of overall immune response, especially in vulnerable populations including immunocompromised and elderly people who are at particularly high risk of hospitalization and death from Covid-19 infection. One possible solution to address these challenges is the use of various proteinaceous antigenic preparations associated with the virus, including, for example, the so-called viral spike protein, the acetylcholine (ACE-2) receptor binding domain (RBD) or the full-length stabilized trimeric spike protein (S-protein), in combination with adjuvants.

[0003] Several different adjuvants are currently used in US FDA-approved vaccines. Aluminum-based adjuvants, including amorphous aluminum hydroxyphosphate sulfate (AAHS), aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate (Alum), may be used in combination with other vaccines to treat 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 (PedvaxHIB), HPV (Gardasil 9), Japanese encephalitis (Ixiaro), MenB (Bexsero, Trumenba), and pneumococcus (Prevnar). 13), Td(Tenivac), Td(Mass Biologics), Tdap(Adacel), and Tdap(Boostrix).

[0004] MF59, a squalene-containing oil-in-water emulsion adjuvant, is included in Fluad, a trivalent and / or quadrivalent inactivated seasonal influenza vaccine licensed for use in adults over 65 years of age. AS01 is a liposome-based adjuvant used in Shingrix, a varicella-zoster vaccine, that contains two immunostimulants, 3-O-desacyl-4'-monophosphoryl lipid A (MPL), a non-toxic derivative of lipopolysaccharide from Salmonella minnesota, and QS-21, a saponin fraction extracted from Quillaja saponaria Molina. AS03 is an adjuvant system composed of α-tocopherol, squalene, and polysorbate 80 in an oil-in-water emulsion and is used in the influenza vaccines Pandemrix and Arepanrix for the A / H1N1 pandemic influenza strain that emerged in 2009. Cytosine phosphoguanosine (CpG) 1018, a Toll-like receptor 9 (TLR9) agonist, is used in the Heplisav-B vaccine. It is a synthetic form of DNA composed of cytosine phosphoguanine (CpG) motifs that mimic the genetic material of bacteria and viruses, stimulating inflammatory cytokines and Th1-driven immune responses.

[0005] Adjuvants are typically used to promote the enhancement of antibody and cellular immune responses against antigens that are poorly immunogenic by themselves. Adjuvants variably induce biased Th1 or Th2-type immune responses. However, limitations exist with respect to these currently available vaccine adjuvants in terms of promoting long-lasting and cross-reactive immunity, especially in at-risk populations, especially immunocompromised populations including the elderly, which are similarly demonstrated in the currently approved Covid vaccines based on mRNA vaccines and adenovirus vector DNA vaccine technologies. However, despite the previous and widespread use of adjuvants, the exact molecular mechanisms by which available adjuvants actually work in humans are not well understood and vary from antigen to antigen among various adjuvants, necessitating empirical trial and error in the selection of formulations and vaccine development strategies. Summary of the Invention

[0006] The present disclosure provides an adjuvant-containing SARS-CoV-2 vaccine. The SARS-CoV-2 proteinaceous antigen is formulated as a water-in-oil nanoglobule emulsion vaccine composition comprising an aqueous phase and an oil phase. The buffered aqueous phase comprises one or more SARS-CoV-2 antigens and is in the form of aqueous nanovesicles having a median diameter of about 0.3 μm to about 1 μm, and constitutes 25% to 35% by weight of the emulsion. The oil phase constitutes 65% to 75% by weight of the emulsion and includes 85% to 90% squalene and squalane, respectively, 9% to 12% mannide monooleate, and 0.5% to 0.7% polyoxyl-40-hydrogenated castor oil 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 two or more SARS-CoV-2 antigens. In some embodiments, the SARS-CoV-2 antigen comprises components derived from two or more SARS-CoV-2 strains. In some embodiments, the SARS-CoV-2 antigen comprises SARS-CoV-2 antigens derived from two or more SARS-CoV-2 strains.

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

[0009] In some embodiments, the median vesicle 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 solubilizer. In some embodiments, the protein solubilizer is urea or DMSO.

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

[0011] The present disclosure further provides a method of long-term SARS-CoV-2 prevention comprising administering a vaccine composition described herein to a patient in need thereof. The present disclosure further provides an immunotherapy method for attenuating one or more symptoms of long-term SARS-CoV-2 infection comprising administering a therapeutic vaccine composition described herein to a patient in need thereof. The present disclosure further provides a method of generating a protective immune response against SARS-CoV-2 infection for a long period of time comprising administering a vaccine composition described herein to a patient in need thereof. The present disclosure further provides a method of generating a protective immune response against SARS-CoV-2 infection for a long period of time comprising administering a vaccine composition described herein to a patient in need thereof, the vaccine composition simultaneously stimulating both a sustained antibody response against viral fragments and a balanced anti-inflammatory response against the patient's long-term hyperinflammatory response. The present disclosure further provides a method of increasing the titer and / or persistence of an immune response against a SARS-CoV-2 antigen comprising administering an antigen in a water-in-oil nanoemulsion vaccine composition described herein.

[0012] The disclosure further provides a kit for point-of-use administration of a water-in-oil nanoemulsion vaccine against the SARS-CoV-2 infectious agent, comprising a vial of adjuvant oil comprising mannide 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 injection needle, and a vial for storing the formulated water-in-oil nanoemulsion vaccine. In some embodiments, the kit is for point-of-use administration of a nanoparticulate water-in-oil emulsion vaccine against the SARS-CoV-2 infectious agent, comprising a vial of adjuvant oil comprising mannide monooleate, squalene, and squalane, a vial of aqueous PBS for combination with the antigen, two syringes, a lipid-resistant three-way stopcock, two injection needles, and a vial for storing the formulated water-in-oil nanoemulsion vaccine. In some embodiments, the nanoparticulate water-in-oil emulsion vaccine produced by the combination of the kit components is a composition described herein. The present disclosure further provides water-in-oil nanoemulsion vaccine compositions produced by the above-mentioned kit components.

[0013] Vaccine emulsions can be prepared in bulk by batch homogenization or pulse-continuous flow-through homogenization procedures, or manually at the point-of-use (POU) in single-dose or multi-dose vials. POU preparation of vaccine emulsions allows for conserving supplies of antigen and oil phase and facilitates adjustment of vaccine formulations as the pandemic evolves as a result of viral mutations producing emerging VOCs that may evade existing vaccine-conferred or prior infection-derived immunity.

[0014] The vaccine can be administered as a single or multiple primary dose to immunologically naive individuals. Alternatively, it can be administered as a booster dose to individuals who have already been vaccinated with a heterologous vaccine, such as mRNA- or DNA-based. Additionally, the vaccine can be administered immunotherapeutically to individuals with long-term symptoms (Long Haul Covid), thereby suppressing hyperinflammatory symptoms in favor of a Th1 / Th1 balanced humoral and cellular immune profile. [Brief description of the drawings]

[0015] [Figure 1] Figure 1A lists the Mean Minimum Log End-point Titers up to 168 days post-vaccination (d0, d28) for Group 3 (CoV-2 RBD adsorbed to 10 μg alum), Group 7 (CoV-2 RBD in 10 μg MAS-1), and Group 9 (CoV-1 RBD in 10 μg MAS-1). [Diagram 2] Figure 2A lists the Individual Minimum Log End-point Titers up to 168 days (d0, d28) post vaccination, Group 3 10 μg CoV-2 RBD alum-adsorbed positive control. Figure 2B lists the Individual Minimum Log End-point Titers up to 168 days (d0, d28) post vaccination, Group 7 10 μg CoV-2 RBD in MAS-1. Figure 2C lists the Individual Minimum Log End-point Titers up to 168 days (d0, d28) post vaccination, Group 9 10 μg CoV-1 RBD in MAS-1. [Diagram 3]Figure 3A depicts the mean minimum log titers for alum-adsorbed CoV-2 RBD:CoV-2 RBD compared to cross-reactive titers for CoV-1 RBD for Group 3. Figure 3B depicts the mean minimum log titers for MAS-1 adjuvanted CoV-2 RBD:CoV-2 RBD compared to cross-reactive titers for CoV-1 RBD for Group 7. Figure 3C depicts the mean minimum log titers for MAS-1 adjuvanted CoV-1 RBD:CoV-1 RBD compared to cross-reactive titers for CoV-2 RBD for Group 9. [Figure 4] Figure 4A describes an in vitro virus neutralization assay of the wild type (WT-DG614G) CoV-2 strain. Figure 4B describes an in vitro virus neutralization assay of the BetaVOC (B.1.351) CoV-2 strain. [Diagram 5] Figure 5A depicts alum-adsorbed CoV-2 RBD from Group 3: mean log end point titers and a comparison of IgG isotypes IgG1, IgG2a, IgG2b, and IgG3 against CoV-2 RBD. Figure 5B depicts MAS-1 adjuvanted CoV-2 RBD from Group 7: mean log end point titers and a comparison of IgG isotypes IgG1, IgG2a, IgG2b, and IgG3 against CoV-2 RBD. Figure 5C depicts MAS-1 adjuvanted CoV-1 RBD from Group 9: mean log end point titers and a comparison of IgG isotypes IgG1, IgG2a, IgG2b, and IgG3 against CoV-2 RBD. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The vaccine composition of the present disclosure is comprised of a water-in-oil nanoemulsion in which 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 an aqueous phase is dispersed in a continuous oil phase in the form of aqueous nanovesicles having a median diameter of about 0.3 μm to about 1 μm. 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 and facilitates viral entry into mammalian cells after membrane fusion.

[0017] The term spike protein refers to the intact Covid virus protein carrying the RBD.

[0018] The vaccines of the present invention can be produced as "point-of-use" or "bulk-filled" finished pharmaceutical products.

[0019] The vaccine emulsion is aseptically prepared in bulk using sterile-filtered components, either by repeated batch homogenization or using a pulsed continuous flow-through homogenization procedure, and aseptically filled into single- or multi-use vials that can be stored at refrigerated temperatures (2-8°C) for up to 3 years. As a "point-of-use" product, the vaccine is formulated as a nanoparticle emulsion at room temperature and produced using a simple, yet robust and reproducible hand-mixing procedure. This is explained by the emulsion vesicle size diameter 50% distribution results (D(v,0.5) determined by laser light diffraction among multiple operators and the stability at room temperature. At T zero, the vesicle size distributions of "point-of-use" emulsions prepared by four operators at the lower and upper limits of mixing of this method were, respectively, N=29; mean D(v,0.5) ± SEM = 0.44 ± 0.03 μm and N=26; mean D(v,0.5) ± SEM = 0.66 ± 0.03 μm. Vesicle size was measured 2 h, 18-24 h, and 48 h after preparation, and no significant changes were observed in this parameter over a 2-day storage period. In maintaining sterility, the vaccine emulsion can be kept at room temperature for up to 4 h before injection and repeated doses of up to 0.3 mL per POU vial of vaccine emulsion can be administered to multiple subjects.

[0020] The components of the oil adjuvant vehicle suitable for use in the present invention include a first sugar ester emulsifier, such as mannide monooleate (MMO) or sorbitan monooleate, a second emulsifier, such as a hydrogenated castor oil, e.g., polyoxyl-40-hydrogenated castor oil (POCO), and a naturally derived, metabolizable oil, preferably squalene and squalane. The metabolizable oil typically comprises about 85% to about 90% by weight of the oil, the first sugar ester emulsifier comprises about 6% to about 15% by weight of the oil, i.e., about 9% to about 12% by weight, or about 10% to about 11% by weight, and the second emulsifier comprises about 0.1% to about 1.1% by weight of the oil, i.e., 0.2% to about 1% by weight, 0.4 to about 0.8%, 0.5 to about 0.7%, or about 0.6%. The metabolizable oil component may be from about 10% to about 90% by weight squalene, and from about 10% to about 90% by weight squalane, hi one embodiment, the weight ratio of squalene to squalene is 1:1.

[0021] The components of the oil vehicle, including their starting materials, may be derived from animals or plants, or a combination thereof, and are all commercially available from multiple sources. Preferably, the components of the oil vehicle, including the starting materials, are derived from plants to avoid the risk of transmissible spongiform encephalitis (TSE) contamination. MAS-1 is available from Mercia Pharma, Inc, Scarsdale, NY (www.merciapharma.com).

[0022] In addition to MMO, suitable sugar esters as first emulsifiers include polysorbates, especially sorbitan monooleate. In addition to POCO as a second emulsifier, sorbitan esters such as sorbitan monopalmitate, polysorbates such as the Tween family of emulsifiers, and Hypermers B239 and B246 may be useful.

[0023] The nanoparticulate vaccine emulsions of the invention typically comprise about 65% to about 75% by weight of the adjuvant oil vehicle and about 25% to about 35% by weight of an aqueous phase containing the proteinaceous antigen. In one embodiment of the invention, the aqueous phase comprises about 27% to about 33% by weight of the vaccine emulsion.

[0024] Water-in-oil vaccine emulsions for use in the present invention should be formulated so that the aqueous vesicles in the emulsion carrying the antigen have a median diameter of less than 1 micron, with median diameters ranging from about 100 nanometers to about 1 micron, typically having an average diameter of about 300 nanometers. The oil component of the adjuvant is preferably a metabolisable, naturally derived biological oil, as opposed to the mineral oil that constitutes the oil phase of the well-known Freund's adjuvant (in both incomplete and complete formulations).

[0025] The vaccine emulsion of the present invention should tolerate high concentrations of antigen, such as 0.1 mg / mL to 20 mg / mL, and be compatible with commonly used protein solubilizers (e.g., 4 M urea, 30% DMSO). Unlike IFA emulsions, it should be compatible with a wide range of aqueous phase pH, i.e., about 4 to 9, preferably 6 to 8, and should be unaffected over a wide range of salt concentrations. Unlike IFA emulsions, which are highly viscous (>1,500 cP), the vaccine emulsion of the present invention should be low viscosity (<100 cP), providing a free-flowing emulsion that allows for precise low-volume (0.05 mL) dosing.

[0026] In some preferred embodiments, the vaccines of the present disclosure use 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 pandemic preparedness stockpiling and facilitating distribution without significant cold chain concerns. The shelf life of the MAS-1 adjuvant-containing vaccine emulsion manufactured in bulk is 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 the antigen by a well-established point-of-use (POU) manual method that is rapid (90-120 seconds), robust, and reproducible.

[0027] MAS-1 adjuvant-containing emulsions contain antigen-containing aqueous vesicles (diameter 300 nm) dispersed in a continuous oil phase. The emulsions are free-flowing (viscosity <100 cP) and allow accurate dispensing of low volume doses of 0.05-0.5 mL, preferably 0.05-0.3 mL.

[0028] Thus, in certain embodiments, a water-in-oil nanoemulsion vaccine composition of the present disclosure comprises an aqueous phase and an oil phase, wherein: the aqueous phase is 25% to 35% by weight of the emulsion and is in the form of aqueous nanovesicles having a median diameter of about 0.3 μm to about 1 μm; and comprising one or more SARS-CoV-2 antigens; and the oil phase is 65% to 75% by weight of the emulsion, the oil phase comprising 85% to 90% squalene and squalane, 9% to 12% mannide monooleate, and 0.5% to 0.7% polyoxyl-40-hydrogenated castor oil, respectively, by weight of the oil phase; For example, a MAS-1 adjuvanted vaccine.

[0029] In certain embodiments, the water-in-oil nanoemulsion vaccine composition of the present disclosure comprises an oil phase, which is 65% to 75% by weight of the emulsion, which comprises 85% to 90% squalene and squalane, 9% to 12% mannide monooleate, and 0.5% to 0.7% polyoxyl-40-hydrogenated castor oil, respectively, by weight of the oil phase; For example, MAS-1 adjuvant oil vehicle.

[0030] The invention is further described in the following examples, which are merely illustrative and are not intended to limit in any way the scope of the invention as described and claimed. EXAMPLES

[0031] Working Example

[0032] overall purpose To evaluate the potential of MAS-1-adjuvanted Covid vaccines to stimulate robust, cross-reactive, durable, and balanced Th1 / Th2-type immune responses against SARS CoV-2 virus.

[0033] Limitations of current Covid-19 vaccines:

[0034] SARS CoV-2 is responsible for the Covid-19 infection that first emerged in Wuhan, China in late 2019, while SARS CoV-1 was responsible for the original SARS infection that emerged in Southeast Asia in 2003. While SARS CoV-1 caused severe illness in a high percentage of infected individuals and as many as 10% died, CoV-1 was not highly infectious. However, SARS CoV-2, while less lethal than SARS CoV-1, is a highly infectious virus that has caused severe illness and death in many infected individuals worldwide. SARS CoV-2 is particularly problematic in immunocompromised and elderly populations. In addition, SARS CoV-2 is highly mutagenic, and many mutant strains have emerged and continue to emerge in a short period of time since it was first identified. Each mutant strain has the potential to evade the host immune system, with greater or lesser infectivity, greater or lesser pathogenicity, and greater or lesser degree of mutation, either by natural exposure to CoV-2 or after immunization with a CoV-2 vaccine derived from an existing strain. Moreover, the persistence of immune responses after natural exposure to coronaviruses in general, including CoV-2, or after currently available CoV-2 vaccinations, is limited, requiring repeated boosts as frequently as after 4–6 months for mRNA vaccines and 2 months after the first dose for adenoviral vaccines. Moreover, SARS CoV-2 infections leading to severe Covid-19 infections are correlated with a hyperimmune Th1-type inflammatory response characterized by a so-called inflammatory “cytokine storm” induced by the host, leading to severe disease and death in infected individuals. Therefore, vaccination strategies that promote a more balanced Th1 / Th2 immune response are likely to provide a more regulated protective immune response that stimulates the host immune system and reduces the likelihood of a life-threatening host Th1-type hyperimmune response. A further complication associated with SARS CoV-2 infection is that many patients, including those who initially showed mild to moderate symptoms after infection, develop complex and diverse long-term symptoms, the so-called “Long Haul” Covid.Although the underlying causes of long-term symptoms are still poorly understood, to the extent that this syndrome is triggered or propagated by Covid-19 infection and / or the host immune response to residual viral particles and / or antigens, immunological interventions with preventive or therapeutic vaccines stimulating a more balanced Th1 / Th2 immune response may play a beneficial role by controlling the proinflammatory Th1 response towards a more balanced Th1 / Th2 state. Moreover, preventive or therapeutic vaccines stimulating a sustained humoral response may also have the potential to eliminate residual viral fragments responsible for stimulating the host inflammatory response for a long time.

[0035] Therefore, the development of new and improved Covid-19 vaccines should ideally address the challenges posed by current Covid-19 virus strains, respond to the emergence of new variants by exhibiting broader cross-reactive protection, and provide a more durable response, thus requiring less frequent booster doses, as well as an effective response in high-risk populations, such as immunocompromised patients, such as cancer patients, and elderly populations, whose immune recovery is compromised by immunosenescence. Furthermore, improved Covid-19 vaccines should stimulate a balanced immune response of the Th1 / Th2 type to counteract the possibility of breakthrough infections promoting the hyperinflammatory "cytokine storm" associated with severe Covid-19 infection and Covid-19 mortality. Furthermore, a vaccine that stimulates a balanced Th1 / Th2 type immune response at humoral and cellular levels may rebalance the host inflammatory profile, thereby playing a role in the prevention and / or therapeutic reversal of the onset and / or transmission of "long-term" Covid symptoms.

[0036] Overall Strategy

[0037] To address these concerns, we evaluated the ability of the MAS-1 adjuvant to enhance immunity to SARS Cov-1 and SARS CoV-2 RBD antigens in mouse models for a minimum of 6 months after vaccination, assessing both the potency and durability of the immune response.

[0038] To assess the potential for improved cross-reactivity against future variants, we assessed the ability of antisera raised with MAS-1 / CoV-2 RBD to cross-react with SARS CoV-1 RBD and conversely, assessed the ability of antisera raised with MAS-1 / CoV-1 RBD to cross-react with SARS CoV-2 RBD. The amino acid sequence of SARS CoV-1 differs from that of SARS CoV-2 by 30%, far greater than the differences among currently emerging SARS CoV-2 variants of concern (VOCs), including alpha, beta, delta, and omicron, and therefore represents an extremely challenging model for assessing the potential of MAS-1 to induce cross-reactive protection against Covid-19 variants that have yet to emerge.

[0039] To evaluate the vaccine's potential to stimulate a balanced Th1 / Th2 immune response to prevent a potential hyperinflammatory "cytokine storm" and / or prevent a host inflammatory response that could lead to "long" Covid, symptoms and / or transmission, we assessed the IgG isotype responses and cytokine profiles stimulated by a MAS-1-adjuvanted Covid vaccine.

[0040] Study Overview: A total of nine groups of nine female BALB / cJ mice were immunized with the test agents on days 0 and 28 as shown in Table 1. Each test agent was administered im into the posterior thigh muscle at a dose of 2 × 0.05 mL. Serum was collected on days 0 (pre-dose 1), 14, 28 (pre-dose 2), 42, 56, 84, and 169. Two mice per group were euthanized by cardiac puncture on days 42 and 84, respectively, to harvest spleens for splenocyte preparation. Serum from these cardiac bleeds was used as in-assay serum controls for ELISA assays on all plates. The remaining five mice per group were euthanized by cardiac puncture on day 169 to harvest six months of serum to assess persistence, spleens to assess cellular immune responses, and various formalin- and cryopreserved tissues for future toxicological evaluation to assess product safety. In addition, to assess the overall safety profile of the MAS-1-adjuvanted Covid vaccine formulation, animals' general condition was monitored and body weights were measured weekly throughout the study period.

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

[0042] For reasons of efficiency and to facilitate evaluation, the nine subjects were enrolled in three cohorts of three: cohort 1: groups 2, 3, 4; cohort 2: groups 5, 6, 7; cohort 3: groups 1, 8, 9.

[0043] ELISA assay To establish the optimal initial dilution for evaluating antibody titers for the various test sera, sera from mice euthanized on day 42 were serially diluted and titers were preliminarily estimated. For groups 3, 4, and 8, the initial "optimal" dilution was determined to be 1:20. For groups 7 and 9, the initial "optimal" dilution was determined to be 1:5,000. End point titer estimations were performed both per animal and for all nine animals.

[0044] In each case, the baseline "threshold" was based on the mean baseline + 3xSD of OD450 (where "baseline" in this case was the OD450 value obtained when serum samples from non-immunized or MAS-1 placebo-immunized mice were assayed in the same ELISA assay format as the test samples). The "maximum" log titer was the dilution that first exceeded the baseline "threshold" and the "intermediate" log titer was the dilution that first exceeded the baseline "threshold". The "minimum" log endpoint titers reported herein were estimated as the serum dilution 3x lower than the dilution that first exceeded the baseline "threshold" OD450 value in order to conservatively estimate the minimum titer induced by each test drug.

[0045] Pseudovirus assay for measuring neutralizing antibodies

[0046] The day before the assay, HEK-293T-hACE2 cells were cultured in 100 μL of DMEM medium at 1.5 × 10 4The pseudoviruses were incubated with serial dilutions of serum samples at 37°C for 1 h. Mouse serum samples were first diluted 50-fold, then serially diluted 3-fold, for a total of 7 dilutions. Rat serum samples were first diluted 25-fold, then serially diluted 3-fold, for a total of 7 dilutions.

[0047] 100 μL of serum-pseudovirus was added to 293T-hACE2 cells in 96-well poly-D-lysine-coated culture plates. After 48 h of incubation at 37°C and 5% CO2, cells were lysed with 100 μL of Promega Glo Lysis buffer for 15 min at RT. Finally, 50 μL of luciferase substrate (Promega Luciferase Assay System) was added to 50 μL of lysate. The amount of luciferase was quantified by luminescence (relative luminescence units (RLU)) using a luminometer (Biosynergy H4).

[0048] Conditions were tested in duplicate wells on each plate, and a virus control (VC = no serum) and a cell control (CC = no pseudovirus) were included on each plate to determine values ​​of 0% and 100% neutralization, respectively. The infection inhibition percentage of each dilution of sample was calculated according to the RLU value as follows: Inhibition percentage = [1-(mean RLU of sample-mean RLU of CC) / (mean RLU of VC-mean RLU of CC)] × 100%. The 50% inhibition dilution (EC50) was further defined as the serum dilution at which the relative luminescence units (RLU) were reduced by 50% compared to the virus control wells (virus + cells) after subtracting the background RLU of the control group with cells only. 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 used as a positive control.

[0050] Test results

[0051] Example 1: Potency and persistence of mean log-minimum endpoint titers with MAS-1

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

[0053] Example 2: Robustness of Mean Log Minimum Endpoint Titers

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

[0055] To maintain inter-assay robustness, positive control reference sera from mice euthanized on day 42 in groups 7 and 9 were measured on each ELISA plate, respectively, as shown in FIG. 1A and FIG. 2A, 2B, and 2C. Similarly, MAS-1 placebo day 42 sera were run on each ELISA plate as a negative control. Additionally, for each animal, day 42 sera were re-assayed as a control along with day 84 sera, and day 84 titers were compared and normalized against day 42 repeat titers. Similarly, for each animal, day 84 sera were re-assayed as a control along with day 168 sera, and day 168 titers were compared and normalized against day 84 repeat titers. The minimum log endpoint titers assigned to these positive controls and baseline thresholds assigned to the negative controls were highly consistent across all ELISA plates and across different days assayed throughout the study up to day 168, confirming the robustness of the study design itself.

[0056] Alum-adsorbed positive control animals in group 3 (Figure 2A) showed high variability between individual animals, and generally positive titers were only observed after the second dose administered on day 28, suggesting a lack of robustness of the immune response to the CoV-2 RBD alum-adsorbed positive control. End point titers of CoV-2 RBD in group 7 on MAS-1 were robust with all nine animals showing a minimum of 4 log titers by pre-dose 2 on day 14 and day 28 after only a single dose of CoV-2 RBD on MAS-1 (Figure 2B), suggesting that a robust immunization regimen is possible with just a single dose of CoV-2 RBD that may provide effective protection against Covid-19. Similarly, although to a lesser extent than Group 7, the minimum log endpoint titers of CoV-1 RBD in MAS-1 in Group 9 also demonstrated a robust response after a single dose of CoV-1 RBD in MAS-1 with all animals developing titers by day 14 and day 28 of pre-dose 2 (Figure 2C). Animals in Groups 7 and 9 showed a further increase in minimum log endpoint titers by day 42 after the second dose of MAS-1-adjuvanted CoV-2 and CoV-1 RBD that was maintained without decline until day 168, whereas individual animal variability in Group 3 was evident until day 168 for alum-adsorbed CoV-2 RBD.

[0057] The data from groups 7 and 9 further suggest that even a single dose regimen of MAS-1 adjuvanted CoV-2 RBD (and CoV-1 RBD) is likely to be clinically effective even as a first-line dose. Furthermore, these data suggest that MAS-1 adjuvanted CoV-2 or CoV-1 RBD would be highly effective as a single dose for annual revaccination in Covid-19 endemic areas, as is currently done with seasonal influenza vaccines.

[0058] The minimum log titers by day 84 for animals in group 4 immunized with alum-adsorbed CoV-2 formulated in MAS-1 at a dose of 5 μg RBD were comparable but slightly lower than those for the 10 μg dose of alum-adsorbed CoV-2 RBD in group 3. The minimum log end point titers for group 8 with alum-adsorbed CoV-1 at a dose of 5 μg RBD were comparable but slightly higher than those for the 10 μg alum-adsorbed CoV-2 RBD positive control in group 3 (data not shown). Due to the relatively poor response compared to groups 7 and 9, sera from groups 4 and 8 as well as groups 5 and 6 were not further analyzed.

[0059] Inter-assay reproducibility across time points was assessed by using common positive and negative controls included on each microtiter plate. The positive control reference sera used on each ELISA plate were derived from day 42 serum samples from one euthanized mouse each from MAS-1 group 7 CoV-2 RBD and MAS-1 group 9 CoV-1 RBD, and the negative control from day 42 MAS-1 placebo used on each ELISA plate showed a high degree of inter-assay agreement for titers assigned to the positive reference sera and the mean baseline OD ± 3 × SD “threshold” for the MAS-1 placebo negative control across all assays over 168 days (Figures 1A, 2A, 2B, and 2C).

[0060] Summary: The data in Examples 1 and 2 show that sera from alum-adsorbed CoV-2 RBD group 3 showed a greater degree of variability between individual mice than either MAS-1-adjuvanted groups 7 and 9. Also, at the same 10 μg of CoV-2 RBD, the mean minimum log endpoint titers of MAS-1-adjuvanted group 7 remained nearly 2 logs higher than those of alum-adjuvanted group 3 for the remaining 5 mice per group, with values ​​of 5.60 vs. 3.78, respectively, at day 168, and a similar response was observed with MAS-1-adjuvanted CoV-1 RBD. Thus, MAS-1 promotes a robust and sustained humoral immune response against CoV-2 RBD or CoV-1 RBD without any decline up to day 168.

[0061] This data suggests that the disclosed MAS-1 adjuvanted vaccines may improve the durability of immune responses to Covid infection. Significantly, currently approved SARS-CoV-2 vaccines show a decline in titers within months, with the CDC-recommended booster dose required 4-6 months after two doses of mRNA-based Covid-19 vaccines and only 2 months after one dose of adenoviral vectored Covid-19 vaccines (CDC, January 4, 2022). More recently, a fourth dose of mRNA vaccines has been recommended for people over 50 years of age to offset further declines in booster recipients' immunity against VOCs, especially omicron VOCs (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). Thus, there is a dire need for improved Covid-19 vaccines with more durable responses, especially against VOCs.

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

[0063] The evolution of the Covid-19 virus presents a significant challenge for ongoing vaccine development. Since the initial outbreak of the Covid-19 pandemic in Wuhan, the SARS CoV-2 virus has evolved and given rise to many viruses 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 alpha variant (B.1.1.7 strain), were the dominant strains circulating in the US, UK, and elsewhere at the start of the Covid19 pandemic in early 2020. The beta VOC (B.1.351 strain) became dominant in South Africa in autumn 2020 and early 2021. Both of these strains have been supplanted by VOCs that emerged later in 2021. First the delta VOC (B.1.617.2 strain), followed by the Omicron VOC (B.1.1.529BA.1 strain and more recently BA.2), which first emerged in South Africa and is now the dominant VOC worldwide. Sera isolated from individuals infected with the B.1.1.7 and B.1.351 variants or immunized with the Pfizer BNT mRNA vaccine have been reported to have significantly reduced antibody titers against the B.1.351 beta variant compared to antibody titers against the B.1.1.7 strain (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 to neutralizing antibodies"). While the efficacy of sera against the D614[G] and B.1.1.7 variants was similar, neutralizing titers of convalescent sera against B.1.351 VOCs were reduced at least six-fold, and neutralizing titers of sera from individuals immunized with the Pfizer mRNA vaccine were reduced 14-fold.The beta variant compared to the WT D614[G] strain has five notable mutations in the receptor binding domain (RBD) and three in the N-terminal domain (NTD) of the spike protein that are associated with antibody escape (BL Sievers et al., Jan 13, 2022 Sci. Transl. Med. 10.1126 / scitranslmed.abn7842). The delta (B.1.617.2) variant has one important mutation (L452R) relative to the D614G WT virus that is associated with antibody escape, whereas the Omicron 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, the Omicron variant, like the beta variant, is predicted to exhibit antibody escape compared to the WT D614G strain. Sievers et al. reported that convalescent sera from individuals infected with WT D614 were as reactive to delta as WT D614[G], but significantly reduced to beta and omicron variants. Similarly, in subjects vaccinated with Pfizer and Moderna mRNA vaccines, both based on the WT D614[G] spike protein, antibody responses to beta and omicron variants were reduced compared to those to WT D614[G] and delta variants. Nevertheless, despite reduced relative titers to beta and omicron, the vaccines were effective in protecting against severe infection and death caused by beta and omicron variants, especially after the third (and even fourth booster) dose. However, as 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) argue, for future protection, Covid vaccines will need to have both a broader and more durable immune response to combat future variants.

[0064] Coronavirus spike proteins bind to the ACE2 receptor and initiate the infection of human cells by the virus. The RBDs of the spike proteins of the original SARS CoV-1 and the current SARS CoV-2 that causes Covid-19 infection represent relatively extreme examples of the range of possible structural differences between functional coronavirus spike proteins. Therefore, evaluation of the cross-reactivity of CoV-2 RBD-specific antisera with CoV-1 RBD and vice versa provides an indication to evaluate the potential of a particular Covid-19 vaccine to induce broadly cross-reactive protection against emerging CoV-2 VOCs.

[0065] The reactivity of sera from groups 3 and 7 was assessed by ELISA against vaccine-matched CoV-2 RBD target antigens and cross-reactivity against CoV-1 RBD heterologous antigens. Similarly, sera from group 9 were assessed against matched CoV-1 RBD and for cross-reactivity against CoV-2 RBD.

[0066] Figure 3A shows the mean minimum log endpoint titers to matched CoV-2 RBD and cross-reactivity to CoV-1 RBD for alum-adjuvanted CoV-2 RBD positive control sera from group 3. Figure 3B shows the mean minimum log endpoint titers to matched CoV-2 RBD and cross-reactivity to CoV-1 RBD for MAS-1-adjuvanted CoV-2 RBD from group 7. Figure 3C shows the mean minimum log endpoint titers to matched CoV-1 RBD and cross-reactivity to CoV-2 RBD for MAS-1-adjuvanted CoV-1 RBD sera from group 9.

[0067] The mean minimum log endpoint titers of sera from group 3 on days 42, 56, and 84 show that 10 μg alum-adsorbed CoV-2 RBD had cross-reactive titers against CoV-1 RBD that were approximately 2 logs lower than those against the “vaccine-matched” CoV-2 RBD target antigens, at 1.9 log and by day 85 at 3.9 log (Figure 3A).

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

[0069] The mean minimum log end point titers of sera from group 9 on days 42, 56, and 84 indicated that 10 μg MAS-1-adjuvanted CoV-1 RBD induced cross-reactive titers against CoV-2 RBD at 4.4 log, approximately 1 log lower than those expressed against the “vaccine-matched” CoV-1 RBD target antigen at 5.4 log on day 85 (Figure 3C), but still higher than those induced by alum-adjuvanted CoV-2 RBD against the matched CoV-2 RBD at 3.9 log on day 85 (Figure 3A), indicating that MAS-1, when coupled to both CoV-1 and CoV-2 RBD antigens, promotes meaningful cross-reactive immunity and shows great potential for the development of Covid vaccine protection against emerging VOCs.

[0070] The day 42 reference standard sera from groups 7 and 9 run on the respective microtiter plates were highly reproducible and provided confidence in the titer assessments shown in Figures 3A, 3B, and 3C. To the extent that MAS-1-adjuvanted CoV-2 and CoV-1 RBDs induce neutralizing titers against live coronaviruses, these cross-reactive ELISA data support that MAS-1-adjuvanted CoV-2 RBDs may provide a robust and durable response against emerging genetic variants of SARS CoV-2 viruses. These data suggest that MAS-1-adjuvanted vaccines combining both CoV-1 and CoV-2 RBDs (or their trimeric spike proteins) may provide robust broad-spectrum and sustained protective immunity.

[0071] Summary: MAS-1 induces antibody responses with CoV-2 and CoV-1 RBDs, with robust and broad cross-reactivity to their respective CoV RBDs. To the extent that MAS-1-adjuvanted CoV-2 and CoV-1 RBDs induce neutralizing titers against live coronaviruses, these cross-reactivity data support that MAS-1-adjuvanted CoV-2 RBDs offer great potential to provide robust and durable responses against emerging genetic variants of the virus resulting from mutations, reducing the chance of immune evasion, and thereby extending the useful lifespan of vaccines.

[0072] Example 4: Evaluation of in vitro pseudovirus neutralization activity and cross-neutralization activity against Covid-19 VOCs by MAS-1 Viral neutralization assays (VNA) were performed at the Baylor College of Medicine and Tropical Medicine to evaluate pooled sera from groups 3, 7, 9, and the 1:1 combination of groups 7+9 for their potential to neutralize the ability of the virus to infect human cells in vitro on days 42 and 84. Procedure: To this end, serial dilutions of antisera from each test group containing MAS-1 adjuvant 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 betaVOC (CoV-2 strain B.1.351) first reported in South Africa, to assess the potential of MAS-1 adjuvant to enhance long-lasting cross-reactive antibody responses against VOC. Results: Data shown in Figure 4A show neutralization titers against the WT-D614G strain, the predominant strain circulating in the US, UK and elsewhere early in the 2020 Covid-19 pandemic. Data shown in Figure 4B show neutralization titers against the Beta (B.1.351) VOC strain that became predominant in South Africa in the fall of 2020. Titers from convalescent sera are included as a positive control for both assays. Neutralization titers are summarized below: Neutralization titer against CoV-2 WT-D614G strain: Convalescent plasma (control) = 3.69E+03 Group 3 CoV-2 RBD-Alum (positive control) = 1.0E+01 Group 7 MAS-1 CoV-2 RBD=4.62E+03 Group 9 CoV-1 RBD in MAS-1=<1.0E+01 Neutralization titer against CoV-2 SA B.1.351 strain: Convalescent plasma (control) = 1.47E+03 Group 3 CoV-2 RBD-Alum (positive control) = <1.0E+01 Group 7 MAS-1 CoV-2 RBD=5.13E+03 Group 9 CoV-1 RBD in MAS-1=<1.0E+01

[0073] The CoV-2 RBD used in this study is from the original WT D614G strain, as are the spikes and RBDs used in all currently approved Covid vaccines. The CoV-2 RBD in serum from MAS-1 (group 7) showed superior neutralizing activity against both the WT D614G and beta strains (SA B.1.351) compared to convalescent sera. The beta mutant strain (B.1.351 strain) reported by Sievers et al. (BL Sievers et al., Jan 13, 2022 Sci. Transl. Med. 10.1126 / scitranslmed.abn7842) was significantly less reactive to antibodies raised by currently approved Covid-19 vaccines, all of which are based on the WT D614[G] spike protein or its RBD. Consistent with the results reported by Sievers et al., the susceptibility of betaB.1.351 VOCs to the convalescent control sera mentioned above was significantly reduced compared to the susceptibility to the WT D614G strain, 3.69E+03 vs. 1.47E+03, respectively. In contrast, betaVOCs were similarly susceptible to MAS-1-adjuvanted CoV-2 RBD sera from group 7 as the WT D614G strain, with neutralization titers of 5.13E+03 vs. 4.62E+03, respectively. These in vitro cross-neutralization assay data for WT DG614G and betaB.1.351 VOCs shown in Figure 4A and Figure 4B are consistent with the high cross-reactivity ELISA data of group 7 MAS-1-adjuvanted CoV-2 RBD sera against the heterologous CoV-1 RBD, indicating that MAS-1-derived Covid vaccines may exhibit a high degree of cross-reactivity with Covid VOCs.

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

[0075] Table 1 below shows the log IC50 neutralizing antibody titers of SARS CoV-2RBD / MAS-1 and convalescent plasma induced by the original Wuhan strain in vaccinated mice at day 168 after prime vaccination with Wuhan, Omicron, Delta, Beta, and SARS CoV-1: [Table 1] These data indicate that MAS-1 induced strong and durable cross-reactive IgG responses against CoV-2 RBD antigens in BALB / c mice, with no decline in neutralizing titers over 6 months after vaccination. Moreover, the neutralizing titers cross-reactive with mutant strains, including beta-VOCs, were comparable to or higher than those induced by the original Wuhan strain in human convalescent sera.

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

[0077] Although it is difficult to predict the outcome of a combination of a particular antigen with a particular adjuvant, MAS-1-adjuvanted seasonal inactivated influenza virus (IIV) antigen improved the robustness and durability of immune protection against both vaccine and non-vaccine virus strains in clinical trials in both the general adult population (Phase 1A; 18-49 years) and in at-risk elderly subjects (Phase 1B / 1B expansion; ≥65 years), a population also at risk for particularly severe Covid-19 infection. See U.S. Patent Application Publication No. 2012 / 0219605, which is incorporated 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 elderly). Thus, the CoV-2 and CoV-1 RBD data in MAS-1 described herein suggest that MAS-1 may improve the durability and cross-reactivity of protection against Covid-19 in human subjects, including elderly subjects, who are most vulnerable to severe infection from both Covid-19 and influenza virus infections.

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

[0079] Context: Infection with Covid-19, transmitted by SARS Cov-2, but also associated with a range of other infections including influenza, is a severe infection that often leads to death, especially in vulnerable at-risk populations including the elderly, demonstrating the critical role for an effective host immune response and the severe consequences of host immune dysregulation and the so-called "cytokine storm" on both short-term 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 innate immunity via type 1 interferon (IFN) after infection is a critical event for defense, and viral interference with this process may promote viral replication and cause host hyperinflammation and cytokine storm (Tan 18). Adaptive immunity, including activation of antibody-producing B cells, CD4+ and CD8+ T cells, are then stimulated to control pathogenic infection, respectively. 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 responses and disease severity (Tan 43, 45). It is unclear to what extent Long Covid (or long-term) symptoms are due to a dysfunctional host immune response to Covid19 infection, but the wide range of symptoms associated with Long Covid suggests that there may be a common link via the host immune response similar to an autoimmune state, and perhaps blood-brain barrier damage and microglial activation may also contribute to the neurological symptoms (Tan 81, 95, 96). Various attempts have been made to block the action of these cytokines to mitigate the effects of hyperinflammation and the associated cytokine storm, but the window of opportunity for these interventions is limited in the early stages of hyperinflammation. Furthermore, it has been proposed that hyperinflammation, especially CD8 hyperinflammation and microglial hyperinflammation, may be involved in the triggering of Long Covid.See Mesa, N., "Multiple Possible Causes of Long COVID Come into Focus;" The Scientist, 10 / 28 / 22. These observations suggest that a Covid-19 vaccine that promotes a more balanced immune response may encourage the host immune response to respond to infection with a more balanced humoral and cellular response, thereby reducing the risk of developing host-mediated hyperinflammation and its subsequent sequelae. To the extent that host inflammatory responses are relevant for Long Covid, vaccination that stimulates a balanced immune response that reduces the risk of Th1 / Th17-mediated hyperinflammation may reduce the risk of developing Long Covid after a "breakthrough" infection. The impact of currently approved vaccines on the frequency of Long Covid incidence has been reported to be modest at best or nonexistent (Ledford, H "How Vaccination Effects the Risk of Long Covid" Nov 23, 2021 Nature 599 pp 546-547).

[0080] Adjuvants are frequently used to enhance immune responses to antigens that are poorly immunogenic by themselves. Alum adjuvants are the most clinically used adjuvants and induce primarily Th2 responses. MF59 and AS03, oil-in-water adjuvants, are both used in influenza vaccine Fluad (El Sahly H. MF59 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-Roels G, 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), respectively, and 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 shift the immune balance, for example by stimulating the expression of type 1 interferon (IFN) 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] The immune responses promoted by MAS-1 carrying the CoV-2 RBD and spike protein were compared with those promoted by alum-adjuvanted CoV-2 RBD as a control.

[0082] Results: Specificity of immune responses by IgG isotype analysis

[0083] IgG isotype responses (Th2 types IgG1 and IgG2b; Th1 types IgG2a and IgG3) of groups 3, 7, and 9 were measured on days 14, 28 (pre-dose 2), 42, 56, and 84 to evaluate the Th2 / Th1 balance of humoral immune responses. IgG isotype analysis was performed on pooled sera from groups 3, 7, and 9 up to day 85. IgG isotype log minimum end-point titers of sera from group 3 against CoV-2 RBD are shown in Figure 5A. Alum-adsorbed CoV-2 RBD induced a Th2-dominated IgG response, with a log titer of 4.5 for IgG1 on day 42 (14 days after dosing). IgG2a, IgG2b, and IgG3 titers were similar, with log titers of approximately 2.0 on day 42. All isotype titers remained constant until day 85, but the combined mean log endpoint titer continued to increase until day 85, with a mean log titer of 3.7. The Th2 / Th1 balance based on relative IgG1 / IgG2a titers on day 85 was 4.5:2.0=2.27.

[0084] The IgG isotype minimum mean log endpoint titers of group 7 sera against CoV-2 RBD are shown in Figure 5B. MAS-1-adjuvanted CoV-2 RBD induced a Th2-biased IgG response, with IgG1 reaching approximately 6.0 by day 42 (14 days after dosing) and rising to 6.6 by day 85. IgG2a and IgG2b achieved log titers of approximately 4.0 by day 42 and remained constant through day 85. IgG3 titers were slightly lower, achieving log titers of 2.3 to 3.0 between days 42 and 85. All isotype titers remained constant through day 85, but total IgG titers peaked on day 56 and remained constant thereafter with a mean 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 the response to the alum-adsorbed CoV-2 RBD control in group 3.

[0085] The IgG isotype minimum mean log endpoint titers of group 9 sera against CoV-1 RBD are shown in Figure 5C. MAS-1 adjuvanted CoV-1 RBD induced a Th2-biased IgG response with IgG1 log titers of approximately 5.40 on day 42 (14 days after dose 2) and rising to 5.65 on day 85. IgG2a and IgG2b achieved log titers of approximately 4.0 by day 42 and remained constant through day 85. IgG3 titers were slightly lower, with log titers ranging from 2.74 to 3.35 from day 42 to day 85. All isotype titers remained constant through day 85, while total IgG titers continued to rise through day 85, achieving a mean log titer of 5.50 on day 85. The Th2 / Th1 balance based on IgG1 / IgG2a on day 85 was 5.65:4.20=1.35.

[0086] Summary: IgG isotype analysis indicates that alum-adsorbed CoV-2 RBD induced a Th2-dominated response with a Th2 / Th1 log ratio of 2.27, whereas MAS-1-adjuvanted CoV-2 and CoV-1 RBD induced balanced isotype profiles with Th2 / Th1 log ratios of 1.67 and 1.35, respectively. MAS-1-adjuvanted CoV-2 RBD induced a Th1 IgG2a minimum log endpoint titer of 3.94 by day 42 that remained stable through day 85, exceeding the total log titer induced by alum-adsorbed CoV-2 RBD, which increased from 2.63 to 3.70 from days 42 to 85, respectively. Similarly, MAS-1-adjuvanted CoV-1 RBD induced Th1 IgG2a log titers of 3.95 and 4.20 from days 42 to 85, which were similar to the Th1 IgG2a log titer of 3.95 induced by MAS-1-adjuvanted CoV-2 RBD.

[0087] Example 6 Comparative adjuvant study: MAS-1 adjuvanted CoV-2 RBD (dose 3 μg) and spike (dose 1 μg) compared with AS03 and MF59 adjuvants. As part of an NIH-supported comparative adjuvant evaluation conducted at the University of Montana, in a collaborative study with different strains of mice (C57BL / 6J), MAS-1 adjuvant with a) CoV-2 RBD antigen (original Wuhan strain) and b) full-length trimer spike ("spike") antigen (original Wuhan strain) was compared with ASO3-like and MF59 (Addavax). End-point titers determined against the target RBD were evaluated for the various treatment groups. Evaluation of the specificity of the response was performed by IgG isotype analysis of Th2 IgG1 and Th1 IgG2c titers. Cellular responses stimulated by the adjuvants were evaluated by determining the cytokine profile expressed in isolated splenocytes and adjacent lymph nodes.

[0088] Procedure: Mice received two injections (100 μl im, 50 μl each into the hind calf muscle) containing 3 μg RBD or 1 μg spike, 21 days apart. ELISA assays for RBD- or spike-specific serum antibodies were performed 21 days after the second injection. Spleen and draining LN cells were harvested 21 days after the second injection and cultured with RBD or spike for 72 hours. Culture supernatants were tested for cytokines using multiplex ELISA kits (MesoScale Discovery).

[0089] Results: Humoral response profile results are shown in Table 2 below: [Table 2]

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

[0091] **The MF59 used in this study is the animal version known as Addavax.

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

[0093] Results showed that MAS-1 induced potent IgG titers against CoV-2 RBD and spike antigens in C57BL / 6J mice superior to either AS03 or MF59 oil-in-water adjuvants, more so for the RBD than for the inherently more immunogenic spike antigen.

[0094] MAS-1 adjuvanted CoV-2 RBD generated the highest levels of anti-RBD IgG and anti-spike, surpassing ASO3 and MF59 (Addavax). As can be seen in Table 2, unadjuvanted RBD and spike induced a Th2-biased IgG response, while both AS03 and MF59 promoted a more Th2-biased IgG response. In contrast, based on the isotype profile, MAS-1 was observed to induce a relatively balanced humoral response of Th2-type IgG1 and Th1-type IgG2c, compared to AS03 and MF59, which were dominated by Th2-type IgG1 responses.

[0095] The results of the cellular response profile are shown in Table 3 below: Table 3 Cellular response profiles

[0096] [Table 3]

[0097] The data assessed the cellular immune response based on the expression of the cytokines IFNγ, IL-5, IL-10, IL-17, TNF-α, and IL-2. These data indicate that MAS-1 induces a Th1-type cellular response and induces more IFNg than AS03 and MF59 with RBD and spike. In addition, MAS-1 did not stimulate IL-17, which is associated with a proinflammatory cytokine storm. MAS-1 also slightly induced IL-10, and MAS-1 did not induce IL-5, whereas both AS03 and MF59 induced IL-5, which is associated with eosinophil activation.

[0098] Cytokine profiles indicate that MAS-1 enhances IFNγ-producing cells indicative of Th1 cell development, but does not induce Th17 cell development associated with Th1 / Th17 hyperinflammatory responses and cytokine storm.MAS-1-adjuvanted RBD stimulated a modest increase in IL-10, which, together with IFNγ expression, is consistent with stimulating a balanced immune response rather than a response dominated by inflammatory Th1 or Th17 cytokines.

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

[0100] In addition, MAS-1 showed significant dose-sparing, enhanced durability, and cross-reactive protection against Covid viral strains, and MAS-1 can suppress hyperinflammatory responses by restoring balance to the immune system, demonstrating its potential as an immunotherapy for certain autoimmune and inflammatory conditions (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 bulk allows for versatile formulation of vaccines with any candidate SARS-CoV-2 viral antigen without the need for large quantities of valuable candidate antigen in a formulation that may not result in "protective" immunity. One vial of sterile MAS-1 adjuvant vehicle is typically mixed with 0.5 mL of sterile aqueous antigen solution to generate 1.9 mL of vaccine emulsion. Dosing should be up to 0.3 mL for elderly subjects and 0.2 mL or occasionally 0.1 mL for young adults, with at least 4 doses per vial for 0.3 mL doses, at least 6-7 doses per vial for 0.2 mL doses, or 10-12 repeated doses for 0.1 mL doses. According to formulation validation studies submitted to the FDA, the physical-chemical integrity of the emulsion is stable for a minimum of 48 hours at room temperature, but to minimize the risk of microbial contamination, doses from one vial of POU prepared vaccine emulsion are administered within 4 hours after emulsification.

[0103] Although various embodiments of the present invention have been shown and described herein, it is emphasized that such embodiments are provided by way of example only. Numerous variations, modifications, and substitutions can be made in various embodiments thereof without departing from the invention herein. Specifically, when any range is described herein, the range includes all values ​​within the range and all subranges within the range, unless expressly stated otherwise.

[0104] Or more generally, following the disclosure, discussion, examples, and embodiments herein, one of ordinary skill in the art may employ conventional molecular biology, cell biology, microbiology, and recombinant DNA techniques. Materials incorporated by reference herein are for the respective contents and teachings described therein. Such incorporation is for at least the specific teachings and / or other purposes described in citing the reference herein. If no specific teachings and / or other purposes are so noted, the published resource is specifically incorporated for the teachings indicated by one or more of the title, abstract, and / or summary of the document. If no such specifically identified teachings and / or other purposes may be so relevant, the published resource is incorporated to more fully describe the state of the art to which the invention pertains and / or to provide such teachings as are generally known to those of ordinary skill in the art. However, it is expressly stated that the citation of a published resource herein should not be construed as an admission that it is prior art to the present invention. In addition, in the event that one or more of the incorporated public resources differs or conflicts with this application, including but not limited to defined terms, term usage, described techniques, etc., this application will control as the preferred embodiment and the conflicting may be deemed the alternative embodiment.

[0105] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It will be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

Claims

[Claim 1] The invention described herein and in the drawings.

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