Mucosal vaccines against respiratory syncytial virus

Abstract

Embodiments of the present disclosure pertain to a composition that includes a particle, an antigen, and a modulator. The modulator may include, without limitation, an agonist, an activator of the immune system, or combinations thereof. The antigen may be operable to elicit an immune response in a subject against respiratory syncytial virus (RSV). Additional embodiments of the present disclosure pertain to methods of treating or preventing a respiratory infection in a subject by administering a composition of the present disclosure to the subject.

Description

TITLEMUCOSAL VACCINES AGAINST RESPIRATORY SYNCYTIAL VIRUSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 566,024, filed on March 15, 2024. The entirety of the aforementioned application is incorporated herein by reference.SEQUENCE DISCLOSURE STATEMENT

[0002] Pursuant to 37 C.F.R. § 1.834, Applicant has submitted a sequence listing in XML format (“Sequence Listing”). The name of the file containing the Sequence Listing is “AF23853.P207WO.xml”. The date of the creation of the Sequence Listing is March 15, 2025. The size of the Sequence Listing is 6,000 bytes. Applicant hereby incorporates by reference the material in the Sequence Listing.BACKGROUND

[0003] A need exists for more effective compositions, vaccines, and methods for treating and preventing various respiratory infections. Numerous embodiments of the present disclosure aim to address this need.SUMMARY

[0004] In some embodiments, the present disclosure pertains to a composition that includes a particle, an antigen, and a modulator. In some embodiments, the modulator includes, without limitation, an agonist, an activator of the immune system, or combinations thereof. In some embodiments, the antigen is operable to elicit an immune response in a subject against respiratory syncytial virus (RSV).

[0005] In some embodiments, the modulator is encapsulated within the particle. In some embodiments, the modulator includes an endogenous agonist of the stimulator of interferon genes (STING) pathway. In some embodiments, the modulator includes 2’, 3 ’-cyclic guanosine monophosphate-adenosine monophosphate (2’,3’-cGAMP).

[0006] In some embodiments, the antigen is associated with an outer surface of the particle. In some embodiments, the antigen includes a protein or peptide derived from respiratory syncytial virus (RSV), or a nucleotide sequence encoding the protein or peptide.

[0007] In some embodiments, the antigen includes, without limitation, DS-CaVl, sc9-10 DS-CaVl, SC-TM, a full protein thereof, a protein fragment thereof, a peptide fragment thereof, or combinations thereof. In some embodiments, the particle includes a lipid-based particle. In some embodiments, thelipid-based particle is in the form of a liposome. In some embodiments, the composition is suitable for use in treating or preventing a respiratory infection.

[0008] Additional embodiments of the present disclosure pertain to methods of treating or preventing a respiratory infection in a subject by administering a composition of the present disclosure to the subject. In some embodiments, the methods of the present disclosure are used to prevent respiratory infection in the subject. In some embodiments, the methods of the present disclosure are used to treat respiratory infection in the subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1A provides a depiction of a composition that includes a particle, a modulator, and an antigen for treating or preventing a respiratory infection in accordance with various embodiments of the present disclosure.

[0010] FIGS. IB and 1C illustrate methods of treating or preventing a respiratory infection in a subject.

[0011] FIGS. 2A-2C provide data and illustrations regarding the expression and characterization of a respiratory syncytial virus (RSV) fusion protein-based vaccine. FIG. 2A provides a construct design for RSV fusion protein variants DS-CaVl, sc9-10 and SCTM. FIG. 2B shows anti-RSV-F Western blot of cell culture supernatant electrophoresed in a denaturing gel. Lane 1 : DS-CaVl (45 kDa), Lane 2: SCTM (55 kDa), Lane 3: sc9-10 (55 kDa). FIG. 2C shows a distribution of liposomal particle sizes in NanoSTING and NanoSTING-DSCaVl by dynamic light scattering (DLS).

[0012] FIGS. 3A-3G show that the intranasal administration of NanoSTING adjuvanted vaccines induces robust humoral and cellular immunity in mice. FIG. 3A shows that an intranasal vaccine was formulated by incubating the purified recombinant protein and adjuvant. Applicant immunized six groups (n=4-5 / group) of animals intranasally (IN) with PBS, DS-CaVl, NanoSTING-DS-CaVl, cGAMP-SCTM, NanoSTING-SCTM, and NanoSTING-sc9-10 vaccine formulations. Four weeks after the vaccination, NanoSTING-sc9-10 and NanoSTING-SCTM groups were boosted intranasally (IN). On day 48, the boosted animals were euthanized to harvest lung and spleen for ELISpot. FIG. 3B shows RSV prefusion protein-specific serum IgG endpoint titers in the vaccinated animals at days 7, 14, 21, and 48. Data are expressed as mean (± SEM) of 4-5 animals. Mann-Whitney t-test p values; ns=p>0.05, *p < 0.05,**p<0.01. FIGS. 3C-3E show RSV prefusion protein-specific IgA titers inserum (FIG. 3C), BALF (FIG. 3D), and nasal wash (FIG. 3E) on day 48. p values were calculated using the Mann-Whitney t-test (**p<0.01). FIGS. 3F-3G show IFNy and IL-4 ELISPOT from lung (FIG. 3F) and spleen (FIG. 3G) cells re-stimulated ex vivo with RSVB fusion protein peptide pools 20 days after the IN booster dose. Data are expressed as the mean (± SEM) of 4-5 animals assayed. Mann-Whitney t-test p values; *p < 0.05,***p<0.001.

[0013] FIGS. 4A-4G show that NanoSTING-adjuvanted vaccines induce virus-neutralizing antibodies in cotton rats and attenuate viral replication in the lung and nasal tissue. FIG. 4A illustrates the immunization of 7-8 female cotton rats IN four weeks apart following a prime-boost vaccination regimen. Vaccinated cotton rats were challenged with 105p.f.u. of RSV A2 at day 48. Applicant also had a group of five cotton rats previously infected with RSV A2 as a positive control. Four days after the challenge, all animals were euthanized to collect lung and nasal tissues for measurement of viral loads. FIGS. 4B-4C shows RSV prefusion protein-specific serum IgG (FIG. 4B) and IgA (FIG. 4C) titers in vaccinated cotton rats three weeks post-booster dose. Mann-Whitney t-test p value; ***p<0.001. FIG. 4D shows an RSV A2 virus neutralization titers in serum collected (day 48) from vaccinated cotton rats. The limit of detection is shown as a dotted line in the figure. Mann-Whitney t- test p values; *p<0.05, ***p<0.001. FIG. 4E shows RSV B virus neutralization titers in serum collected (day 48) from vaccinated cotton rats. The limit of detection is shown as a dotted line in the figure. Mann-Whitney t-test p value; ***p<0.001. FIGS. 4F-4G show RSV virus load in the lung (FIG. 4F) and nasal (FIG. 4G) homogenates collected from challenged cotton rats four days after the viral challenge. Mann-Whitney t-test p values; **p<0.01, ****p<0.0001.DETAILED DESCRIPTION

[0014] It is to be understood that both the foregoing general description and the following detailed description are illustrative and explanatory, and are not restrictive of the subject matter, as claimed. In this application, the use of the singular includes the plural, the word “a” or “an” means “at least one”, and the use of “or” means “and / or”, unless specifically stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements or components comprising one unit and elements or components that include more than one unit unless specifically stated otherwise.

[0015] The section headings used herein are for organizational purposes and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.

[0016] The human respiratory syncytial virus (RSV) is a highly infectious and ubiquitous virus responsible for severe respiratory tract diseases in children and the elderly. About one-third of deaths resulting from acute lower respiratory infection (ALRI) are caused by the virus in the first year of life, and nearly all children have been infected by RSV at least once by 24 months of age. A recent study estimated that, in 2019, there were 33 million RSV-associated lower respiratory tract (LRT) infections and 101,400 RSV-related deaths worldwide in children under 5 years of age. RSV infection is also a leading cause of disease among adults older than 65 years, with approximately 177,000 hospitalizations and 14,000 deaths each year in the United States. Effective vaccination against RSV can help relieve the tremendous burden of RSV on infants and older individuals.

[0017] Following over 60 years of global research efforts, two vaccines, Abrysvo (Pfizer) and Arexvy (GSK), were recently approved for use in adults aged 60 years or older. Both these vaccines are based on RSV prefusion protein immunogens well known for their ability to elicit neutralizing antibodies against the Q epitope. A phase 3 clinical trial reported vaccine efficacy of 85.7% for Abrysvo against RSV-associated LRT illness with at least three symptoms. Arexvy was reported to have an efficacy of 83% against RSV-related LRT disease over a period of 6.7 months. Follow-up phase 3 studies concluded that Abrysvo’ s efficacy against LRT illness with at least three symptoms in older adults dropped to 77.8% over two seasons. At the same time, Arexvy’s efficacy against RSV-related LRT disease over two seasons dropped to 67.2%. A recent test-negative design analysis found vaccination efficacy against RSV-associated hospitalizations to be 73% in immunocompromised, and 80% in adults without any immunocompromising conditions.

[0018] Approved vaccines and others in phase 3 clinical trials are all administered through the parenteral route. However, as RSV enters the body through the eyes, nose, or mouth, vaccination through the mucosal route can elicit mucosal immunity that protects at the viral port of entry. Notsurprisingly, the current vaccines have not been evaluated on their ability to stop or reduce viral transmission from one infected individual to another.

[0019] Considering the current scenario, newer vaccination strategies are required to realize the full potential of RSV vaccines against disease and transmission. The most efficient route to induce broad and effective mucosal immunity is through the nasal mucosa. Despite their appeal, developing a mucosal vaccine against RSV has been challenging due to a lack of safe and potent adjuvants for RSV fusion protein immunogens.

[0020] As such, a need exists for more effective compositions, vaccines, and methods for treating and preventing various respiratory infections, such as respiratory infections caused by RSV. Numerous embodiments of the present disclosure aim to address this need.

[0021] Compositions

[0022] In some embodiments, the present disclosure pertains to a composition. In some embodiments, the composition includes a particle. In some embodiments, the composition also includes a modulator. In some embodiments, the composition also includes an antigen. As set forth in more detail herein, the compositions of the present disclosure can include various types of particles, modulators, and antigens in various arrangements.

[0023] Modulators

[0024] The compositions of the present disclosure can include various modulators. For instance, in some embodiments, the modulator includes, without limitation, an agonist, an activator of the immune system, or combinations thereof.

[0025] In some embodiments, the modulator is an agonist. In some embodiments, the agonist is a modulator of the stimulator of interferon genes (STING) pathway. In some embodiments, the agonist is an agonist of the stimulator of interferon genes (STING) pathway. In some embodiments, the agonist is an endogenous agonist of the stimulator of interferon genes (STING) pathway. In some embodiments, the modulator includes, without limitation, an endogenous agonist of the stimulator of interferon genes (STING) pathway, bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP), cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), 2’,3’-cyclic guanosine monophosphate-adenosine monophosphate (2 ’,3 ’-cGAMP), amidobenzimidazole, derivatives of amidobenzimidazole, nucleotide modulators, plasmid DNA modulators, CF501 (CAS No. 2408723- 12-4), MSA-2 (CAS No. 129425-81-6), SHR1032 (structure disclosed in Chunying Song, et al. SciRep. 2022 May 20;12(l):8579), C-178 (CAS No. 329198-87-0), H-151 (CAS No. 941987-60-6), divalent cations, or combinations thereof. In some embodiments, the modulator includes 2’,3’-cyclic guanosine monophosphate-adenosine monophosphate (2’,3’-cGAMP).

[0026] The modulators of the present disclosure may be associated with particles in various manners. For instance, in some embodiments, the modulators of the present disclosure are encapsulated within the particle. In some embodiments, the modulators of the present disclosure are on a surface of a particle.

[0027] In some embodiments, encapsulating one or more modulators within a particle as described herein can increase uptake efficiency, tissue targeting, stability, and / or efficacy of the composition or a method of its use, such as by controlling (e.g., maintaining) spatial concentration of the one or more modulators and / or by presenting a recognition signal (e.g., an antigen) to a target cell. In some embodiments, compositions that include one or more modulators without a particle or antigen (or that include one or more modulators outside of and unassociated with a particle of the composition) can elicit a more immediate response from a target tissue or cell and / or decrease the cost and / or complexity of manufacture of the composition.

[0028] The compositions of the present disclosure can include various amounts of modulators. For instance, in some embodiments, a composition of the present disclosure can include 0.1 micrograms to 200 micrograms of a modulator (e.g., a STING pathway activator (e.g., a STING agonist)).

[0029] Antigens

[0030] The compositions of the present disclosure can also include various antigens. For instance, in some embodiments, the antigen is operable to elicit an immune response in a subject against respiratory syncytial virus (RSV). In some embodiments, the antigen includes a protein or peptide derived from respiratory syncytial virus (RSV) (e.g., recombinant proteins or peptides), or a nucleotide sequence encoding the protein or peptide (e.g., a plasmid DNA molecule and / or minigenes expressing the protein or peptide). In some embodiments, the antigen includes a protein or peptide derived from respiratory syncytial virus (RSV). In some embodiments, the antigen includes a nucleotide sequence encoding a protein or peptide derived from respiratory syncytial virus (RSV).

[0031] In some embodiments, the antigen includes a protein derived from respiratory syncytial virus (RSV), or a nucleotide sequence encoding the protein. In some embodiments, the protein is in the form of a fusion protein, a protein fragment, a full protein, a prefusion protein, or combinations thereof.

[0032] In some embodiments, the antigen includes, without limitation, small-hydrophobic protein (SH), attachment protein (G), fusion protein (F), engineered or modified versions of the prefusion F protein, DS-CaVl, sc9-10 DS-CaVl, SC-TM, a full protein thereof, a protein fragment thereof, a peptide fragment thereof, a fusion protein thereof, a prefusion protein thereof, or combinations thereof.

[0033] In some embodiments, the antigen includes a prefusion protein of respiratory syncytial virus (RSV), or a nucleotide encoding the prefusion protein or its variants. In some embodiments, the prefusion protein includes, without limitation, DS-CaVl, sc9-10 DS-CaVl, SC-TM, a full protein thereof, a protein fragment thereof, a peptide fragment thereof, or combinations thereof.

[0034] In some embodiments, the antigen includes SC-TM. In some embodiments, SC-TM includes SEQ ID NO: 1 . In some embodiments, SC-TM includes a sequence with at least 65% sequence identity to SEQ ID NO: 1. In some embodiments, SC-TM includes a sequence with at least 70% sequence identity to SEQ ID NO: 1. In some embodiments, SC-TM includes a sequence with at least 75% sequence identity to SEQ ID NO: 1. In some embodiments, SC-TM includes a sequence with at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, SC-TM includes a sequence with at least 85% sequence identity to SEQ ID NO: 1. In some embodiments, SC-TM includes a sequence with at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, SC-TM includes a sequence with at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, SC-TM includes a sequence with at least 99% sequence identity to SEQ ID NO: 1.

[0035] In some embodiments, the antigen includes DS-CaVl. In some embodiments, DS-CaVl includes SEQ ID NO: 2. In some embodiments, DS-CaVl includes a sequence with at least 65% sequence identity to SEQ ID NO: 2. In some embodiments, DS-CaVl includes a sequence with at least 70% sequence identity to SEQ ID NO: 2. In some embodiments, DS-CaVl includes a sequence with at least 75% sequence identity to SEQ ID NO: 2. In some embodiments, DS-CaVl includes a sequence with at least 80% sequence identity to SEQ ID NO: 2. In some embodiments, DS-CaVl includes a sequence with at least 85% sequence identity to SEQ ID NO: 2. In some embodiments, DS-CaVl includes a sequence with at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, DS-CaVl includes a sequence with at least 95% sequence identity to SEQ ID NO: 2. In some embodiments, DS-CaVl includes a sequence with at least 99% sequence identity to SEQ ID NO: 2.

[0036] In some embodiments, the antigen includes sc9-10 DS-CaVl. In some embodiments, sc9-10 DS-CaVl includes SEQ ID NO: 3. In some embodiments, sc9-10 DS-CaVl includes a sequence with at least 65% sequence identity to SEQ ID NO: 3. In some embodiments, sc9-10 DS-CaVl includes a sequence with at least 70% sequence identity to SEQ ID NO: 3. In some embodiments, sc9-10 DS- CaVl includes a sequence with at least 75% sequence identity to SEQ ID NO: 3. In some embodiments, sc9-10 DS-CaVl includes a sequence with at least 80% sequence identity to SEQ ID NO: 3. In some embodiments, sc9-10 DS-CaVl includes a sequence with at least 85% sequence identity to SEQ ID NO: 3. In some embodiments, sc9-10 DS-CaVl includes a sequence with at least 90% sequence identity to SEQ ID NO: 3. In some embodiments, sc9-10 DS-CaVl includes a sequence with at least 95% sequence identity to SEQ ID NO: 3. In some embodiments, sc9-10 DS-CaVl includes a sequence with at least 99% sequence identity to SEQ ID NO: 3.

[0037] The antigens of the present disclosure may be associated with the particles of the present disclosure in various manners. For instance, in some embodiments, the antigen is associated with an outer surface of the particle. In some embodiments, the antigen is encapsulated within the particle.

[0038] In some embodiments, association of an antigen with a particle (e.g., on an outer surface of a particle) that is associated with a modulator (e.g., an encapsulated modulator) can significantly increase the targeting and / or delivery of the modulator to a target tissue of interest (e.g., an intranasal compartment and / or a lung compartment of a subject). In some embodiments, the association of an antigen with a particle of the present disclosure may increase the efficacy of the composition in treating and / or preventing a respiratory infection in a subject, such as by spatially concentrating an antigen and a modulator of the composition (e g., at a target tissue).

[0039] The compositions of the present disclosure can include various amounts of antigens. For instance, in some embodiments, a composition of the present disclosure can include 0.1 micrograms to 200 micrograms of an antigen.

[0040] Particles

[0041] The compositions of the present disclosure can include various particles. For instance, in some embodiments, the particles include, without limitation, a lipid-based particle, a carbon-based particle, a metal-based particle, or combinations thereof. In some embodiments, the particles of the present disclosure include a lipid-based particle. In some embodiments, the lipid-based particle is in the form of a liposome. In some embodiments, the lipid-based particle includes one or more lipids. In someembodiments, the one or more lipids include, without limitation, l,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), l,2-dipalmitoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (DPPG), cholesterol, l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly ethylene glycol)- 2000] DPPE-PEG2000, or combinations thereof. In some embodiments, the lipid-based particle includes DPPC, DPPG, DPPE-PEG2000 at a molar ratio of 10: 1 : 1 : 1.

[0042] In some embodiments, a particle can include a membrane or wall. In some embodiments, a membrane or wall of a particle can define an interior space. In some embodiments, an interior space of a particle can include one or more modulators. In some embodiments, one or more antigens can be associated with a membrane or wall of the particle (e.g., an outer surface of a membrane or wall of the particle).

[0043] In some embodiments, the particles of the present disclosure include a plurality of molecules. In some embodiments, the particles of the present disclosure or a portion thereof can be anionic (e.g., can include an anionic membrane or one or more anionic lipids). For example, in some embodiments, a lipid-based particle of the present disclosure can include an anionic lipid (e g., DPPE, DPPE- PEG2000, DPPC, DPPG) or a neutral lipid (e.g., cholesterol). In some embodiments, a particle of the present disclosure may include no cationic lipids. In some embodiments, a particle of the present disclosure can be cationic.

[0044] In some embodiments, a particle of the present disclosure can include a cationic (e.g., positively charged) lipid. For example, in some embodiments, a particle of the present disclosure can include, without limitation, l,2-dipalmitoyl-3-trimethylammonium-propane chloride (DPTAP) or 1,2- dioleoyl-3-trimethylammonium propane (DOTAP).

[0045] In some embodiments, a particle of the present disclosure can be zwitterionic. In some embodiments, a particle of the present disclosure or a portion thereof can have a net zero charge. In some embodiments, a particle of the present disclosure or a portion thereof can be uncharged. In some embodiments, a particle of the present disclosure can include dipalmitoylphosphatidylcholine, dipalymitoylphosphatidylglycerol, l,2-bis(diphenylphosphino)ethane (DPPE), cholesterol, or a combination thereof.

[0046] In some embodiments, a particle of the present disclosure can include a poly(ethyleneglycol)- lipid (e.g., a PEG-lipid). In some embodiments, a particle of the present disclosure can include DPPC (l,2-dipalmitoyl-sn-glycero-3-phosphocholine), DPPG (l,2-dipalmitoyl-sn-glycero-3-phospho-(l'-rac-glycerol)), DPPE-PEG2000 (l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(poly ethylene glycol)-2000]), cholesterol, l,2-dipalmitoyl-3-trimethylammonium-propane chloride (DPTAP), l,2-dioleoyl-3 -trimethylammonium propane (DOTAP), or a combination thereof.

[0047] In some embodiments, a particle of the present disclosure can include a combination of DPPC and DPPG, for example, at a molar ratio of about 10: 1. In some embodiments, a particle of the present disclosure can include a combination of DPPC and cholesterol, for example, at a molar ratio of about 10: 1. In some embodiments, a particle of the present disclosure can include a combination of DPPC and DPPE-2000, for example, at a molar ratio of about 10: 1. In some embodiments, a particle of the present disclosure can include a combination of DPPG and cholesterol, for example, at a molar ratio of about 1 : 1. In some embodiments, a particle of the present disclosure can include a combination of cholesterol and DPPE-PEG2000, for example, at a molar ratio of about 1: 1. In some embodiments, a particle of the present disclosure can include a combination of DPPG and DPPE-PEG2000, for example, at a molar ratio of about 1 : 1.

[0048] In some embodiments, a particle of the present disclosure can include DPPC, DPPG, cholesterol, and DPPE-PEG2000. In some embodiments, a particle of the present disclosure can be composed of a molar ratio of 10: 1 : 1 : 1 of DPPC, DPPG, cholesterol, and DPPE-PEG2000, respectively. In some embodiments, a particle of the present disclosure can be composed of a molar ratio of 20: 1: 1 : 1 of DPPC, DPPG, cholesterol, and DPPE-PEG2000, respectively. In some embodiments, a particle of the present disclosure can be composed of a molar ratio of 5 : 1 : 1 : 1 of DPPC, DPPG, cholesterol, and DPPE-PEG2000, respectively. In some embodiments, a particle of the present disclosure can be composed of a molar ratio of 10:2: 1 : 1 of DPPC, DPPG, cholesterol, and DPPE- PEG2000, respectively. In some embodiments, a particle of the present disclosure can be composed of a molar ratio of 10: 1 :2: 1 of DPPC, DPPG, cholesterol, and DPPE-PEG2000, respectively.

[0049] In some embodiments, a particle of the present disclosure can be composed of a molar ratio of 10: 1 : 1 :2 of DPPC, DPPG, cholesterol, and DPPE-PEG2000, respectively. In some embodiments, a particle of the present disclosure can be composed of a molar ratio of 10:2:2: 1 of DPPC, DPPG, cholesterol, and DPPE-PEG2000, respectively. In some embodiments, a particle of the present disclosure can be composed of a molar ratio of 10: 1 :2:2 of DPPC, DPPG, cholesterol, and DPPE- PEG2000, respectively. In some embodiments, a particle of the present disclosure can be composed of a molar ratio of 10:2:1 :2 of DPPC, DPPG, cholesterol, and DPPE-PEG2000, respectively.

[0050] In some embodiments, the particles of the present disclosure can include a nanoparticle. In some embodiments, the nanoparticles have particle diameter (e.g., mean hydrodynamic particle diameter) of less than 300 nanometers (nm), less than 200 nm, less than 150 nm, less than 120 nm, less than 115 nm, less than 111 nm, less than 110 nm, less than 105 nm, less than 100 nm, less than 95 nm, less than 90 nm, less than 85 nm, or less than 80 nm.

[0051] In some embodiments, the particles of the present disclosure have an outer diameter of 1 nanometer to 500 nanometers, 1 nanometer to 750 nanometers, or 1 nanometer to 1,000 nanometers. In some embodiments, the particles of the present disclosure can have an outer diameter of 1 nanometer to 10 nanometers, 1 nanometer to 15 nanometers, 1 nanometer to 20 nanometers, 1 nanometer to 30 nanometers, 1 nanometer to 50 nanometers, 1 nanometer to 75 nanometers, 1 nanometer to 100 nanometers, 1 nanometer to 150 nanometers, 1 nanometer to 200 nanometers, 1 nanometer to 250 nanometers, 1 nanometer to 300 nanometers, 1 nanometer to 400 nanometers, 1 nanometer to 500 nanometers, 10 nanometers to 15 nanometers, 10 nanometers to 20 nanometers, 10 nanometers to 30 nanometers, 10 nanometers to 50 nanometers, 10 nanometers to 75 nanometers, 10 nanometers to 100 nanometers, 10 nanometers to 150 nanometers, 10 nanometers to 200 nanometers, 10 nanometers to 250 nanometers, 10 nanometers to 300 nanometers, 15 nanometers to 20 nanometers, 15 nanometers to 30 nanometers, 15 nanometers to 50 nanometers, 15 nanometers to 75 nanometers, 15 nanometers to 100 nanometers, 15 nanometers to 150 nanometers, 15 nanometers to 200 nanometers, 15 nanometers to 250 nanometers, 15 nanometers to 300 nanometers, 20 nanometers to 30 nanometers, 20 nanometers to 50 nanometers, 20 nanometers to 75 nanometers, 20 nanometers to 100 nanometers, 20 nanometers to 150 nanometers, 20 nanometers to 200 nanometers, 20 nanometers to 250 nanometers, 20 nanometers to 300 nanometers, 30 nanometers to 50 nanometers, 30 nanometers to 75 nanometers, 30 nanometers to 100 nanometers, 30 nanometers to 150 nanometers, 30 nanometers to 200 nanometers, 30 nanometers to 250 nanometers, 30 nanometers to 300 nanometers, 50 nanometers to 75 nanometers, 50 nanometers to 100 nanometers, 50 nanometers to 150 nanometers, 50 nanometers to 200 nanometers, 50 nanometers to 250 nanometers, 50 nanometers to 300 nanometers, 75 nanometers to 100 nanometers, 75 nanometers to 150 nanometers, 75 nanometers to 200 nanometers, 75 nanometers to 250 nanometers, 75 nanometers to 300 nanometers, 100 nanometers to 150 nanometers, 100 nanometers to 200 nanometers, 100 nanometers to 250 nanometers, 100 nanometers to 300 nanometers, 150 nanometers to 200 nanometers, 150 nanometers to 250nanometers, 150 nanometers to 300 nanometers, 200 nanometers to 250 nanometers, 200 nanometers to 300 nanometers, or 250 nanometers to 300 nanometers. In some embodiments, a particle can have an outer diameter of 1 nanometer, 10 nanometers, 15 nanometers, 20 nanometers, 30 nanometers, 50 nanometers, 75 nanometers, 100 nanometers, 150 nanometers, 200 nanometers, 250 nanometers, 300 nanometers, 400 nanometers, or 500 nanometers.

[0052] In some embodiments, the particles of the present disclosure have an outer diameter of at least 1 nanometer, 10 nanometers, 15 nanometers, 20 nanometers, 30 nanometers, 50 nanometers, 75 nanometers, 100 nanometers, 150 nanometers, 200 nanometers, 250 nanometers, 300 nanometers, 400 nanometers, or 500 nanometers. In some embodiments, the particles of the present disclosure can have an outer diameter of at most 1 nanometer, 10 nanometers, 15 nanometers, 20 nanometers, 30 nanometers, 50 nanometers, 75 nanometers, 100 nanometers, 150 nanometers, 200 nanometers, 250 nanometers, 300 nanometers, 400 nanometers, 500 nanometers, 750 nanometers, or 1,000 nanometers.

[0053] A modulator and / or an antigen of the present disclosure may be associated with the particles of the present disclosure in various manners. For instance, in some embodiments, the antigens and modulators of the present disclosure can be positioned on different regions of the particles of the present disclosure. In some embodiments, a modulator of the present disclosure can be encapsulated in a particle. In some embodiments, all or a portion of an antigen of the present disclosure can be associated with (e.g., attached to, adhered to, adsorbed onto, electrostatically interacted with, covalently bound to, noncovalently bound to, integrated into, or formulated onto) a surface of the particle. In some embodiments, all or a portion of an antigen is encapsulated within the particle. In some embodiments, the association (e.g., adsorption) of an antigen with a surface of a particle can increase stability of the particle and / or increase delivery efficiency (e g., to a target tissue) after administration.

[0054] In some embodiments, one or more antigens of the present disclosure can be encapsulated within a particle of the present disclosure. In some embodiments, one or more modulators of the present disclosure can be associated with an outer surface of the particle. In some embodiments, one or more antigens of the present disclosure can be encapsulated in a particle of the present disclosure while one or more modulators of the present disclosure are associated with an outer surface of the particle. In some embodiments, one or more antigens of the present disclosure can be associated withan outer surface of a particle of the present disclosure. In some embodiments, one or more modulators of the present disclosure can be encapsulated within the particles of the present disclosure.

[0055] In some embodiments, one or more antigens of the present disclosure can be associated with an outer surface of a particle of the present disclosure while one or more modulators of the present disclosure are encapsulated within the particle. In some embodiments, one or more antigens and one or more modulators of the present disclosure can both be encapsulated within a particle of the present disclosure. In some embodiments, one or more antigens and one or more modulators of the present disclosure may both be associated with a surface of a particle of the present disclosure. In some embodiments, one or more antigens of the present disclosure can be integrated into a membrane of a particle of the present disclosure. In some embodiments, one or more modulators of the present disclosure can be integrated into a membrane of a particle of the present disclosure. In some embodiments, one or more antigens of the present disclosure can be encapsulated within a particle of the present disclosure while one or more modulators are integrated into a membrane of a particle of the present disclosure. In some embodiments, one or more antigens of the present disclosure can be associated with an outer surface of a particle of the present disclosure while one or more modulators are integrated into a membrane of a particle of the present disclosure.

[0056] In some embodiments, one or more modulators of the present disclosure can be encapsulated within a particle of the present disclosure while one or more antigens of the present disclosure are integrated into a membrane of the particle. In some embodiments, one or more modulators can be associated with an outer surface of a particle of the present disclosure while one or more antigens of the present disclosure are integrated into a membrane of the particle. In some embodiments, the incorporation of both an antigen and a modulator of the present disclosure within a particle can facilitate coordinated cytosolic delivery.

[0057] The compositions of the present disclosure can include various amounts of particles. For instance, in some embodiments, a composition of the present disclosure can include 0.1 micrograms to 200 micrograms of a particle of the present disclosure.

[0058] Composition forms

[0059] The compositions of the present disclosure may be in various forms. For instance, in some embodiments, the compositions of the present disclosure may be formulated for intranasal delivery. In some embodiments, the composition is lyophilized. In some embodiments, the composition is inliquid form. In some embodiments, the composition is suitable for use in treating or preventing a respiratory infection, such as a respiratory infection caused by respiratory syncytial virus (RSV).

[0060] An example of a composition of the present disclosure is illustrated in FIG. 1A as composition 10, which includes a lipid-based particle 12 (e.g., a liposome), a modulator 16 (e.g., a STING agonist, such as cGAMP, and one or more antigens 14 (e.g., one or more prefusion proteins of respiratory syncytial virus (RSV), such as DS-CaVl, sc9-10 DS-CaVl, and / or SC-TM). In this example, modulator 16 is encapsulated within lipid-based particle 12 while antigens 14 are associated with the outer surface of lipid-based particle 12.

[0061] In some embodiments, it can be advantageous to formulate the compositions of the present disclosure within a desired pH range. For example, formulation of a composition described herein (e.g., for intranasal delivery) between pH 4.0 and pH 7.5 (e.g., pH 4.5 to pH 6.5 or pH 5.5 to pH 6.5) can avoid irritation and / or histological damage to the intranasal tissue, which may occur if extremely acidic or basic formulations are used for intranasal delivery (e.g., because nasal cavity pH can be from about 5.5 to 6.5). In some embodiments, a composition of the present disclosure can have a pH of 4.0 to 7.5.

[0062] In some embodiments, it can be advantageous to formulate a composition of the present disclosure within a desired osmolarity range. For example, formulation of a composition of the present disclosure (e.g., for intranasal delivery) having an osmolarity from 50 to 900 mOsm / kg can improve absorption while avoiding potential epithelial damage (e.g., which may occur from compositions with very low osmolarities) and avoiding increases to mucosal secretions (e.g., which may occur from compositions with very high osmolarities) (e.g., because nasal cavity osmolarity can be about 280 mOsm / kg). In some embodiments, a composition of the present disclosure can have an osmolarity of 50 mOsm / kg to 900 mOsm / kg.

[0063] In some embodiments, the compositions of the present disclosure can be advantageous to formulate compositions described herein within a desired viscosity range. For example, formulation of a composition of the present disclosure (e.g., for intranasal delivery) having a viscosity, for example, from 1.1 cP (centipoise) to 50 cP (e.g., 1.5 cP to 50 cP) can increase residence time in the nasal cavity while adversely affecting droplet size (e.g., which can affect spray pattern and / or distribution within the nasal cavity). In some embodiments, a composition of the present disclosure can have a viscosity of 1 cP (centipoise) to 100 cP.

[0064] The compositions of the present disclosure can be in various forms. For instance, in some embodiments, the compositions of the present disclosure can be in the form of a solubilized liquid. In some embodiments, the compositions of the present disclosure are suitable for intranasal and / or inhalational administration to a subject. In some embodiments, intranasal delivery of the compositions of the present disclosure can be used to target intranasal compartment tissues. In some embodiments, inhalational administration of the compositions of the present disclosure can be used to target lung compartment tissues.

[0065] In some embodiments, the compositions of the present disclosure also include one or more stabilizers. In some embodiments, the stabilizers include, without limitation, anti-oxidants, sequestrants, ultraviolet stabilizers, or combinations thereof.

[0066] In some embodiments, the compositions of the present disclosure also include one or more surfactants. In some embodiments, the surfactants include, without limitation, anionic surfactants, sugars, cationic surfactants, zwitterionic surfactants, non-ionic surfactants, or combinations thereof.

[0067] In some embodiments, the compositions of the present disclosure also include one or more excipients. In some embodiments, the excipients include, without limitation, lactose, sucrose, starch powder, cellulose esters of alkanoic acids, trehalose, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum, trehalose, sodium alginate, polyvinylpyrrolidone, polyvinyl alcohol, or combinations thereof.

[0068] In some embodiments, a composition of the present disclosure is in lyophilized form (e.g., freeze-dried). Lyophilization of a composition of the present disclosure (or a component thereof) can increase the storage stability (e.g., shelf stability) of a composition. In some embodiments, one or more components of a composition of the present disclosure can be lyophilized.

[0069] In some embodiments, lyophilization of a composition of the present disclosure (or a component thereof) can allow for easy preparation of the composition. In some embodiments, such lyophilization allows for use of the compositions of the present disclosure (e.g., administration to a subject) in regions without easy access to material preparation facilities. For instance, a modulator, a lipid-based particle, and / or an antigen described herein can be lyophilized and then rehydrated and mixed (e.g., as described herein) to formulate a composition described herein at a site of administration to a subject that is remote from a permanent medical or pharmaceutical facility.

[0070] In some embodiments, a lyophilized composition of the present disclosure (or a portion thereof, such as a lyophilized particle or a lyophilized antigen or a lyophilized modulator) can be stored at 4 °C. In some embodiments, a lyophilized composition of the present disclosure (or a portion thereof) can be stored at 4 °C and used for up to 1 week, up to 2 weeks, up to 3 weeks, up to 1 month, up to 2 months, up to 3 months, up to 4 months, up to 5 months, up to 6 months, up to 7 months, up to 8 months, up to 9 months, up to 10 months, or up to 12 months.

[0071] In some embodiments, the compositions of the present disclosure may be in liquid form. In some embodiments, the compositions of the present disclosure may be in solid form.

[0072] In some embodiments, a composition of the present disclosure can include a divalent ion, such as a divalent cation. In some embodiments, a composition of the present disclosure includes a divalent cation encapsulated within, adsorbed onto, covalently coupled to, electrostatically interacted with, or formulated onto a membrane of a particle of the present disclosure. In some embodiments, a composition can include a divalent cation that includes, without limitation, Mn2+, Mg2+, Ca2+, and Zn2+. For example, a composition of the present disclosure can include a modulator (e.g., a STING agonist), a particle (e.g., a lipid-based nanoparticle), and a divalent cation, such as Mn2+, Mg2+, Ca2+, or Zn2+.

[0073] Methods of treating or preventing a respiratory infection

[0074] Additional embodiments of the present disclosure pertain to methods of treating or preventing a respiratory infection in a subject. As illustrated in FIG. IB, such methods generally include administering a composition of the present disclosure to the subject (step 20) to result in the treatment or prevention of a respiratory infection in the subject (step 22). As set forth in more detail herein, the methods of the present disclosure can have numerous uses and embodiments.

[0075] Subjects

[0076] The methods of the present disclosure may be utilized to treat or prevent a respiratory infection in various subjects. For instance, in some embodiments, the subject does not exhibit symptoms of a respiratory infection. In some embodiments, the subject exhibits symptoms of a respiratory infection.

[0077] The compositions of the present disclosure may be administered to various subjects. For instance, in some embodiments, the subject is a mammal (e.g., a human). In some embodiments, the subject is a human being. In some embodiments, the subject is a domesticated animal. For example, in some embodiments, the subject can be a dog or a cat. In some embodiments, a subject can be a cow,a horse, a non-human primate, a mouse, a rat, a rabbit, a guinea pig, a goat, a sheep, a giraffe, a zebra, a lion, a tiger, or a bear.

[0078] In some embodiments, the subject can be vulnerable to or suffering from a respiratory infection. For example, in some embodiments, the compositions of the present disclosure may be administered to a subject who has been exposed to or who has been infected with respiratory syncytial virus (RSV). In some embodiments, a subject can be selected for treatment as a result of exhibiting one or more symptoms of a respiratory infection. For example, a subject may be selected for treatment based on having one or more symptoms, including persistent coughing, elevated body temperature (e g., greater than 100.4 ° C by forehead skin measurement), body chills, achy joints, difficulty breathing or catching one’s breath, fluid in the lungs, fatigue, headache, loss of taste or smell, or a positive a respiratory infection test, such as a PCR test.

[0079] In some embodiments, a subject may be a subject vulnerable to a respiratory infection. For instance, in some embodiments, the subject may be selected for treatment with a composition of the present disclosure based on a demographic risk factor, such as obesity, advanced age (e.g., 65 years old or older), immune impairment, or pregnancy. In some embodiments, a subject may be selected for treatment based on a risk of infection by respiratory syncytial virus (RSV), for instance, if the subject has an occupation involving close interaction with customers, frequent interaction with at-risk populations, handling of biological samples, or close contact with potentially infected individuals.

[0080] Administration

[0081] The compositions of the present disclosure may be administered to subjects in various manners. For instance, in some embodiments, the composition is administered in at least one dose. In some embodiments, the composition is administered in one dose. In some embodiments, the composition is administered in two doses.

[0082] The compositions of the present disclosure may be administered to subjects by various methods. For instance, in some embodiments, the administration occurs by methods that include, without limitation, intravenous administration, intramuscular administration, intradermal administration, intraperitoneal administration, subcutaneous administration, spray-based administration, aerosol-based administration, in ovo administration, oral administration, intraocular administration, intratracheal administration, intranasal administration, inhalational administration, parenteral administration, mucosal administration, or combinations thereof. In some embodiments,the compositions of the present disclosure are administered through intranasal administration, inhalational administration, intravenous administration, or combinations thereof. In some embodiments, the compositions of the present disclosure are administered through intranasal administration. In some embodiments, the compositions of the present disclosure are administered through inhalational administration. In some embodiments, the compositions of the present disclosure are administered through parenteral administration. In some embodiments, the compositions of the present disclosure are administered through mucosal administration.

[0083] In some embodiments, a composition of the present disclosure may be administered to a subject before exposure to respiratory syncytial virus (RSV), for example, to prevent the subject from acquiring a respiratory infection (e.g., as illustrated in FIG. IB). In some embodiments, a composition of the present disclosure may be administered in the form of a vaccine.

[0084] In some embodiments, a method of the present disclosure can include administering a dose of a composition of the present disclosure (e.g., via intranasal administration) to a subject (e.g., an animal subject) after exposure or suspected exposure to respiratory syncytial virus (RSV). In some embodiments, a composition of the present disclosure may be administered to a subject after exposure to respiratory syncytial virus (RSV), for example, to treat (e.g., ameliorate or, in some embodiments, cure) a respiratory infection, for example, after the subject has acquired a respiratory infection (e.g., as illustrated in FIG. 1C). In some embodiments, the compositions of the present disclosure may be administered to a subject at least 1 minute, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 14 days, or at least 28 days after exposure or suspected exposure to respiratory syncytial virus (RSV).

[0085] In some embodiments, the compositions of the present disclosure can be administered in combination with other therapeutic treatments. In some embodiments, the other therapeutic treatments include, without limitation, antibiotic treatment, targeted inhibition, or combinations thereof. In some embodiments, a composition of the present disclosure can include and / or can be administered in a treatment regimen (e.g., administered concurrently or non-concurrently) with an adjuvant (e g., one or more antibodies, one or more vaccines, one or more small molecules, one or more nucleic acids, and / or one or more peptides or proteins, such as an interleukin (e.g., IL-21)).

[0086] Treatment or prevention of a respiratory infection

[0087] The methods of the present disclosure may have various uses. For instance, in some embodiments, the methods of the present disclosure may be used to treat a respiratory infection in a subject. In some embodiments, the methods of the present disclosure may be used to prevent a respiratory infection in a subject. For instance, in some embodiments, the methods of the present disclosure may be used to prevent the establishment of a respiratory infection in the subject, to prevent progression of a respiratory infection in the subject, to prevent the transmission of a respiratory infection to a second subject, or combinations thereof. In some embodiments, the methods of the present disclosure may initiate an innate immune response that leads to associated adaptive immunity for a respiratory infection.

[0088] In some embodiments, the respiratory infection may be caused by respiratory syncytial virus (RSV). In some embodiments, the respiratory infection may include a severe lower respiratory tract infection (LRTI).

[0089] In some embodiments, the administered compositions of the present disclosure can rapidly activate a subject’s innate immune system to facilitate a broad-spectrum response against respiratory syncytial virus (RSV). For instance, in some embodiments, a single intranasal dose of a composition of the present disclosure can: (1) treat infections throughout the respiratory tree and minimize symptoms, (2) reduce transmission by decreasing infectious in the nasal passage, (3) protect against respiratory syncytial virus (RSV), and / or (4) provide durable protection against reinfection by stimulating adaptive immunity.

[0090] In some embodiments, administered compositions of the present disclosure can enable sustained release of modulators (e.g., cGAMP) to both the nasal compartment and the lung over a period of 48 hours or more following administration to a subject. In some embodiments, the administered compositions of the present disclosure can activate multiple pathways and facilitate a type-I interferon (IFN-I) mediated response in a subject.

[0091] In some embodiments, the compositions of the present disclosure can elicit an immune response in the subject against a respiratory infection. In some embodiments, the compositions of the present disclosure can elicit such immunity through at least one of innate immunity, mucosal immunity, systemic immunity, cellular immunity, humoral immunity, T-cell immunity, production of systemic neutralizing antibodies, induction of IgG responses, induction of IgA responses, induction ofIgM responses, induction of T-cell responses, induction of mucosal IgA responses in lung and nasal compartments, induction of Thl T-cell responses, induction of CD8+ T-cell responses, induction of CD4+ T cell responses, induction of NK cell responses, activation or inhibition of the stimulator of interferon genes (STING) pathway, or combinations thereof.

[0092] Additional embodiments

[0093] Reference will now be made to more specific embodiments of the present disclosure and experimental results that provide support for such embodiments. However, Applicant notes that the disclosure below is for illustrative purposes only and is not intended to limit the scope of the claimed subject matter in any way.

[0094] Example 1. Mucosal vaccines with STING-agonist liposomal formulations inhibit RSV (respiratory syncytial virus) replication in cotton rats

[0095] Respiratory syncytial virus (RSV) is responsible for severe lower respiratory tract infections (LRTI) in immunocompromised individuals. While recent breakthroughs in vaccine design have led to approved vaccines for the elderly, these vaccines are all administered through the parenteral route. Administration through the mucosal route could protect the viral route of entry and can be advantageous over injected vaccines. There is, however, a lack of safe and efficacious mucosal adjuvants that can facilitate both mucosal and systemic immune responses.

[0096] Recently, stimulator of interferon gene (STING)-agonists like 2’-3’ cyclic GMP-AMP (cGAMP) have emerged as promising adjuvants that can enhance mucosal immune response of protein-based vaccines against respiratory pathogens. cGAMP activates the cGAS / STING cytosolic DNA sensing pathway that results in the secretion of immunostimulatory type-I and type-III interferons. In this Example, Applicant presents preclinical data based on liposomal nanoparticles, NanoSTING, that encapsulate the endogenous STING-agonist 2’ 3 ’-cGAMP (cyclic guanosine adenosine monophosphate) as an adjuvant for prefusion protein-based intranasal vaccine against RSV. NanoSTING significantly increased the immunogenicity of well-documented RSV prefusion protein antigens DS-CaVl, sc9-10 DS-CaVl, and SC-TM after a single intranasal dose, when compared to the protein-only and naked-cGAMP adjuvanted groups. Two doses of NanoSTING adjuvanted vaccines yielded robust secretory IgA titers at the mucosal surfaces and induced potent Thl T-cell responses in the lungs of vaccinated mice.

[0097] Both NanoSTING-sc9-10 DS-CaVl and NanoSTING-SCTM vaccines protect against viral replication at the upper (nose) and lower (lung) respiratory tract of RSV-challenged cotton rats. The ability of Applicant’s mucosal vaccines against RSV to elicit immunity in the respiratory tract can prevent the establishment of infection in individuals and potentially prevent disease transmission.

[0098] Example 1.1. Expression and purification prefusion proteins

[0099] Applicant obtained DSCaVl, sc9-10 DSCaVl, and SCTM protein-expressing plasmids. Applicant used the Expi293™ expression system (Thermo Scientific, MA, USA) to express all the protein variants. Applicant cultured Expi293™ cells in a shaking incubator at 37°C and 8% CO2, according to manufacturer recommendations, until the desired cell density of 3-5 x 106cells / ml was obtained. Plasmid DNA encoding the prefusion proteins was transiently transfected into the cells using the ExpiFectamine™ 293 transfection kit. Applicant collected the culture supernatants containing the expressed RSV fusion proteins five days after the transfection by centrifuging the cell suspension at 5000 x g for 20 mins. The supernatant was sterile-filtered using a 0.22 pm filter and stored at -80 °C until further use.

[0100] Applicant purified the 6xhis tagged DS-CaVl and sc9-10 DSCaVl proteins using a two-step purification protocol applying immobilized metal affinity (IMAC) and size-exclusion chromatography (SEC) on an AKTA™ UV-900, P-900, pH / C-900, Frac-950 purification system (GE healthcare life sciences, IL, USA). For affinity chromatography, nine volumes of harvested supernatant were mixed with one volume of 10 x loading buffer (50 mM NaFEPCh, 300 mM NaCl, 10 mM Imidazole, and 0.01% Tween20, pH = 8.0). The supernatant was passed over an XK 16 / 20 column packed with 5 ml Ni Sepharose 6 Fast Flow to load the protein on the resin. Next, Applicant washed the column with 10 CV (column volume) of wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM Imidazole, and 0.01% Tween-20, pH = 8.0) and eluted the protein with 2 CV elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM Imidazole and 0.01% Tween-20, pH = 8.0).

[0101] The eluted fractions were promptly exchanged into PBS using a 3 kDa molecular weight cut-off Slide-A-Lyzer dialysis cassette (ThermoFisher Scientific, MA, USA). Applicant concentrated the eluted fractions and purified them on a Superdex-200 (GE Healthcare Life Sciences, IL, USA) SEC column using PBS as a running buffer.

[0102] The SCTM protein was purified using a combination of ion exchange chromatography (IEX) and SEC steps. Applicant diluted the culture supernatant with two volumes of 50 mM NaOAc,pH= 5.0, and loaded the protein on a 5 ml SP Sepharose cation exchange resin (Cytiva, MA, USA). Applicant washed the column with 10 CV of wash buffer (20 mM NaOAc, 50 mM NaCl, 0.01% Tween20, pH=5.0) and eluted the protein using a linear gradient of NaCl from 100 mM to IM. Applicant concentrated the eluate and purified it further using SEC, following similar steps as for DSCaVl and sc9-10 DSCaVl.

[0103] Example 1.2. Western blot analysis

[0104] Applicant analyzed the expressed and purified proteins by electrophoresing through 4- 15% Mini-PROTEAN TGX (BioRad, CA, USA) gels under reducing and denaturing conditions. Applicant added 2-mercaptoethanol (Sigma Life Science, Burlington, MA) to the protein samples to disrupt the disulfide bonds and incubated the samples at 95°C for 5 minutes to denature the protein. Vertical electrophoresis was performed with 1 x tris / glycine / SDS running buffer for 2 hours at 90 volts. Immediately after the electrophoresis, the protein was blotted on a polyvinylidene fluoride (PVDF) membrane using IX tris / glycine / methanol transfer buffer for 1 hour at 90 volts. Applicant incubated the membrane in IX tris-buffered saline / Tween20 (TBST) (J.T. Baker / Fisher Bioreagents; PA, USA; Sigma, MO, USA) containing 5% skim milk to block active binding sites of the protein. Following 2 hours of blocking, Applicant incubated the membranes overnight in the primary antibody solution of 2.5 % bovine serum albumin (BSA) (ThermoFisher Scientific, MA, USA) in TBST. The primary antibodies used for the detection of RSV fusion proteins were the anti-RSV antibody MAB8599 (MilliporeSigma, MA, USA) and the anti-his tag antibody (Clone: J095G46) (BioLegend, CA, USA). Applicant incubated the membrane overnight with an anti-mouse IgG HRP (Cell Signalling Technology, MA, USA) secondary antibody for 1 hour. Finally, the membranes were washed three times with TBST buffer and developed using Pierce 1-Step Ultra TMB Blotting Solution (ThermoFisher Scientific) as per manufacturer recommendations. Images were taken using cell phone cameras and analyzed using Image! software.

[0105] Example 1.3. SEC-MALS analysis

[0106] The molar mass of the RSV F proteins was determined by SEC-MALS analysis. The protein samples were filtered using a 0.22 pm nylon syringe filter (MicroSolv, Wilmington, NC) prior to analysis. Separation was achieved on a Superdex 200 Increase 10 / 300 GL SEC column (GE Healthcare, Chicago, IL) for 100 pL sample injection volumes (Agilent 1290 Infinity autosampler, Agilent Technologies Santa Clara, CA). The mobile phase was 0.22 pm-filtered phosphate-bufferedsaline (PBS) at a flow rate of 0.5 mL / min (Agilent 1290 Infinity binary pump). Concentration detection of the eluting protein was provided by an Agilent 1260 Infinity UV-vis diode array detector at 280 nm wavelength using a UV extinction coefficient of 0.95 mL mg1cm1for RSV F protein. Light scattering data were collected on a Wyatt HELEOS II MALS detector (Wyatt Technologies, Santa Barbara, CA), using a Ist-order Zimm analysis as implemented in the Wyatt ASTRA 7.3.2 software for molar mass determination. Detector alignment, band broadening corrections, and normalization of the MALS detectors across the various scattering angles were performed using a sample of bovine serum albumin (BSA) at 1 g / L, which was run before the RSV F analyses. It is noted that a Wyatt OptiLab T-rEX differential refractive index (dRI) detector was also used and yielded consistent molar mass results to the UV-vis detector for a refractive index increment (dn / dc) of 0.185 mL / g for the protein.

[0107] Example 1.4, Mouse studies

[0108] Applicant performed all mouse experiments in accordance with the guidelines provided by the Institutional Animal Care and Use Committee (IACUC). Applicant purchased six- to ten-week- old female Balb / c mice from The Jackson Laboratory (ME, USA). Applicant housed them at the institutional animal facility of the University of Houston for at least 10 days before the start of the experiments. The animals were vaccinated intranasally using these vaccine formulations: 1) PBS, 2) 10 pg DSCaVl, 3) 10 pg DSCaVl + 20 pg NanoSTING, 4) 10 pg SCTM + 20 pg cGAMP, 5) 10 pg SCTM + 20 pg NanoSTING, 6) 10 pg sc9-10 DSCaVl + 20 pg NanoSTING. Three weeks after the first dose, the mice were boosted with the corresponding prime dosage. Mice were bled every week to record antibody response. All animals were euthanized five weeks after the first vaccination, and tissue (lung and spleen) samples were collected for ELISpot.

[0109] Example 1.5, ELISA

[0110] Using ELISA, Applicant tested the magnitude of vaccine-induced antibody response in serum against the fusion proteins. In short, Applicant incubated high protein binding ELISA plates (Corning, NY, USA) with RSV prefusion proteins at 0.5 pg / ml in phosphate-buffered saline (PBS) overnight at 4 °C or 2 hours at 37 °C. Applicant washed the plates with PBS+0.05% Tween20 (PBST) to remove unbound protein. The plates were blocked with PBS + 1% BSA (Fisher Scientific, PA, USA) + 0.1% Tween20 for 2 hours at room temperature. Following three more washes with PBST, Applicant added the serum samples at different dilutions to the plate. To detect RSV prefusion bindingantibodies, Applicant washed the plates with PBST and added HRP-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, 1 :6000; PA, USA) or Biotin-conjugated Goat anti-mouse IgA (Southern Biotech, 1 :5000; AL, USA). Streptavidin-HRP (Vector Laboratories, 1:2500, CA, USA) detected an anti-IgA secondary antibody. Finally, Applicant developed the plates using 1-Step™ TMB ELISA substrate (ThermoFisher Scientific).

[0111] Example 1.6, ELISpot

[0112] Applicant harvested lung and spleen cells from vaccinated mice to test antigen-specific T-cell responses. To perform the ELISpot assays, Applicant incubated plates (Ref: MSIPS4W10, Millipore, MA, USA) with anti-IFNy antibody (1 pg / ml, Ref: 3321-3-250, Mabtech, VA, USA) at 4 °C overnight. Applicant washed the plates with sterile PBS five times the next day and added lung lymphocytes and splenocytes. Applicant treated the cells from mice with 1) R10 media (negative control), 2) 12-myristate 13-acetate (PMA) (Sigma, St. Louis, MI, USA), and 1 ug / mL of ionomycin (positive control), and 3) RSV fusion protein peptide pool (2 pg / ml / peptide, JPT, Germany). For positive control, l * 104cells were stimulated in triplicates, whereas Applicant stimulated 3* 1O5cells in the rest of the wells. Following overnight stimulation of the cells, Applicant washed the plates with PBS and added a biotinylated anti-IFNy (1 pg / ml, Ref: 3321-6-250, Mabtech) detection antibody. After 1 hour of incubation at 37 °C, Applicant rewashed the plates and added diluted Extravidin-ALP conjugate (1 :30,000, Sigma, St. Louis, MI, USA). Finally, the spots were developed by adding BCIP / NBT-plus substrate (Ref: 3650-10, Mabtech). Applicant rinsed off the substrate with water and imaged the spots using Cytation 7 (BioTek Instruments, Inc.) imaging plate reader. The spots were enumerated using Gen5 (BioTek) software.

[0113] Example 1.7, Virus cultivation

[0114] Respiratory Syncytial Virus strain A / A2 (RSV A / A2) (ATCC, Manassas, VA) was propagated in HEp-2 cells after serial plaque-purification to reduce defective-interfering particles. A pool of virus designated as hRSVLot# 092215 SSM containing approximately 3.0 x 108pfu / mL in sucrose stabilizing media was used in this in vivo experiment. Virus stock was stored at -80°C and has been characterized in vivo using the cotton rat model and validated for upper and lower respiratory tract replication.

[0115] Example 1.8, Cotton rat studies

[0116] Twenty -five (25) inbred, 5-7 weeks-old, Sigmodon hispidus female cotton rats (source:Sigmovir Biosystems, Inc., Rockville, MD) were maintained and handled under veterinary supervision in accordance with the National Institutes of Health guidelines and Sigmovir Institutional Animal Care and Use Committee’s approved animal study protocol (IACUC Protocol #15). Cotton rats were housed in clear polycarbonate cages and provided with standard rodent chow (Harlan #7004) and tap water ad lib. The cotton rats were intranasally immunized twice (four weeks apart) in groups of 5-8 animals with 1) PBS, 2) 20 pg sc9-10 + 40 pg NanoSTING, and 3) 20 pg SCTM+ 40 pg NanoSTING. A group of five cotton rats were intramuscularly immunized with FI-RSV as control animals. Three weeks after the booster dose, the animals were challenged with 105plaque-forming units (p.f.u.) of RSV A2 virus. Four days after the challenge, Applicant collected the lung and nasal tissues to evaluate viral titers.

[0117] Example 1.9, RSV-specific microneutralization assay

[0118] Heat-inactivated sera samples were diluted 1 : 10 with EMEM and serially diluted further 1 :4. Applicant incubated the diluted sera samples with RSV A2 (25-50 PFU) for 1 hour at 25 °C and inoculated duplicates onto confluent HEp-2 monolayers in 24-well plates. After one hour incubation at 37°C in a 5% CO2 incubator, the wells were overlayed with 0.75% Methylcellulose medium. After 4 days of incubation, the overlay was removed, and the cells were fixed with 0.1% crystal violet stain for one hour and then rinsed and air dried. Serum neutralizing antibody to RSV was assayed by measuring 60% plaque reduction on Hep-2 cell monolayers. RSV B-specific virusneutralizing titers were determined using HEp-2 cell monolayers infected with RSV / B / 18537. Applicant defined RSV B neutralizing titers as the highest serum dilution at which Applicant observed at least a 50% reduction in viral replication. Applicant assigned a value of 2 log2 for any serum sample exhibiting neutralizing titers less than the lower limit of detection (2.5 log2).

[0119] Example 1.10. Lung and nose viral titration

[0120] Applicant clarified the lung and nose homogenates by centrifugation and diluted them in EMEM media. Confluent HEp-2 monolayers were infected in duplicates with diluted homogenates in 24-well plates. After one hour incubation at 37 °C in a 5% CO2 incubator, the wells were overlayed with 0.75% Methylcellulose medium. After 4 days of incubation, Applicant removed the overlays and fixed the cells with a 0.1% crystal violet stain for one hour. The wells were properly rinsed and air-dried. Plaques were counted, and virus titer was expressed as plaque-forming units per gram of tissue. Viral titers are calculated as geometric mean ± standard error for all animals in a group at a given time.

[0121] Exampl e 1.11. Stati sti cal anal v si s

[0122] Applicant presented all data as mean values, and error bars represent ±SEM (standard error of the mean). All statistical analyses were performed using GraphPad Prism (V8). Applicant compared two groups using the Mann-Whitney t-test, while comparisons between multiple groups were performed using Tukey’s multiple tests for repeated measures analysis.

[0123] Example 1.12, Purification, preparation, and characterization of prefusion proteinbased vaccines

[0124] Most RSV-neutralizing antibodies are directed against the prefusion protein. However, due to the propensity of the fusion protein to spontaneously refold into the postfusion form, rational immunogen designs have been successfully employed to arrest the fusion protein in a stable prefusion state. Applicant has adopted the well-characterized prefusion-stabilized RSV fusion proteins as immunogens in Applicant’s intranasal vaccine formulations.

[0125] The amino acid substitutions that stabilize the prefusion proteins DS-CaVl, sc9-10, and SCTM are annotated in FIG. 2A. Applicant expressed these proteins in an Expi293 suspensionculture expression system and verified their expression in the supernatant using anti-RSV F western blot (FIG. 2B).

[0126] The molecular weight of the purified proteins was analyzed using SEC-MALS. The SEC chromatogram shows a major peak corresponding to the trimeric protein. Applicant analyzed the multi-angle light-scattering data, which confirmed the size of the sc9-10 and SCTM trimeric proteins in the expected molecular weight range (-160-180 kDa). Notably, the sc9-10 DS-CaVl chromatogram also indicates the presence of a minor species corresponding to a 320-400 kDa protein, which could have formed due to the association of two molecules of the trimeric protein. The final yields of the prefusion trimers were determined using an AM14-prefusion protein-specific ELISA. AM14 is a monoclonal antibody that recognizes an epitope spanning two protomers of the RSV prefusion protein trimer, and previous studies have reported that this antibody does not bind to the monomeric RSV fusion protein. Applicant observed prefusion trimer yields ranging from 1 mg / L for DS-CaVl to 50 mg / L for SCTM in Expi293 expression system (FIG. 2C).

[0127] To synthesize the adjuvant, Applicant encapsulated 2’3’-cGAMP in lipid-based nanoparticles (NanoSTING), as described previously. i Science, vol. 24, no. 9, p. 103037, Sep. 2021. Applicant mixed the purified prefusion proteins with NanoSTING at 25 °C for 15 minutes in a single- step “mix and immunize” approach to formulate the vaccines for animal studies. Applicant performed dynamic light scattering (DLS) on the nanoparticles before (116 ± 7 nm) and after (121 ± 3 nm) mixing with proteins (FIG. 2C). DLS did not indicate any signs of protein aggregation in the vaccines, at least for the duration of immunizations (30 min- 1 hour). Applicant proceeded to use these vaccines for in vivo studies.

[0128] Example 1.13, NanoSTING adjuvanted vaccines improve immunogenicity of RSV prefusion proteins in mice

[0129] Applicant evaluated the immunogenicity of NanoSTING adjuvanted intranasal vaccines in mice. Groups of Balb / c mice were immunized with a single dose of: 1) PBS, 2) DS-CaVl (no adjuvant), 3) NanoSTING-DS-CaVl, 4) cGAMP-SCTM (no liposomes), 5) NanoSTING-SCTM, and 6) NanoSTING-sc9-10 vaccines. Prefusion protein-specific antibody titers increased from day 7 to day 21 for all the groups and were no different when comparing the three antigens immunized with NanoSTING as an adjuvant (FIG. 3B). Notably, serum IgG titers were significantly lower (p-value= 0.006) in the unadjuvanted DS-CaVl immunized (4 ±l x 102) group compared to NanoSTING-DS- CaVl (6 ± 2 x io3). Additionally, naked 2’3’-cGAMP adjuvanted SCTM immunized mice also exhibited much lower prefusion protein specific titers ( 1.5 ± 0.6 x 103, / ?-value= 0.04) compared to NanoSTING-SCTM (5 ± 1 x io3). To evaluate the durability of vaccine-induced immunity, Applicant immunized a group of mice with NanoSTING-DS-CaVl and tested prefusion protein-specific antibody titers in serum six months after immunization. The serum IgG endpoint titers 180 days postimmunization (2.6 x 103± 4 x 102) were comparable to day 21 titers, suggesting durable humoral immunity.

[0130] Unlike the serum IgG titers, the serum IgA titers were detectable but modest following a single intranasal dose of the vaccines. Next, Applicant aimed to test the nature of cellular immunity induced by NanoSTING-adjuvanted vaccines. Extensive studies on failed RSV vaccines have associated vaccine-induced enhanced respiratory disease (ERD) with Th2 response, and a Thl T-cell response is desirable for safe and protective cellular immunity. Applicant performed ELISpot to quantify T-cell responses stimulated by RSV A2 fusion protein peptides. The antigen-specific T-cellresponses in the lung / spleen showed a Thl bias and were detectable but variable across the different antigens adjuvanted with NanoSTING (e.g. NanoSTING-DS-CaVl, lung: 8 ± 2 * 102, spleen: 6 ± 1 x 102). Collectively, these results established that, while liposomal NanoSTING is required for robust serum IgG responses, a single dose vaccine yields only modest mucosal responses that is dependent on the antigen.

[0131] To improve the mucosal immune response elicited upon vaccination, Applicant prioritized the NanoSTING-sc9-10 and NanoSTING-SCTM vaccines and utilized a prime-boost vaccination strategy (FIG. 3A). Following the booster dose, Applicant collected serum from vaccinated animals on day 48 to evaluate the IgG and IgA responses. Prefusion protein-specific serum IgG increased in both NanoSTING-sc9-10 (1 ± 0. 3 x IIP on day 48 vs 5 ± 1 x 103on day 21) and NanoSTING-SCTM (1 ± 0. 5 x 105on day 48 vs 5 ± 1 x 103on day 21) groups following the booster (FIG. 3B). RSV-specific nasal IgA correlates with protection against RSV infection, and antigenspecific serum IgA can be a predictor for secretory IgA (slgA) in the nasal mucosa. Applicant observed significant antigen-specific serum IgA in NanoSTING-sc9-10 (mean endpoint titer: 4 ± 1 x 104) and NanoSTING-SCTM (4 ± 1 x io3) vaccinated mice compared to unvaccinated animals ( / ?- value = 0.004) on day 48 (FIG. 3C).

[0132] To directly assay mucosal IgA, Applicant tested for prefusion protein-specific IgA in the bronchoalveolar lavage fluid (BALF) and nasal wash samples by ELISA. Both vaccines induce significant IgA titers after the booster dose in the BALF (NanoSTING-sc9-10: 4 ± 2 x io3, NanoSTING-SCTM: 3 ± 2 x io3±) (FIG. 3D) and nasal wash (NanoSTING-sc9-10: 1 ± 0.2x 103, NanoSTING-SCTM: 1 ± 0.4 x 103) (FIG. 3E) compared to PBS-vaccinated mice (BALF: / ?-value = 0.004, nasal wash: / ?-value= 0.002).

[0133] Applicant next evaluated the magnitude of the cellular response by tracking the T cell responses in the spleen (systemic cellular immunity) and the lung (site of disease). Prime-boost immunization with NanoSTING-sc9-10 yielded significantly higher IFNy secreting T cells in the lung after the second dose [900 ± 400 spot-forming cells (SFC), day 48] in comparison to the primary dose (21 ± 5 SFC, day 28) (y>-value = 0.0003, FIG. 3F). This significant increase in IFNy secreting T cells was also observed in the spleen after two doses of immunization (370 ± 120 SFC, day 48; vs. 20 ± 7, day 28, / ?-value = 0.0003) (FIG. 3G). Similar significant increases in both the lung-resident (1800 ± 200 SFC, day 48; vs. 9 ± 6 SFC, day 28, y>-value = 0.04) and systemic T cell IFNy responses (520 ±90 SFC, day 48; vs. 8 ± 4 SFC, day 28. / ?-value = 0.02) were observed with dual dose immunization with the NanoSTING-SCTM vaccine.

[0134] Importantly, both vaccines elicit significantly higher fENy-secreting T cells compared to IL-4 secreting T-cells in the lung (p-value = 0.002) and the spleen ( - value = 0.002), confirming a Thl-biased T-cell response. Taken together, these data suggest that dual-dose NanoSTING- adjuvanted vaccines induce robust antigen-specific antibodies and a Thl / Tcl T-cell response in immunized mice, regardless of the antigen used for immunization.

[0135] Example 1.14, NanoSTING-adjuvanted vaccines induce neutralizing antibodies in cotton rats and protect against viral challenge

[0136] Applicant had confirmed the immunogenicity of NanoSTING-adjuvanted vaccines and aimed to evaluate whether the vaccine formulations in this Example could induce neutralizing antibodies and protect against viral challenge. Applicant conducted these studies in the standard RSV vaccine preclinical model, cotton rats (Sigmodon hispidus), since they are more susceptible to viral infection compared to Balb / c mice. Applicant immunized groups of cotton rats intranasally with 1) PBS, 2) NanoSTING-sc9-10, and 3) NanoSTING-SCTM. The animals were boosted with an intranasal dose four weeks after the prime dose (FIG. 4A). Applicant collected serum samples three weeks after the booster dose to evaluate antigen-specific immune responses. RSV prefusion proteinspecific ELISAs indicated high serum IgG (NanoSTING-sc9-10: 18 ± 6 104, NanoSTING-SCTM: 7 ± 2 x io4) (FIG. 4B) and IgA (NanoSTING-sc9-10: 2 ± 0.7* 103, NanoSTING-SCTM: 1.5 ± 0.4 x 103) (FIG. 4C) titers in vaccinated groups compared to the PBS-administered group (PBS IgG: 100, / ?-value = 0.0001 ; PBS IgA: 100, / ?-value = 0.0001).

[0137] Applicant’ s experimental vaccines show high serum antibody titers against RSV fusion protein. However, neutralizing antibody titers are a better correlation of protection, as not all binding antibodies can neutralize viral infection. Therefore, Applicant tested the serum collected from the cotton rats on day 49 in virus neutralization assays against both strains of RSV (A and B). Both NanoSTING-sc9-10 and NanoSTING-SCTM induce neutralizing antibodies. However, neutralizing titers were significantly higher (p-value = 0.003) for NanoSTING-SCTM (RSV-A2, mean reciprocal of highest dilution 2.5 ± 0.5 x 104) compared to NanoSTING-sc9-10 (RSV-A2, 2 ± 0.4 102) (FIG. 4D). The higher titers were also observed with RSV-B [NanoSTING-SCTM (4 ± 1 x 102) compared to NanoSTING-sc9-10 (6 ± 2 x 10'), / i-value = 0.0025], Thus, while immunogenicity studies (serumor BALF IgG, mucosal IgA or lung Thl / Tcl responses) showed no difference between NanoSTING- DS-CaVl and NanoSTING-SCTM, viral neutralization tests demonstrated that NanoSTING-SCTM yielded superior responses compared to NanoSTING-DS-CaVl (FIG. 4D).

[0138] To verify the efficacy of Applicant’s experimental vaccines in vivo, Applicant challenged the cotton rats with 105plaque-forming units (p.f.u.) RSV A2 virus three weeks after the booster dose. Applicant harvested the lung and nasal tissues four days post-challenge to evaluate viral load at the lower and upper respiratory tract of the cotton rats. Despite a >100-fold difference in neutralizing antibody titers (FIG. 4D) both Nano S TING- sc9- 10 and NanoSTING-SCTM vaccines completely inhibit viral replication in the lung of vaccinated animals (FIG. 4F). Quantification of the viral titers in the nasal compartment upon vaccination yielded surprising results.

[0139] While NanoSTING-sc9-10 immunization attenuated viral replication in the nasal compartment (viral load- NanoSTING-sc9-10: 2 ± 1 x 104, PBS: 8 ± 1 x IO5, / -value = 0.002), immunization with NanoSTING-SCTM completely eliminated viral replication (FIG. 4G). These results indicate that NanoSTING adjuvanted vaccines induce robust immunity in vaccinated cotton rats that protect against viral challenge.

[0140] Recent studies have identified cross-protective monoclonal antibodies that bind to RSV and human metapneumovirus (hMPV) fusion proteins and neutralize them both. Despite having only -35% amino acid residue identity, the structural similarity of RSV and hMPV prefusion proteins gives rise to cross-protective epitopes. As Applicant utilizes the RSV prefusion protein as an immunogen in this Example, Applicant aimed to test the cross-neutralizing potential of these vaccines against hMPV. In this regard, Applicant tested the day 49 serum collected from cotton rats for antibodies against hMPV prefusion protein. Applicant observed that both NanoSTING-sc9-10 (mean endpoint titer, NanoSTING-sc9-10: 3 ± I x 105, PBS 50) and NanoSTING-SCTM (4 ± 2 x io4) induce high levels of hMPV prefusion protein cross-reactive antibodies. Applicant also conducted hMPV neutralization assays using day 49 serum from NanoSTING-sc9-10 and NanoSTING-SCTM vaccinated cotton rats. Despite high binding titers, Applicant noted that cross-reactive antibodies elicited by these vaccines could not neutralize the hMPV virus in vitro.

[0141] Example 1.15, Discussion

[0142] In this Example, Applicant formulated an RSV prefusion protein-based intranasal vaccine that utilizes a cGAMP-encapsulating nanoparticles (NanoSTING) as an adjuvant.NanoSTING is a lipid-based cationic adjuvant that protects cGAMP from rapid extracellular degradation and enhances its half-life in vivo. Additionally, NanoSTING facilitates the decoration of multiple copies of protein antigens on the nanoparticle surface, leading to enhanced immunogenicity and multi-factorial protection against respiratory viruses.

[0143] Applicant hereby reports that NanoSTING adjuvanted vaccines induce robust humoral and cellular immunity against RSV, resulting in protection from viral replication in the respiratory tract. Applicant’ s data indicates that NanoSTING enhances the immunogenicity of established protein antigens and could be adopted as an adjuvant platform for developing mucosal vaccines.

[0144] After the initial failures with the formalin-inactivated RSV vaccine, protein-based vaccines have been prioritized due to their well-documented history of safety. The RSV virion presents three proteins on the viral membrane that can be targeted for vaccine development: smallhydrophobic protein (SH), attachment protein (G), and fusion protein (F). The SH protein is important for viral replication in vivo, and helps the virus evade host immune response. SH protein elicits binding antibodies that cannot neutralize the virus. However, these antibodies can protect RSV- challenged mice against viral replication via the Fc-mediated ADCC (antibody-mediated cellular cytotoxicity) pathway.

[0145] G protein, the 90 kDa type-II membrane protein, presents conserved domains in the central region of the protein that is targeted by neutralizing antibodies. However, G protein exhibits a high degree of genetic variability giving rise to the virus subtypes. Several early-stage clinical trials have demonstrated the safety and efficacy of G protein-based vaccines. However, cases of type III hypersensitivity in healthy adults related to the vaccines have raised serious concerns about G protein as an immunogen for a safe RSV vaccine. In comparison, the RSV F protein is highly conserved among the virus subtypes and have been safely tested in clinical trials that led to the approval of two fusion protein-based vaccines for people over the age of 65. The 574 amino acid protein is composed of two subunits (Fl and F2) joined together by disulfide bonds. Three such protomers then assemble on the viral membrane to form the trimeric fusion protein. The native fusion protein is metastable and spontaneously refolds into the postfusion form. Six antibody-neutralizing epitopes have been discovered on the fusion protein (I, II, III, IV, V, and ). Most potent neutralizing antibodies are directed against epitope CD, which is only present on the prefusion protein. Thus, RSV prefusion protein has served as an attractive choice as the immunogen for vaccine design.

[0146] Several successful attempts at designing a stable RSV prefusion protein in the past decade have led to approved vaccines like Abrysvo (Pfizer) and Arexvy (GSK). Structure-based protein engineering efforts at the NTH introduced a disulfide bond (DS) and two cavity-filling (CaVl) mutations that resulted in prefusion stabilized trimer DS-CaVl, which was adopted in GSK’s FDA- approved vaccine. Further improvements in the DS-CaVl protein resulted in a second generation of stabilized proteins with better physical stability, such as sc9-10 DS-CaVl. Interprotomer dityrosine crosslinking by introducing mutations in the Fl subdomain also imparts stability to fusion proteins in their native state. Three unique mutations (single-chain triple mutant; SCTM) were previously identified that prevent the refolding of the protein into the postfusion form. The SCTM immunogen was reported to have better expression yields and thermal stability than DS-CaV 1. However, another study halted phase-3 studies due to changes in the RSV vaccine landscape. Immunogenicity and protective efficacy of the prefusion F variants DS-CaVl (DS-CaVl), sc9-10 DS-CaVl (sc9-10), and SCTM (SCTM) in parenteral vaccines have been reported in multiple preclinical and clinical studies. Thus, Applicant adopted these immunogens to benchmark the effectiveness of Applicant’s mucosal adjuvant (NanoSTING) in intranasal vaccines against RSV.

[0147] In this Example, Applicant demonstrates the efficacy of NanoSTING as an adjuvant in prefusion-protein-based vaccines against respiratory syncytial virus (RSV). Applicant tested these vaccines in the cotton rat model, as lung pathology in cotton rats mimics that of humans after RSV infection. Both sc9-10 DS-CaV 1 and SCTM-based vaccines exhibit neutralizing antibody (nAb) titers against RSV in virus neutralization assays. However, NanoSTING-SCTM induces higher nAb titers than NanoSTING-sc9-10 against both virus subtypes (RSV A and RSV B) (FIGS. 4D-4E).

[0148] A prior study compared the antibody responses by sc9-10 and SCTM-based parenteral vaccines in mice. The study reported that following two doses of 10 pg protein administered with AddaVax adjuvant intradermally, sc9-10 elicits higher neutralizing antibody titers than SCTM. The contrast between Applicant’s findings and prior study suggests that, despite using the same antigens, the route of administration and the adjuvant play critical roles in determining the quality and quantity of the immune response.

[0149] Applicant challenged the vaccinated cotton rats with live RSV A2 virus and evaluated viral titers in the lung and nasal tissue. NanoSTING-SCTM eliminates RSV below the limit of detection in both the upper (nose) and lower (lung) respiratory tract (FIGS. 4F-4G). NanoSTING-sc9-10 DS-CaVl fully protected the lung and only partially protected the nose against viral replication (FIGS. 4F-4G) The difference in protection at the upper respiratory tract between the vaccines may result from the higher neutralization titers elicited by NanoSTING-SCTM. However, serum nAb titers, while necessary, are not a singular correlate of protection against infection. Antigen-specific binding antibodies, T-cells, and mucosal immunity (IgA and T-cells) are known to complement the protective effects of neutralizing antibodies.

[0150] NanoSTING adjuvanted vaccines produce similar serum IgG and IgA responses in mice (FIGS. 3B-3C) and cotton rats (FIGS. 4B-4C). T-cell responses (FIGS. 3F-3G) and mucosal immune correlates (BALF and nasal wash IgA) from mice (FIGS. 3D-3E) further highlight the comparable immunogenicity of both sc9-10 DS-CaVl and SCTM-based vaccines. Similar immunogenicity profiles and yet variations in nAb titers leading to differential protection observed at the upper respiratory tract may be linked to the inherent stability of the antigen in the vaccine formulation.

[0151] NanoSTING facilitates the adsorption of proteins on the nanoparticle surface, increasing their retention time in the nasal mucosa and promoting uptake by antigen-presenting cells. The physiochemical properties that lead to the adsorption of proteins on nanoparticles can impact the stability of antigens in vaccine formulation. It is possible that adsorption onto NanoSTING stabilizes the SCTM antigen better than the sc9-10 DS-Cavl antigen. While the SEC-MALS data suggests differences in the stability of the SCTM and sc9-10 DS-CaVl proteins, a more thorough investigation into the stability of these antigens in the context of NanoSTING needs to be undertaken.

[0152] Due to past cases of vaccine-induced enhanced disease (VED), safety is a major concern in RSV vaccine development. In a clinical trial in the 1960s, a vaccine formulated with formalin-inactivated RSV (FI-RSV) did not elicit protection against natural infections and led to increased disease severity among children vaccinated with FI-RSV compared to children who were not vaccinated. Later, it was discovered that the immune response induced by FI-RSV vaccine is Th2- skewed, and the antibodies induced by the vaccine have low neutralization potential, causing immune- complex formation in the mucosa. Applicant’s data indicates that NanoSTING adjuvanted prefusion protein vaccines elicit a Thl-skewed response in the lung and spleen of vaccinated animals (FIGS. 3F-3G), confirming the safety of vaccine-induced immunity.

[0153] There is no established animal model to test whether vaccination can prevent transmission of RSV from infected animals to uninfected ones. A prior study demonstrated that RSV- infected ferrets can transmit viruses to cohoused naive ferrets. However, infected ferrets did not show any sign of weight loss.

[0154] In summary, Applicant has demonstrated the immunogenic and protective efficacy of NanoSTING as an adjuvant for intranasal vaccine against RSV. Applicant’s study highlights the potential of NanoSTING to boost both mucosal and cellular immune responses, offering a promising approach for developing intranasal vaccines against RSV, with possible implications in preventing viral transmission.

[0155] Without further elaboration, it is believed that one skilled in the art can, using the description herein, utilize the present disclosure to its fullest extent. The embodiments described herein are to be construed as illustrative and not as constraining the remainder of the disclosure in any way whatsoever. While the embodiments have been shown and described, many variations and modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of the invention. Accordingly, the scope of protection is not limited by the description set out above, but is only limited by the claims, including all equivalents of the subject matter of the claims. The disclosures of all patents, patent applications and publications cited herein are hereby incorporated herein by reference, to the extent that they provide procedural or other details consistent with and supplementary to those set forth herein.

Claims

CLAIMS1. A composition comprising: a particle, an antigen, and a modulator encapsulated within the particle, wherein the modulator is selected from the group consisting of an agonist, an activator of the immune system, or combinations thereof, and wherein the antigen is operable to elicit an immune response in a subject against respiratory syncytial virus (RSV).2 The composition of claim 1, wherein the modulator comprises an endogenous agonist of the stimulator of interferon genes (STING) pathway.3 The composition of claim 1, wherein the modulator is selected from the group consisting of an endogenous agonist of the stimulator of interferon genes (STING) pathway, bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP), cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), 2’,3’-cyclic guanosine monophosphate-adenosine monophosphate (2’,3’-cG MP), amidobenzimidazole, derivatives of amidobenzimidazole, nucleotide modulators, plasmid DNA modulators, CF501, MSA-2, SHR1032, C-178, H-151, divalent cations or combinations thereof.4 The composition of claim 1, wherein the modulator comprises 2’,3’-cyclic guanosine monophosphate-adenosine monophosphate (2 ’,3 ’-cGAMP).5 The composition of claim 1, wherein the antigen comprises a protein or peptide derived from respiratory syncytial virus (RSV), or a nucleotide sequence encoding the protein or peptide.6 The composition of claim 5, wherein the antigen is selected from the group consisting of smallhydrophobic protein (SH), attachment protein (G), fusion protein (F), a prefusion F protein, DS-CaVl, sc9-10 DS-CaVl, SC-TM, a full protein thereof, a protein fragment thereof, a peptide fragment thereof, a fusion protein thereof, a prefusion protein thereof, or combinations thereof.

7. The composition of claim 1, wherein the antigen comprises a protein or peptide derived from respiratory syncytial virus (RSV).

8. The composition of claim 1, wherein the antigen comprises a nucleotide sequence encoding a protein or peptide derived from respiratory syncytial virus (RSV).9 The composition of claim 1, wherein the antigen comprises a prefusion protein of respiratory syncytial virus (RSV), or a nucleotide encoding the prefusion protein.10 The composition of claim 9, wherein the prefusion protein is selected from the group consisting of DS-CaVl, sc9-10 DS-CaVl, SC-TM, a full protein thereof, a protein fragment thereof, a peptide fragment thereof, or combinations thereof.11 The composition of claim 1, wherein the antigen comprises SC-TM, wherein SC-TM comprises SEQ ID NO: 1 or a sequence with at least 65% sequence identity to SEQ ID NO: 1.12 The composition of claim 1, wherein the antigen comprises DS-CaVl, wherein DS-CaVl comprises SEQ ID NO: 2 or a sequence with at least 65% sequence identity to SEQ ID NO: 2.13 The composition of claim 1, wherein the antigen comprises sc9-10 DS-CaVl, wherein sc9-10 DS- CaVl comprises SEQ ID NO: 3 or a sequence with at least 65% sequence identity to SEQ ID NO: 3.14 The composition of claim 1, wherein the antigen is associated with an outer surface of the particle.15 The composition of claim 1, wherein the composition comprises a lipid-based particle.16 The composition of claim 15, wherein the lipid-based particle is in the form of a liposome.17 The composition of claim 1, wherein the composition is suitable for use in treating or preventing a respiratory infection in a subject.18 The composition of claim 17, wherein the respiratory infection is caused by respiratory syncytial virus (RSV).

19. A method of treating or preventing a respiratory infection in a subject, said method comprising: administering a composition to the subject, wherein the composition comprises: a particle, an antigen, and a modulator encapsulated within the particle, wherein the modulator is selected from the group consisting of an agonist, an activator of the immune system, or combinations thereof, and wherein the antigen is operable to elicit an immune response in the subject against respiratory syncytial virus (RSV).

20. The method of claim 19, wherein the method is used to prevent the respiratory infection in the subject.

21. The method of claim 19, wherein the method is used to treat the respiratory infection in the subj ect.

22. The method of claim 19, wherein the subject is a human being.

23. The method of claim 19, wherein the respiratory infection is caused by respiratory syncytial virus (RSV).

24. The method of claim 19, wherein the respiratory infection comprises a severe lower respiratory tract infection (LRTI).

25. The method of claim 19, wherein the composition is administered through intranasal administration, inhalational administration, intravenous administration, or combinations thereof.

26. The method of claim 19, wherein the modulator comprises an endogenous agonist of the stimulator of interferon genes (STING) pathway.

27. The method of claim 19, wherein the modulator is selected from the group consisting of an endogenous agonist of the stimulator of interferon genes (STING) pathway, bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP), cyclic guanosine monophosphate-adenosine monophosphate(cGAMP), 2’,3’-cyclic guanosine monophosphate-adenosine monophosphate (2’,3’-cGAMP), amidobenzimidazole, derivatives of amidobenzimidazole, nucleotide modulators, plasmid DNA modulators, CF501, MSA-2, SHR1032, C-178, H-151, divalent cations or combinations thereof.

28. The method of claim 19, wherein the modulator comprises 2’,3’-cyclic guanosine monophosphate-adenosine monophosphate (2’,3’-cGAMP).

29. The method of claim 19, wherein the antigen comprises a protein or peptide derived from respiratory syncytial virus (RSV), or a nucleotide sequence encoding the protein or peptide.

30. The method of claim 29, wherein the antigen is selected from the group consisting of smallhydrophobic protein (SH), attachment protein (G), fusion protein (F), prefusion F protein, DS-CaVl, sc9-10 DS-CaVl, SC-TM, a full protein thereof, a protein fragment thereof, a peptide fragment thereof, a fusion protein thereof, a prefusion protein thereof, or combinations thereof.

31. The method of claim 19, wherein the antigen comprises a protein or peptide derived from respiratory syncytial virus (RSV).

32. The method of claim 19, wherein the antigen comprises a nucleotide sequence encoding a protein or peptide derived from respiratory syncytial virus (RSV).

33. The method of claim 19, wherein the antigen comprises a prefusion protein of respiratory syncytial virus (RSV), or a nucleotide encoding the prefusion protein.

34. The method of claim 33, wherein the prefusion protein is selected from the group consisting of DS-CaVl, sc9-10 DS-CaVl, SC-TM, a full protein thereof, a protein fragment thereof, a peptide fragment thereof, or combinations thereof.

35. The method of claim 19, wherein the antigen comprises SC-TM, wherein SC-TM comprises SEQ ID NO: 1 or a sequence with at least 65% sequence identity to SEQ ID NO: 1.

36. The method of claim 19, wherein the antigen comprises DS-CaVl, wherein DS-CaVl comprises SEQ ID NO: 2 or a sequence with at least 65% sequence identity to SEQ ID NO: 2.

37. The method of claim 19, wherein the antigen comprises sc9-10 DS-CaVl, wherein sc9-10 DS- CaVl comprises SEQ ID NO: 3 or a sequence with at least 65% sequence identity to SEQ ID NO: 3.

38. The method of claim 19, wherein the antigen is associated with an outer surface of the particle.

39. The method of claim 19, wherein the composition comprises a lipid-based particle.

40. The method of claim 34, wherein the lipid-based particle is in the form of a liposome.

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