Delivery systems for intracellular adjuvants

Abstract

The present disclosure relates to lipid nanoparticle (LNP) compositions including at least one ionizable cationic lipid and Polyinosinic:polycytidylic acid (poly(I:C)), and compositions and formulations incorporating such LNP compositions. The disclosure also provides methods of using these compositions to elicit an immune response, including for the prevention or treatment of infectious diseases.

Description

Atorney Docket No. 770075: MTST-553PCDELIVERY SYSTEMS FOR INTRACELLULAR ADJUVANTSRELATED APPLICATIONS

[0001] The present patent application is related to and claims priority benefit of U.S. Provisional Patent Application No. 63 / 700,612, filed September 27, 2024, and is hereby incorporated by reference in their entirety into this disclosure.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grant no. 75N93021C00014 awarded by the National Institute of Allergy and Infectious Diseases (NIAID) / National Institutes of Health (NIH). The Government has certain rights in the invention.BACKGROUND

[0003] Recombinant protein and peptide subunit antigens are often weakly immunogenic and unable to mount protective humoral and cellular immune responses. Therefore, most subunit antigens require co-formulation with adjuvants. Aluminum salts (alum) and oil-in-water emulsions are the most widely used adjuvants in vaccine formulations to date and some mainly elicit a T helper cell type-2 (Th2) skewed immune response. A need exists for specific adjuvants to specifically direct responses to IgG2a or IgGl, hence inducing Thl / Th2 responses.BRIEF SUMMARY

[0004] One aspect of the disclosure is a lipid nanoparticle (LNP) composition comprising at least one ionizable cationic lipid and Polyinosinic:polycytidylic acid (poly(I:C)).

[0005] In an aspect, the ionizable cationic lipid comprises an azepanyl moiety. In an aspect, the ionizable cationic lipid comprises S-Ac7-Dog or K-Ac7-Dsa. In an aspect, the LNP composition further comprises phospholipid, cholesterol, and / or PEG-lipid. In an aspect, the Poly(I:C) is encapsulated by the LNP.

[0006] One aspect of the disclosure is an adjuvant formulation comprising the LNP composition.81099325. v1 1Atorney Docket No. 770075: MTST-553PC

[0007] One aspect of the disclosure is an immunogenic composition comprising the adjuvant formulation and an immunogen or antigen. In an aspect, the immunogen or antigen is capable of eliciting immune response against an infectious disease. In an aspect, the infectious disease is selected from the group consisting of COVID-19, Influenza, Pneumococcal pneumonia, RSV, and combinations thereof. In an aspect, the immunogenic composition is in the form of lyophilized powder, aqueous solution, suspension, microemulsion, or dispersion. In an aspect, the immunogenic composition is suitable for intravenous administration, subcutaneous administration, intramuscular administration, parenteral administration, rectal administration, spinal cord administration, epidermal administration, infusion administration, or intraperitoneal administration. In an aspect, the immunogenic composition is a vaccine or a therapeutic composition.

[0008] One aspect of the disclosure is a method of inducing an immune response, the method comprising administering an effective amount of the immunogenic composition to a subject in need thereof.

[0009] One aspect of the disclosure is a method of preventing or treating an infectious disease or, the method comprising administering an effective amount of the immunogenic composition of any to a subject in need thereof.

[0010] One aspect of the disclosure is the use of the immunogenic composition for inducing an immune response in a subject in need thereof.

[0011] One aspect of the disclosure is the use of the immunogenic composition for preventing or treating an infectious disease in a subject in need thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0013] FIG 1 is a Chemical structure of in-house synthesized ionizable lipids- S-Ac7-Dog and K-Ac7-Dsa lipids, comprising a disulfide bond that can be cleaved by reduction and a ketal bond that can be cleaved by acidic pH, respectively.81099325. v1 2Atorney Docket No. 770075: MTST-553PC

[0014] FIGs. 2Aand 2B show the structure and characterization of LNPs consisting of K-Ac7- Dsa or S-Ac7-Dog lipids. FIG. 2A is a diagrammatic representation of LNP structure encapsulating SDI. FIG. 2B is an intensity-based size distribution curves measured by dynamic light scatering (DLS) of empty and SDI-incorporating S-Ac7-Dog and K-Ac7-Dsa LNP formulations.

[0015] FIGs. 3A -3C show S-Ac7-Dog (- or SDI) and K-ac7-Dsa (- or SDI) in combination with IMDQ-PEG-Chol define IgG subtype profile. FIG. 3A is a study outline. FIG. 3B are graphs showing ELISA end point titers calculated based on OD 450 ELISA values against serum dilutions for total IgG, IgGl, and IgG2a (n = 5 per group), represented as geometric mean. The statistical analysis was performed using one-way ANOVA with a Dunnett’s multiple comparison test, and the p-values shown are calculated in reference to the unadjuvanted QIV group, which received neither adjuvant nor LNP formulations. FIG. 3C is a heatmap showing the ratio of end point titers of IgG2a to IgGl and represented as geometric mean of IgG2a / IgGl ratios for all animals in each group.

[0016] FIG. 4 is a heatmap showing mean OD450 ELISA values 572 (n=5 per group) plotted against serum dilutions for total IgG, IgGl and IgG2a. 6-8-week female BALB / c mice were vaccinated with QIV with and without IMDQ-PEG-Chol and formulated into empty or SDI encapsulating S or K LNPs. Serum was collected 3 weeks post-vaccination by submandibular bleed. Total IgG, IgGl and IgG2a titers were quantified by ELISA with 3 -fold serum dilutions starting with 1 : 100, for Hl HA specific antibodies.

[0017] FIG. 5 shows QIV adjuvanted with IMDQ-PEG-Chol exhibits a better control over virus infection when formulated in S-Kc7-Dog LNPs. The sera collected from all vaccinated animals 3 weeks post-vaccination were tested for HAI titers, using 4 HA units of the IVR-180 virus. The HAI titers are represented as geometric mean ± geometric SD for n = 4 animals per group. The samples with un-detectable HAI titers were set as 1 and the limit of detection was set to 2, corresponding to the lowest detectable HAI units. The statistical analysis was performed using one-way ANOVA with a Dunnett’s multiple comparison test. The p-values shown are calculated in reference to the QIV group, which received neither adjuvant nor LNP formulations.

[0018] FIGs. 6A-6C show QIV formulated in S-Ac7-Dog(SDI) and K-Ac7-Dsa(SDI) LNPs efficiently induces T-cell responses when combined with IMDQ-PEG-Chol. FIG. 6A shows81099325. v1 3Atorney Docket No. 770075: MTST-553PC the experiment outline. FIGs. 6B and 6C show Six- to 8-week BALB / c mice were vaccinated with QIV with and without IMDQ-PEG-Chol and formulated into SAc7-Dog(- or SDI) or K- Ac7-Dsa(- or SDI) LNPs. The spleens were harvested at 10 DPV to examine the T-cell activation by IFN-g (FIG. 6B) and IL-4 (FIG. 6C) ELIspots, upon restimulation with Hl -HA shortoverlapping peptides or live IVR-180 (A / Singapore / GP1908 / 2015 H1N1) virus. The results are represented as IFN-g- or IL-4-producing cells per million splenocytes (geometric mean ± geometric SD) for n = 4 animals per group. The cutoff was set to 10, which indicates one spot in any well. The wells with no spots were given the value 1. The statistical analysis was performed using one-way ANOVA with a Dunnets multiple comparison test and the p-values shown are calculated in reference to the respective PBS group.

[0019] FIGs. 7A and 7B show QIV formulated in S-Ac7-Dog(SDI) and K-Ac7-Dsa(SDI) LNPs contributes to reduction in IVR-180 virus replication in lungs of vaccinated animals irrespective of combination with IMDQ-PEG-Chol. All unvaccinated and QIV ± IMDQ-PEG-Chol ± S-Ac7- Dog(SDI) or K-Ac7-Dsa(SDI) vaccinated animals were intranasally challenged with 18,000 plaque-forming units (PFU) of IVR-180 virus per animal. FIG. 7A shows the body weight of each animal in all groups was recorded every day until the day of harvest and represented as percentage of initial body weight for each group (n = 6) (geometric mean ± geometric SD). FIG. 7B shows the viral lung titers were quantified at 5 DPI by plaque assays on pre-seeded MDCK cells and are represented as PFU / mL for n = 6 animals per group (geometric mean ± geometric SD). Each data point represents one animal in the respective group. Statistical analysis was performed using one-way ANOVA with a Dunnet’s multiple comparison test. The p-values shown are calculated in reference to the unadj uvanted QIV group, which received neither adjuvant nor LNP formulations.

[0020] FIGs 8A and 8B are heatmaps showing cytokine profile in lungs of vaccinated mice upon intranasal challenge with IVR-180. All unvaccinated and vaccinated animals were intranasally challenged with 18,000 PFU of IVR-180 virus. The lungs were collected at 5 DPI and the cytokine levels were quantified by multiplex ELISA. Levels of IL-lb, IL-5, IL-13, TNF- a, IL-12 p70, IL-4, IL-6, IFN-g, IL-18, and GMCSF for n = 6 animals per group are represented as median of z-scores (FIG. 8A) and PCA plots (FIG. 8B) for all animals in each group.

[0021] FIGs. 9A-9J are graphs from all lungs from unvaccinated and vaccinated animals were collected at 5 days post infection and the cytokine levels were quantified by multiplex ELISA.81099325. v1 4Atorney Docket No. 770075: MTST-553PCLevels of (FIG. 9A) IL- Ip, (FIG. 9B) IL-5, (FIG. 9C) IL-13, (FIG. 9D) TNF-a, (FIG. 9E) IL- 12 p70, (FIG. 9F) IL-4, (FIG. 9G) IL-6, (FIG. 9H) IFN-y, (FIG. 91) IL- 18 and (FIG. 9 J) GMCSF, for n=6 animals per group are represented as geometric mean ±geometric SD, where each data point corresponds to individual mouse. Statistical analysis was performed using two- sided Mann Whitney U test. The p values shown are calculated in reference to the virus- challenged unvaccinated group (denoted as PBS).

[0022] FIG. 10 is a simplified schematic representation of the signaling cascade in response to poly(I:C)-induced endosomal TLR3 and cytoplasmic RLR triggering.

[0023] FIG. 11 is a schematic representation lipid nanoparticles (LNP) encapsulating poly(LC).

[0024] FIG. 12 is intensity-based size distribution curves measured by DLS of LNP(poly(I:C)) and LNP formulations.

[0025] FIGs. 13A-13D are in vitro characterization of LNP(poly(I:C)). FIG. 13A shows fluorescence emission intensity ( ex: 535 nm / Aem: 670 nm) of poly(I:C)RFTO and LNP(poly(I:C)RFTO) (n = 2, mean±SD). FIG. 13B shows flow cytometry analysis of DC2.4 pulsed with LNP(poly(I:C)RFTO) and unformulated soluble poly(I:C)RFTO: histograms and corresponding mean fluorescence intensity (MFI values), (n = 3, mean ±SD; / -test **: p < 0.01). FIG. 13C shows confocal microscopy images of DC2.4 cells treated with LNP(poly(I:C)RFTO) and poly(I:C)RFTO. Cells were counterstained with LysoTrackerGreen. Scale bar represents 30 jim. FIG. 13D Innate immune activation by LNP(poly(I:C)) and poly(I:C) in TLR3- and ISG- reporter cell lines (n = 6, mean ± SD). Note that a soluble, unformulated, poly(I:C) could be tested at concentrations that cannot be reached for LNP(poly(I:C)).

[0026] FIGs. 14A-14F show li vivo innate immune activation by LNP(poly(I:C)). FIG. 14A) Bioluminescence imaging of BALB / c IFN ?+ / A ?-luc mice at 0, 4, and 24 h after receiving intramuscular injection into the quadricep of a 25 jig dose of poly(I:C) in soluble unformulated and in LNP-formulated form. An equivalent dose of empty LNP was administered (n = 3). FIG. 14B) Region of interest (ROI) analysis quantifying total flux (photons s- 1) of the full body, injection site, spleen of the corresponding samples (n = 3, mean ± SD). FIG. 14C shows bio-Plex analysis of cytokine levels in serum at 6 h post-injection (n = 3, mean ±SD; / -test *:p < 0.05; **: p < 0.01). FIG. 14D shows flow cytometry analysis of the uptake of poly(I:C)RFTO in immune81099325. v1 5Atorney Docket No. 770075: MTST-553PC cell subsets in draining popliteal lymph node and the spleen at 24 h after receiving intramuscular injection into the quadri cep of a 25 jig dose of poly(I:C)RFTO in soluble unformulated and in LNP -formulated form. An equivalent dose of empty LNP was administered. FIG. 14E-14F shows flow cytometry analysis of immune cell activation and maturation in FIG. 14E) the draining popliteal lymph node and FIG. 14F) the spleen at 24 h after receiving intramuscular injection into the quadricep of a 25 pg dose of poly(I:C) in soluble unformulated and in LNP- formulated form. An equivalent dose of empty LNP was administered (n = 3 per group, mean ±SD; one-way analysis of variance (ANOVA) *: p < 0.05; **: p < 0.01; ***: p < 0.001; and ****:p< 0.0001).

[0027] FIGs. 15A-15F show in vivo adjuvanticity of LNP(poly(I:C)). FIG. 15A is an experimental outline. FIG. 15B are ELISA titers for Bl) IgGl, B2) IgG2a, B3) total IgGin mice sera collected 3 weeks postprime (left) and postboost (right) vaccination (graph represents area under the OD(450 nm) ELISA curve against serum dilutions for individual groups as well as area under curve (AUC) calculated based on OD450 curves (mean ± SD) of the individual serum samples). FIG. 15C shows IgGl / IgG2a serum antibody ratio. FIG. 15D shows half maximal inhibitory dilution ID50 of serum containing virus-neutralizing antibodies postprime and postboost vaccination (mean ±SD) in microneutralization assay using 350 tissue culture infectious dose 50 (TCID50) of USA-WA1 / 2020 SARS-CoV-2. The limit of detection was 30, corresponding to the lowest starting serum dilution used in microneutralization assays. FIG. 15E shows Viral lung titers and FIG. 15F shows viral nasal turbinate titers.

[0028] FIG. 16 A and 16B show In vivo adjuvanticity of LNP(poly(I:C)) is abrogated in MAVS- / - KO mice. A) ELISA titers for total IgG in MAVS- / - mice sera collected 3 weeks postprime and postboost vaccination. The graph represents area under curve (AUC) calculated based on OD (450 nm) curve against serum dilutions for individual animals (n = 5, mean ± SD; one-way ANOVA *: p < 0.05; **: p < 0.01). B) In vitro microneutralization titers observed in serum from vaccinated MAVS- / - mice (n = 5, mean ±SD; one-way ANOVA **: p < 0.01) represented as ID50 of sera postprime and postboost vaccination (mean ± SD, one-way ANOVA) using 350TCID50 ofUSA-WAl / 2020 SARS-CoV-2. represented as plaque-forming units (PFU)mL-l (mean ± SD) after challenge with 105 PFU per animal of mouse-adapted SARS-CoV-2. The limit of detection was 67 PFU for plaque assays and the undetectable titers were given a value 10 (below detection limit) (n = 5, mean ±SD; one-way ANOVA *: p < 0.05; **p < 0.01; ***p < o.ooi; ****: / ?< 0.0001).81099325. v1 6Atorney Docket No. 770075: MTST-553PCDETAILED DESCRIPTIONI. Introduction

[0029] After decades of research into influenza virus vaccines, the respiratory virus is still a major global health concern, causing thousands of cases of severe medical illness in humans every year. Several licensed influenza vaccine candidates, including recombinant, inactivated, and split influenza vaccines, have been developed and eventually licensed for use in the human population. Despite the availability of licensed vaccines, the need to update and vaccinate people every year remains a challenge as the circulating influenza viruses can escape host immunity provided by antibodies that target the immunodominant but ever-changing antigenic sites on the hemagglutination (HA) protein. Vaccination against both seasonal influenza A virus (IAV) and influenza B virus (IBV) has been effective in controlling virus related disease severities. However, the protection provided by humoral immunity induced by these vaccines is reported as antigenically constricted and short term. Moreover, the vaccine induced neutralizing antibody titers drop over time, rendering the immunity less effective against an antigenically different strain of virus in the subsequent seasons. Therefore, to combat the need of a seasonal vaccine, a better cost-effective approach is required in vaccine development that can provide a broader and longterm immune response that lasts for multiple seasons.

[0030] Quadrivalent inactivated vaccines (QIV) are the most commonly used influenza vaccines. They consist of two IAV and two IBV strain components (representing the Yamagata and Victoria linages). QIV can induce strain-specific antibody responses with high serum IgG levels in vivo but are poor inducers of cell-mediated immunity and, therefore, provide limited protection against antigenically drifted virus strains. Owing to the continuous acquisition of mutations in antigenic sites of the viral hemagglutination, the protective effect of currently licensed seasonal influenza virus vaccines is time confined.

[0031] Novel vaccine concepts that aim at inducing broader, long-lasting immunity against influenza virus infection are based on enhancing vaccine-induced B- and T-cell responses that can recognize multiple antigens from vaccine components, with special focus on targeting the conserved viral epitopes. While natural infection typically results in the induction of type 1 responses, characterized by Thl and in BALB / c mice class switching to serum IgG2a antibodies to clear viral infection, inactivated split virus influenza vaccines typically induce high IgGl levels correlating with Th2-type immune response. Therefore, many studies, have been focusing on81099325. v1 7Atorney Docket No. 770075: MTST-553PC combining commercially available vaccines with specific adjuvants to specifically direct responses to IgG2a or IgGl, hence inducing Thl / Th2 responses. Eventually, an efficiently balanced humoral response with enhanced T-cell activation post-vaccination is desired to be protective.

[0032] Lipid nanoparticles (LNPs) are non-viral vectors that are widely used in formulating vaccines and / or adjuvants to enhance their antigenicity and improve immune responses. LNPs have already shown promising outcomes in formulating antigen-encoding mRNA, such as SARS-CoV-2 mRNA vaccines. These mRNA vaccine-LNP formulations have also successfully demonstrated the role of an LNP -based vaccine platform for an efficient induction of humoral and cell-mediated immunity. Moreover, LNPs can also be used for formulating molecular adjuvants, such as RIG-I or TLR agonists, and facilitate uptake by actively phagocytosing innate immune cell subsets. Nevertheless, the composition of LNP is crucial to achieve the optimal uptake by innate immune cells and efficient humoral responses. A typical LNP consists of four main components: an ionizable lipid, a phospholipid, a cholesterol moiety, and a polyethylene glycol (PEG) lipid. The ionizable lipids consist of ionizable positively charged lipids that can effectively interact with negatively charged mRNA molecules. Phospholipids and cholesterol provide structural stability to LNPs and facilitate endosomal escape, thus enhancing efficient delivery of mRNA into the cytosol of cells.

[0033] The PEG lipids prolong the circulation of LNPs consisting of vaccines / adjuvants in circulation by increasing their half-life. Additionally, the surface molecules of LNPs can also be modified to target specific innate immune cells and facilitate uptake for efficient antigen presentation. Overall, LNPs present as an efficient in vivo vaccine-adjuvant delivery system.

[0034] As disclosed herein, the inventors investigated and compared the efficiency of various LNP formulations. The inventors observed adjuvant specific differences in B- and T-cell responses, which not only were driven by the presence of different adjuvants but also depended on the type of ionizable / cationic lipid composition of the LNPs.

[0035] Some implementations herein relate to a method. For example, a lipid nanoparticle (Inp) composition at least one ionizable cationic lipid and polyinosinic may include poly cytidylic acid (poly(I:. A lipid nanoparticle (Inp) composition at least one ionizable cationic lipid and polyinosinic may also include. Other embodiments of this aspect include81099325. v1 8Atorney Docket No. 770075: MTST-553PC corresponding computer systems, apparatus, and computer programs recorded on one or more computer storage devices, each configured to perform the actions of the methods.

[0036] The described aspects may also include one or more of the following features. A lipid nanoparticle (LNP) composition including at least one ionizable cationic lipid and Polyinosinic:polycytidylic acid (poly(I:C)). The Poly(I:C) may be encapsulated by the LNP. Then encapsulation may be full or partial. The encapsulation efficiency may be about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0037] In certain embodiments, the ionizable cationic lipid may include an azepanyl moiety. The ionizable cationic lipid may also include S-Ac7-Dog or K-Ac7-Dsa. The LNP composition may further include phospholipid, cholesterol, and / or PEG-lipid.

[0038] In certain embodiments, the LNP composition may be formulated as an adjuvant formulation. In certain embodiments, the adjuvant formulation may be combined with an immunogen or antigen to generate an immunogenic composition. The immunogen or antigen may be capable of eliciting immune response against an infectious disease. In certain embodiments, the infectious disease is COVID-19, Influenza, Pneumococcal pneumonia, or Respiratory Syncytial Virus (RSV).

[0039] COVID-19, Influenza, Pneumococcal pneumonia, and RSV are all respiratory illnesses that range in severity and affect vulnerable populations. COVID-19, caused by SARS-CoV-2, can lead to mild to severe symptoms and long-term complications. Influenza is a seasonal viral infection that causes fever, cough, and fatigue, and can result in serious outcomes for the elderly and chronically ill. Pneumococcal pneumonia, a bacterial infection, leads to lung inflammation and can cause life-threatening complications like meningitis, especially in older adults and young children. RSV typically causes mild cold-like symptoms but can be dangerous for infants and seniors.

[0040] In certain embodiments, the immunogenic composition is in the form of lyophilized powder, aqueous solution, suspension, microemulsion, or dispersion. In certain embodiments the immunogenic composition is suitable for intravenous administration, subcutaneous administration, intramuscular administration, parenteral administration, rectal administration,81099325. v1 9Atorney Docket No. 770075: MTST-553PC spinal cord administration, epidermal administration, infusion administration, or intraperitoneal administration. The immunogenic composition may also be formulated as a vaccine or a therapeutic composition.

[0041] In certain embodiments, the immunogenic composition may be used to induce an immune response, or prevent or treat an infectious disease.II. Definitions

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods described herein belong. Any reference to standard methods refers to the most recent available version of the method at the time of filing of this disclosure unless otherwise indicated.

[0043] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

[0044] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0045] The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

[0046] The term "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims. Such terms will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0047] By "consisting of' is meant including, and limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of' indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of' is meant including any elements listed after the phrase, and limited to other elements that do not interfere81099325. v1 10Atorney Docket No. 770075: MTST-553PC with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of' indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0048] The singular form "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. These articles refer to one or to more than one (i.e., to at least one). As used herein, the term "or" is generally employed in its usual sense including "and / or" unless the content clearly dictates otherwise. The term "and / or" means any one or more of the items in the list joined by "and / or". As an example, "x and / or y" means any element of the three-element set {(x), (y), (x, y)}. In other words, "x and / or y" means "one or both of x and y". As another example, "x, y, and / or z" means any element of the seven-element set {(x), (y), (z), (x, y), (x, z), (y, z), (x, y, z)}. In other words, "x, y and / or z" means "one or more of x, y and z".

[0049] Where ranges are given, endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Herein, "up to" a number (for example, up to 50) includes the number (for example, 50). The term "in the range" or "within a range" (and similar statements) includes the endpoints of the stated range.

[0050] Reference throughout this specification to "one aspect,” "an aspect,” "certain aspects," or "some aspects," etc., means that a particular feature, configuration, composition, or characteristic described in connection with the aspect is included in at least one aspect of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more aspects.

[0051] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." As used herein in connection with a measured quantity, the term "about" refers to that variation in the measured quantity as would81099325. v1 11Atorney Docket No. 770075: MTST-553PC be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. The term "about" as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is + / -10%. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0052] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0053] The term "exemplary" means serving as a non-limiting example, instance, or illustration. As utilized herein, the terms "e.g.," and "for example" set off lists of one or more non-limiting aspects, examples, instances, or illustrations.

[0054] As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. Biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For example, "substantially" may refer to being within at least about 20%, alternatively at least about 10%, alternatively at least about 5% of a characteristic or property of interest.

[0055] A "disease", as used herein, is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated, the subject's health continues to deteriorate. A "disorder" is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease81099325. v1 12Atorney Docket No. 770075: MTST-553PC in the subject's state of health. A “syndrome” is a recognizable complex of symptoms and physical findings that occur together and suggest the presence of a certain disease or disorder or an increased chance of developing the disease or disorder. A disease, disorder, or syndrome is "alleviated" if the severity of a sign or symptom of the disease, disorder, or syndrome, or the frequency with which such a sign or symptom is experienced by a subject, or both, is reduced.

[0056] As used herein, the terms “subject”, “individual”, and “patient” are interchangeable, and relate to vertebrates, preferably mammals. For example, mammals in the context of the disclosure are humans, non-human primates, domesticated animals such as dogs, cats, sheep, catle, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., as well as animals in captivity such as animals in zoos. The term "animal" as used herein includes humans. The term "subject" may also include a patient, i.e., an animal, having a disease. In exemplary aspects, a subject, individual, or patient refers to a human (e.g., a man, a woman, or a child).

[0057] The terms “treat”, “treating”, or “treatment” refer to administering to a subject a compound or pharmaceutical composition disclosed herein to partially or completely alleviate, inhibit, ameliorate, or relieve the disease or disorder from which the subject is suffering. This means any manner in which one or more of the symptoms of a disease or disorder are ameliorated or otherwise beneficially altered. As used herein, amelioration of the symptoms of a particular disease or disorder refers to any lessening, whether permanent or temporary, lasting or transient, that can be atributed to or associated with treatment by the compounds, compositions, and methods of the present disclosure. For example, treating a subject can mean eliminating or reducing the clinical signs of a disease or disorder in the subject; arrest, inhibit, or slow the progression of the disease or disorder in the subject; and / or decrease the number, frequency, or severity of clinical symptoms and / or recurrence of the disease or disorder in the subject who currently has or who previously had the disease or disorder. In particular, the terms “treatment of a disease” and “treating a disease” include curing, shortening in duration, ameliorating, slowing down, inhibiting progression or worsening, or delaying the onset of clinical symptoms in a subject who has the disease or disorder.

[0058] The terms “prophylactic”, “preventive”, “preventing”, and “prevention” refer to a decrease in the occurrence of a disease or disorder, or a decrease in the risk of acquiring a disease or its associated symptoms in a subject. The prevention can be complete, e.g., the total81099325. v1 13Atorney Docket No. 770075: MTST-553PC absence of the disease or disorder) or partial, e.g., the occurrence of the disease or disorder in a subject is less than, occurs later than, or develops more slowly than that which would have occurred without the disclosed compounds, compositions, and methods.

[0059] As used herein, the term “preventing a disease” in a subject means, for example, to stop the development of one or more clinical symptoms of a disease or disorder in a subject before they occur or are detectable. Preferably, the disease or disorder does not develop at all, i.e., no symptoms of the disease or disorder are detectable. In some aspects, it can also mean delaying or slowing of the development of one or more symptoms of the disease or disorder. Alternatively, or in addition, it can mean decreasing the severity of one or more subsequently developed symptoms.

[0060] The term “administered” as used herein, means administration of an effective amount of, for example, at least one nucleic acid encoding a spike epitope-loaded single-chain trimer MHC I molecule and any another other additional agent for treatment.

[0061] An "effective amount" includes a "therapeutically effective amount" and a "prophylactically effective amount." The term "therapeutically effective amount" refers to an amount effective in treating and / or ameliorating a disease or condition in a subject. The term "prophylactically effective amount" refers to an amount effective in preventing and / or substantially lessening the chances of a disease or condition in a subject.

[0062] The invention is defined in the claims. However, below is a non-exhaustive listing of non-limiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein.III. Lipid Nanoparticle Formulations for Adjuvant Delivery

[0063] QIV, consisting of two IAV and two IBV strain components, are one of the licensed influenza vaccines, and require annual updates to provide immunity against circulating influenza viruses in the human population. Several studies have focused on improving split vaccines’ efficiency by combining them with adjuvants, including our recent study on SDI RNA and IMDQ-PEG-Chol (RIG-I and TLR7 / 8 agonists, respectively, and both potent inducers of innate immune responses), as adjuvants for QIV. Then again, the stability of these81099325. v1 14Atorney Docket No. 770075: MTST-553PC vaccines ± adjuvants, including SDI RNA, and their efficient in vivo delivery are still challenging. To overcome this, LNPs have emerged as promising vehicles for in vivo delivery systems. Their ability to encapsulate, stably carry, and efficiently deliver the molecules of interest has provided an effective platform in pharmaceutical and vaccine fields. One such example is highly efficient current mRNA vaccines for SARS-CoV-2 that use LNPbased formulations. Nevertheless, these vaccines still need multiple booster doses to be sufficiently effective against emerging virus strains and therefore are limited in inducing an antigenically broader immune response. Therefore, optimization of LNP dose and composition needs to be tailored together with vaccine and / or adjuvant combinations in order to get the desired vaccination outcomes in terms of both humoral and cellular responses, as well as providing protection against viral infections.

[0064] In one study, the inventors tested adjuvant formulations for QIV [2018-2019 season; with A / Singapore / GP 1908 / 2015 IVR-180 (H1N1) as one of the two IAV components] with two different LNP formulations using either S-Ac7-Dog or K-Ac7-Dsa cationic lipids. Since the lipids are positively charged, they can stably interact with negatively charged biomolecules. The vaccine-LNP combinations were further adjuvanted with either one or both of SDI-RNA and IMDQ-PEG-Chol to explore the outcomes of the vaccine ± adjuvant ± LNP formulations in the context of antibody responses, antibody class switching, T-cell responses, protection against in vivo IVR-18O(H1N1) virus challenge, and inflammatory responses in the infected lungs.

[0065] The unadjuvanted QIV vaccine induces modest serum IgGl and IgG2a titers as well as low HAI titers 3 weeks after a single dose of intramuscular vaccination in mice. This is consistent with our previous findings using the same mouse vaccination model. The low antibody titers for unadjuvanted QIV correlated well with low T-cell responses (IL-4 and IFN- g ELIspots) as well as inadequate protection against virus infection, implied by high levels of replicating virus in lungs upon intranasal IVR-180 challenge. These animals also showed the most body weight loss and high levels of inflammatory cytokines in their lungs postinfection, suggesting the recruitment of immune cells to aid in the control / clearance of the virus. QIV formulated with either of the two LNPs and / or further combined with either SDI and / or IMDQPEG-Chol as adjuvant show a boosted total IgG response and a beter control over virus replication by 5 DPI. However, the administration of individual or combination adjuvants skewed the B-cell class switch as well as T-cell responses in vaccinated mice. Upon a single81099325. v1 15Atorney Docket No. 770075: MTST-553PC vaccination, SDI induced a balanced IgGl / IgG2a response, IMDQ-PEG-Chol directed more towards an IgG2a response, and the combination of the two skewed completely towards IgG2a, when formulated into S or K LNPs, suggesting very strong class switching events driven by this combination of adjuvants and consistent with our previous findings. Interestingly, the combination of the two adjuvants SDI and IMDQ-PEG-Chol induces a balanced type I and II T-cell response as suggested by both IL-4 and IFN-g release upon antigenic restimulation. Despite the fact that the combination of IMDQ-PEG-Chol and SDI in LNP formulations results in a balanced Thl / Th2, a very strong type 1 skewing driven by IgG2a is seen when antibody responses are considered.

[0066] Interestingly, S-Ac7-Dog LNPs were found to be more immunogenic than K-Ac7-Dsa LNPs in inducing both humoral and cellular responses in the corresponding groups. This can also be explained by the smaller size of S-Ac7-Dog LNPs than K-Ac7-Dsa LNPs, as the size of LNPs plays an important role in vaccine efficacy. Yet, vaccination with empty S-Ac7-Dog LNPs resulted in lack of protection from morbidity in challenged mice. Remarkably, SAc7- Dog(-) + IMDQ-PEG-Chol vaccination also resulted in lack of protection from morbidity after infection, with body weight loss comparable to unvaccinated / PBS control animals. This lack of protection from morbidity was accompanied by enhanced inflammatory cytokine responses including interleukins IL-4, IL-5, IL-6, and IL-13, interferon gamma (IFN-g), as well as chemokine GMCSF. In contrast, when S-Ac7-Dog LNPs were combined with SDI, inflammation is relatively reduced in infected lungs as suggested by the chemokine / cytokine levels. The differences might be attributed to the lower stability of S-Ac7-Dog than K-Ac7-Dsa lipids in the respective LNPs, which might be stabilized by the addition of oppositely charged SDI molecules.

[0067] Overall, the inventors compared different lipid compositions in LNP formulations, empty or loaded with individual or combination adjuvants. Different combinations affected both B- and T-cell responses along with vaccine / adjuvant / LNP-dependent inflammation in single vaccinated mice upon virus infection. The negatively charged SDI might stably interact with cationic lipid moieties, providing more stability to the entire structure and thereby reducing inflammation in infected animals. The immunogenicity and protection data in mice combined with the cytokine / chemokine induction indicate that lipid composition of LNPs used in vaccines is important and can skew host immune responses to subsequent infection, and therefore is important for vaccine safety and efficacy.81099325. v1 16Atorney Docket No. 770075: MTST-553PC

[0068] Preparation and characterization of SDI-RNA and IMDO-PEGChol LNPs

[0069] LNP formulations were prepared by mixing an aqueous solution containing the in vitro transcribed SDI-RNA (or SDI) with an ethanolic solution containing (1) ionizable lipids, either K-Ac7-Dsa (comprising a ketal bond) or S-Ac7-Dog (comprising a disulfide bond; chemical structure outlined in FIG. 1) to interact with negatively charged SDI and mediate endosomal escape; (2) cholesterol, for structural stability; (3) dioleoylphosphatidylethanolamine (DOPE) phospholipid, as helper lipids to aid in nanoparticle formation; and (4) 1,2-distearoyl-racglycero- 3-methylpolyethylene glycol (DSG-PEG; 2kDa PEG) to provide steric hindrance and thus avoiding aggregation and promoting mobility in vivo. The structure and composition of LNP incorporating SDI RNA is schematically represented in FIG. 2A and Table 1, respectively.

[0070] Table 1. Composition ratio of different components of LNPs, with and without SDI

[0071] The molar ratio of ionizable lipid (KAc7- Dsa or S-Ac7-Dog):cholesterol:DOPE : DSG- PEG was chosen to be 50:38.5:10: 1.5, based on literature. Empty LNPs that did not contain SDI were prepared as control and hence referred to as empty LNP or LNP(-). Both LNPs encapsulating SDI [S-Ac7-Dog(SDI) and K-Ac7-Dsa (SDI)] and corresponding control empty LNPs [S-Ac7-Dog(-) and K-Ac7-Dsa (-)] were fabricated and characterized for their size and zeta potential (ZP) (Figures 1C, D). While K-Ac7-Dsa (-) and K-Ac7-Dsa(SDI) LNPs showed some differences in their size (78 and 189 nm, respectively), the S-Ac7-Dog(-) and S-Ac7-Dog (SDI) LNPs showed similar size distributions (91 and 101 nm, respectively) with low81099325. v1 17Atorney Docket No. 770075: MTST-553PC polydispersity indices (PDI < 0.25; as shown in FIG. 2B) and a positive ZP of approximately 4-6.5 mV at physiological pH (Table 2).

[0072] Table 2. LNPs zeta potential measured by electrophoretic light scattering (ELS)

[0073] LNP formulations with adjuvanted QIV define IgG subtype profile, with S-Ac7-Dog LNPs inducing higher antibody titers than K-Ac7-Dsa LNPs

[0074] The inventors evaluated the potential of empty and SDI-incorporating SAc7- Dog and K-Ac7-Dsa LNPs to adjuvant a licensed QIV, with and without combination with IMDQ-PEG- Chol adjuvant, using our previously well-established preclinical vaccination-infection animal model (17, 30). The study is outlined in FIG. 3A.The inventors vaccinated 6-to 8-week female BALB / c mice with unadjuvanted QIV or in combination with SDI or IMDQ-PEG-Chol, or both, while mock animals received PBS. The inventors further tested the adjuvant effect for QIV upon co-administration of SDI and / or IMDQ-PEG-Chol combined with one of the two LNPs containing different cationic lipids (S-Ac7- Dog or K-Ac7-Dsa) as described in the previous section. LNPs either were empty (-) or had SDI incorporated. The rationale of this setup is that LNP -formulated SDI can, besides endosomes, also be delivered to the cytosol, thereby promoting more efficient RIG-I-mediated innate immune sensing. On the other hand, IMDQ-PEG-Chol is expected to incorporate efficiently in LNPs via its cholesterol moiety. The animals were vaccinated only once via the intramuscular (IM) route. The serum collected 3 weeks post-vaccination was examined for the presence of Hl HA-specific IgG antibodies using enzyme linked immunosorbent assay (ELISA) for total IgG, IgGl, and IgG2a (FIG. 3B).

[0075] No virus-specific antibody titers were detected in the serum from the mock PBS group. The group that received unadjuvanted QIV was used as the reference to compare the IgG responses of other groups.81099325. v1 18Atorney Docket No. 770075: MTST-553PC

[0076] QIV formulated with S-Ac7-Dog LNP (-) or K-Ac7-Dsa LNP(-), corresponding to empty S-Ac7-Dog and empty K-Ac7-Dsa LNPs, respectively, showed higher IgGl titers but the lowest IgG2a titers compared with the corresponding S-Ac7-Dog LNP(SDI) or K-Ac7- Dsa LNP(SDI) groups (FIG. 3B; FIG. 4), thereby illustrating the intrinsic adjuvant effect of LNPs. Additionally, QI formulated with SDI-incorporated LNPs [S-Ac7-Dog LNP(SDI) and K-Ac7- Dsa LNP(SDI)] can induce a balanced IgGl and IgG2a antibody response. Overall, the total IgG levels were found similar among all adjuvanted and LNP -formulated groups post-prime vaccination. However, the S-Ac7-Dog LNP(SDI) seemed to induce slightly higher total IgG compared to K-Ac7-Dsa LNP (SDI) in the corresponding SDI ± IMDQ-PEG-Chol combination adjuvanted groups. A similar observation was made for IgGl antibody titers with S-Ac7-Dog LNPs inducing higher IgGl titers than respective KAc7- Dsa LNP groups. Consistent with our previous findings, IMDQ-PEG-Chol administration, with either empty or SDI incorporated S- Ac7-Dog or K-Ac7-Dsa LNPs, skews the antibody responses towards IgG2a with a significant reduction in IgGl titers (FIG. 3C). QIV+SDI and QIV+IMDQ-PEG-Chol groups, with no LNP formulations, were used as control vaccination groups for the study and to correlate with a previous study.

[0077] QIV adjuvanted with IMDQ-PEG-Chol exhibit better control over virus neutralization when formulated in S-Ac7- Dog LNPs than in K-Ac7-Dsa LNPs.

[0078] Neutralizing antibodies are important defense mechanisms during viral infection. These antibodies bind to the viral antigens and change the conformation, thereby blocking the viral attachment to cells. As a surrogate for virus neutralizing antibody levels, the inventors performed hemagglutination inhibition (HAI) assays with postvaccination sera collected from all vaccinated animals at 3 weeks post-vaccination. As shown in FIG. 5, the mice that received unadjuvanted QIV showed low HAI titers, which were not significantly different from the groups that received QIV formulated in either empty or SDI-containing S-Ac7-Dog or KAc7- Dsa LNPs. The group administered with QIV+IMDQ-PEGChol, without LNP formulations, showed significantly higher HAI titers compared with the unadjuvanted QIV group. HAI titers were significantly higher for the groups that received empty or SDI containing S-Ac7-Dog in the LNP formulation when combined with the IMDQ-PEG-Chol adjuvant. However, the corresponding K-Ac7-Dsa LNP-formulated groups did not show any significant differences in HAI titers compared with the unadjuvanted or unformulated QIV group.81099325. v1 19Atorney Docket No. 770075: MTST-553PC

[0079] QIV formulated in either SDI-containing S-Ac7-Dog or K-Ac7-Dsa LNPs efficiently induce T-cell responses when combined with IMDO-PEG-Chol

[0080] Helper CD4+ and cytotoxic CD8+ T cells play an important part in vaccination-mediated humoral and cellular responses by facilitating Ig class switch during B-cell maturation and direct killing of infected cells, respectively. T cells can recognize foreign antigens presented on the major histocompatibility complex (MHC) molecules on infected cells followed by the release of various cytokines, including IFN-g and IL-4, two major cytokines corresponding to helper type 1 (Thl) and type 2 (Th2) T cells, respectively. IFN-g and IL-4 can modulate class switching of B cells to IgG2a or IgGl. To study the correlation between the two cytokines and antibody responses in our vaccination model, the inventors examined the release of IFN-g and IL-4 from splenocytes obtained from the mice 10 days post-vaccination (DPV) (outlined in FIG. 6A), in the presence and absence of

[0081] specific antigen (IVR-180 virus or Hl HA peptide) using enzyme-linked immunosorbent spot (ELISpot) assays.

[0082] As shown in FIGs. 6A-6C, antigen-specific IFN-g (Figure 4B) or IL-4 (FIG. 6C) release from the splenocytes was very low in the absence of a stimulant, except for QIV+S- Ac7-Dog(-) or QIV+K-Ac7-Dsa (-) groups, suggesting a basal level of non-specific stimulation after vaccination by the empty LNPs. Upon stimulation with specific Hl -HA peptide or IVR-180 virus, the number of splenocytes producing antigen-specific IFN-g as well as IL-4 were found to be higher in the mice that received QIV+IMDQ-PEG-Chol formulated in S-Ac7-Dog(SDI) or K-Ac7-Dsa(SDI) LNPs, suggesting an induction in both Thl and Th2 immune response. The group administered with QIV+IMDQ-PEG-Chol, without LNP formulations, showed high IFN-g release compared with the unadjuvanted QIV group, consistent with our previous study. Mice that received PBS were used as a reference for statistical comparison.

[0083] S-Ac7-Dog and K-Ac7-Dsa LNP formulations with SDI and / or IMDO-PEGChol potentiates QlV-mediated protection against viral challenge with homologous influenza virus81099325. v1 20Atorney Docket No. 770075: MTST-553PC

[0084] To further correlate B- and T-cell responses with the extent of protection against virus replication in vivo, all vaccinated and unvaccinated mice were given a high-titer virus challenge with 18,000 plaque-forming units (PFU) of IVR-180 virus per animal. A single dose of vaccination was found effective in conferring protection from severe morbidity in challenged animals compared with mock challenged mice in the initial 5 days of virus challenge. As shown in FIG. 7A, the groups receiving unadjuvanted QIV and QIV with KAc7- Dsa(- or SDI) LNPs showed less than 10% body weight loss over 5 days post-infection. The unvaccinated PBS group lost approximately 20% body weight by 4 DPI. Interestingly, the mice vaccinated with SAc7- Dog (-), irrespective of the combination with IMDQ-PEG-Chol, showed drastic weight loss over 5 days, almost comparable to the unvaccinated PBS group. This is especially important to note because the same S-Ac7-Dog LNPs incorporating SDI did not show such extensive weight loss in vaccinated / challenged animals, suggesting higher morbidity in virus-infected lungs in case of empty S-Ac7-Dog LNP formulations.

[0085] Irrespective of the body weight loss differences atributed by LNPs, all groups that received adjuvanted QIV showed a lower amount of replicating virus in their lungs at 5 DPI (FIG. 7B), compared with the unadjuvanted QIV group. QIV formulated in either of the two empty LNPs [S-Ac7-Dog(-) or K-Ac7-Dsa(-)] did not provide significant protection in the initial days of infection compared with the unadjuvanted QIV group, contrary to the higher IgGl induction. The groups that received QIV with either S-Ac7-Dog(- or SDI) or K-Ac7-Dsa(- or SDI) LNPs, irrespective of IMDQ-PEG-Chol combination, resulted in significantly beter control of lung virus replication, with very low detectable titers in their lungs, and, therefore, correlated with enhanced vaccine responses observed in mice. Interestingly, S-Ac7-Dog LNPs resulted in lower body weight loss in the first 5 days of infection, compared to K-Ac7-Dsa LNPs in animals when compared between corresponding adjuvant groups.

[0086] Incorporation of SDI as an adjuvant improves the cytokine profile in lungs of infected animals causing less morbidity as compared to empty S-Ac7-Dog LNP formulations

[0087] The body weight data over 5 DPI suggested that the administration of empty S-Ac7-Dog LNP as vaccine ± adjuvant formulation, although protective, resulted in higher body weight loss, comparable to PBS-vaccinated control mice. The lungs of all infected animals were examined for their cytokine profiles at 5 DPI. As shown in FIGs. 8A, 8B, 9A-9J; the cytokine profiles in the infected lungs of animals from vaccinated or unvaccinated / PBS groups were different. The81099325. v1 21Atorney Docket No. 770075: MTST-553PCPBS group showed high levels of inflammatory cytokines including IL-6, IL-18, IL-12 p70, IFN-g,and TNF-a as well as chemokine GMCSF, which may suggest enhanced vascular permeability and increased infiltration of innate immune cells in response to infection in unvaccinated animals. As shown in FIG. 8A, these cytokine levels were significantly lower in all vaccinated groups, implying a better control over inflammation and morbidity in response to the virus. Some of the cytokines were very low in the PBS group, especially the signatures for T-cell responses such as IL-4, IL-5, and IL-13, which is in line with the typical type 1 skewed host immune response to influenza infection. Interestingly, pro-inflammatory cytokines and chemokines, including IL- lb, GMCSF, and the type 2 cytokines IL-4, IL-5, IL-6, and IL-13, were significantly elevated in unadjuvanted QIV and QIV± IMDQ-PEG-Chol formulated with empty LNPs. This was not the case for the corresponding groups with LNP(SDI), especially for those LNP groups with S-Ac7-Dog lipids. The PCA plot in FIG. 8B clusters different vaccinated and unvaccinated groups based on their differences in the production of inflammatory cytokines and chemokines in lungs post-infection. The unvaccinated PBS group clusters far away from all vaccinated animals, corresponding to high inflammation and chemokine production in lungs post-infection with lower interleukins. Moreover, the QIV+ S-Ac7-Dog(-) vaccinated group clustered separately from the other vaccinated groups. Additionally, the group that received QIV+ S-Ac7-Dog(-) combined with IMDQPEG-Chol is clustered together with unadjuvanted QIV, suggesting that the use of empty S-Ac7-Dog is disadvantageous as it reduces the protective effect of IMDQ-PEG-Chol when used without any lipid formulation. The other vaccinated groups showing low inflammation in lungs are clustered together.IV. Polyinosinic: poly cytidylic acid (poly(I:C)) Encapsulated LNPs

[0088] Polyinosinic:polycytidylic acid (poly(I:C)) is a synthetic analog of double stranded RNA (dsRNA) that can be recognized as a pathogen-associated molecular pattern (PAMP) and can trigger several receptors in endosomes and cytoplasm of innate immune cells. In the endosomes of antigen-presenting cells, poly(I:C) activates Toll-like receptor 3 (TLR3) while in the cytoplasm poly(I:C) can trigger retinoic acid-inducible gene-like receptors (RLRs) like retinoic acid-inducible gene I (RIG-I) and the melanoma differentiation-associated 5 (MDA- 5) receptors. Innate immune responses following TLR3 activation rely on TIR-domain- containing adapter-inducing interferon- / 3 (TRIF)-mediated signaling, whereas triggering of RLRs will result in RLR oligomerization, exposure of the N-terminal Caspase recruitment domains (CARD) domains and translocation to the mitochondria for further downstream81099325. v1 22Atorney Docket No. 770075: MTST-553PC signaling via mitochondrial antiviral signaling (MAVS) protein, resulting in the induction of strong types I and III interferon (IFN), which can promote type 1 cytokine responses (FIG. 10). This is of great relevance in the context of antiviral and anticancer vaccination, by instigating robust levels of cytotoxic T cells and antibody isotypes with neutralizing and innate effector killing capacity.

[0089] In vivo, in soluble form, poly(I:C) is prone to degradation by nucleases and poor uptake by antigen presenting cells in immune-inducing lymphoid tissues. Furthermore, in its soluble form, poly(I:C) cannot reach into the cellular cytoplasm, and thus cannot efficiently activate RLRs. To improve its uptake by active phagocytic cells such as dendritic cells (DCs) and macrophages, poly(I:C) has been formulated into films, hydrogels, microparticles, and nanoparticles. However,

[0089] achieving cytoplasmic delivery remains very inefficient with most nanoparticles, especially in vivo. Hence, the therapeutic potential of poly(I:C) is currently underdeveloped.

[0090] The inventors hypothesized that encapsulating poly(I:C) into lipid nanoparticles (LNPs) (FIG. 11) through electrostatic complexation with an ionizable cationic lipid could overcome these limitations. LNPs are currently the most performing RNA delivery systems, as demonstrated by the recent success of the messenger ribonucleic acid (mRNA) LNP COVID-19 vaccines.

[0091] The LNPs used in this study are composed of an ionizable cationic lipid that, at low pH, can electrostatically complex with the anionic inosinic and cytidylic acid residues in the poly(I:C) backbone. LNPs protect nucleic acids from degradation in the extracellular medium and can be efficiently taken up by actively phagocytic cells in lymphoid tissues. LNPs facilitate cytoplasmic delivery of a nucleic acid payload by destabilizing endosomal membranes. This is particularly relevant for accessing RLR, as these innate immune sensors are typically located in the cytoplasm of cells, which are generally inaccessible to soluble, unformulated poly(I:C). Furthermore, nanoparticulate delivery systems mediate efficiently translocation to immune- inducing sites in lymphoid tissue, which is of great relevance in the context of vaccine application.

[0092] Here the inventors demonstrate that encapsulating poly(I:C) in LNP greatly enhances the innate immune response to poly(I:C), both in vitro and in vivo. While mere TLR3 triggering resulted in relatively poor adjuvanticity, combined TLR3 and RLR triggering81099325. v1 23Atorney Docket No. 770075: MTST-553PC induced robust innate and adaptive immune responses. Following intramuscular (IM) injection in mice, LNP-encapsulated poly(I:C) was delivered to DC subsets in draining lymph nodes and in the spleen, resulting in the activation of a broad range of myeloid and lymphoid immune cell subsets. Furthermore, when recombinant trimeric full-length spike protein of the SARS- CoV-2 virus was admixed with LNP-encapsulated poly(I:C), it induced robust antigenspecific neutralizing antibody responses superior to soluble poly(I:C) and generated protection against viral challenge in vivo.

[0093] In this study, the inventors investigated the encapsulation of the synthetic doublestranded RNA TLR3 agonist, poly(I:C), in LNPs via electrostatic interaction between the phosphate anions in the poly(I:C) backbone and a cationic amine of an ionizable lipid. Our results showed that LNP encapsulation maintained the capacity of poly(I:C) to trigger TLR3 in endosomes, while greatly amplifying its capacity to trigger RLRs by mediating delivery of poly(I:C) into the cytoplasm. This hypothesis is supported by literature data reporting in vitro experiments that show direct cytoplasmic delivery of poly(I:C) by transfection can activate RLRs and convert the TLR3-mediated induction of apoptosis into an immunoadjuvant effect driven by IFN- ? production.

[0094] In vivo in mice, LNP formulation altered the pharmacokinetic profile of poly(I:C) upon intramuscular administration, resulting in vastly amplified innate immune activation in distal tissues, including liver and spleen. Immunization of mice with LNP(poly(I:C)) adjuvanted recombinant Spike protein from the SARS-CoV-2 virus yielded robust antigen-specific antibody titers, with LNP(poly(I:C)) vastly outperforming soluble unformulated poly(I:C), which exhibited rather weak adjuvant properties. Additionally, immune serum from mice receiving a prime and booster immunization of LNP(poly(I:C)) adjuvanted Spike protein strongly reduced viral infection in vitro, while prime and boost immunization of LNP(poly(I:C)) adjuvanted Spike protein fully protected against a mouse-adapted SARS-CoV-2 viral challenge in vivo.

[0095] The inventors confirmed the importance of the innate signaling adapter molecule MAVS[4] for the adjuvant effect of LNP(poly(I:C)), although the inventors were unable to discriminate the contributions of different RLRs (RIG-I or MDA5) to the observed adjuvant effect, as both signal through MAVS. Both RIG-I and MDA5 are known to be activated by81099325. v1 24Atorney Docket No. 770075: MTST-553PC poly(I:C) with the help of zinc finger protein ZCCHC3 but are also reported to have different preferential binding depending on molecular weight of the dsRNA.

[0096] The efficiency of innate immune activation by poly(I:C) depends on molecular mass of poly(I:C).

[0049] For these studies, the inventors have used low molecular mass (LMM) poly(I:C). It has been suggested that MDA5 is mainly activated by high-molecular-weight (3 kb) poly(I:C), whereas RIG-I would typically sense low-molecular weight (<1.5 kb) poly(I:C) as well as in vitro transcribed 5' phosphorylated dsRNAs like the Sendai virus defective interfering RNA. The inventors anticipate that LNP(poly(I:C)) could find application in the context of vaccine design against viral infection and cancer, as well as in the context of cancer immunotherapy to mount immunity against neo- and self-antigens. Overall, our findings suggest that LNP-formulation is a scalable and translational feasible approach that greatly amplifies the adjuvant properties of poly(I:C) by triggering both TLR3 and RLRs and altering its pharmacokinetic and pharmacodynamic properties in vivo. Notably, LNPs, including LNPs containing S-Ac7-DOG as an ionizable lipid, have been amenable to lyophilization, which enables long-term storage and thereby facilitates their use in vaccine formulations.

[0097] Encapsulation of Poly(I:C) in LNP

[0098] Poly(I:C) was encapsulated into LNP (FIG. 11) using a rapid mixing method. An aqueous buffer (5 mm, pH 4) containing poly(I:C) was mixed vigorously with an ethanolic solution containing an ionizable lipid (S-Ac7-DOG;), cholesterol, l,2-dioleoyl- w-glycero-3- phosphoethanolamine (DOPE), and distearoyl-rac-glycerolpoly(ethylene glycol) (DSG-PEG; 2 kDa PEG). Cholesterol and DOPE served to facilitate nanoparticle formation and endosomal escape. The PEG-lipid served for colloidal stabilization of the LNP. The reduction-sensitive ionizable lipid S-Ac7-DOG was recently developed in our laboratory as part of a combinatorial synthesis campaign aimed at identifying novel ionizable lipids for nucleic acid drug delivery. ‘Low molecular weight’ (LMW) poly(I:C), with a claimed size of 0.2-1 kilobases (kb), was used. The inventors selected a molar ratio of ionizable lipid: cholesterol :DOPE:DSG- PEG of 50:38.5:10:1.5, based on established ratios used in mRNA delivery.

[0099] LNP encapsulating poly(I:C), further referred to as LNP(poly(I:C)), were prepared at an NP ratio of 5 : 1 (N: molar fraction of ionizable amines in the ionizable lipid; P: molar fraction of anionic phosphate in poly(I:C)). LNP lacking poly(I:C), i.e., empty LNP, was prepared as a control.81099325. v1 25Atorney Docket No. 770075: MTST-553PC

[0100] The size, electrophoretic mobility, and encapsulation efficiency of the LNP formulations were characterized using dynamic light scattering (DLS), electrophoretic mobility measurements, and a RiboGreen assay, as depicted in FIG. 12 and Table 3.

[0101] Table 3. Physicochemical characterization of LNP (poly(I:C)) and empty control LNPa)Measured in (4-(2-hydroxyethyl)-l-piperazoneethanesulfonic acid) (HEPES) buffer pH7.4

[0102] Both LNP(poly(I:C)) and empty LNP had a hydrodynamic diameter ranging from 90 to 110 nm, with a low polydispersity index (PDI) below 0.2, indicating a homogeneous population. RiboGreen assay confirmed complete encapsulation of poly(I:C) in the LNP. The zeta potential of both formulations was slightly positive at the physiological pH of 7.4. Notably, the zeta potential of nanoparticles is a measure of the electrostatic potential at the boundary between the dispersing medium and the “slipping plane” of the particle. Therefore, it assesses the properties of the LNP surface rather than the LNP core. The ionizable lipid, specifically S-Ac7-DOG, in our LNP formulations has a pK of 6.74 and thus will have reduced ionization at the physiological pH of 7.4, at which the inventors measured the zeta potential of the LNP. The inventors used an NP ratio of 5:1. Consequently, the ionizable lipid is in excess and likely ensures encapsulation of poly(I:C) inside the core of the LNP. Therefore, the presence of poly(I:C) might not significantly alter the surface properties as it is shielded from the external environment. Moreover, the PEG coating can “shield” the surface charge, further making the zeta potential of the LNP closer to neutral.

[0103] In Vitro Cellular Uptake and Innate Activation Pathway of LNPfPolyfTC))

[0104] Fluorescently-labeled rhodamine-conjugated poly(I:C) (poly(LC)RHO) was employed to evaluate the uptake of LNP by antigen-presenting cells in vitro. Interestingly, the fluorescence emission intensity of poly(I:C)RHO was higher in LNP-formulated form than in unformulated form in solution (cfr. the experimental section for exact details how this was assessed). The inventors then used the immortalized DC2.4 mouse dendritic cell line as a81099325. v1 26Atorney Docket No. 770075: MTST-553PC model for antigen-presenting cells. The DC2.4 cells were treated with LNP(poly(I:C)RHO) and unformulated soluble poly(I:C)RHO for 12 h, respectively, to assess their uptake efficiency. Flow cytometry analysis (Figure 4B) revealed that the uptake of LNP(poly(I:C)RHO) and poly(LC)RHO) by DC2.4 cells was dose dependent and was slightly higher in case of LNP(poly(I:C)RHO). However, the difference in fluorescence emission intensity between poly(LC)RHO) and LNP(poly(I:C)RHO) induces a bias in the quantitative interpretation of these data.

[0105] Confocal microscopy imaging (FIG. 13C) confirmed the cellular internalization of both unformulated soluble poly(I:C)RHO and LNP(poly(I:C)RHO), showing a punctate intracellular pattern in cells. Counterstaining with LysoTracker demonstrated strong colocalization, suggesting that both unformulated soluble poly(I:C)RHO and LNP(poly(I:C)RHO) are primarily stored in intracellular acidic vesicles such as endosomes and lysosomes. It should be noted that endosomal escape with simultaneous cytoplasmic delivery of free poly(I:C)RHO is expected to happen only for a small fraction of LNP(poly(I:C)RHO), which makes it difficult to detect due to the strong signal from poly(I:C)RHO in endosomes.

[0106] Next, the inventors utilized a panel of commercially available reporter cell lines to examine the impact of LNP encapsulation on the ability of poly(I:C) to activate endosomal and cytoplasmic innate immune sensors (FIG. 13D). HEK-Blue mTLR3 cells are HEK293 cells that have been genetically modified through cotransfection of the murine TLR3 gene and an inducible secreted embryonic alkaline phosphatase (SEAP)-reporter gene. The SEAP gene is driven by the IFN- ? minimal promoter fused to NF-K and activator protein 1 (AP-1) binding sites. Activation of TLR3 by poly(I:C) triggers NF-K and AP-1, leading to SEAP production, which can be quantified by a simple colorimetric assay. RAW-Lucia ISG cells are RAW264.7 macrophages that have been genetically engineered to carry an interferon regulatory factor (IRF)-inducible luciferase-reporter construct. RAW264.7 cells express dsRNA RLRs. Activation of RLRs by poly(I:C) in the cytosol triggers IRF,[4] leading to luciferase production, which can be measured by bioluminescence detection. Additionally, the inventors tested LNP(poly(I:C)) and poly(I:C) on RAW-Lucia ISG cells in which the signaling proteins involved in TLR3- and RLRmediated innate activation, TRIF and MAVS, respectively, were either knocked out or present. In TLR3-reporter cells, LNP encapsulation resulted in a significant increase in TLR3 activation by poly(I:C). In ISG-reporter cells, LNP encapsulation81099325. v1 27Atorney Docket No. 770075: MTST-553PC led to a remarkable increase in ISG activation by poly(I:C). This response wasmaintained in TRIFKO ISG-reporter cells, but was completely abolished in MAVSKO ISG-reporter cells. Notably, all three ISGreporter cell lines required high concentrations of unformulated, soluble poly(I:C) to induce a measurable response. However, the maximum concentration of poly(I:C) in TLR3-reporter cells was below 104 ng mL-1, the ISG-reporter cells required a poly(I:C) concentration of up to 105-106 ng mL-1. At such high concentrations, the samples containing LNP(poly(I:C)) induced severe cytotoxicity, making proper assessment at these levels unfeasible. Overall, our findings support the hypothesis that LNP encapsulation enhances the ability of poly(I:C) to strongly activate RIG-I and / or MDA-5, and it confirms that LNP can facilitate cytoplasmic delivery of poly(I:C).

[0107] LNP Formulation Strongly Increases Innate Activation Capacity of Poly(I:C) In Vivo

[0108] The influence of LNP formulation on the biodistribution and amplitude of the poly(I:C)-induced type I IFN response in vivo at the macroscopic tissue level was investigated using an interferon beta (IFN- / ?) luciferase-reporter mouse model (IFN / ?+ / A / ?-luc). This transgenic mouse model links the expression of IFN- / ? to the firefly luciferase gene, enabling spatiotemporal analysis of nanocarrier-mediated IFN- / ? induction through bioluminescence imaging.

[0040] IFN / ?+ / A / ?-luc mice were given an intramuscular injection into the quadriceps of an equivalent high dose of 25 pg poly(I:C) in unformulated soluble and LNP-formulated form.

[0109] The inventors deliberately selected a high dose to fully capture the effect of LNP formulation on the pharmacokinetic and pharmacodynamic profile of poly(I:C). Bioluminescence imaging was performed immediately upon injection, 4 h, and 24 h postinjection (FIGs. 14A and 14B) Unformulated soluble poly(I:C) induced IFN- / ? expression at the site of injection 4 h postinjection, which declined strongly after 24 h. IFN- / ? expression was also observed in the urinary tract and mouth, indicating renal secretion (and reingestion) of poly(I:C). LNP(poly(I:C)) induced a similar signal at the injection site 4 h postinjection, but also induced strong IFN- / ? expression in the liver and spleen. The magnitude of the IFN- / ? expression further increased after 24 h. Empty LNP did not induce a detectable response. To analyze cytokine levels in the blood, the inventors performed Bio-Plex assays on cytokines (IL-lb, IL-4, IL-6, IL-10, IL-12(p70), IFN-y, monocyte chemoattractant protein 1 (MCP1), TNF-ct) at 6 h postinjection (FIG. 14C). MCP1 and IL-6 were increased in mice that81099325. v1 28Atorney Docket No. 770075: MTST-553PC received LNP(poly(I:C)), while cytokine levels in mice injected with unformulated soluble poly(I:C) remained below the detection level. Empty LNP also induced IL-6, but not MCP1, and IL-6 levels were lower than those induced by LNP(poly(I:C)). These findings are consistent with recent observations by Alameh et al., who reported on the ability of empty LNP to induce IL-6 in vivo in mice, suggesting that the adjuvant activity of empty LNP observed in an immunization setting may be attributed to its ability to induce IL-6. The absence of a clear pro-inflammatory cytokine profile shortly after injection suggests that the LNP formulations are safe.

[0109] The inventors analyzed the effect of LNP formulation on the biodistribution of poly(I:C) (25 pg dose) on a cellular level by flow cytometry in the draining iliac lymph node and spleen at 24 h post IMinj ection in the quadricep. For fluorescent tracking, poly(I:C)RHO was used. In the draining lymph node, poly(I:C)RHO was detected in cDCl and cDC2 dendritic cell subsets and, to a lesser extent, also in B cells. LNP formulation did not have a major impact on the delivery of poly(I:C)RHO to DCs (cDCl and cDC2 DC subsets) and B cells (FIG. 14D). In the spleen, by contrast, LNP(poly(I:C)RHO) showed, relative to unformulated soluble poly(I:C)RHO, a vast increase in delivery to cDCl and cDC2 dendritic cells and, to a lesser extent, also to B cells.

[0110] The draining iliac lymph node and spleen were analyzed by flow cytometry, 24 h post IM injection, to investigate the influence of LNP formulation on the cellular biodistribution of poly(I:C). Poly(I:C)RHO was used to enable fluorescent tracking. In the draining lymph node, poly(I:C)RHO was detected in cDCl and cDC2 dendritic cell subsets, and to a lesser extent in B cells. The LNP formulation did not have a significant impact on the delivery of poly(I:C)RHO to DCs (cDCl and cDC2 DC subsets) and B cells (Figure 5C). However, in the spleen, LNP(poly(I:C)RHO) showed enhanced delivery to cDCl and cDC2 dendritic cells compared to unformulated soluble poly(I:C)RHO. Also, B cells received a higher dose of poly(I:C) when poly(I:C) was formulated in LNP.

[0111] Next, the inventors analyzed the effect of LNP formulation on innate immune activation on a cellular level by flow cytometry in the draining iliac lymph node and spleen at 24 h postintramuscular injection. In the draining lymph node, both unformulated soluble poly(I:C) and LNP(poly(I:C)) induced upregulation of the maturation markers CCR7, CD80, and CD86 (FIG. 14E). LNP(poly(I:C)) and unformulated soluble poly(I:C) were equally effective in upregulating CCR7 expression. However, LNP(poly(I:C)) was more potent in upregulating CD80 and CD86 expressions. Empty LNP induced intermediate maturation above background81099325. v1 29Atorney Docket No. 770075: MTST-553PC levels. In the draining lymph node, LNP(poly(I:C)) induced upregulation of the activation marker CD69 in B and T cells to a much higher extent than unformulated soluble poly(I:C). LNP did not induce activation of B and T cells. In the spleen, LNP(poly(I:C)) strongly induced maturation of cDCl and cDC2 dendritic cells and induced strong activation of B and T cells (FIG. 14F). In the spleen, unformulated soluble poly(I:C) induced slight maturation of cDCl and cDC2 dendritic cells but did not induce activation of B and T cells. Empty LNP had no effect on dendritic cells or B and T cells in the spleen. Taken together, the data provides clear evidence that the LNP formulation strongly amplifies the innate immune activation capacity of poly(I:C).

[0112] Uptake of LNP(poly(I:C)) by dendritic cells is likely to directly induce maturation of these cells. The data showed that in the draining lymph node, there was no significant difference in the uptake of LNP(poly(I:C)) and unformulated soluble poly(I:C) by DCs. However, in the spleen, LNP(poly(I:C))was delivered to DCs at a much higher rate than unformulated soluble poly(I:C). Band T-cell activation in both the lymph node and spleen likely occurs as a bystander effect in response to cytokine secretion by dendritic cells or other TLR3- and RLR-expressing cells, such as muscle cells and epithelial cells that internalized LNP(poly(I:C)). The ability of LNP to deliver poly(I:C) to both endosomal vesicles (location of TLR3) and the cytoplasm (location of RLR) likely plays a major role in contributing to the strong innate immune activating capacity of LNP(poly(I:C)), as suggested by our in vitro data (FIG. 14C) Moreover, the inherent properties of LNPs themselves, such as their ability to induce systemic levels of IL-6 and dendritic cell maturation in lymphoid tissue, might also contribute to the potency of LNP(poly(I:C)).

[0113] LNP Formulation Strongly Alters the Vaccine Adjuvant Properties Of Poly(I:C)

[0114] The inventors investigated the adjuvant properties of LNP(poly(I:C)) by admixing them with recombinant full-length trimeric SARSCoV-2 spike protein (further abbreviated as S protein). Well adjuvanted S protein is able to mount protective responses in mice as shown before by us and others. 6-8 week old female 129Slmice were immunized intramuscularly with a primeboost schedule using 5 pg of recombinant S protein, either unadjuvanted or adjuvanted with LNP(poly(I:C)), LNP, or AddaVax, an MF59-like water-in-oil established control vaccine adjuvant. To limit the systemic immune activation, a fivefold lower dose of poly(I:C) was used in this experiment. The experimental outline is depicted in FIG. 15A, and81099325. v1 30Atorney Docket No. 770075: MTST-553PC blood was collected 22 days postprime and 21 days postboost vaccination for analysis of S protein-specific total immunoglobulin G (IgG), IgGl, and IgG2a isotype / subclass titers by enzyme-linked immunosorbent assay (ELISA) (FIG. 15B). Booster immunization increased antigen specific antibody titers across all cohorts and isotype classes. The highest antigenspecific total IgG titers were measured in the LNP(poly(I:C)) adjuvanted cohorts. After prime, antigen-specific IgGl titers were highest in mice immunized with antigen adjuvanted with empty LNP, whereas after boost, the AddaVaxadjuvanted cohort had the highest IgGl titers, confirming its type 2 skewing effect, and empty LNP and LNP(poly(I:C)) adjuvanted cohorts were on par. Antigen-specific IgG2a titers were greatly increased in LNP(poly(I:C)) adjuvanted cohorts, relative to all other cohorts, both after prime and boost. It is noteworthy that the adjuvanticity of soluble unformulated poly(I:C) was found to be very weak and barely increased antigen-specific antibody titers beyond those of nonadjuvanted antigen. Empty LNP also exhibited an adjuvant effect, which is in line with recent reports in the literature. The high IgG2a / IgGl (FIG. 15C) ratio observed in LNP(poly(I:C)) adjuvanted cohorts suggests that LNP(poly(I:C)) skews toward a Thl immune response and class switching toward an IgG2a isotype.

[0115] The inventors then investigated the capacity of sera from immunized mice to neutralize the ancestral USA-WA1 / 2020 virus, aWuhanlike SARS-CoV-2 strain, in vitro using a microneutralization assay (FIG. 15D). Sera collected postprime and postbooster immunization were tested, and only serum from mice immunized with LNP(poly(I:C)) adjuvanted antigen induced viral neutralization

[0116] beyond the background level from the postprime sera. All postboost sera from mice immunized with adjuvanted antigen were able to neutralize the virus to some extent, and LNP(poly(I:C)), LNP, and AddaVax-adjuvanted sera were on par. Immunization with unadjuvanted antigen or antigen adjuvanted with soluble unformulated poly(I:C) was unable to neutralize the virus in vitro, suggesting an absence of detectable neutralizing antibody titers in these groups postprime as well as postboost. After booster immunization, mice received an intranasal dose of a mouse-adapted SARS-CoV-2 strain to determine the correlation between observed vaccine responses and protection from viral challenge. The mouse-adapted SARS-CoV-2 strain was derived from the ancestral Wuhan-like USA-WA1 / 2020 SARS-CoV-2 virus, antigenically matching the vaccine antigen, except for the N501 Y mutation in its receptor-binding domain, which allows the virus to bind to mouse ACE2 more efficiently, resulting in infection of laboratory strains of wild-type mice. Four days81099325. v1 31Atorney Docket No. 770075: MTST-553PC post challenge, lungs and nasal turbinates were harvested, and the residual viral load in these tissues was quantified by a plaque assay.

[0116] Immunization with antigen adjuvanted with LNP(poly(I:C)) and AddaVax conferred sterilizing immunity, with plaque numbers below the detection limit in both lungs and nasal turbinates (FIG. 15E-15F). Unadjuvanted antigen and antigen adjuvanted with LNP and soluble unformulated poly(I:C) were unable to fully control viral infection.

[0117] The inventors repeated the vaccination study using trimeric recombinant SARS-CoV- 2 spike protein in MAVS- / - mice. MAVS is an innate signaling adapter molecule that is involved in RLR signaling, but not TLR3 signaling. The inventors observed that the dominant adjuvant effect of LNP(poly(I:C)) was abolished in MAVS- / - mice, with the highest ELISA binding titers being observed in the group that received soluble poly(I:C) (FIG. 16A). This was also reflected in the microneutralization assay, where the LNP(poly(I:C)) group had lower titers compared to the soluble poly(I:C) group, and overall low titers in MAVS- / -mice (FIG. 16B) These results confirm

[0119] our hypothesis that, also in vivo, induction of immunity by LNP(poly(I:C)) is primarilymediated by cytoplasmic RLR triggering, rather than TRL3 triggering.

[0118] The presently described technology and its advantages will be better understood by reference to the following examples. These examples are provided to describe specific implementations of the present technology. By providing these specific examples, it is not intended limit the scope and spirit of the present technology. It will be understood by those skilled in the art that the full scope of the presently described technology encompasses the subject matter defined by the claims appending this specification, and any alterations, modifications, or equivalents of those claims.

[0119] Example 1: Materials and Methods for preparation and characterization of SDI- RNA and IMDQ-PEGChol LNPs

[0120] Cell lines

[0121] The Madin-Darby canine kidney (MDCK) cell line was maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1 x antibiotics (penicillin / streptomycin).81099325. v1 32Atorney Docket No. 770075: MTST-553PC

[0122] QIV vaccine

[0123] Quadrivalent inactivated influenza vaccine (FLUCALVEX 2018 / 2019 season Lot 252681) was obtained from Seqirus. The vaccine consists of MDCK-grown two IAV and two IBV-A / Singapore / GP 1908 / 2015 IVR-180 (H1N1) (A / Michigan / 45 / 2015-like virus), A / North Carolina / 04 / 2016 (H3N2) (A / Singapore / INFIMH- 16-0019 / 2016-like virus), B / Iowa / 06 / 2017 (B / Colorado / 06 / 2017-like virus), and B / Singapore / INFTT-16-0610 / 2016 (B / Phuket / 3073 / 2013 -like virus).

[0124] SDI-RNA

[0125] The SDI-RNA (or SDI) was in vitro transcribed as described in our recent study (Jangra S, Laghlali G, Choi A, Rathnasinghe R, Chen Y, Yildiz S, et al. RIG-I and TLR-7 / 8 agonists as combination adjuvant shapes unique antibody and cellular vaccine responses to seasonal influenza vaccine. Front Immunol. (2022) 13:974016. doi:10.3389 / fimmu.2022.974016).

[0126] Lipid nanoparticle fabrication

[0127] SDI or SDI equivalent (1 mg) was encapsulated in LNPs by rapid mixing under vigorous stirring of an acetate buffer (5 mM, pH 4.5) containing SDI with an ethanolic solution containing the ionizable lipid S-Ac7-Dog or K-Ac7-Dsa (to obtain S-Ac7-Dog and K-Ac7- Dsa LNPs respectively), cholesterol, l,2-dioleoyl-n-glycero-3 -phosphoethanolamine (DOPE), and a poly(ethylene glycol)-lipid (DSG-PEG; PEG had an MW of 2 kDa) at a 50:38.5: 10: 1.5 ratio.

[0128] After mixing, LNPs were dialyzed against 1 x PBS to get rid of ethanol and the pH was adjusted to 7.4. An N / P (ionizable nitrogen atoms of the ionizable lipid to anionic phosphor atoms of SDI) molar ratio of 5: 1 was targeted.

[0129] LNP characterization

[0130] The diameter and poly dispersity index (PDI) of LNPs were measured with dynamic light scattering (DLS) at physiological pH. Each sample was measured in triplicate and a cumulative average of z average and PDI was calculated. For ELS, each sample was81099325. v1 33Atorney Docket No. 770075: MTST-553PC appropriately diluted in HEPES buffer and measurements were taken in triplicate. The Zeta potential was calculated for all samples based on the Smoluchowski equation.

[0131] Vaccine-adjuvant preparation and administration

[0132] For each animal, 1.5 mg of HA equivalent of QIV was mixed with empty or SDI- encapsulating S-Ac7-Dog or K-Ac7-Dsa LNPs, with or without 100 mg of IMDQ-PEG-Chol (equivalent to 10 mg of core IMDQ), and vortexed for 30 s. Unadjuvanted or adjuvanted QIV, with or without LNP formulations, was administered intramuscularly in a total of 50 mL per mouse, in the right hind leg. The control group was administered with equal volume of PBS instead of vaccine or vaccine ± adjuvant ± LNP mixture. All animals received only one dose of vaccine without any further boosters.

[0133] IVR-180 virus

[0134] A / Singapore / GP 1908 / 2015 IVR-180 (H1N1) was grown in 8-day-old embryonated chicken eggs and was titrated by plaque assay on pre-seeded MDCK cells.

[0135] Mouse model for vaccination

[0136] The study was performed on 6- to 8-week-old female BALB / c mice strains obtained from Jackson Laboratories, CT. The mice were housed with food and water ad libitum in a pathogen-free facility at Icahn School of Medicine at Mount Sinai, New York. All mice were vaccinated intramuscularly (50 mL; hamstring muscles of the left hind leg; per mouse) and infected intranasally (in 50 mL total volume per mouse) under ketamine / xylazine anesthesia. All procedures were performed according to the protocols approved by the Icahn School of Medicine at Mount Sinai Institutional Animal Care and Use Commitee (IACUC- PROT0202100007).

[0137] Serum collection for serology

[0138] Mice blood was collected by submandibular bleed 3 weeks postvaccination from all animals. The blood was allowed to clot at 4°C for overnight. The serum was collected after a brief centrifugation and was heat inactivated at 56°C for 30 min. The samples were stored at -20°C until further use.81099325. v1 34Atorney Docket No. 770075: MTST-553PC

[0139] Enzyme-linked immunosorbent assay

[0140] ELISA was performed to quantify the vaccine-specific IgG titers in mice sera. Briefly, ELISA plates were coated with recombinant trimeric HA derived from the A / Michigan / 45 / 2015 H1N1 virus, which is closely related to IVR-180, equivalent to 2 pg HINl-HA / mL, in bicarbonate buffer and left overnight at 4°C. Plates were washed three times with l x PBS and incubated in 100 ml of blocking buffer per well [5% fat-free milk in PBST (1 x PBS + 0.1% Tween20)] for 1 h at room temperature (RT).

[0141] In the meantime, the serum samples were serially diluted 3-fold, starting with 1: 100 dilution, in blocking buffer and 50 mL of each sample dilution was incubated on HA-coated ELISA plates overnight at 4°C. The following day, the plates were washed three times with PBST and incubated with 100 mL of diluted horseradish peroxidase (HRP)-conjugated anti -mouse secondary total IgG (1:5,000) or IgGl (1:2,000) or IgG2a (1:2,000) antibodies, for 1 h at RT. Finally, the plates were washed three times in PBST and incubated with 100 pl of tetramethylbenzidine (TMB) substrate at RT until the blue color appeared. The reaction was terminated with 50 pl of 1 M sulfuric acid (H2SO4), and the absorbance was measured at 450 nm and 650 nm wavelengths using BIOTEK ELISA plate reader.

[0142] Hemagglutination inhibition assay

[0143] Mice sera collected 3 weeks post-vaccination were treated with four volumes of receptor destroying enzyme (RDE) at 37°C overnight, followed by treatment with five volumes of 1.5% sodium citrate at 56°C, 30 min. The thus obtained l :20-diluted serum samples were further serially diluted in a transparent Vbottom 96-well plate and incubated with 4 HA units per well of IVR-180 virus for 30 min at RT, followed by the addition of 0.5% chicken red blood cells for 40 min at 4°C. The results were recorded as HAI titers.

[0144] Enzyme-linked immunosorbent spot

[0145] Mice were vaccinated with different combinations of QIV ± adjuvant ± LNPs and spleens were harvested at 10 DPV from all vaccinated as well as unvaccinated animals. Spleens were collected in 5 mL of RPMI-1640 media supplemented with 2% FBS and lxpenicillin / streptomycin and kept on ice. A single-cell suspension of splenocytes was obtained by homogenizing the spleens against a 70- mm strainer. Interferon gamma (IFN-g) or interleukin-481099325. v1 35Atorney Docket No. 770075: MTST-553PC(IL-4) ELIspot assays were performed using 105 splenocytes per well in a 96-well poly vinylidene difluoride (PVDF) ELIspot plates provided in the ELIspot kits, precoated with IFN-g or IL-4 capture antibodies, respectively, according to the manufacturer’s protocol. Splenocytes were left unstimulated or restimulated either with hemagglutination (HA-H1N1) overlapping 15-mer peptides or whole live IVR-180 (H1N1) virus and incubated overnight in 37°C incubator. The wells were washed thrice with water to get rid of cells and incubated with 100 mL of biotinylated polyclonal detection antibody against IFN-g or IL-4 for 1.5 h at RT, followed by an incubation with streptavidin-HRPconjugated antibody for 1 h at RT. The plates were finally incubated with 100 mL of the substrate for 1 h in dark, followed by thorough washing under tap water multiple times. The plates were air-dried in the dark and the number of spots in each well was manually counted using a dissection microscope. The results were represented as number of IFN-g- or IL-4-producing splenocytes per million splenocytes.

[0146] Virus challenge

[0147] A high-titer dose of IVR-180 H1N1 virus of 18,000 PFU, which corresponds to 100* lethal dose that kills 50% of female BALB / c mice, per animal was used for intranasal infection in a final volume of 50 pL per mouse. The virus challenge was performed under mild anesthesia with ketamine / xylazine (intraperitoneal) as recommended by IACUC. The unvaccinated but challenged group was used as a control in the experiment. Body weights were recorded every day post-infection until lung harvest. The lungs were collected at 5 DPI in 500 mL of 1 x PBS and homogenized using a tissue homogenizer. The lysate thus obtained was stored at -80°C for viral titrations.

[0148] Plaque assay

[0149] Virus titrations were performed by plaque assays to quantify the replicating virus titers in the lungs of vaccinated versus unvaccinated mice. The lung homogenate (or lysate) was 10- fold serially diluted in l x PBS and incubated on pre-seeded and prewashed monolayers of MDCK cells for 1 h in an incubator, at 37°C, 5% CO2 with gentle shaking every 5 min. The diluted samples were then removed, and the monolayers were again briefly washed with 1 mL of l x PBS. Lastly, 1 mL of the overlay mixture [2% oxoid agar and 2x minimal essential medium (MEM) supplemented with 1% diethyl-aminoethyl (DEAE)-dextran and 1 mg / mL tosylamide-2 -phenylethyl chloromethyl ketone (TPCK)-treated trypsin] was added on top of the monolayers and incubated for 48 h in the incubator, at 37°C, 5% CO2. The plates were finally81099325. v1 36Atorney Docket No. 770075: MTST-553PC fixed in 4% formaldehyde. The overlay was removed, and the plaques were immune-stained with IVR-180-postchallenge polyclonal serum, diluted 1: 1,000 in blocking buffer. The plates were washed and incubated with 1:5,000 dilution of HRP-conjugated anti -mouse secondary antibody for 1 h at RT with gentle shaking. Followed by a brief washing in 1 x PBST, the plaques were finally visualized with True Blue substrate and the number of plaques was counted and presented as PFU / mL.

[0150] Multiplex cytokine ELISA

[0151] Luminex-based cytokine ELISA was performed for simultaneous measurements of different cytokines in the lung homogenates from IVR-180-infected mice using the Thl / Th2 Cytokine 11-Plex Mouse ProcartaPlexTM kit (Invitrogen; EPX110- 20820-901). Lungs were harvested at 5 DPI, homogenized in 500 mL of PBS, and centrifuged at 5,000 g for 5 min. Twenty-five microliters of each lysate was used for the assay. The following cytokines were measured: Granulocyte macrophage colony-stimulating factor (GMCSF), interleukin (IL)-lb, IL- 4, IL-5, IL-6, IL-12p70, IL-13, IL-18, and interferon gamma (IFN-g). The assay was performed according to the manufacturer’s guidelines and the readings were recorded using the Luminex 100 / 200 system.

[0152] Software

[0153] The schematic figures were created with BioRender.com. GraphPad Prism version 10 was used for data visualization, analysis, graph plotting, and statistical analysis. Principal component analysis was performed using the statistical software package R: R Core Team (2023). R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. R-project.org

[0154] Example 2: Materials and Methods for preparation and characterization of poly(I:C) encapsulated LNPs

[0155] Materials

[0156] Unless otherwise stated, all chemicals were purchased from Sigma Aldrich. Cell culture medium and supplements, phosphate buffered saline (PBS) (lx), penicillin / streptomycin ( 100 / ), sodium pyruvate ( 100 / ), and Quanti-iT RiboGreen RNA assay kit were purchased from Thermo Fischer (USA). HEK-Blue-mTLR3 cells as well as HEK-Blue Detection, Normocin,81099325. v1 37Atorney Docket No. 770075: MTST-553PC poly(I:C) LMW, poly(I:C) LMW rhodamine, and AddaVax were obtained from Invivogen. DOPE and DSG-PEG2000 were purchased from Avanti Polar Lipids. Antimouse SARS-CoV- 2 nucleoprotein and antimouse SARS-CoV-2 S protein antibodies were obtained from the Center for Therapeutic Antibody Development at the Icahn School of Medicine at Mount Sinai, New York. The secondary antibodies with conjugated horseradish peroxidase (HRP) were obtained from Abeam.

[0157] Cell Lines

[0158] The DC2.4 cell line was a kind gift from Dr. Kenneth Rock (University of Massachusetts, Boston, USA). HEK-Blue mTLR3 cells, RAW-Lucia ISG cells, RAW-Lucia ISGO-KO-TRIF cells, and RAW-Lucia ISGO-KO-MAVS cells were purchased from InvivoGen. Vero-E6, obtained from ATCC, and Vero-E6-TMPRSS2 cells (stably expressing TMPRSS2) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 1 * penicillin / streptomycin, and 1 * nonessential amino acids (Gibco), and further with puromycin for Vero- E6-TMPRSS2.

[0159] Mice

[0160] 6-8 week old 129S mice were obtained from JAX Laboratories, MA. Mice were housed with food and water ad libitum in a specified pathogen-free animal facility at Icahn School of Medicine at Mount Sinai. Mice were vaccinated intramuscularly (50 pL per hind leg per mouse) and infected via the intranasal route (105 PFU (plaque forming units) mouse adapted SARS-CoV-2 virus in 50 pL PBS per mouse) under ketamine / xylazine anesthesia. All procedures were approved by the Icahn School of Medicine at Mount Sinai Institutional Animal Care and Use Committee (IACUC-2013-1408). IPS-1 (MAVS- / -)-deficient mice were kindly provided by Dr. Matthias J. Schnell, Ph.D. Homozygous MAVS- / - were bred and housed in specific pathogen free (SPF) facilities maintained by the Center for Comparative Medicine and Surgery at the Icahn School of Medicine at Mount Sinai. All experiments were performed with sex matched mice at 6- 8 weeks of age. All studies were performed in accordance with the principles described by the Animal Welfare Act and the National Institutes of Health guidelines for the care and use of laboratory animals in biomedical research. The protocols for performing mice studies were reviewed and approved by Institutional Animal Care and Use committee (IACUC) at ISMMS.81099325. v1 38Atorney Docket No. 770075: MTST-553PC

[0161] Virus

[0162] The WA1-USA / 2020 SARS-CoV-2 virus was obtained from BEI resources (NR- 52281). The mouse-adapted SARS-CoV-2 virus as obtained after several serial passages of WA1-USA / 2020 SARSCoV-2 virus in mice with different genetic backgrounds, at the Animal Biosafety level 3 (ABSL3) facility at Icahn school of Medicine at Mount Sinai. The virus was further propagated and titrated on Vero E6 cells and diluted appropriately in l x PBS for intranasal infection in animals.

[0163] Instrumentation

[0164] Both DLS and electrophoretic light scatering (ELS) measurements were performed on a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK) equipped with a HeNe laser (A = 633 nm) and detection at a scatering angle of 173°. Flow cytometry analysis was performed on a BD LSRFortessa and BD FACSCanto.Data were processed using the FlowJo software package.

[0165] Samples were prepared in 100 pL PBS and transferred to Eppendorf tubes prior to analysis. Confocal microscopy images were taken via a Leica DMI6000B microscope (63 x 1.40 numerical aperture (NA) objective) coupled to an AndorDSD2 confocal scanner and a Zyla5.5 CMOS camera. Images were processed with Imaged software package.

[0166] LNP Formulation

[0167] Aqueous solutions of poly(I:C) were made by adding 250 pL of a poly(I:C) LMW stock solution (1 mg mL-1 in PBS) to 3.083 mL 5 mm acetate buffer (pH 4). Ethanol solutions (1.667 mL) consisted of S-Ac7-DOg, DOPE, cholesterol, DSG-PEG2000. LNPs were fabricated by solvent displacement, mixing rapidly with a vortex the ethanolic solution of lipids to the aqueous solution of poly(I:C). To remove ethanol, the formed LNP suspensions were dialyzed overnight with PBS using Slide- ALyzer cassettes (cut-off 3.5 kDa) (Thermo Fischer). Next, the LNP suspensions were concentrated by using an Amicon Ultra 10k Centrifugal Filter (MilliporeSigma, USA) (cut-off 3.5 kDa) to yield a poly(I:C) concentration of 0.5 mg mL-1.

[0168] LNP(Poly(I:C)RHO) Formulation81099325. v1 39Atorney Docket No. 770075: MTST-553PC

[0169] Poly(I:C)RHO was formulated into LNP(poly(I:C)RHO) as mentioned above but not dialyzed. To serve as a proper control, poly(I:C)RHO was subjected to identical conditions (e.g., addition of ethanol). Fluorescence emission intensities of poly(I:C)RHO and LNP(poly(I:C)RHO), respectively, were recorded on an Ensight Multimode Microplate reader (PerkinElmer, USA) (Aex = 530 nm; Aem = 560 nm).

[0170] PLS and ELS

[0171] 100 pL of each sample was individually measured thrice via DLS. Cumulants analysis of the data gave the z-average and PDI (data provided as average). For ELS, each sample was diluted 10-fold in 5 mm HEPES buffer (pH 7.4) and was individually measured thrice. Zeta potential values were calculated based on the Smoluchowski equation and were provided as average ± standard deviation (SD).

[0172] Determination of Encapsulation Efficiency: RiboGreen Assay:

[0173] Quant-iT RiboGreen RNA Assay kit (Thermo Fisher, USA) was used to determine poly(I:C) encapsulation efficiency after LNP production. LNP samples were diluted in Tris- EDTA (TE) buffer to obtain a (theoretical) concentration of 1 pg mL-1 poly(I:C) (= working solutions). 50 pL of these working solutions were two-fold diluted in duplicate in a black 96-well plate with TE buffer. Similar samples were prepared with TE buffer supplemented with 2% Triton X-100 to lyse the LNPs. Next, 100 pL of RiboGreen solution (1 : 100 diluted RiboGreen reagent in TE buffer) was added to each well. Fluorescence was measured using an Ensight Multimode Microplate reader (PerkinElmer, USA) (lex = 485 nm; 2em = 528 nm). The resulting fluorescence values were subtracted by the fluorescence value of the reagent blank. The encapsulation efficiency (%) was calculated according to the equation (Ttotal-Ifree) / total x 100, where / total is the fluorescence intensity of samples measured total poly(I:C) in presence of 2% Triton X-100 and / free is the fluorescence intensity of samples measured free or unencapsulated poly(I:C) in absence of 2% Triton X-100.

[0174] In Vitro Cellular Uptake: Flow Cytometry

[0175] DC2.4 cells were seeded in a 24-well plate at a concentration of 200 000 cells per well in 450 pL of culture medium and allowed to adhere overnight at 37 °C (5% CO2). The cells were pulsed overnight, at 37 °C (5%CO2), with 50 pL of poly(I:C)RHO and81099325. v1 40Atorney Docket No. 770075: MTST-553PCLNP(poly(I:C)RHO), respectively, resulting in poly(I:C)RHO concentrations of 0.5 and 1 pg mL-1, respectively. After overnight incubation, the culture medium was aspirated and washed with PBS. Next, 500 pL cell dissociation buffer was added and incubated for 15 min at 37 °C (5% CO2) to detach the cells. The cell suspensions were transferred to Eppendorf tubes and centrifuged (5 min, 300 G, 4 °C). After aspiration of the supernatant, the cell pellets were resuspended in 200 pL PBS and analyzed using a BD LSRFortessa flow cytometer. Data was processed by FlowJo software package.

[0176] In Vitro Cellular Uptake: Confocal Microscopy

[0177] DC2.4 cells were seeded in WillCo-Dish glass bottom at a concentration of 50 000 cells in 180 pL culture medium and allowed to adhere overnight at 37 °C (5% CO2). The cells were pulsed overnight with 20 pL of poly(I:C)-Rho LNP suspension (LNP(poly(I:C)-Rho)) in PBS (50 pg mL-1 poly(I:C)-Rho) or soluble poly(I:C)-Rho (poly(I:C)-Rho) (50 pg mL-1 in PBS) at 37 °C (5% CO2). After overnight incubation, the culture medium was aspirated and the cells were fixated with 4% PFA for 15 min at 37 °C (5%CO2) followed by washing with PBS. A staining solution was prepared by adding 10 pL Hoechst (1 mg mL-1 stock solution in dimethylsulfoxide (DMSO) to 2 mL PBS supplemented with 1% bovine serum albumin (BSA). 200 pL of this staining solution was added to the fixed cells and incubated for 40min at 37 °C (5%CO2). Finally, the samples were washed with PBS followed by confocal imaging using a Leica DMI6000B microscope (63 x 1.40 NA objective) coupled to an AndorDSD2 confocal scanner and a Zyla5.5 CMOS camera. Images were processed with Imaged software package.

[0178] HEK-Blue mTLR3 Innate Immune Activation Assay

[0179] HEK-Blue mTLR3 cells were seeded in a flat-bottom 96-well plate at a concentration of 50 000 cells per well in 180 pL HEK-Blue detection medium (prepared per manufacturer instruction). The cells were pulsed overnight (6-16 h) with 20 pL of poly(I:C) LNP suspension (LNP(poly(I:C))), empty LNP (LNP(- )), soluble poly(I:C) or sterile endotoxine-free water (negative control) at 37 °C (5% CO2) at concentrations of 3.333, 1.667, 0.333, 0.033, and 0.003 pg mL-1 poly(I:C). After overnight incubation, SEAP levels were determined by measuring optical density at 620 nm using an Ensight Multimode Microplate reader (PerkinElmer, USA). Note that colorimetric quantification of the samples was obtained relative to the negative control and each concentration was performed in fivefold.81099325. v1 41Atorney Docket No. 770075: MTST-553PC

[0180] Bio-Plex Cytokine Assay

[0181] Cytokines (IL-lb, IL-4, IL-6, IL-10, IL-12(p70), IFN-y, MCP1, and TNF-a) in the mice serum samples were measured on a Luminex Bio-Plex suspension array system (BioRad, USA) according to the manufacturer’s instructions. Briefly, fourfold diluted serum samples were added to magnetic capture beads (Bio-Rad) and incubated for 2 h at room temperature. After incubation, the beads were washed and a detection antibody (Bio-Rad) was added to the wells. After 1 h incubation the beads were washed again and incubated for 30 min with streptavidin-PE (Bio-Rad). Finally, the samples were measured by Bio-Plex 200 System (Bio-Rad). Cytokine concentrations were analyzed using standard curves and expressed as pg mL-1.

[0182] Anti-SARS-CoV-2 S Protein ELISA

[0183] Anti-SARS-CoV-2 S protein ELISA was performed to estimate S-specific antibody responses upon vaccination as described before. Briefly, Maxisorp Nunc 96-well microtiter plates were coated with 50 pl . per well of recombinant trimeric S protein, diluted to a concentration of 2 pg mL-1 in carbonate / bicarbonate buffer and incubated with 50 pL per well overnight at 4 °C. 50 pL per well of threefold serially diluted serum samples, starting from 1 : 100, were added to the antigen-coated plates followed by overnight incubation at 4 °C. The plates were then washed in 1 * PBS + 0.01% Tween20 and again incubated with appropriate HRP-conjugated secondary antibodies targeting total IgG, IgGl, or IgG2a antibodies at room temperature for 1 h. The plates were washed and developed with 100 pL of tetramethyl benzidine (TMB) substrate per well until blue color appeared. The reaction was terminated with 50 pL 1 m H2SO4 and the absorbance was measured at 450 nm with 650 nm as a reference.

[0184] In Vivo Immune Activation Imaging

[0185] Luciferase-reporter mice (IFN / ?+ / A / ?-luc) with a BALB / c background, aged 7-9 weeks, were housed in individual ventilated cages and given ad libitum access to food and water. 50 pL of poly(LC) LNP suspension (LNP(poly(I:C))), empty LNP (LNP(-)), soluble poly(I:C), or PBS (untreated) were injected intramuscularly in the quadriceps (n = 3) at an equivalent dose of 25 pg poly(I:C). About 6 h post injection, blood was collected from mice for Bio-Plex cytokine analysis (see above). For in vivo imaging at the given time points (0, 4, and 24 h), mice were81099325. v1 42Atorney Docket No. 770075: MTST-553PC injected subcutaneously with 200 pL d-luciferin and in vivo luminescence imaging was recorded 12 min later using the IVIS Lumina II imaging system. Local (injection site and spleen) luminescence and full body luminescence were quantified using the Living Image 4.4 software.

[0186] Analysis of In Vivo Lymphocyte Targeting and Activation

[0187] After the last time point (24 h) of the immune activation imaging (as described above) luciferase-reporter mice were sacrificed, and iliac lymph nodes and spleens were isolated. Next, single cell suspensions were prepared from the dissected lymph nodes and spleens for flow cytometry analysis. Isolated lymph nodes and spleens were collected in ice cold PBS, smashed through 70 pm cell strainers, washed with PBS and stained for 30 min at 4 °C with following primary labeled antibodies: CD3, CD20, CDl lc, MHCII, CD86, CD80, CD40, CD69, and CCR7. Live-dead ratios were determined by staining with fixable dead / live staining and 123 count ebeads were added to determine cellularity prior to analysis by a BD FACSCan to flow cytometer. Data were processed using the FlowJo software package.

[0188] In Vivo Cellular Uptake by Immune Cell Subsets

[0189] Luciferase-reporter mice (IFN / ?+ / A / ?-luc) with a B ALB / c background, aged 7-9 weeks, were housed in individual ventilated cages and given ad libitum access to food and water. 50 pL poly(I:C)-Rhodamine LNP suspension (LNP(poly(I:C)-Rho)), empty LNP (LNP(-)), soluble poly(I:C)-Rhodamine (soluble poly(I:C)-Rho), or PBS (untreated) were injected intramuscularly in the quadriceps (n = 3) at an equivalent poly(I:C) dose of 25 pg. About 24 h post injection, mice were sacrificed and iliac lymph nodes and spleens were isolated. Next, single cell suspensions were prepared from the dissected iliac lymph nodes and spleens for analysis by flow cytometry. Isolated lymph nodes and spleens were collected in ice cold PBS, smashed through 70 pm cell strainers, washed with PBS and stained for 30 min at 4 °C with DAPI and with the following primary labeled antibodies: CD11c, B220, and CD169. 123 count ebeads were added to determine cellularity prior to analysis by a BD FACSCanto flow cytometer. Data were processed using the FlowJo software package.

[0190] Trimeric recombinant SARS-CoV-2 S protein vaccination

[0191] Trimeric full length recombinant S protein was produced as followed: only the ectodomain of the S protein (GenBank: MN908947.3) was cloned into a mammalian expression81099325. v1 43Atorney Docket No. 770075: MTST-553PC plasmid and the cleavage site was removed and stabilizing prolines were added at positions 986 and 987 (24- 26). A hexahistidine tag as well as a T4 fold on trimerization domain was present in the plasmid for ease of purification. The S protein was expressed in 293F cells, using the ExpiFectamine 293 Transfection Kit (Thermo Fisher). Supernatant was collected on day 3 post transfection and Ni-NTA agarose (Qiagen) was used to purify the protein. S protein (5 jig per mouse) was used as such or mixed with adjuvant as described below and injected following a prime-boost schedule via the TM route with a BD 300 pL insulin syringe in the hamstring muscles of one hind leg (50 pL per mouse).

[0192] Adjuvants

[0193] Soluble poly(T:C), LNP(poly(T:C)), LNP(-), and AddaVax (0.2 mg mL-1 poly(T:C) or AddaVax) were mixed 1 : 1 volume ratio with S protein (0.2 mg mL-1) to yield an equivalent dose of 5 gg S protein and poly(T:C) per mouse.

[0194] Tn Vitro Microneutralization Assay

[0195] To measure the neutralizing potential of SARS-CoV-2 vaccine-induced sera, an in vitro microneutralization assay was performed. Briefly, the S protein ± adjuvant- vaccinated mice sera were inactivated at 56 °C for 30 min. Serum samples were serially diluted threefold starting from 1:30 dilution in infection medium (DMEM + 2% Fetal Bovine Serum (FBS) + l x nonessential amino acids). The samples were incubated with 350 TCTD50 of SARS-CoV-2 isolate USAWA1 /

[0197] 2020 (BET resources; NR-52281) for 1 h in an incubator at 37 °C (5% CO2) and then transferred on preseeded Vero E6-TMPRSS2 cells in 96- well cell culture plates. The plates were incubated at 37 °C for 48 h and fixed in 4% formaldehyde. The cells were permeabilized with 0.1% Triton X-100 for 15 min at room temperature (RT) and washed three times with 1XPBS + 0.1% Tween 20 (PBST). The cells were then blocked in 5% milk in PBST for 1 h at RT. After blocking, the cells were incubated with anti-SARSCoV- 2 nucleoprotein and anti-SARS-CoV-2 S protein monoclonal antibodies, mixed in a 1: 1 volume ratio, for 1.5 h at RT. The cells were washed again and incubated with HRP-conjugated antimouse TgG secondary antibody for 1 h at room temperature followed by a brief PBS wash. Finally, 100 pL of TMB substrate was added and incubated until blue color appeared and the reaction was terminated with 50 pL 1 m H2SO4. Absorbance at 450 nm was recorded and percentage inhibition calculated.

[0196] Lung Virus Titration81099325. v1 44Atorney Docket No. 770075: MTST-553PC

[0197] Plaque assays were performed to quantify and compare the replicating lung viral titers in vaccinated versus unvaccinated mice. Whole lungs and nasal turbinates were harvested from the mice 4 days post infection and homogenized in 500 pL 1 * PBS. After brief centrifugation, the tissue debris was discarded and the supernatant was tenfold serially diluted starting from 1 : 10 dilution. Pre-seeded Vero-E6 cells were incubated with diluted lung and nasal turbinate homogenates for 1 h at room temperature and then overlaid with 1 mL mixture of 2% oxoid agar and 2* minimal essential medium (MEM) supplemented with 2% FBS. After 72 h of incubation at 37 °C (5% CO2) the plates were fixed in 4% formaldehyde, followed by immune-staining of infected cells with antimouse SARS-CoV-2 nucleoprotein and antimouse SARS-CoV-2 S protein monoclonal antibodies. After incubation with primary antibodies, HRPconjugated antimouse secondary antibody was added for 1 h. Finally, the plaques were developed with TrueBlue substrate (KPL-Seracare). The final viral titers were calculated in terms of PFU mL-1.

[0198] Various embodiments are also contemplated:1. A lipid nanoparticle (LNP) comprising at least one ionizable cationic lipid.2. A lipid nanoparticle (LNP) composition comprising at least one ionizable cationic lipid and Polyinosinic:polycytidylic acid (poly(I:C)).3. The LNP composition of embodiment 1 or embodiment 2, wherein the ionizable cationic lipid comprises an azepanyl moiety.4. The LNP composition of any one of embodiments 1 to 3, wherein the ionizable cationic lipid comprises S-Ac7-Dog or K-Ac7-Dsa5. The LNP composition of any one of embodiments 1 to 4, wherein the LNP composition further comprises phospholipid, cholesterol, and / or PEG-lipid.6. The LNP composition of any one of embodiments 1 to 5, wherein the Poly(I:C) is encapsulated by the LNP.7. An adjuvant formulation comprising the LNP composition of any one of embodiments1 to 6.81099325. v1 45Atorney Docket No. 770075: MTST-553PC8. An immunogenic composition comprising the adjuvant formulation of embodiment 7 and an immunogen or antigen.9. The immunogenic composition of embodiment 8, wherein the immunogen or antigen is capable of eliciting immune response against an infectious disease.10. The immunogenic composition of embodiment 9, wherein the infectious disease is selected from the group consisting of CO VID-19, Influenza, Pneumococcal pneumonia, RSV, and combinations thereof.11. The immunogenic composition of any one of embodiments 8 to 10, wherein the immunogenic composition is in the form of lyophilized powder, aqueous solution, suspension, microemulsion, or dispersion.12. The immunogenic composition of any one of embodiments 8 to 11, wherein the immunogenic composition is suitable for intravenous administration, subcutaneous administration, intramuscular administration, parenteral administration, rectal administration, spinal cord administration, epidermal administration, infusion administration, or intraperitoneal administration.13. The immunogenic composition of any one of embodiments 8 to 12, wherein the immunogenic composition is a vaccine or a therapeutic composition.14. A method of inducing an immune response, the method comprising administering an effective amount of the immunogenic composition of any one of embodiments 8 to 13 to a subject in need thereof.15. A method of preventing or treating an infectious disease or, the method comprising administering an effective amount of the immunogenic composition of any one of embodiments 8 to 13 to a subject in need thereof.16. The use of the immunogenic composition of any one of embodiments 8 to 13 for inducing an immune response in a subject in need thereof.17. The use of the immunogenic composition of any one of embodiments 8 to 13 for preventing or treating an infectious disease in a subject in need thereof.81099325. v1 46Attorney Docket No. 770075: MTST-553PC

[0199] All publications, patents, and patent applications cited herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0200] It will be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.81099325. v1 47

Claims

Attorney Docket No. 770075: MTST-553PCCLAIMS1. A lipid nanoparticle (LNP) composition comprising at least one ionizable cationic lipid and Polyinosinic:polycytidylic acid (poly(I:C)).

2. The LNP composition of claim 1, wherein the ionizable cationic lipid comprises an azepanyl moiety.

3. The LNP composition of claim 1 or claim 2, wherein the ionizable cationic lipid comprises S-Ac7-Dog or K-Ac7-Dsa4. The LNP composition of any one of claims 1 to 3, wherein the LNP composition further comprises phospholipid, cholesterol, and / or PEG-lipid.

5. The LNP composition of any one of claims 1 to 4, wherein the Poly(I:C) is encapsulated by the LNP.

6. An adjuvant formulation comprising the LNP composition of any one of claims 1 to 5.

7. An immunogenic composition comprising the adjuvant formulation of claim 6 and an immunogen or antigen.

8. The immunogenic composition of claim 7, wherein the immunogen or antigen is capable of eliciting immune response against an infectious disease.

9. The immunogenic composition of claim 8, wherein the infectious disease is selected from the group consisting of COVID-19, Influenza, Pneumococcal pneumonia, RSV, and combinations thereof.

10. The immunogenic composition of any one of claims 7 to 9, wherein the immunogenic composition is in the form of lyophilized powder, aqueous solution, suspension, microemulsion, or dispersion.

11. The immunogenic composition of any one of claims 7 to 10, wherein the immunogenic composition is suitable for intravenous administration, subcutaneous81099325. v1 48Attorney Docket No. 770075: MTST-553PC administration, intramuscular administration, parenteral administration, rectal administration, spinal cord administration, epidermal administration, infusion administration, or intraperitoneal administration.

12. The immunogenic composition of any one of claims 7 to 11, wherein the immunogenic composition is a vaccine or a therapeutic composition.

13. A method of inducing an immune response, the method comprising administering an effective amount of the immunogenic composition of any one of claims 7 to 12 to a subject in need thereof.

14. A method of preventing or treating an infectious disease or, the method comprising administering an effective amount of the immunogenic composition of any one of claims 7 to 12 to a subject in need thereof.

15. A use of the immunogenic composition of any one of claims 7 to 12 for inducing an immune response in a subject in need thereof.

16. A use of the immunogenic composition of any one of claims 7 to 12 for preventing or treating an infectious disease in a subject in need thereof.81099325. v1 49

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