Vaccine targeting to mucosal lymphoid tissues in the gastrointestinal tract

Abstract

Viruses, bacteria, and parasites frequently cause infections in the gastrointestinal (GI) tract, but traditional vaccination strategies typically elicit little or no mucosal antibody responses. Disclosed herein is a vaccine comprising a nanoemulsion and an immunogen non-covalently conjugated to the surface of the nanoemulsion by an amphiphilic linker, wherein the vaccine is suitable for inducing a mucosal antibody response in the gastrointestinal tract. Also disclosed are methods of using the vaccine to immunize a subject by intraperitoneal administration of an effective amount of the vaccine, alone or with an adjuvant.

Description

[0001] VACCINE TARGETING TO MUCOSAL LYMPHOID TISSUES IN THE

[0002] GASTROINTESTINAL TRACT

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0004]

[0001] This invention was made with government support under UM 1 Al 144462 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0005] REFERENCE TO RELATED APPLICATIONS

[0006]

[0002] This application claims priority to U.S. Provisional Application No. 63 / 572,715, filed on April 1, 2024. The entire contents of the aforementioned application are expressly incorporated herein by reference.

[0007] REFERENCE TO SEQUENCE LISTING

[0008]

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on March 31, 2025, is named 127299-03820-SL.XML and is 5,608 bytes in size.

[0009] INCORPORATION BY REFERENCE

[0010]

[0004] All documents cited or referenced herein and all documents cited or referenced in the herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated by reference, and may be employed in the practice of the invention.

[0011] BACKGROUND OF THE INVENTION

[0012]

[0005] To combat long-standing epidemics such as HIV and emerging threats such as SARS- CoV-2, immunization strategies are needed that can elicit systemic antibody responses and humoral immunity at mucosal portals of entry in tandem. Many pathogens including HIV, SARS-CoV-2, influenza, rotavirus, and cholera infect the host through mucosal surfaces and thus are thought to require engagement of both systemic and mucosal branches of the immune system, employing a combination of IgG and IgA antibodies, for effective management and protection. Secretory IgA (SIgA) is the main humoral defense at mucosal tissue sites and plays a particularly important role in providing protection through mechanisms such as immune exclusion, inhibition of transcytosis, and direct neutralization of virus. Establishment of antigen- specific SIgA antibodies at mucosal surfaces provides a frontline defense that can help prevent infection and transmission. With HIV, where 90% of transmissions occur via mucosal routes, induction of mucosal IgA responses (in combination with systemic IgG) has been found to be effective in promoting protection against mucosal SHIV challenge in primates. Similarly, SARS-CoV-2 clinical studies have shown that mucosal IgA exhibits potent neutralization and is a strong correlate of protection against the virus, which primarily infects cells in the upper and lower respiratory mucosa.

[0013]

[0006] Traditional parenteral immunization regimens typically elicit poor mucosal immunity. By contrast, vaccination at mucosal surfaces, which initiates immune responses in mucosa associated lymphoid tissues (MALT), is known to be a very effective strategy to promote protective immunity at barrier tissues due to programming of mucosa- specific lymphocyte function and tissue homing at these sites. Priming of mucosal T and B lymphocytes takes place in MALT inductive sites, such as the nasal- associated lymphoid tissue (NALT) and gut-associated lymphoid tissue (GALT). Through a property of the ‘common mucosal immune system’, antigen priming can induce expression of homing markers that lead activated antigen specific T cells, B cells, and plasma cells to migrate to other local or distal mucosal effector sites. The location of antigen exposure determines which homing markers are expressed, dictating the homing destination and ultimate effector site. Typically, the strongest response is elicited at the site of antigen exposure and in the most anatomically adjacent mucosal tissue.

[0014]

[0007] Although well-motivated by the biology of mucosal immunity, delivery of vaccine components across mucosal barriers has been a major challenge for mucosal vaccine development. Vaccine uptake into the underlying mucosal immune compartment is impeded by multiple factors, including potential rapid antigen loss due to degradation by proteolytic enzymes and acidic conditions at mucosal surfaces, high rates of mucociliary clearance, and the lack of diffusive uptake across the tight junctions of the epithelial monolayer. In fact, only a small number of mucosal vaccines have reached licensure, all of which except the inactivated oral cholera vaccine are based on live attenuated pathogens that naturally infect mucosal surfaces, such as the oral polio vaccine (OPV) or the intranasal influenza type A / B vaccine (EluMist). However, live attenuated vaccines often face manufacturing challenges, poor stability, and safety concerns. These challenges have been addressed in parenteral vaccines by a focus on recombinant protein- or polysaccharide-based subunit vaccines that are safe, stable, and highly manufacturable, but subunit vaccines have historically exhibited poor immunogenicity and short-lived responses when applied to mucosal barriers due in large part to challenges of delivery and poor uptake.

[0015]

[0008] Gut mucosal immune responses are programmed in the GALT and mesenteric lymph nodes (mesLNs), but delivery of vaccines to these inductive sites is a major challenge.

[0016] Antigen uptake directly from the gut lumen can directly enter Peyer’s patches or traffic in lymph to the mesenteric LNs, but orally-administered vaccines must survive the proteolytic environment of the GI tract, penetrate the mucus overlying the gut epithelium, and overcome the tolerogenic state favored for gut-derived antigens. Development of technologies to overcome barriers to mucosal delivery in the GI tract while meeting safety and efficacy requirements of prophylactic vaccines remains an urgent unmet need.

[0017] SUMMARY OF THE INVENTION

[0018]

[0009] This Summary introduces a selection of concepts in simplified form that are described further below in the Detailed Description. This Summary neither identifies key or essential features, nor limits the scope, of the claimed subject matter.

[0019]

[0010] In one aspect, the present disclosure provides a vaccine comprising: a nanoemulsion comprising a surfactant shell and a lipophilic core; and an immunogen non-covalently conjugated to the surface of the nanoemulsion by an amphiphilic linker.

[0020] [Oil] In some embodiments, the vaccine is capable of inducing a mucosal antibody response in the gastrointestinal tract.

[0021]

[0012] In some embodiments, the amphiphilic linker is capable of dissociating from the nanoemulsion, e.g., to transfer the immunogen to a cell.

[0022]

[0013] In some embodiments, the amphiphilic linker comprises a lipid covalently linked to a hydrophilic group,

[0023]

[0014] In some embodiments, the lipid of the amphiphilic linker is embedded in the surfactant shell of the nanoemulsion.

[0024]

[0015] In some embodiments, the immunogen is covalently linked to the hydrophilic group of the amphiphilic linker.

[0025]

[0016] In some embodiments, the lipid of the amphiphilic linker is a monoacyl lipid or a diacyl lipid.

[0017] In some embodiments, the monoacyl lipid or diacyl lipid has at least 8 carbons in the hydrocarbon tail(s).

[0026]

[0018] In some embodiments, the monoacyl lipid or diacyl lipid has between 8 and 18 carbons in the hydrocarbon tail(s).

[0027]

[0019] In some embodiments, the lipid is l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

[0028]

[0020] In some embodiments, the lipid comprises a palmitoyl group.

[0029]

[0021] In some embodiments, the lipid comprises cholesterol or the lipid is cholesterol.

[0030]

[0022] In some embodiments, the hydrophilic group comprises a polyethylene glycol (PEG), a polysaccharide, a polyvinyl alcohol, or a polypeptide.

[0031]

[0023] In some embodiments, the hydrophilic group is a PEG. In some embodiments, the PEG comprises 2 to 96 ethylene glycol subunits (PEG2-PEG96). In some embodiments, the PEG is PEG24. In some embodiments, the amphiphilic linker comprises cholesterol-PEG24 or DSPE-PEG24.

[0032]

[0024] In some embodiments, the nanoemulsion is an oil-in-water nanoemulsion.

[0033]

[0025] In some embodiments, the lipophilic core comprises a biodegradable oil.

[0034]

[0026] In some embodiments, the biodegradable oil is an animal oil or plant oil.

[0035]

[0027] In some embodiments, the biodegradable oil is selected from the group consisting of squalene oil, castor oil and sesame oil.

[0036]

[0028] In some embodiments, the surfactant shell comprises one or more surfactants selected from the group consisting of Span® 85, Span® 80, Tween® 80, and Tween® 20.

[0037]

[0029] In some embodiments, the nanoemulsion is (i) smaller than 200 nm in diameter; or

[0030] (ii) about 1-200 nm, about 1-100 nm, about 1-50 nm, about 1-30 nm, about 1-20 nm, about 5-50 nm, about 5-30 nm, or about 5-20 nm in diameter.

[0038]

[0031] In some embodiments, the immunogen is derived from a disease-causing organism selected from a bacteria, fungus, parasite, and virus.

[0039]

[0032] In some embodiments, the immunogen comprises a peptide antigen or a protein antigen.

[0040]

[0033] In some embodiments, the immunogen is selected from the group consisting of a human immunodeficiency virus (HIV) antigen, a SARS-CoV-2 antigen, an influenza antigen, a rotavirus antigen, a cytomegalovirus (CMV) antigen, an Epstein-Barr virus (EBV) antigen, a respiratory syncytial virus (RSV) antigen, and a cholera antigen.

[0034] In some embodiments, the HIV antigen comprises HIV gpl20 engineered outer domain-germ line-targeting immunogen 8 (eOD-GT8),

[0041]

[0035] In some embodiments, the SARS-CoV-2 antigen comprises an antigen from the receptor-binding domain (RBD) of SARS-CoV-2 spike protein.

[0042]

[0036] In some embodiments, the immunogen further comprises a pan human leukocyte antigen DR-binding epitope (PADRE) peptide, a T-helper epitope from tetanus toxoid, or a T-helper peptide from diphtheria toxoid.

[0043]

[0037] In some embodiments, the immunogen comprises a polysaccharide, optionally wherein the polysaccharide is a bacterial polysaccharide.

[0044]

[0038] In some embodiments, the nanoemulsion further comprises an adjuvant in the lipophilic core.

[0045]

[0039] In some embodiments, the adjuvant is lipophilic.

[0046]

[0040] In some embodiments, the adjuvant comprises a TLR7 / 8 agonist, optionally wherein the TLR7 / 8 agonist comprises 3M-052.

[0047]

[0041] In some embodiments, the adjuvant comprises CpG-cholesterol or 3D(6-acyl)- PHAD™.

[0048]

[0042] In some embodiments, intraperitoneal (i.p.) administration of the vaccine elicits or enhances production of antibodies that bind to the immunogen.

[0049]

[0043] In some embodiments, the antibodies comprise IgA antibodies, IgG antibodies, or IgA and IgG antibodies.

[0050]

[0044] In some embodiments, the antibodies are neutralizing antibodies.

[0051]

[0045] In another aspect, the present disclosure provides composition comprising the vaccine described herein and a pharmaceutically acceptable carrier.

[0052]

[0046] In some embodiments, the composition further comprises an adjuvant.

[0053]

[0047] In some embodiments, the adjuvant is polyinosinic-polycytidylic acid (poly-IC) or a STING agonist, optionally wherein the STING agonist comprises cyclic di-guanylate (c-di- GMP).

[0054]

[0048] In another aspect, the present disclosure provides a method of vaccinating a subject, comprising administering to the subject an effective amount of the vaccine described herein or the composition described herein, thereby vaccinating the subject.

[0055]

[0049] In yet another aspect, the present disclosure provides a method of immunizing a subject, comprising administering to the subject an effective amount of the vaccine described herein or the composition of described herein, thereby immunizing the subject.

[0050] In some embodiments, the vaccine or the composition is administered via intraperitoneal (i.p.) injection.

[0056]

[0051] In some embodiments, administration of the vaccine or the composition elicits or enhances production of antibodies that bind to the immunogen in the gastrointestinal tract.

[0052] In some embodiments, administration of the vaccine or the composition elicits or enhances production of IgA antibodies that bind to the immunogen in the subject.

[0057]

[0053] In another aspect, the present disclosure provides a method of eliciting antiimmunogen IgA antibodies in a subject, comprising administering via intraperitoneal (i.p.) injection to the subject an effective amount of the vaccine described herein or the composition described herein, thereby eliciting anti-immunogen IgA antibodies in the subject.

[0058]

[0054] In another aspect, the present disclosure provides a method of eliciting a mucosal antibody response in the gastrointestinal tract of a subject, comprising administering via intraperitoneal (i.p.) injection to the subject an effective amount of the vaccine described herein or the composition described herein, thereby eliciting a mucosal antibody response.

[0055] In some embodiments, the nanoemulsion traffics to the mesenteric lymph node, mediastinal lymph node, or both the mesenteric lymph node and mediastinal lymph node.

[0056] In some embodiments, the immunogen accumulates at the mesenteric lymph node, mediastinal lymph node, or both the mesenteric lymph node and mediastinal lymph node.

[0059]

[0057] In some embodiments, the vaccine or the composition is administered in one or more doses.

[0060]

[0058] In some embodiments, the vaccine or the composition is administered in at least 2 doses.

[0061]

[0059] In some embodiments, doses of the vaccine are administered about 2, 3, 4, 5, 6, 7, or 8 weeks apart.

[0062]

[0060] In some embodiments, each dose comprises about 5-300 pg of the immunogen.

[0063]

[0061] In another aspect, the present disclosure provides a composition comprising a nanoemulsion (NE) carrier; a protein antigen; a PEG-lipid surfactant, wherein the PEG- lipid antigen is covalently attached to the protein antigen; and a toll-like receptor (TLR) agonist.

[0064]

[0062] In some embodiments, the NE carrier comprises squalene oil, span® 85 and Tween® 80.

[0063] In some embodiments, the NE carrier further comprises azide-functionalized PEG- lipid surfactants.

[0065]

[0064] In some embodiments, the TLR agonist comprises TLR7 / 8 agonist 3M-052.

[0066]

[0065] In some embodiments, the NE carrier further comprises the TLR agonist.

[0067]

[0066] In some embodiments, the azide-functionalized polyethyleneglycol (PEG)-lipid surfactants comprise cholesterol-PEG24-azide or l,2-distearoyl-sn-glycero-3- phosphoethanolamine-PEG24-azide (DSPE-PEG24-azide).

[0068]

[0067] In some embodiments, the azide-functionalized polyethyleneglycol (PEG)-lipid surfactants comprise cholesterol-PEG24-azide.

[0069]

[0068] In some embodiments, the protein antigen is covalently attached to pan human leukocyte antigen DR-binding epitope (PADRE) peptide.

[0070]

[0069] In some embodiments, the PADRE peptide is attached at the C terminus of the protein antigen.

[0071]

[0070] In some embodiments, a terminal free cysteine is covalently attached at the N- terminus of the protein antigen.

[0072]

[0071] In some embodiments, the terminal free cysteine is coupled to dibenzocyclooctyne (DBCO)-derivatized-PEG12-maleimide.

[0073]

[0072] In some embodiments, the NE comprises nanoparticles of 12-20 nm size in diameter.

[0074]

[0073] In some embodiments, the protein antigen is derived from a disease-causing organism selected from a bacteria, fungus, parasite, and virus.

[0075]

[0074] In some aspects, the present disclosure provides a method of preventing or treating a disease, comprising administering to a subject in need thereof the composition described herein.

[0076]

[0075] In some embodiments, the administering to a subject comprises an intraperitoneal injection.

[0077]

[0076] In some embodiments, the method further comprises administering a booster injection.

[0078]

[0077] In another aspect, the present disclosure provides a method of eliciting anti-protein antigen IgA antibodies, the method comprising administering to a subject in need thereof the composition described herein.

[0079]

[0078] In another aspect, the present disclosure provides a method of eliciting a mucosal antibody response, the method comprising administering to a subject in need thereof the composition described herein.

[0079] In another aspect, the present disclosure provides a vaccine comprising the composition of one any of claims 1-13.

[0080]

[0080] In another aspect, the present disclosure provides a kit comprising the vaccine described herein. In some embodiments, the kit further comprises instructions for the method described herein.

[0081]

[0081] The following Detailed Description references the accompanying drawings which form a part this application, and which show, by way of illustration, specific example implementations. Other implementations may be made without departing from the scope of the disclosure.

[0082] BRIEF DESCRIPTION OF THE DRAWINGS

[0083]

[0082] FIGs. 1A-1G show synthesis and characterization of antigen-coupled nanoemulsions. FIG. 1A: schematic of Nanoemulsion-eOD-GT8 conjugate. The amphiphilic linker is cholesterol-PEG24-azide (eOD-Chol-NE) or DSPE-PEG24-azide (eOD-DSPE-NE). FIG. IB: Size exclusion chromatography (SEC) profiles of eOD-Chol-NE and eOD-DSPE-NE together with unconjugated eOD-GT8. SEC experiments were conducted with fluorescent NE carrying BODIPY-cholesteryl ester and AlexaFluor 647- conjugated eOD-GT8. FIG. 1C: Dynamic light scattering (DLS) analysis (size distribution by volume) eOD-Chol-NE and eOD-DSPE-NE together with unconjugated eOD-GT8 and NEs. FIG. ID: Representative Cryo-TEM images of the eOD-Chol-NE and eOD-DSPE-NE.

[0083] FIGs. 2A-2J: Nanoemulsions target cholesterol-anchored antigen to mesenteric lymph nodes. Representative in-vivo imaging system (IVIS) images (FIGs 2A, 2C, 2E) and quantification (FIGs. 2B, 2D, 2F) of fluorescence from Alexa647-labeled eOD in excised LNs 6 hr following i.v. (FIGs. 2A, 2B), s.c. (FIGs. 2C, 2D), or i.p. (FIGs. 2E, 2F) injection of eOD-Chol-NE, eOD-DSPE-NE or eOD / NE (10 pg eOD, 5 pg 3M-052, 0.5 pg squalene NE per vaccine dose, n=4 mice / group). Statistical significance was determinedd by one-way ANOVA followed by Tukey’s post hoc test. FIGs. 2G-2J: Fluorophore-labeled (eOD- AlexaFluor647 and NE-Bodipy Cholesterylester) eOD-Chol-NE, eOD-DSPE-NE and eOD / NE formulations were injected i.p. in balb / C mice (n=4 animals / group, same dose as in FIGs. 2A-2F) and at different time points, mesLNs were isolated for IVIS imaging. Shown are representative fluorescence images of NE (oil phase, FIG. 2G) and eOD antigen (FIG. 21) accumulation, and mean NE fluorescence signals of NE (FIG. 2H) and eOD antigen (FIG. 2J) over time. Statistical significance was determined by two-way ANOVA followed by Tukey’s post hoc test, ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0084]

[0084] FIGs. 3A-3J: Cholesterol-conjugated antigen transfers from nanoemulsions to lymphocyte membranes in vitro and in vivo. FIGs. 3A-3D: Splenocytes were incubated with fluorescently labeled eOD-Chol-NE, eOD-DSPE-NE or eOD / NE at the indicated concentrations for 1 hour in RPMI medium with 5% fetal bovine serum at 37 °C, then washed and stained with VRC01-PE antibody (n=3 samples / group). Shown are representative flow cytometry plots of eOD-GT8 and NE uptake by splenocytes (at 75nM eOD-GT8) (FIG. 3A), the percentage of eOD-GT8 (left) or NE positive cells (right) as a function of eOD- GT8 concentration (FIG. 3B), representative flow cytometry plots of eOD-GT8 signal and VRC01 binding (FIG. 3C), and quantification of the mean frequency of eOD-GT8+VRC01+ double positive cells as a function of eOD-GT8 concentration (FIG. 3D). Statistical significance was determined by two-way ANOVA followed by Tukey’s post hoc test, ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. FIGs. 3E-3J: Balb / c mice (n=4 mice / group) were injected i.p. with fluorescent eOD-Chol-NE, eOD-DSPE-NE and eOD / NE (10 pg eOD, 5 pg 3M-052, 0.1 pg squalene NE), mesLNs were collected 24 hr later, and eOD-GT8 / nanoemulsion uptake by the cells was analyzed by flow cytometry. Shown are representative flow cytometry plots showing eOD-GT8 and NE uptake by dendritic cells (FIG. 3E), macrophages (FIG. 3G), and B cells (FIG. 31); and quantification of mean frequencies of eOD or NE-positive cells (FIGs. 3F, 3H, 3J). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test, ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0085]

[0085] FIGs. 4A-4L: Targeting of vaccines to mesLNs using nanoemulsions amplifies mucosal germinal center and antibody responses. FIGs. 4A-4H: Balb / c mice (n=5 mice / group) were immunized with eOD-Chol-NE, eOD-DSPE-NE or eoD / NE (10 pg eOD- GT8, 5 pg 3M-052, 0.1 pg squalene NE) and GC responses in mesLNs analyzed by flow cytometry at day 12. Shown are representative flow cytometry gating of CXCR5+PD-1+ Tfh cells (FIG. 4A), total GC B cells (FIG. 4C), eOD-GT8-binding GC B cells (FIG. 4E), and IgA+ eOD-GT8-binding GC B cells (FIG. 4G), as well as mean frequencies of Tfh (FIG. 4B), GC B cells (FIG. 4D), eOD- GT8-binding GC B cells (FIG. 4F), and IgA+eOD-GT8+ GC B cells (FIG. 4H). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test, ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. FIGs. 4I-4L: Balb / C mice (n=5 animals / group) were immunized with eOD-Chol- NE, eOD-DSPE-NE or eoD / NE (-10 pg eOD-GT8, 5 pg 3M-052, 0.1 pg squalene NE), boosted 4 weeks later, and antibody titers in serum IgG titers (FIG. 41), serum IgA titers (FIG. 4J), fecal IgG titers (FIG. 4K), and fecal IgA titers (FIG. 4L). Statistical significance was d by two-way ANOVA followed by Tukey’s post hoc test, ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0086]

[0086] FIGs. 5A-5N: Nanoemulsion RBD vaccines promote cross-reactive SARS-CoV-2 systemic and gut mucosal antibody responses. Balb / C mice (n=5 mice / group) were immunized with RBDJ-Chol-NE, RBDJ-DSPE-NE or RBDJ / NE (10 pg RBD, 5 pg 3M-052, 0.1 pg squalene NE) and boosted 4 weeks later. Shown are serum IgG titers (FIGs. 5A, 5E, 51), serum IgA titers (FIGs. 5B, 5F, 5J), fecal IgG titers (FIGs. 5C, 5G, 5K) and fecal IgA titers (FIGs. 5D, 5H, 5L) at week 6 against RBD J, the SI protein of the SARS-CoV-2 D614G variant and RBD protein of the SARS-CoV-2 Omicron variant, respectively. FIGs. 5M-5N: Serum (FIG. 5M) and feces (FIG. 5N) SARS- CoV-2 D614G variant pseudo-virus ID50 neutralizing titers. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test, ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0087]

[0087] FIGs. 6A-6B: Synthesis and characterization of DBCO-PEG12-Maleimide modified eOD-GT8. FIG. 6A: A schematic of eOD-GT8 conjugation protocol to DBCO- PEG12-Maleimide. FIG. 6B: Absorbance curves before and after the conjugation.

[0088]

[0088] FIGs. 7A-7H: Synthesis and characterization of antigen conjugated NEs. FIGs. 7A-7B: Schematic of 3M-052-loaded Cholesterol-PEG24-Azide (Chol-NE, FIG. 7A) or DSPE-PEG24-Azide (DSPE-NE, FIG. 7B). FIGs. 7C-7D: Absorbance curves of the Chol-NE (FIG. 7C) and DSPE-NE (FIG. 7D) before and after 3M-052 loading and washing to remove unloaded TLR7 agonist. The peak at -320 nm is specific to 3M-052. FIGs. 7E-7F: Size exclusion chromatography (SEC) profiles of Chol-NE (FIG. 7E) and DSPE-NE (FIG. 7F) showing 3M-052 incorporation in the NEs. For SEC analysis, BODIPY-cholesteryl ester was encapsulated in the NEs and absorbance wavelength of the dye (at 510 nm) was used to trace the NEs together with the absorbance wavelength of 3M-052. FIG. 7G: A schematic of DBCO modified eOD-GT8 conjugation to the NEs. FIG. 7H: DLS analysis (size distribution by intensity) of eOD-Chol-NE and eOD-DSPE-NE.

[0089]

[0089] FIGs. 8A-8C: Schematic, SEC profile and in-vivo trafficking of the eOD-GT8 with cholesterol tail. FIG. 8A: A schematic of eOD-GT8 conjugated to cholesterol-PEG24- azide. FIG. 8B: Size exclusion chromatography (SEC) profiles of the conjugate and free eOD-GT8. FIG. 8C: Fluorophore-labeled conjugate and eOD-Chol-NE injected IP and 6h later tissues were collected. Representative IVIS images showing eOD-GT8 accumulation in the mesenteric lymph nodes and respective fluorescence quantification. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. **P < 0.01; ***P < 0.001. All data show means ± SEM.

[0090]

[0090] FIGs 9A-9B: In-vitro antigen transfer from NEs to the splenocytes in the FBS- free RPMI medium. Splenocytes were incubated with eOD-Chol-NE, eOD-DSPE-NE or eOD / NE for 1 hour, in FBS-free medium at 37°C at a range of concentrations, then washed and stained with VRC01-PE antibody. FIG. 9A: Representative flow cytometry plots are shown of eOD-GT8 uptake in FBS free RPMI medium and VRC01 binding to the cells (at 75nM of eOD-GT8). FIG. 9B: The percentage of eOD-GT8 and VRC01 positive cells were quantified as a function of eOD-GT8 concentration. Statistical significance was determined by two-way ANOVA followed by Tukey’s post hoc test. ****P < 0.0001. All data show means ± SEM.

[0091]

[0091] FIGs. 10A-10C: Flow cytometry gating strategy for eOD-GT8 / NE uptake by lymphocytes in mesenteric LNs and eOD-GT8 / NE uptake by T cells. FIG. 10A: Mice were immunized with Fluorescent eOD-Chol-NE, eOD-DSPE-NE or eOD / NE. Mesenteric lymph nodes were harvested 24h later for flow cytometry analysis of antigen uptake. Schematic shows gating strategy to identify AF647-eOD-GT8 and NE uptake in macrophages, B cells, and dendritic cells. FIGs. 10B-10C: Shown are representative flow cytometry plots showing eOD-GT8 and NE uptake by T cells (FIG. 10B) and quantification of mean frequencies of eOD or NE-positive cells (FIG. 10C). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test, ns, P > 0.05; **P < 0.01; ****P < 0.0001. All data show means ± SEM.

[0092]

[0092] FIGs. 11A-11B: Flow cytometry gating strategy for GC B cells and Tfh cells.

[0093] Mice (n = 5 per group) were immunized with eOD-Chol-NE, eOD-DSPE-NE or eOD / NE. 12 days after, Tfh (FIG. 11A) and GC B cell (FIG. 11B) responses in mesenteric LNs were analyzed by flow cytometry. Representative gating strategy is shown.

[0094]

[0093] FIGs. 12A-12D: ELISA absorbance curves from FIGs. 4A-4L. Mice (n = 5 per group) were immunized with eOD-Chol-NE, eOD-DSPE-NE or eOD / NE and boosted 4 weeks later. Shown are ELISA absorbance vs dilution curves at week 6 for serum IgG (FIG. 12A), serum IgA (FIG. 12B), fecal IgG (FIG. 12C) and fecal IgA (FIG. 12D). All data show means ± SEM.

[0094] FIGs. 13A-13D. Comparison of i.p. and s.c. immunization routes. Mice (n = 5 per group) were immunized with eOD-Chol-NE and boosted 4 weeks later by i.p. or s.c. routes. Shown are serum IgG (FIG. 13A) and IgA (FIG. 13B) titers, fecal IgG (FIG. 13C) and IgA (FIG. 13D) titers. Statistical significance was determined by two-way ANOVA followed by Tukey’s post hoc test, ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. All data show means ± SEM.

[0095]

[0095] FIGs. 14A-14G: Comparison of eOD-Chol-NE immunizations with and without 3M-052. (FIGs. 14A-14C) Mice (n = 5 per group) were immunized with eOD-Chol-NE with or without telratolimod. Shown are percentage of GC B cells (FIG. 14A), eOD-GT8+ GC B cells (FIG. 14B), IgA+ eOD-GT8+ GC B cells (FIG. 14C) at day 12. FIGs. 14D-14G: Mice (n = 5 per group) were immunized with eOD-Chol-NE with or without 3M-052 and boosted 4 weeks later. Shown are serum IgG (FIGs. 14D) and IgA (FIGs. 14E) titers, fecal IgG (FIGs. 14F) and IgA (FIGs. 14G) titers, at week 6. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. ****P < 0.0001. All data show means ± SEM.

[0096]

[0096] FIGs. 15A-15B: BAL fluid (BALf) titers following the RBDJ immunizations.

[0097] Mice (n = 5 per group) were immunized with RBDJ-Chol-NE, RBDJ-DSPE-NE or RBDJ / NE and boosted 4 weeks later. Shown are (BALF IgG (FIGs. 15A) and IgA (FIGs. 15B) titers at week 8 against WT RBDJ. All data show means ± SEM.

[0098] DETAILED DESCRIPTION

[0099]

[0097] Humoral immune (antibody) response is desired both systemically and at localized mucosal surfaces to combat infectious pathogens that infect a host through mucosal transmission.

[0100]

[0098] The present invention is based on the surprising findings that vaccines comprising a nanoemulsion (NE) carrying an immunogen, wherein the immunogen is non-covalently conjugated to the nanoemulsion via an amphiphilic linker, can elicit humoral immunity to the immunogen (such as, for example HIV and SARS-CoV-2 antigen) in the gastrointestinal tract significantly more effectively than free immunogen after administration. It was surprisingly discovered that, upon intraperitoneal (i.p.) administration, the NEs accumulated in gut-draining mesenteric lymph nodes, and the amphiphilic linker-immunogen conjugates carried by the NEs were able to dissociate from the NEs and transfer to cell membranes of antigen presenting cells in the lymph nodes, particularly dendritic cells, macrophages and B cells. Further, immunization with the immunogen-conjugated NEs elicited enhanced systemic IgG response as well as far stronger mucosal IgG and IgA responses compared to immunization with free immunogen mixed with NEs. The amount of immunogen used to effectively produce an immune response to the immunogen using immunogen-conjugated NEs was comparable to the amount used for vaccination through subcutanenous injection, which is much lower than the immunogen amount that is generally required for oral vaccine administration.

[0101]

[0099] Thus, the present disclosure provides vaccines suitable for gastrointestinal transmucosal administration, and methods of use thereof to induce an immune response or immunity (e.g., involving a humoral antibody response) against an infections pathogen.

[0102] I. Definitions

[0103]

[0100] Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0104]

[0101] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0105]

[0102] As used herein, "about" will be understood by persons of ordinary skill and will vary to some extent depending on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill given the context in which it is used, "about" will mean up to plus or minus 10% of the particular value.

[0106]

[0103] As used herein, the term "adjuvant" refers to a compound that, with a specific immunogen or antigen e.g., of a vaccine), will augment or otherwise alter or modify the resultant immune response. Modification of the immune response includes intensification or broadening the specificity of either or both antibody and cellular immune responses. Modification of the immune response can also mean decreasing or suppressing certain antigen-specific immune responses. In some embodiments, the adjuvant is administered prior to, concurrently, or after administration of a vaccine, or composition comprising the vaccine. In some embodiments, the adjuvant is co-formulated in the same composition as a vaccine. In some embodiments, the adjuvant is comprised within a vaccine, e.g., within a vaccine that comprises a nanoemulsion, and, for example, the adjuvant is encapsulated in the lipophilic core of the nanoemulsion of the vaccine.

[0104] ‘ ‘Amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y- carboxyglutamate, and O -phospho serine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.

[0107]

[0105] Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, can be referred to by their commonly accepted single-letter codes.

[0108]

[0106] An “amino acid substitution” refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence (an amino acid sequence of a starting polypeptide) with a second, different "replacement" amino acid residue. An “amino acid insertion” refers to the incorporation of at least one additional amino acid into a predetermined amino acid sequence. While the insertion will usually consist of the insertion of one or two amino acid residues, larger “peptide insertions,” can be made, e.g. insertion of about three to about five or even up to about ten, fifteen, or twenty amino acid residues. The inserted residue(s) may be naturally occurring or non-naturally occurring as disclosed above. An “amino acid deletion” refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.

[0109]

[0107] As used herein, "amphiphile" or “amphiphilic” refers to a molecule, e.g., a linker, comprising a hydrophilic head group and a hydrophobic tail. In some embodiments, an amphiphilic linker comprises one or more hydrophobic lipid tails. In some embodiments, the amphiphilic linker comprises a hydrophilic polymer (e.g., polyethylene glycol), wherein the hydrophilic polymer is covalently conjugated to the one or more hydrophobic lipid tails.

[0108] The term "ameliorating" refers to any therapeutically beneficial result in the treatment of a disease state, e.g., cancer, including prophylaxis, lessening in the severity or progression, remission, or cure thereof.

[0110]

[0109] As used herein, the term "antibody" refers to an immunoglobulin molecule comprising four polypeptide chains, two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds. An antibody consists of two structural regions: a variable fragment (Fab) that mediates antigen binding and a constant fragment (Fc) that mediates downstream effector functions.

[0111]

[0110] There are five immunoglobulin classes (isotypes) of antibody molecules found in serum: IgG, IgM, IgA, IgE, and IgD. They are distinguished by the type of heavy chain they contain. IgG molecules possess heavy chains known as y-chains; IgMs have p-chains; IgAs have a-chains; IgEs have s-chains; and IgDs have 5-chains. The variation in heavy chain polypeptides allows each immunoglobulin class to function in a different type of immune response or during a different stage of the body’s defense. The amino acid sequences that confer these functional differences are located mainly within the Fc domain. IgG (immunoglobulin G) is expressed on the surface of mature B cells, and is also the most prevalent Ig in serum and extravascular spaces. IgG has 4 subtypes: IgGl, IgG2, IgG3 and IgG4. IgA (immunoglobulin A) plays a pivotal role in mucosal homeostasis in the gastrointestinal, respiratory, and genitourinary tracts, functioning as the dominant antibody of immunity in this role. IgA has two subtypes: IgAl and IgA2.

[0112]

[0111] Immunoglobulin class switching, also known as isotype switching, is a biological mechanism that changes a B cell's production of immunoglobulin from one type to another. Class switching occurs rapidly after activation of mature naive B cells, resulting in a switch from expressing IgM and IgD to expression of IgG, IgE, or IgA; this switch improves the ability of antibodies to remove the pathogen that induces the humoral immune response.

[0113]

[0112] As used herein, the terms "antigen" or “immunogen” refer to molecule which, when administered to a vertebrate, especially a mammal, will induce an immune response. Common immunogens includes peptides, proteins, and polysaccharides. Other types of molecules, including lipids and nucleotides, can also be immunogens.

[0114]

[0113] The terms "antigenic peptide" or “peptide antigen”, used interchangeably herein, refer to a peptide which, when administered to a vertebrate, especially a mammal, will induce an immune response, e.g., a cell-mediated immune response.

[0114] The terms “antigenic protein” or “protein antigen”, as used herein, refer to a protein which, when administered to a vertebrate, especially a mammal, will induce an immune response, e.g., a humoral antibody mediated immune response.

[0115]

[0115] The term “antigen presenting cell” or “APC” is a cell that displays foreign antigen complexed with MHC on its surface. T cells recognize this complex using T cell receptor (TCR). Examples of APCs include, but are not limited to, dendritic cells (DCs), peripheral blood mononuclear cells (PBMC), monocytes (such as THP-1), B lymphoblastoid cells (such as C1R.A2, 1518 B-LCL) and monocyte-derived dendritic cells (DCs). Some APCs internalize antigens either by phagocytosis or by receptor-mediated endocytosis.

[0116]

[0116] The term “B cells” refers to a type of lymphocytes that are responsible for mediating the production of antigen- specific immunoglobulin (Ig) directed against invasive pathogens that are typically known as antibodies.

[0117]

[0117] As used herein, "CG oligodeoxynucleotides (CG ODNs)", also referred to as "CpG ODNs", are short single- stranded synthetic DNA molecules that contain a cytosine nucleotide (C) followed by a guanine nucleotide (G). In certain embodiments, the immuno stimulatory oligonucleotide is a CG ODN.

[0118]

[0118] A polypeptide or amino acid sequence "derived from" a designated polypeptide or protein refers to the origin of the polypeptide. Preferably, the polypeptide or amino acid sequence which is derived from a particular sequence has an amino acid sequence that is essentially identical to that sequence or a portion thereof, wherein the portion consists of at least 10-20 amino acids, preferably at least 20-30 amino acids, more preferably at least 30-50 amino acids, or which is otherwise identifiable to one of ordinary skill in the art as having its origin in the sequence.

[0119]

[0119] Polypeptides derived from another peptide may have one or more mutations relative to the starting polypeptide, e.g., one or more amino acid residues which have been substituted with another amino acid residue or which has one or more amino acid residue insertions or deletions.

[0120]

[0120] A polypeptide can comprise an amino acid sequence which is not naturally occurring. Such variants necessarily have less than 100% sequence identity or similarity with the starting molecule. In a preferred embodiment, the variant will have an amino acid sequence from about 75% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide, more preferably from about 80% to less than 100%, more preferably from about 85% to less than 100%, more preferably from about 90% to less than 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) and most preferably from about 95% to less than 100%, e.g., over the length of the variant molecule.

[0121]

[0121] In one embodiment, there is one amino acid difference between a starting polypeptide sequence and the sequence derived therefrom. Identity or similarity with respect to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e., same residue) with the starting amino acid residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.

[0122]

[0122] As used herein, the term antigen “cross-presentation” refers to presentation of exogenous protein antigens to T cells via MHC class I and class II molecules on APCs.

[0123]

[0123] As used herein, the term “cytotoxic T lymphocyte (CTL) response” refers to an immune response induced by cytotoxic T cells. CTL responses are mediated primarily by CD8+ T cells.

[0124]

[0124] As used herein, the term “effective amount” or “effective dose” is defined as an amount sufficient to achieve or at least partially achieve the desired effect, such as e.g., inducing or enhancing an immune response, or providing immunity, to an immunogen. The term “therapeutically effective amount” or “therapeutically effective dose” is defined as an amount that is effective to ameliorate a symptom of a disease. A therapeutically effective amount can be “prophylactically effective amount” as prophylaxis can be considered therapy.

[0125]

[0125] As used herein, the term “effector cell” or “effector immune cell” refers to a cell involved in an immune response, e.g., in the promotion of an immune effector response. In some embodiments, immune effector cells specifically recognize an antigen. Examples of immune effector cells include, but are not limited to, Natural Killer (NK) cells, B cells, monocytes, macrophages, T cells (e.g., cytotoxic T lymphocytes (CTLs)). In some embodiments, the effector cell is a T cell.

[0126]

[0126] As used herein, “enteral administration” refers to a route of administration wherein a drug (e.g., vaccine) is absorbed via the gastrointestinal (GI) tract. Enteral administration includes oral administration, rectal administration, or administration through an enteral tube to bypass the mouth, such as via the nasopharynx (e.g., nasogastric (NG) or nasojejunal (NJ) tube), or via direct access to the GI tract through the skin, for example gastrostomy or jejunostomy tubes.

[0127]

[0127] As used herein the term “humoral immune response” is an immune response mediated by antibody molecules that are secreted by B cells. The presence of antigens triggers B cell activation and differentiation into antibody- secreting plasma cells and usually requires helper T cells (which are CD4+ T cells).

[0128]

[0128] As used herein, the term “immune effector function” or “immune effector response” refers to a function or response of an immune effector cell that promotes an immune response to a target.

[0129]

[0129] As used herein, “immune cell” is a cell of hematopoietic origin and that plays a role in the immune response. Immune cells include lymphocytes (e.g., B cells and T cells), natural killer cells, and myeloid cells (e.g., monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes).

[0130]

[0130] As used herein, an "immunostimulatory oligonucleotide" is an oligonucleotide that can stimulate (e.g., induce or enhance) an immune response.

[0131]

[0131] The terms “inducing an immune response” and “enhancing an immune response” are used interchangeably and refer to the stimulation of an immune response (i.e., either passive or adaptive) to a particular antigen. The term “induce” as used with respect to inducing CDC or ADCC refer to the stimulation of particular direct cell killing mechanisms.

[0132]

[0132] As used herein, a subject “in need of prevention,” “in need of treatment,” “in need of immunization”, or “in need thereof,” refers to one, who by the judgment of an appropriate medical practitioner (e.g., a doctor, a nurse, or a nurse practitioner in the case of humans; a veterinarian in the case of non-human mammals), would reasonably benefit from a given treatment (such as treatment with a a vaccine for immunization against an immunogen).

[0133]

[0133] As used herein, “intraperitoneal administration” refers to administering a drug or other substance directly to the peritoneum (body cavity) of a subject, such as through injection.

[0134]

[0134] The term "in vivo" refers to processes that occur in a living organism.

[0135]

[0135] As used herein, the terms “linked”, “operably linked,” “fused”, or “fusion”, are used interchangeably. These terms refer to the joining together of two more elements or components or domains, by an appropriate means including chemical conjugation or recombinant DNA technology. Methods of chemical conjugation (e.g., using heterobifunctional crosslinking agents or using “click” chemistry) are known in the art as are methods of recombinant DNA technology.

[0136]

[0136] The term “lipid” refers to a biomolecule that is soluble in nonpolar solvents and insoluble in water. Lipids are often described as hydrophobic or amphiphilic molecules which allows them to form structures such as vesicles or membranes in aqueous environments. Lipids include fatty acids, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids (including cholesterol), prenol lipids, saccharolipids, and polyketides. In some embodiments, the lipid suitable for the amphiphilic linkers of the disclosure can be embedded in the surfactant shell of a nanoemulsion. In some embodiments, the lipid suitable for the amphiphilic linker of the disclosure inserts into a cell membrane under physiological conditions. In some embodiments, the lipid can transfer between a nanoemulsion and a cell membrane under physiological conditions. In some embodiments, the lipid is a cholesterol. In some embodiments, the lipid is a monoacyl lipid. In some embodiments, the lipid is a diacyl lipid..

[0137]

[0137] As used herein, a “nanoemulsion” refers to emulsions with droplet size in the nanometer range that are made from two immiscible fluids. Nanoemulsions (NEs) typically have sizes of about 2-500 nm in diameter. A typical nanoemulsion contains oil, water and an emulsifier. An emulsifier is crucial in creation of the emulsion droplets as it decreases the interfacial tension between the oil and the water phases of the emulsion.

[0138]

[0138] Depending on the relative composition and dispersal of the fluid phases, nanoemulsions are categorized into biphasic (water-in-oil or oil-in-water) or multiple nanoemulsions. Water-in-oil nanoemulsions have an aqueous core, and oil-in-water nanoemulsions have an lipophilic core. The emulsifier forms a shell surrounding the core of the nanoemulsions. Surfactants are typically used as the emulsifier, but proteins and lipids have also been effective in preparation of nanoemulsions.

[0139]

[0139] Nanoemulsions are kinetically stable, and their small size leads to useful properties such as high surface area per unit volume, robust stability, optically transparent appearance, and tunable rheology. Nanoemulsions are finding application in diverse areas such as drug delivery, food, cosmetics, and pharmaceuticals. Methods of making nanoemulsions are broadly classified into two primary categories: high-energy and low-energy methods. High energy methods such as high pressure homogenization (HPH) and ultrasonication consume significant energy (~1O8-1O10W kg'1) to make small droplets. On the other hand, low energy methods exploit specific system properties to make small droplets without consuming significant energy (~103W kg'1). Methods of making nanoemulsions for drug delivery purposes are well-researched and well-known (see, e.g., Gupta et al. “Nanoemulsions: formation, properties and applications”, Soft Matter 2016,12: 2826-2841; Singh et al. “Nanoemulsions: Concepts, development and applications in drug delivery” Journal of Controlled Release 2017, 252:28-49; Preeti et al. “Nanoemulsion: An Emerging Novel Technology for Improving the Bioavailability of Drugs” Scientifica 2023, 2023:6640103; incorporated herein by reference).

[0140]

[0140] As used herein, “neutralizing antibody” refers to an antibody that not only binds to a pathogen (e.g., a virus, a bacteria) but also binds in a manner that prevents infection. For example, a neutralizing antibody may block interaction of a viral capsid protein with a receptor on a host cell, thereby preventing the virus from entering a host cell. Only a small subset of antibodies that bind a pathogen are capable of neutralization. After an infection, it can take some time for a subject to produce highly effective neutralizing antibodies, but these can persist to protect against future encounters with the agent.

[0141]

[0141] ‘ ‘Nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double- stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985); and Cassol et al., 1992; Rossolini et al., Mol. Cell. Probes 8:91-98, 1994). For arginine and leucine, modifications at the second base can also be conservative. The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0142]

[0142] In some embodiments, the peptides of the invention are encoded by a nucleotide sequence. Nucleotide sequences of the invention can be useful for a number of applications, including: cloning, gene therapy, protein expression and purification, mutation introduction, DNA vaccination of a host in need thereof, antibody generation for, e.g., passive immunization, PCR, primer and probe generation, and the like.

[0143]

[0143] As used herein, “parenteral administration,” “administered parenterally,” and other grammatically equivalent phrases, refer to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intranasal, intraocular, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid and intrasternal injection and infusion.

[0144]

[0144] As generally used herein, “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and / or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0145]

[0145] As used herein, the term “physiological conditions” refers to the in vivo condition of a subject (e.g., human). In some embodiments, physiological condition refers to a neutral pH (e.g., pH between 6-8).

[0146]

[0146] "Polypeptide," "peptide", and "protein" refer to a polymer of amino acid residues. The terms apply to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer amino acid polymers, including in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid.

[0147]

[0147] As used herein, “protein” refers to a molecule that comprises or consists of more than 50 amino acids. As used herein, “peptide” refers to a molecule that consists of between 2 and 50 amino acids. An “oligopeptide” refers to a molecule that consists of between 2 and about 20 amino acids.

[0148]

[0148] As used herein, a "small molecule" is a molecule with a molecular weight below about 500 Daltons.

[0149]

[0149] As used herein, a “surfactant” (also known as “surface-active agent”) is a molecule which reduces the surface tension of a liquid in which it is dissolved, or reduces the interfacial tension between two liquids, a liquid and a gas, or a liquid and a solid. Surfactant are highly versatile products in the chemical industry (e.g., detergents are surfactants), and a very wide range of chemical compounds are used as surfactants. Surfactants are amphiphilic molecules with distinct hydrophobic and hydrophilic components. The hydrophobic tail can be a hydrocarbon, fluorocarbon, or siloxane. Surfactants are generally categorized based on their polar head groups: anionic surfactants (having negatively charged head group); cationic surfactants (having positively charged headgroup), nonionic surfactants (lack electrical charge in the head group), and zwitterionic surfactants (containing both positive and negative charges within the same molecule). Due to their amphiphilic nature, surfactants typically set themselves in the interface of two immiscible phases (e.g., water and oil interface). At the interface, surfactants align such that the hydrophobic part is in the hydrophobic phase e.g., oil), and the hydrophilic part is in the hydrophilic phase (e.g., water).

[0150]

[0150] As used herein, the term “subject” includes any human or non-human animal. The term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, canines, felines, murines, bovines, equines, porcines, sheep, chickens, amphibians, or reptiles.

[0151]

[0151] The term "sufficient amount" or "amount sufficient to" means an amount sufficient to produce a desired effect, e.g., an amount sufficient to immunize a subject against an immunogen.

[0152]

[0152] The term “T cell” refers to a type of white blood cell that can be distinguished from other white blood cells by the presence of a T cell receptor on the cell surface. There are several subsets of T cells, including, but not limited to, T helper cells (a.k.a. TH cells or CD4+ T cells) and subtypes, including TH1, TH2, TH3, TH17, TH9, and TFH cells, cytotoxic T cells (i.e., TC cells, CD8+ T cells, cytotoxic T lymphocytes, T-killer cells, killer T cells), memory T cells and subtypes, including central memory T cells (TCM cells), effector memory T cells (TEM and TEMRA cells), and resident memory T cells (TRM cells), regulatory T cells (a.k.a. Treg cells or suppressor T cells) and subtypes, including CD4+ FOXP3+ Treg cells, CD4+FOXP3- Treg cells, Tri cells, Th3 cells, and Tregl7 cells, natural killer T cells (a.k.a. NKT cells), mucosal associated invariant T cells (MAITs), and gamma delta T cells (y5 T cells), including Vy9 / V52 T cells. Any one or more of the aforementioned or unmentioned T cells may be the target cell type for a method of use of the invention.

[0153]

[0153] As used herein, the term “T cell activation” or “activation of T cells” refers to a cellular process in which mature T cells, which express antigen- specific T cell receptors on their surfaces, recognize their cognate antigens and respond by entering the cell cycle, secreting cytokines or lytic enzymes, and initiating or becoming competent to perform cellbased effector functions. T cell activation requires at least two signals to become fully activated. The first occurs after engagement of the T cell antigen- specific receptor (TCR) by the antigen-major histocompatibility complex (MHC), and the second by subsequent engagement of co-stimulatory molecules (e.g., CD28). These signals are transmitted to the nucleus and result in clonal expansion of T cells, upregulation of activation markers on the cell surface, differentiation into effector cells, induction of cytotoxicity or cytokine secretion, induction of apoptosis, or a combination thereof.

[0154] As used herein, the term “T cell-mediated response” refers to any response mediated by T cells, including, but not limited to, effector T cells (e.g., CD8+ cells) and helper T cells (e.g., CD4+ cells). T cell mediated responses include, for example, T cell cytotoxicity and proliferation.

[0154]

[0155] The term “T cell cytotoxicity” includes any immune response that is mediated by CD8+ T cell activation. Exemplary immune responses include cytokine production, CD8+ T cell proliferation, granzyme or perforin production, and clearance of an infectious agent.

[0155]

[0156] As used herein, “transmucosal administration” refers to a route of drug administration wherein a drug (e.g., vaccine) enters through or across a mucosal epithelium to underlying tissue. In some embodiments, a drug administered transmucosally enters systemic circulation. In embodiments, transmucosal administration provides local delivery of the drug. In some embodiments, transmucosal administration provides both local and systemic delivery of the drug.

[0156]

[0157] The terms “treat,” “treating,” and “treatment,” as used herein, refer to therapeutic or preventative measures described herein. The methods of “treatment” employ administration to a subject in need of such treatment a vaccine of the present disclosure, for example, a subject at risk of infection with an immunogen. In some embodiments, a vaccine is administered to a subject in need of an enhanced immune response against a particular antigen or a subject who ultimately may acquire such a disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurring disorder.

[0157]

[0158] As used herein, "vaccine" refers to a composition which contains a nanoemulsion and immunogens described herein, which is in a form that is capable of being administered (e.g., intraperitoneally) to a subject, and which is capable of inducing a protective immune response. In some embodiments, the protective immune response is sufficient to induce immunity, and / or to prevent and / or ameliorate an infection or disease, and / or to reduce at least one symptom of an infection or disease, and / or to enhance the efficacy of another dose of the vaccine. Upon introduction into a host, the vaccine provokes an immune response including, but not limited to, for example, the production of antibodies (e.g., IgA and / or IgG antibodies) and / or cytokines and / or the activation of cytotoxic T cells, antigen presenting cells, helper T cells, dendritic cells and / or other cellular responses.

[0158] II. Compositions of the Invention Nanoemulsions

[0159]

[0159] In some aspects, the present disclosure provides a vaccine comprising a nanoemulsion suitable for injection, e.g., intraperitoneal injection, into a subject. In some embodiments, the nanoemulsion is an oil-in-water nanoemulsion. Oil-in-water emulsions, which are composed of oil droplets dispersed in an aqueous continuous phase, are particularly useful in delivering poorly soluble drugs. Oil-in-water nanoemulsions can also be used to stabilize drugs that undergo hydrolytic and oxidative degradation.

[0160]

[0160] Nanoemulsions useful for delivering agents, such as adjuvants for co-administration with an immunogen to boost vaccine response, are generally known in the art (see, e.g., Wilson et al. “Nanoemulsion for drug delivery” Particuology 2022, 64:85-97; Hagan et al. “‘World in motion’ -emulsion adjuvants rising to meet the pandemic challenges” npj Vaccines 2021, 6:158). For example, multiple licensed vaccines employ nanoemulsions to deliver adjuvants and induce distinct immunological responses, such as the MF59-adjuvanted influenza vaccine and the AS03-adjuvanted influenza vaccine.

[0161]

[0161] In some embodiments, the nanoemulsion of the vaccines described herein comprises a lipophilic core and a surfactant shell.

[0162]

[0162] In some embodiments, the lipophilic core comprises an oil, e.g., a biodegradable oil. In some embodiments, the oil is a biocompatible oil.

[0163]

[0163] In some embodiments, the oil is an animal oil (i.e., found in or derived from an animal). In some embodiments, the oil is a plant oil (i.e., found in or derived from a plant).

[0164]

[0164] In some embodiments, the oil is selected from the group consisting of squalene oil, caster oil, soy bean oil, grapeseed oil and sesame oil.

[0165]

[0165] In some embodiments, the oil is squalene, a naturally occurring molecule found in plants and animals, including humans. Squalene can be derived from a natural source (shark liver oil), although alternative sources are being explored and advances have been made using both synthetic biology techniques and plant sources. However, other biodegradable oils are also suitable for the vaccines of the present disclosure.

[0166]

[0166] In some embodiments, the nanoemulsion of the vaccine described herein comprises a surfantant shell comprising one or more biocompatible surfactants. The surfactant is preferably biodegradable. Commonly-used emulsifiers including Pluronics®, Tweens®, Spans®, phospholipids, sucrose fatty acid esters are applicable for the nanoemulsions described herein.

[0167] In some embodiments, the surfactant useful in the nanoemulsions of the present disclosure may comprise one or more commonly-used biocompatible surfactants including, but not limited to, sorbitant monooleate (also known as Span® 80), sorbitan trioleate (also known as Span® 85), polysorbate 80 (also known as Tween® 80), and polysorbate 20 (also known as Tween® 20).

[0167]

[0168] In some embodiments, the surfactant comprises a phospholipid, such as a phospho tidy choline. Egg phosphatidylcholine is commonly used as an emulsifier in formulations administered parenterally. Synthetic phosphatidylcholine emulsifiers are widely available and may be desirable substitutes for egg-derived phospholipids due to stability, purity, and material source considerations, (see, e.g., Fox et al. “Immunomodulatory and Physical Effects of Phospholipid Composition in Vaccine Adjuvant Emulsions” AAPS Pharm Sci Tech 2012, 13(2):498-506; Nii and Ishii, “Properties of various phosphatidylcholines as emulsifiers or dispersing agents in microparticle preparations for drug carriers” Colloids Surf B Biointerfaces 2004, 39( 1 -2):57-63 ; the entire contents of each are incorporated herein by reference). In some embodiments, the surfactant may comprise one or more of l-palmitoyl-2- oleoyl-sn-glycero-3-phosphocholine (POPC; a main component of egg PC), 1,2-dioleoyl-sn- glycero-3-phosphocholine (DOPC), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), l,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), or l,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC).

[0168]

[0169] In some embodiments, the surfactant shell comprises substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, or 99%) one surfactant. In some embodiments, the surfactant shell comprises substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, or 99%) one surfactant selected from the group consisting of Span® 80, Span® 85, Tween® 20 and Tween® 80.

[0169]

[0170] In some other embodiments, the surfactant shell comprises more than one type of surfactant, e.g., a mixture of 2, 3, 4 or more surfactants, e.g., any combination of surfactants disclosed in the present disclosure. For example, the surfactant shell may comprise Span® 20 and Span® 85, Tween® 20 and Tween® 80, Span® 85 and Tween® 80, Span® 80 and Tween® 80, Span® 80 and Tween® 20, or Span® 80 and Tween® 80. In one embodiment, the surfactant shell comprises Span® 85 and Tween® 80.

[0170]

[0171] In some embodiments, the surfactant shell comprises one or more additional emulsifiers, such as Eumulgin Bl or sucrose fatty acid sulfate esters.

[0171]

[0172] Compositions of nanoemulsions applicable for making vaccines and methods of making them are well known in the art and within the skill of the ordinarily skilled person in the art. Table 1 below presents non-limiting examples of compositions of emulsions that are undergoing various stage of clinical trials for delivering vaccine adjuvants, and which can be utilized in the vaccines of the present disclosure.

[0172] Table 1: Emulsions in Clinical Trials for delivering Vaccine Adjuvants

[0173]

[0173] In some embodiments, the nanoemulsion of the vaccines of the present disclosure comprises the components of any one of the emulsions listed in Tables 1-2 or described in Example 6. In some embodiments, the nanoemulsion of the vaccine of the present disclosure comprises the components and the corresponding ratios of any one of the emulsions listed in Tables 1-2 or Example 6.

[0174]

[0174] In some embodiments, small emulsions are more preferable for the vaccines described herein because emulsion droplets that are too big can be trapped in fat tissues at the injection site, and thus preventing or decreasing delivery to the lymph nodes. Additionally, smaller particle sizes allow more particles to be packed into the vaccine volume to be injected into a subject, enabling delivery of more active agent (e.g., immunogen).

[0175]

[0175] In some embodiments, the vaccine of present disclosure comprises nanoemulsions having a diameter of less than 300 nm, or less than 250 nm. In some embodiments, the nanoemulsions are 200 nm or smaller in diameter.

[0176]

[0176] In some embodiments, the nanoemulsion has a size of about 1-200 nm, about 1-150 nm, about 1-100 nm, about 1-50 nm, about 1-30 nm, about 1-20 nm, or about 1-15 nm in diameter. In some embodiments, the nanoemulsion has a size of about 5-200 nm, about 5-150 nm, about 5-100 nm, about 5-50 nm, about 5-30 nm, about 5-20 nm, or about 5-15 nm in diameter. In some embodiments, the nanoemulsion has a size of about 10-200 nm, about 10- 150 nm, about 10-100 nm, about 10-50 nm, about 10-30 nm, about 10-20 nm, or about 10-15 nm in diameter. In some embodiments, the nanoemulsion has a size of about 15-200 nm, about 15-150 nm, about 15-100 nm, about 15-50 nm, about 15-30 nm, or about 15-20 nm in diameter. In some embodiments, the nanoemulsion has a size of about 20-200 nm, about 20- 150 nm, about 20-100 nm, about 20-50 nm, about 20-30 nm in diameter. In some embodiments, the nanoemulsion has a size of about 30-200 nm, about 30-150 nm, about 30- 100 nm, or about 30-50 nm in diameter. In some embodiments, the nanoemulsion has a size of about 50-200 nm, about 50-150 nm, or about 50-100 nm in diameter. In some embodiments, the nanoemulsion has a size of about 100-200 nm, about 100-150 nm, or about 150-200 nm in diameter.

[0177]

[0177] The nanoemulsion of the vaccines of the present disclosure can be prepared using any methods known in the art. Nanoemulsion formulations can be prepared by high-speed mixing the oil phase (e.g., squalene oil) and an aqueous phase followed by sonication or high- pressure homogenization. Emulsion particle size can be determined or confirmed by dynamic light scattering (see, e.g., Fox et al. “Immunomodulatory and physical effects of oil composition in vaccine adjuvant emulsions” Vaccine 2011, 29(51): 9563-72; Akamatsu et al. “Preparedness against pandemic influenza: Production of an oil-in-water emulsion adjuvant in Brazil”, PLoS ONE 15(6): e0233632; the entire contents of each are incorporated herein by reference). The nanoemulsions can be filter-sterilized prior to administration to a subject.

[0178]

[0178] Common techniques to prepare nanoemulsions typically produce droplets of sizes between 45-200 nm. If smaller sizes of oil-in-water nanoemulsions are desired, differing ratios of surfactant(s) and oil can be tested to identify compositions that are optimal to achieve the desired sizes. For example, Shah et al. (“The development of self-emulsifying 1 oil-in-water emulsion adjuvant and an evaluation of the impact of droplet size on performance” J. Pharm. Sci. 2015, 104, 1352-1361; the entire contents are incorporated herein by reference) demonstrates a low shear process to prepare stable reproducible emulsions of sizes less than 30 nm by screening different ratios of oi and surfactants and using only simple emulsifier instruments.

[0179]

[0179] Methods of making nanoemulsions are broadly classified into two primary categories: high-energy and low-energy methods. High energy methods such as high pressure homogenization (HPH) and ultrasonication consume significant energy (~1O8-1O10W kg'1) to make small droplets. On the other hand, low energy methods exploit specific system properties to make small droplets without consuming significant energy (~103W kg'1). Methods of making nanoemulsions for drug delivery purposes are well-researched and well- known (see, e.g., Gupta et al. “Nanoemulsions: formation, properties and applications”, Soft Matter 2016,12: 2826-2841; Singh et al. “Nanoemulsions: Concepts, development and applications in drug delivery” Journal of Controlled Release 2017, 252:28-49; Preeti et al.

[0180] “Nanoemulsion: An Emerging Novel Technology for Improving the Bioavailability of Drugs” Scientifica 2023, 2023:6640103; incorporated herein by reference).

[0181]

[0180] In some embodiments, the nanoemulsions disclosed herein further comprise one or more additional agents, in addition to the primary components that make up the surfactant shell and lipophilic core. For example, in some embodiments, the surfactant shell of the nanoemulsion further comprises an amphiphilic linker which is conjugated to an immunogen. In some embodiments, the lipid component of the amphiphilic linker is associated with, inserted into, or embedded in the surfactant shell. In some embodiments, an additional hydrophobic or lipophilic agent, such as an adjuvant, is encapsulated in the lipophilic / oil core. To incorporate the additional agents into the nanoemulsions, the additional agents may be added into the oil phase prior to mixing with the aqueous phase.

[0182]

[0181] It is noted that the nanoemulsions, surfactants, or oils described herein are nonlimiting examples only. Other nanoemulsions compatible with the properties and methods of use of the vaccine described herein are also intended to be encompassed by the invention of the present disclosure.

[0183]

[0182] In some embodiments, the nanoemulsion of the vaccine further comprises an adjuvant encapsulated in the lipophilic core. As shown in Table 1, a number of vaccines comprising nanoemulsions encapsulating adjuvants have been developed.

[0184]

[0183] In some embodiments, the adjuvant is lipophilic.

[0184] In some embodiments, the adjuvant comprises a TLR7 / 8 agonist. In some embodiments, the TLR7 / 8 agonist comprises imidazoquinolinone 3M-052 (also known as “3M-052”; see, e.g., Smirnov et al. “Vaccine adjuvant activity of 3M-052: an imidazoquinoline designed for local activity without systemic cytokine induction” Vaccine 2011; 29(33):5434-42; Kasturi et al. “3M-052, a synthetic TLR-7 / 8 agonist, induces durable HIV-1 envelope-specific plasma cells and humoral immunity in nonhuman primates” Sci Immunol. 2020; 5(48):eabbl025; the entire contents of each are incorporated herein by reference).

[0185]

[0185] In some embodiments, the adjuvant comprises an oligodeoxynucleotide (ODNs) containing unmethylated CpG motifs that trigger cells that express Toll-like receptor 9 (including dendric cells and B cells) (see Bode et al. “CpG DNA as a vaccine adjuvant” Expert Rev Vaccines 2011; 10(4):499-511); incorporated herein by reference). In some embodiments, the adjuvant comprises a CpG ODN conjugated to a cholesterol, z.e., CpG- cholesterol.

[0186]

[0186] In some embodiments, the adjuvant comprises a monophosphoryl lipid A (MPLA), which is an agonist of TLR4 that activates dendric cells, or an analog thereof. In some embodiments, the adjuvant comprises 3D(6-acyl)-PHAD™, which is a structural analog of MPLA.

[0187]

[0187] In some embodiments, the adnjuvant comprises or consists of any adjuvant disclosed herein.

[0188] Amphiphilic Linker

[0189]

[0188] In some aspects, the present disclosure provides a vaccine comprising an immunogen (e.g., peptide antigen or protein antigen) non-covalently conjugated to the surface of a nanoemulsion by an amphiphilic linker, wherein the vaccine is suitable for administration (e.g., intraperitoneal administration) to induce an immune response (e.g., a cell-mediated immune response or a humoral antibody-mediated immune response), including a mucosal immune response in the gastrointestinal tract. Nanoemulsions encapsulating adjuvants have previously been developed. However, traditional nanoemulsions have been shown not to directly bind to co-formulated antigens, and thus coformulated antigens are not trafficked to the same location in the body (e.g., lymph nodes) with the nanoemulsions. The present disclosure provides vaccines comprising nanoemulsions that successfully carry and deliver immunogen (e.g., peptide antigen or protein antigen) to the lymph nodes to induce strong immunity (e.g., gut-associated mucosal immunity)

[0190]

[0189] A diversity of amphiphilic linker structures are provided, wherein a lipophilic moiety, or “lipid tail” (e.g. cholesterol or DSPE), is covalently linked to a hydrophilic group (e.g., a PEG moiety), and the amphiphilic linker is further conjugated (e.g., covalently linked) to an immunogen, such as a peptide or protein antigen. Without being bound by theory, the amphiphilic linker comprised in the nanoemulsion of the vaccine of the disclosure is believed to dissociate from the nanoemulsion and associate with or transfer to a cell membrane when the nanoemulsion comes into contact with a neighboring cell. It is believed that, when a nanoemulsion carrying the amphiphilic linker and immunogen is injected intraperitoneally, the nanoemulsion is trafficked to gut-associated lymph nodes, including mediastinal and mesenteric lymph nodes. At the lymph nodes, the nanoemulsion comes into contact with cells in the lymphoid tissue, and the amphiphilic linker bound to immunogen is exchanged from the nanoemulsion to the cell membrane of lymphoid-resident cells, such as antigen-presenting cells (APCs). Since the amphilic linker is conjugated to an immunogen (e.g., peptide antigen or protein antigen), the immunogen is also transferred to the APCs and displayed on the surface of the APCs, thereby triggering robust germinal center (GC) B cell response.

[0191]

[0190] In some embodiments, the amphiphilic linker comprises a lipid comprising a hydrophobic tail, wherein the hydrophobic tail associates with or inserts into the surfactant shell of a nanoemulsion. In some embodiments, the amphiphilic linker comprises a lipid comprising a hydrophobic tail, wherein the hydrophobic tail associates with or inserts into a cell membrane. In some embodiments, the lipid or its hydrophobic tail is more thermodynamically stable in a cell membrane than in a surfactant shell of a nanoemulsion. In some embodiments, the lipid or its hydrophobic tail is more kinetically stable in a cell membrane than in a surfactant shell of a nanoemulsion. In some embodiments, the amphiphilic linker is more thermodynamically stable when anchored in a cell membrane than in a surfactant shell of a nanoemulsion. In some embodiments, the amphiphilic linker is more kinetically stable when anchored in a cell membrane than in a surfactant shell of a nanoemulsion. In some embodiments, the lipid and its hydrophobic tail enable the amphiphilic linker to tether to cell membrane (e.g., cell membrane of APCs). Consequently, an immunogen conjugated to the amphiphilic linker is also transferred to the cell (e.g, APC) and retained at lymph node or lymphoid tissue (e.g., mesenteric lymph node). In some embodiments, the lipid, its hydrophobic tail or the amphiphilic linker has similar thermodynamic and / or kinetic stability in cell membrane compared to a surfactant shell of a nanoemulsion, but the amphiphilic linker (and conjugated immunogen) once transferred to a cell membrane (e.g., cell membrane of APCs) in the lymphoid tissue, remains with the cell after the nanoemulsion is trafficked out of the lymph node or lymphoid tissue.

[0192]

[0191] It is well within the knowledge of a person of ordinary skill in the art to determine whether an amphiphilic linker is capable of dissociating from a nanoemulsion and associating with or transferring to a cell membrane. Techniques for determining amphiphile exchange from one composition to another have been described in the art (see, e.g., Estronca et al. “Kinetics and Thermodynamics of Lipid Amphiphile Exchange between Lipoproteins and Albumin in Serum” Biophys J. 2004, 88( l):557-565; the entire contents are incorporated herein by reference).

[0193]

[0192] In one non-limiting example, the capability of a candidate amphiphilic linker to dissociate from a nanoemulsion and associate with or transfer to a cell membrane can be determined by an assay comprising the steps of:

[0194] (i) providing a test nanoemulsion, or population of test nanoemulsions, comprising a candidate amphiphilic linker, wherein the candidate amphiphilic linker is labeled with a tag;

[0195] (ii) incubating the test nanoemulsion, or population of test nanoemulsions, with a population of cells;

[0196] (iii) washing the cells after step (ii); and

[0197] (iv) determining the number of cells or percentage of cells that are labeled with the tag, and / or determining the total signal intensity from the tag possessed by the population of cells after step (iii).

[0198]

[0193] Tags that may be used to label the amphilic linker are well known in the art. In one embodiment, the tag can be a fluorescent tag, which enables direct detection of the tag’s signal (e.g., in step (iv)). Alternatively, the tag may be a molecule e.g., peptide tag or small molecule) detectable using a secondary reagent such as an antibody. The tag may also be an oligonucleotide, wherein the tag is detectable using a complementary oligonucleotide conjugated to a fluorescent molecule.

[0199]

[0194] Any cells may be used in the assay. In some embodiments, the cells are splenocytes.

[0200]

[0195] In some embodiments, at least 1%, at least 2%, at least 3%, at least 4 %, at least 5 %, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of cells receive the amphiphilic linker and / or are labeled with the tag.

[0201]

[0196] In some embodiments, at least 1%, at least 2%, at least 3%, at least 4 %, at least 5 %, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the amphiphilic linkers are transferred from the nanoemulsions to the cells.

[0202]

[0197] In some embodiments, an amphiphilic linker useful in the vaccines of the present disclosure dissociates from a nanoemulsion and associates with or transfers to a cell membrane in vitro (e.g., as assessed by the assay disclosed above) to about the same, or to a greater extent, as the amphiphilic linker comprising l,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE) Iniked to PEG24 (DSPE- PEG24).

[0203] (i) Lipids of Amphiphilic Linker

[0204]

[0198] In some embodiments, the lipid component of the amphiphilic linker of the present disclosure comprises a hydrophobic tail.

[0205]

[0199] In some embodiments, the lipid of the amphiphilic linker of the disclosure is embedded in the surfactant shell of the nanoemulsion. In some embodiments, the hydropohic tail of the lipid is embedded in the surfactant shell of the nanoemulsion. In some embodiments, a lipid suitable for the amphiphilic linker of the disclosure is capable of associating with or inserting into the surfactant shell of the nanoemulsion. In some embodiments, a lipid suitable for the amphiphilic linker of the disclosure is capable of associating with or inserting into a cell membrane under physiological conditions. In some embodiments, the lipid of the amphiphilic linker transfers or is capable of transferring between the nanoemulsion and a cell membrane under physiological conditions.

[0206]

[0200] In some embodiments, the covalent linkage of the lipid to the hydrophilic group of the amphiphilic linker decreases the stability of association of the lipid with the surfactant shell of the nanoemulsion. In some embodiments, the covaleng linkage of the lipid to the hydrophilic group of the amphiphilic linker increases the capability of the lipid to dissociate from the surfactant shell of the nanoemulsion.

[0207]

[0201] Examples of preferred lipids for use in anchoring the immunogen to a nanoemulsion include, but are not limited to, fatty acids with aliphatic tails of about 8-18 carbons including, but not limited to, linear unsaturated and saturated fatty acids, branched saturated and unsaturated fatty acids, and fatty acids derivatives, such as fatty acid esters, fatty acid amides, and fatty acid thioesters, diacyl lipids, cholesterol, cholesterol derivatives, and steroid acids such as bile acids, Lipid A or combinations thereof. In some embodiments, the lipid is saturated. In some embodiments, the lipid is unsaturated.

[0208]

[0202] In some embodiments, the lipid can be linear, branched, or cyclic.

[0209]

[0203] In some embodiments, the lipid comprises or consists of cholesterol. In some embodiments, the lipid is cholesterol. Cholesterol has the structure shown below; cholesterol is often linked to other moieties via the hydroxyl group:

[0210]

[0204] In some embodiments, the lipid is a monoacyl lipid. A monoacyl lipid has only one fatty acyl chain (e.g., one hydrocarbon chain). In some embodiments, the acyl chain of the monoacyl lipid can be saturated or unsaturated.

[0211]

[0205] In some embodiments, the lipid is a diacyl lipid. A diacyl lipid has two fatty acyl chains (e.g., two hydrocarbon chains). The two acyl chains of a diacyl lipid may have the same length, or different lengths. In some embodiments, the acyl chains of the diacyl lipid can be saturated, unsaturated, or combinations thereof.

[0212]

[0206] In some embodiments, the acyl chain of the monoacyl or diacyl lipid comprises at least 8 carbons. In some embodiments, the acyl chain of the monoacyl or diacyl lipid comprises about 8-25 carbons, or about 8-18 carbons.

[0213]

[0207] In some embodiments, the acyl chain of the monoacyl lipid has 8 carbons in length. In some embodiments, the acyl chain of the monoacyl lipid has 9 carbons in length

[0214] In some embodiments, the acyl chain of the monoacyl lipid has 10 carbons in length. In some embodiments, the acyl chain of the monoacyl lipid has 11 carbons in length. In some embodiments, the acyl chain of the monoacyl lipid has 12 carbons in length. In some embodiments, the acyl chain of the monoacyl lipid has 13 carbons in length. In some embodiments, the acyl chain of the monoacyl lipid has 14 carbons in length. In some embodiments, the acyl chain of the monoacyl lipid has 15 carbons in length. In some embodiments, the acyl chain of the monoacyl lipid has 16 carbons in length. In some embodiments, the acyl chain of the monoacyl lipid has 17 carbons in length. In some embodiments, the acyl chain of the monoacyl lipid has 18 carbons in length. In some embodiments, the acyl chain of the monoacyl lipid has more than 18 carbons in length, wherein the amphiphilic linker is capable of dissociating from a nanoemsulsion of the disclosure and associating with or transferring to a cell membrane.

[0215]

[0208] In some embodiments, the acyl chain of the monoacyl lipid is palmitoyl (z.e., the acyl chain of palmitic acid).

[0216]

[0209] In some embodiments, one or both of the acyl chains of the diacyl lipid has 8 carbons in length. In some embodiments, one or both sof the acyl chain of the diacyl lipid has 9 carbons in length In some embodiments, one or both of the acyl chains of the diacyl lipid has 10 carbons in length. In some embodiments, one or both of the acyl chains of the diacyl lipid has 11 carbons in length. In some embodiments, one or both of the acyl chains of the diacyl lipid has 12 carbons in length. In some embodiments, one of the acyl chain of the diacyl lipid has 13 carbons in length. In some embodiments, one or both of the acyl chains of the diacyl lipid has 14 carbons in length. In some embodiments, one or both of the acyl chains of the diacyl lipid has 15 carbons in length. In some embodiments, one or both of the acyl chains of the diacyl lipid has 16 carbons in length. In some embodiments, one or both of the acyl chains of the diacyl lipid has 17 carbons in length. In some embodiments, one or both of the acyl chains of the diacyl lipid has 18 carbons in length. In some embodiments, one or both of the acyl chains of the diacyl lipid has more than 18 carbons in length, wherein the amphiphilic linker is capable of dissociating from a nanoemsulsion of the disclosure and associating with or transferring to a cell membrane.

[0217]

[0210] The acyl tail(s) of the monoacyl or diacyl lipid can be coupled to the head group via ester bond linkages, amide bond linkages, thioester bond linkages, or combinations thereof. In some embodiments, the monoacyl or diacyl lipid is a phosphate lipid, glycolipid, sphingolipid, or a combination thereof.

[0218]

[0211] In some embodiments, the lipid is a diacyl lipid. In some embodiments, the lipid is l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). In some embodiments, a diacyl lipid is synthesized as described in US 9,107,904, the entire contents of which are incorporated herein by reference. In some embodiments, a diacyl lipid is synthesized as provided below:

[0219]

[0220] (ii) Hydrophilic group of Amphiphilic Linker

[0221]

[0212] In various aspects of the present disclosure, the lipid of the amphiphilic linker is covalently linked (e.g., directly covalently linked) to a hydrophilic group. In some embodiments, the immunogen is covalently linked (e.g., directly covalently linked) to the hydrophilic group of the amphiphilic linker.

[0222]

[0213] In some embodiments, the hydrophilic group decreases the stability of association of the lipid component of the amphiphilic linker with the surfactant shell of the nanoemulsion. In some embodiments, the hydrophilic group of the amphiphilic linker increases the capability of the lipid to dissociate from the surfactant shell of the nanoemulsion, thereby increasing the delivery of the immunogen to cell membranes.

[0223]

[0214] One of ordinary skill in the art will recognize that the length and composition of the hydrophilic group can be adjusted based on the lipid and the nanoemulsion selected. Nonlimiting examples of hydrophilic groups applicable for the amphiphilic linker of the present disclosure may be a hydrophilic group described in WO 2019 / 060425, the entire contents of which are incorporated herein by reference.

[0215] In some embodiments, suitable hydrophilic groups include, but are not limited to, oligonucleotides such as those discussed below, a hydrophilic polymer including but not limited to poly(ethylene glycol) (MW: 500 Da to 20,000 Da), polyacrylamide (MW: 500 Da to 20,000 Da), polyacrylic acid, or polyvinyl alcohol; a string of hydrophilic amino acids such as serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or combinations thereof; polysaccharides, including but not limited to, dextran (MW: 1,000 Da to 2,000,000 Da); or combinations thereof.

[0224]

[0216] In some embodiments, the hydrophilic group has a molecular weight of about 300 to about 20,000 Da. In some embodiments, the hydrophilic group has a molecular weight of about 1,000 to about 15,000 Da. In some embodiments, the hydrophilic group has a molecular weight of about 1,500 to about 10,000 Da. In some embodiments, the hydrophilic group has a molecular weight of about 2,000 to about 5,000 Da. In some embodiments, the hydrophilic group has a molecular weight of about 1,000 to about 2,500 Da. In some embodiments, the hydrophilic group has a molecular weight of about 1,000 to about 3,000 Da. In some embodiments, the hydrophilic group has a molecular weight of about 1,000 to about 3,500 Da. In some embodiments, the hydrophilic group has a molecular weight of about 1,000 to about 4,000 Da. In some embodiments, the hydrophilic group has a molecular weight of about 1,000 to about 5,000 Da. In some embodiments, the hydrophilic group has a molecular weight of about 5,000 to about 10,000 Da. In some embodiments, the hydrophilic group has a molecular weight of about 15,000 to about 20,000 Da.

[0225]

[0217] In some embodiments, the lipid and the hydrophilic group are covalently (e.g., directly) linked. In some embodiments, the covalent bond is a non-cleavable linkage or a cleavable linkage. In some embodiments, the non-cleavable linkage includes an amide bond or phosphate bond, and the cleavable linkage includes a disulfide bond, acid-cleavable linkage, ester bond, anhydride bond, biodegradable bond, or enzyme-cleavable linkage.

[0226]

[0218] In certain embodiments, the hydrophilic group comprises one or more ethylene glycol (EG) units, more preferably two or more EG units (z.e., polyethylene glycol (PEG)).

[0227]

[0219] In some embodiments, amphiphilic linkers suitable for use in the compositions and methods disclosed herein contain a lipid linked to a PEG molecule. The PEG molecule is, in turn, covalently linked to an immunogen (e.g., peptide antigen or protein antigen). The precise number of EG units depends on the lipid and the nanoemulsion.

[0228]

[0220] In some embodiments, the hydrophilic group comprises a PEG molecule or other similarly soluble polymer. The PEG molecule is a repeating unit of polyethylene glycol represented as (PEG)n, where n represents the number of repeating PEG monomers (z.e., EG units). In some embodiments, the number of repeating PEG monomers (n) in the PEG molecule can be between about 2 and about 150, between about 2 and about 125, between about 2 and about 100, between about 2 and about 50, between about 50 and about 100, between about 100 and about 150. In some embodiments the number of repeating PEG monomers (ne) in the PEG molecule can be between about 2 and about 96, between about 10 and about 90, between about 20 and about 80, between about 30 and about 70, or between about 40 and about 60 monomers. In certain embodiments, the number of repeating. In certain embodiments, the number of repeating PEG monomers in the PEG linker is between about 45 and 55 monomers. In certain embodiments, the number of repeating PEG monomers in the PEG linker (e.g., first linker) is about 24 monomers.

[0229]

[0221] In some embodiments, the PEG molecule has a molecular weight of about 300 -

[0230] 20,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about

[0231] 1,000-1,300 daltons, In some embodiments, the PEG molecule has a molecular weight of about 1,500 daltons, In some embodiments, the PEG molecule has a molecular weight of about 2,000 daltons, In some embodiments, the PEG molecule has a molecular weight of about 2,500 daltons, In some embodiments, the PEG molecule has a molecular weight of about 3,500 daltons, In some embodiments, the PEG molecule has a molecular weight of about 4,000 daltons, In some embodiments, the PEG molecule has a molecular weight of about 5,000 daltons, In some embodiments, the PEG molecule has a molecular weight of about 6,000 daltons, In some embodiments, the PEG molecule has a molecular weight of about 7,000 daltons, In some embodiments, the PEG molecule has a molecular weight of about 8,000 daltons, In some embodiments, the PEG molecule has a molecular weight of about 9,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 10,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 11,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 12,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 13,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 14,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 15,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 16,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 17,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 18,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 19,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 20,000 daltons.

[0232]

[0222] In certain embodiments, the hydrophilic group comprises or consists of an oligonucleotide. Non-limiting examples of oligonucleotides applicable for the amphiphilic linker of the present disclosure may be found in WO 2019 / 060425, the entire contents of which are incorporated herein by reference. The oligonucleotides can have any sequence, for example, the sequence of the oligonucleotide can be a random sequence, or a sequence specifically chosen for its molecular or biochemical properties (e.g., highly polar). In certain embodiments, the hydrophilic group linker includes one or more series of consecutive adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analog thereof. In certain embodiments, the hydrophilic group linker consists of a series of consecutive adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analog thereof.

[0233]

[0223] In certain embodiments, the hydrophilic group comprises one or more guanines, for example between 1-30 guanines. The number of guanines in the linker can be selected based on the desired stability of an amphiphilic linker in a nanoemulsion.

[0234] Immunogen

[0235]

[0224] Vaccines of the present disclosure comprise an immunogen conjugated to the nanoemulsion via an amphiphilic linker. In some embodiments, the immunogen is covalently linked to the amphiphilic linker. In some embodiments, the immunogen is covalently linked to the hydrophilic group of the amphiphilic linker. In some embodiments, the covalent link is a non-cleavable linkage or a cleavable linkage. In some embodiments, the non-cleavable linkage includes an amide bond or phosphate bond, and the cleavable linkage includes a disulfide bond, acid-cleavable linkage, ester bond, anhydride bond, biodegradable bond, or enzyme-cleavable linkage.

[0236]

[0225] In some embodiments, the immunogen is derived from a disease-causing organism selected from a bacteria, fungus, parasite, and virus. In some embodiments, the immunogen is not a live- attenuated virus.

[0237]

[0226] In some embodiments the immunogen is a peptide antigen (also referred to herein as an antigenic peptide), a protein antigen (also referred to herein as antigenic protein), or a polysaccharide antigen. In some embodiments, the immunogen is an antigenic peptide or a protein antigen. In some embodiments, the immunogen comprises or consists of an peptide antigen or a protein antigen. In some embodiments the immunogen comprises or consists of a polysaccharide antigen.

[0227] In some embodiments, the immunogen is an antigenic peptide. As used herein, an “antigenic peptide” has fewer than 50 amino acids and comprises at least one sequence of amino acids sufficient to elicit an immune response, e.g., cell-mediated immune response.

[0238]

[0228] In some embodiments, the peptide antigen comprises about 4 to about 50 amino acids, about 4 to about 45 amino acids, about 4 to about 40 amino acids, about 4 to about 35 amino acids, about 4 to about 30 amino acids, about 4 to about 25 amino acids, about 4 to about 20 amino acids, about 4 to about 15 amino acids or about 4 to about 10 amino acids. In some embodiments, the peptide antigen comprises about 5 to about 50 amino acids, about 5 to about 45 amino acids, about 5 to about 40 amino acids, about 5 to about 35 amino acids, about 5 to about 30 amino acids, about 5 to about 25 amino acids, about 5 to about 20 amino acids, about 5 to about 15 amino acids, or about 5 to about 10 amino acids. In some embodiments, the peptide antigen comprises about 10 to about 50 amino acids, about 10 to about 45 amino acids, about 10 to about 40 amino acids, about 10 to about 35 amino acids, about 10 to about 30 amino acids, about 10 to about 25 amino acids, about 10 to about 20 amino acids, or about 10 to about 15 amino acids. In some embodiments, the peptide antigen comprises about 15 to about 50 amino acids, about 15 to about 45 amino acids, about 15 to about 40 amino acids, about 15 to about 35 amino acids, about 15 to about 30 amino acids, about 15 to about 25 amino acids, or about 15 to about 20 amino acids.

[0239]

[0229] In some embodiments, the immunogen is not an antigenic peptide.

[0240]

[0230] For many types of infectious diseases that transmit via muscosal routes such as HIV, SARS-CoV-2 and influenza, larger protein antigens that more closely resemble native proteins of the infectious agents are believed to be significantly more effective at inducing an immune response through vaccination than small peptides. Accordingly, in some embodiments, the immunogen is a protein antigen. As used herein, a “protein antigen” comprises at least 50 or more amino acids and includes at least one sequence of amino acids sufficient to elicit an immune response, e.g., a humoral antibody-mediated immune response.

[0241]

[0231] In some embodiments, the protein antigen comprises at least 50 amino acids, at least 51 amino acids, at least 52 amino acids, at least 53 amino acids, at least 54 amino acids, at least 55 amino acids, at least 56 amino acids, at least 57 amino acids, at least 58 amino acids, at least 59 amino acids, at least 60 amino acids, at least 75 amino acids, at least 100 amino acids, at least 125 amino acids, at least 150 amino acids, at least 175 amino acids, at least 200 amino acids, at least 250 amino acids, at least 300 amino acids, at least 350 amino acids, at least 400 amino acids, at least 450 amino acids, at least 500 amino acids, at least 550 amino acids, at least 600 amino acids, at least 650 amino acids, at least 700 amino acids, at least 750 amino acids, at least 800 amino acids, at least 850 amino acids, at least 900 amino acids, at least 950 amino acids, at least 1000 amino acids, at least 1050 amino acids, at least 1100 amino acids, at least 1150 amino acids, at least 1200 amino acids, at least 1250 amino acids, at least 1300 amino acids, at least 1350 amino acids, at least 1400 amino acids, at least 1450 amino acids, at least 1500 amino acids, at least 1550 amino acids, at least 1600 amino acids, at least 1650 amino acids, at least 1700 amino acids, at least 1750 amino acids, at least 1800 amino acids, at least 1850 amino acids, at least 1900 amino acids, at least 1950 amino acids, at least 2000 amino acids, at least 2050 amino acids, at least 2100 amino acids, at least 2150 amino acids, at least 2200 amino acids, at least 2300 amino acids, at least 2400 amino acids, at least 2500 amino acids, at least 2600 amino acids, at least 2700 amino acids, at least 2800 amino acids, at least 2900 amino acids, at least 3000 amino acids, at least 3100 amino acids, at least 3200 amino acids, at least 3300 amino acids, at least 3400 amino acids, at least 3500 amino acids, at least 3600 amino acids, at least 3700 amino acids, at least 3800 amino acids, at least 3900 amino acids, at least 4000 amino acids, at least 4100 amino acids, at least 4200 amino acids, at least 4300 amino acids, at least 4400 amino acids, or at least 4500 amino acids.

[0242]

[0232] In some embodiments, the protein antigen comprises greater than 50 amino acids, greater than 51 amino acids, greater than 52 amino acids, greater than 53 amino acids, greater than 54 amino acids, greater than 55 amino acids, greater than 56 amino acids, greater than 57 amino acids, greater than 58 amino acids, greater than 59 amino acids, greater than 60 amino acids, greater than 75 amino acids, greater than 100 amino acids, greater than 125 amino acids, greater than 150 amino acids, greater than 175 amino acids, greater than 200 amino acids, greater than 250 amino acids, greater than 300 amino acids, greater than 350 amino acids, greater than 400 amino acids, greater than 450 amino acids, greater than 500 amino acids, greater than 550 amino acids, greater than 600 amino acids, greater than 650 amino acids, greater than 700 amino acids, greater than 750 amino acids, greater than 800 amino acids, greater than 850 amino acids, greater than 900 amino acids, greater than 950 amino acids, greater than 1000 amino acids, greater than 1050 amino acids, greater than 1100 amino acids, greater than 1150 amino acids, greater than 1200 amino acids, greater than 1250 amino acids, greater than 1300 amino acids, greater than 1350 amino acids, greater than 1400 amino acids, greater than 1450 amino acids, greater than 1500 amino acids, greater than 1550 amino acids, greater than 1600 amino acids, greater than 1650 amino acids, greater than 1700 amino acids, greater than 1750 amino acids, greater than 1800 amino acids, greater than 1850 amino acids, greater than 1900 amino acids, greater than 1950 amino acids, greater than 2000 amino acids, greater than 2050 amino acids, greater than 2100 amino acids, greater than 2150 amino acids, greater than 2000 amino acids greater than 2300 amino acids, greater than 2400 amino acids, greater than 2500 amino acids, greater than 2600 amino acids, greater than 2700 amino acids, greater than 2800 amino acids, greater than 2900 amino acids, greater than 3000 amino acids, greater than 3100 amino acids, greater than 3200 amino acids, greater than 3300 amino acids, greater than 3400 amino acids, greater than 3500 amino acids, greater than 3600 amino acids, greater than 3700 amino acids, greater than 3800 amino acids, greater than 3900 amino acids, greater than 4000 amino acids, greater than 4100 amino acids, greater than 4200 amino acids, greater than 4300 amino acids, greater than 4400 amino acids, or greater than 4500 amino acids.

[0243]

[0233] In some embodiments the protein antigen comprises about 50 to 5000 amino acids, about 50 to 4500 amino acids, about 50 to 4000 amino acids, about 50 to 3500 amino acids, about 50 to 3000 amino acids, or about 51 to 3000 amino acids. In some embodiments, the protein antigen comprises about 100 to 5000 amino acids, about 100 to 4500 amino acids, about 100 to 4000 amino acids, about 100 to 3500 amino acids, about 100 to 3000 amino acids, about 100 to about 2500 amino acids, about 100 to about 2000 amino acids, about 100 to about 1500 amino acids, about 100 to about 1000 amino acids, about 100 to about 750 amino acids, about 100 to about 500 amino acids, or about 100 to about 300 amino acids. In some embodiments the protein antigen comprises about 200 to 5000 amino acids, about 200 to 4500 amino acids, about 200 to 4000 amino acids, about 200 to 3500 amino acids, about 200 to 3000 amino acids, about 200 to about 2500 amino acids, about 200 to about 2000 amino acids, about 200 to about 1500 amino acids, about 200 to 1000 amino acids, about 300 to about 900 amino acids, about 400 to about 800 amino acids, or about 500 to about 700 amino acids. In some embodiments the protein antigen comprises about 250 to 5000 amino acids, about 500 to 5000 amino acids, about 750 to 5000 amino acids, about 1000 to 5000 amino acids, about 1500 to 5000 amino acids, about 2000 to 5000 amino acids, about 2500 to about 5000 amino acids, about 3000 to about 5000 amino acids, about 3500 to about 5000 amino acids, or about 4000 to 5000 amino acids. In some embodiments, the protein antigen comprises about 100 to about 3000 amino acids, about 250 to about 2750 amino acids, about 400 to about 2500 amino acids, about 500 to about 2500 amino acids, about 750 to about 2500 amino acids, about 1000 to about 2500 amino acids, or about 1500 to about 2500 amino acids.

[0244]

[0234] In some embodiments, the protein antigen has a molecule weight (MW) of about 10 kDa to about 500 kDa. In some embodiments, the protein antigen has a molecule weight (MW) of about 10 kDa to about 500 kDa, about 10 kDa to about 450 kDa, about 10 kDa to about 400 kDa, about 10 kDa to about 350 kDa, about 10 kDa to about 300 kDa, about 10 kDa to about 250 kDa, about 10 kDa to about 200 kDa, about 10 kDa to about 150 kDa, about 10 kDa to about 100 kDa, about 10 kDa to about 50 kDa. In some embodiments, the protein antigen has a MW of about about 20 kDa to about 500 kDa, about 20 kDa to about 450 kDa, 20 kDa to about 400 kDa, 20 kDa to about 350 kDa, about 20 kDa to about 300 kDa, about 20 kDa to about 250 kDa, about 20 kDa to about 200 kDa, about 20 kDa to about 150 kDa, about 20 kDa to about 100 kDa, or about 20 kDa to about 50 kDa. In some embodiments, the protein antigen has a MW of about 30 kDa to about 500 kDa, about 30 kDa to about 450 kDa, about 30 kDa to about 400 kDa, 30 kDa to about 350 kDa, about 30 kDa to about 300 kDa, about 30 kDa to about 250 kDa, or about 30 kDa to about 200 kDa. In some embodiments, the protein antigen has a MW of about about 50 kDa to about 500 kDa, about 50 kDa to about 450 kDa, 50 kDa to about 400 kDa, about 50 kDa to about 350 kDa, about 50 kDa to about 300 kDa, about 50 kDa to about 250 kDa, or about 50 kDa to about 200 kDa, about 50 kDa to about 150 kDa, or about 50 kDa to about 100 kDa. In some embodiments, the protein antigen has a MW of about about 75 kDa to about 500 kDa, about 75 kDa to about 450 kDa, 75 kDa to about 400 kDa, about 75 kDa to about 350 kDa, about 75 kDa to about 300 kDa, about 75 kDa to about 250 kDa, about 75 kDa to about 200 kDa, about 75 kDa to about 150 kDa, or about 75 kDa to about 100 kDa. In some embodiments, the protein antigen has a MW of about about 100 kDa to about 500 kDa, about 100 kDa to about 450 kDa, 100 kDa to about 400 kDa, about 100 kDa to about 350 kDa, about 100 kDa to about 300 kDa, about 100 kDa to about 250 kDa, about 100 kDa to about 200 kDa, or about 100 kDa to about 150 kDa. In some embodiments, the protein antigen has a MW of about about 150 kDa to about 500 kDa, about 150 kDa to about 450 kDa, 150 kDa to about 400 kDa, about 150 kDa to about 350 kDa, about 150 kDa to about 300 kDa, about 150 kDa to about 250 kDa, about 150 kDa to about 200 kDa. In some embodiments, the protein antigen has a MW of about 200 kDa to about 500 kDa, about 200 kDa to about 450 kDa, about 200 kDa to about 400 kDa, about 200 kDa to about 350 kDa, about 200 kDa to about 300 kDa, or about 200 kDa to about 250 kDa. In some embodiments, the protein antigen has a MW of about 250 kDa to about 500 kDa, about 250 kDa to about 450 kDa, about 250 kDa to about 400 kDa, about 250 kDa to about 350 kDa, or about 250 kDa to about 300 kDa. In some embodiments, the protein antigen has a MW of about 300 kDa to about 500 kDa, about 300 kDa to about 450 kDa, about 300 kDa to about 400 kDa, or about 300 kDa to about 350 kDa. In some embodiments, the protein antigen has a MW of about 350 kDa to about 500 kDa, about 350 kDa to about 450 kDa, or about 350 kDa to about 400 kDa. In some embodiments, the protein antigen has a MW of about 400 kDa to about 500 kDa, or about 400 kDa to about 450 kDa.

[0245]

[0235] In some embodiments, the protein antigen is a monomeric antigen (z.e., a single antigenic polypeptide chain).

[0246]

[0236] In some embodiments, the protein antigen is a multimeric antigen, e.g., a dimer, trimer, tetramer, pentamer, hexamer, septamer, octamer, or decamer. In some embodiments, the protein antigen is a dimer antigen. In some embodiments, the protein antigen is a trimer antigen. In some embodiments, the multimeric antigen comprises identical monomer subunits, i.e., repeating sequences of the same antigen, such as two repeating sequences of the same antigen (i.e., a homodimer antigen) or three repeating sequences of the same antigen (i.e., a homotrimer antigen). In some embodiments, the multimeric antigen comprises different monomer subunits, i.e., different protein antigen sequences from the same pathogen, such as two different sequences from the same pathogen (i.e., a heterodimer antigen) or three different sequences from the same pathogen (i.e., a heterotrimer antigen). In some embodiments, two or more of the protein antigen sequences of the monomer subunits of the multimeric antigen are each from different pathogens.

[0247]

[0237] In some embodiments, the protein antigen can be derived from a virus, bacterium, parasite, plant, protozoan, fungus.

[0248]

[0238] In some embodiments the antigen is not a protein antigen.

[0249]

[0239] Suitable antigenic peptides or protein antigens are commonly known in the art and are available from commercial, government, and scientific sources. The antigens may be purified or partially purified polypeptides derived from viral or bacterial sources. The antigens can be recombinant polypeptides produced by expressing DNA encoding the polypeptide antigen in a heterologous expression system.

[0250]

[0240] In some embodiments, antigenic peptide or protein antigen can be from a virus, including but not limited to a virus from any of the following viral families: Arenaviridae, Arterivirus, Astroviridae, Baculoviridae, Badnavirus, Barnaviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Capillovirus, Carlavirus, Caulimovirus, Circoviridae, Closterovirus, Comoviridae, Coronaviridae (e.g., Coronavirus, such as severe acute respiratory syndrome (SARS) virus), Corticoviridae, Cystoviridae, Deltavirus, Dianthovirus, Enamovirus, Filoviridae (e.g., Marburg virus and Ebola virus (e.g., Zaire, Reston, Ivory Coast, or Sudan strain)), Flaviviridae, (e.g., Hepatitis C virus, Dengue virus 1, Dengue virus 2, Dengue virus 3, and Dengue virus 4), Hepadnaviridae, Herpesviridae (e.g., Human herpesvirus 1, 3, 4, 5, and 6, and Cytomegalovirus), Hypoviridae, Iridoviridae, Eeviviridae, Lipothrixviridae, Microviridae, Orthomyxoviridae (e.g., Influenzavirus A and B and C), Papovaviridae, Paramyxoviridae (e.g., measles, mumps, and human respiratory syncytial virus), Parvoviridae, Picomaviridae (e.g., poliovirus, rhinovirus, hepatovirus, and aphthovirus), Poxviridae (e.g., vaccinia and smallpox virus), Reoviridae (e.g., rotavirus), Retroviridae (e.g., lentivirus, such as human immunodeficiency virus (HIV) 1 and HIV 2), Rhabdoviridae (for example, rabies virus, measles virus, respiratory syncytial virus, etc.), Togaviridae (for example, rubella virus, dengue virus, etc.), and Totiviridae. Suitable viral antigens also include all or part of Dengue protein M, Dengue protein E, Dengue D1NS1, Dengue D1NS2, and Dengue D1NS3.

[0251]

[0241] Viral antigens may be derived from a particular strain such as a papilloma virus, a herpes virus, e.g., herpes simplex 1 and 2; a hepatitis virus, for example, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), the delta hepatitis D virus (HDV), hepatitis E virus (HEV) and hepatitis G virus (HGV), the tick-bome encephalitis viruses; parainfluenza, varicella-zoster, cytomeglavirus, Epstein-Barr, rotavirus, rhinovirus, adenovirus, coxsackieviruses, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, and lymphocytic choriomeningitis.

[0252]

[0242] In some embodiments, the antigenic peptide or protein antigen can be from a bacteria, including but not limited to a bacteria from any of the following families: Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Hemophilus influenza type B (HIB), Hyphomicrobium, Eegionella, Eeptspirosis, Listeria, Meningococcus A, B and C, Methanobacterium, Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas, Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum, Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus, and. Treponema, Vibrio, and Yersinia.

[0253]

[0243] In some embodiments, the antigenic peptide or protein antigen can be from a parasite, including but not limited to a parasite from any of the following families: Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis and Schistosoma mansoni. These include Sporozoan antigens, Plasmodian antigens, such as all or part of a Circumsporozoite protein, a Sporozoite surface protein, a liver stage antigen, an apical membrane associated protein, or a Merozoite surface protein.

[0254]

[0244] In some embodiments, the protein antigen or antigenic peptide comprises a human immunodeficiency virus (HIV) antigen, a SARS-CoV-2 antigen, an influenza antigen, a rotavirus antigen, a cytomegalovirus (CMV) antigen, an Epstein-Barr virus antigen, a respiratory syncytial virus (RSV) antigen, or a cholera antigen. In some embodiments, the protein antigen comprises a human immunodeficiency virus (HIV) antigen, a SARS-CoV-2 antigen, an influenza antigen, a rotavirus antigen, a cytomegalovirus (CMV) antigen, an Epstein-Barr virus antigen, a respiratory syncytial virus (RSV) antigen, or a cholera antigen. In some embodiments, the antigenic peptide comprises a human immunodeficiency virus (HIV) antigen, a SARS-CoV-2 antigen, an influenza antigen, a rotavirus antigen, a cytomegalovirus (CMV) antigen, an Epstein-Barr virus antigen, a respiratory syncytial virus (RSV) antigen, or a cholera antigen.

[0255]

[0245] In some embodiments, the protein antigen comprises an HIV antigen. In some embodiments, the protein antigen comprises a SARS-CoV-2 antigen. In some embodiments, the protein antigen comprises an influenza antigen. In some embodiments, the protein antigen comprises a rotavirus antigen. In some embodiments, the protein antigen comprises a CMV antigen. In some embodiments, the protein antigen comprises a cholera antigen. In some embodiments, the antigenic peptide comprises an HIV antigen. In some embodiments, the antigenic peptide comprises a SARS-CoV-2 antigen. In some embodiments, the antigenic peptide comprises an influenza antigen. In some embodiments, the antigenic peptide comprises a rotavirus antigen. In some embodiments, the antigenic peptide comprises a CMV antigen. In some embodiments, the antigenic peptide comprises a cholera antigen.

[0246] In some embodiments, the amino acid sequence of the antigenic peptide or protein antigen may be naturally existing amino acid sequence of the antigen. In some embodiments, the antigenic peptide or protein antigen may be a sequence modified from the naturally existing amino acid sequence of the antigen. The modifications may serve to enhance antigenicity or improve production of the vaccine.

[0256]

[0247] Human immunodeficiency virus (HIV) antigens are commonly known in the art. Non-limiting examples of HIV antigens may be found in Jardine et al (2015) (Priming a broadly neutralizing antibody response to HIV-1 using a germline-targeting immunogen. Science. 349(6244): 156-61); Kim et al (2021) (Current approaches to HIV vaccine development: a narrative review. J Int AIDS Soc., 24: e25793); and Haynes et al (2023) (Strategies for HIV-1 vaccines that induce broadly neutralizing antibodies. Nat Rev Immunol 23, 142-158), the entire contents of each of which are incorporated herein by reference.

[0257]

[0248] In some embodiments, the HIV antigen comprises or consists of an HIV envelope protein (Env) antigen. In some embodiments, the HIV Env antigen is a gpl20 antigen or gpl40 antigen. In some embodiments, the HIV Env antigen is a gpl20 antigen. In some embodiments, the HIV Env antigen is gpl20 engineered outer domain-germ line-targeting immunogen 8 (eOD-GT8). In some embodiments, the eOD-GT8 gpl20 antigen comprises or consist of the amino acid sequence of SEQ ID NO: 1.

[0258]

[0249] In some embodiments, the HIV envelope protein antigen is a native-like trimer antigen (of repeating monomers) that mimics the structure of the virion- associated spike (e.g., HIV MD39 SOSIP).

[0259]

[0250] SARS-CoV-2 antigens are commonly known in the art. Examples of SARS-CoV- 2 antigens used for existing vaccine technology as well as those under testing and experimentation may be found in Poland et al (2020) (SARS-Cov-2 Immunity: Review and Applications to Phase 3 Vaccine Candidates. Lancet 396:1595-606); Dalvie et al (2021) (Engineered SARS-CoV-2 receptor binding domain improves manufacturability in yeast and immunogenicity in mice. Proc. Natl. Acad. Sci. U.S.A. 118, e2106845118), and Jang et al (2020) (A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity. Nature 586, 572 - 577), which are incorporated herein by reference.

[0260]

[0251] In some embodiments, the SARS-CoV-2 antigen comprises a SARS-CoV-2 spike protein (also known as “S protein”), or an antigenic fragment of the spike protein. In some embodiments, the SARS-CoV-2 antigen comprises an antigen from the SI subunit of the spike protein. In some embodiments, the SARS-CoV-2 antigen comprises an antigen from the N-terminal domain of the spike protein. In some embodiments, the SARS-CoV-2 antigen comprises an antigen from the receptor binding domain (RBD) of the spike protein. In some embodiments, the SARS-CoV-2 antigen comprises an antigen of the S2 subunit of the spike protein.

[0261]

[0252] In some embodiments, the protein antigen comprises the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, or an antigen derived from the RBD. In some embodiments, the SARS-CoV-2 RBD protein antigen comprises or consists of the amino acid sequence of SEQ ID NO: 2.

[0262]

[0253] Influenza antigens are commonly known in the art and may be found in Gomez Lorenzo et al (2013) (Immunobiology of influenza vaccines. Chest. 143(2):502-510; Rao et al (2010) Comparative efficacy of hemagglutinin, nucleoprotein, and matrix 2 protein genebased vaccination against H5N1 influenza in mouse and ferret. PLoS One. 5(3):e9812), incorporated herein by reference. In some embodiments, the influenza antigen comprises a hemagglutinin (HA) antigen, a neuraminidase antigen, a nucleoprotein (NP) antigen or an ion channel matrix protein (M2) antigen.

[0263]

[0254] Rotavirus antigens are commonly known in the art. Teachings of antigens known to elicit expression of antibodies, particularly neutralizing antibodies, against rotarovirus may be found in, e.g., US7311918B2, US 6,16431, and Dennehy (2008) (Rotavirus vaccines: an overview. Clin Microbiol Rev. 21(1): 198-208), incorporated herein by reference. In some embodiments, the rotarovirus antigen comprises a VP4 antigen, VP6 antigen, or VP7 antigen.

[0264]

[0255] Cytomegalovirus (CMV) antigens are commonly known in the art. Teachings of CMV antigens may be found at, e.g., Nelson et al (2018) (A new era in cytomegalovirus vaccinology: considerations for rational design of next-generation vaccines to prevent congenital cytomegalovirus infection, npj Vaccines 3, 38), incorporated herein by reference.

[0265] Neutralizing antibodies targeting proteins gB, gH, and UL128-131A of CMV have been found after natural infection. In some embodiments, the CMV antigen comprises a gB antigen, gH antigen, or a UL128-131A antigen.

[0266]

[0256] Currently, no vaccines against Epstein-Barr virus (EBV) have been successfully developed, though EBV antigens that elicit antibody production, particularly neutralizing antibody production, are commonly known in the art. Teachings of known EBV antigens may be found at Cui et al (2021) (Epstein Barr Virus: Development of Vaccines and Immune Cell Therapy for EBV- Associated Diseases. Front. Immunol., Vol 12), incorporated herein by reference. In some embodiments, the EBV antigen comprises a gp35O antigen, a gH antigen, a gL antigen, or gB antigen.

[0267]

[0257] Most recent attempts to generate an respiratory syncytial virus (RSV) vaccine have been based on the F protein of RSV, as the F protein mediates virus entry into host cells and an anti-F antibody has been shown to reduce severe RSV disease in high-risk infants. Other proteins which have been shown to be capable of eliciting neutralizing antibodies include the N and M2-1 protein. Teachings of known RSV antigens may be found at, e.g., Ciconi et al (2020) (First-in-Human Randomized Study to Assess the Safety and Immunogenicity of an Investigational Respiratory Syncytial Virus (RSV) Vaccine Based on Chimpanzee-Adenovirus-155 Viral Vector-Expressing RSV Fusion, Nucleocapsid, and Antitermination Viral Proteins in Healthy Adults, Clinical Infectious Diseases, 70(10): 2073- 2081) and Graham et al (2015) (Novel antigens for RSV vaccines. Curr Opin

[0268] Immunol.35:30-8), incorporated herein by reference. In some embodiments, the RSV antigen comprises a F protein antigen, an N protein antigen, or an M2-1 protein antigen.

[0269]

[0258] Research on immune response to cholera e.g., Vibrio cholerae) infection has focuses primarily on antibodies. Antibody responses have been found against the O-specific polysaccharide of V. cholerae, as well as against the A subunit (CtxA) and B subunit (CtxB) of cholera toxin (see Harris (2018) Cholera: Immunity and Prospects in Vaccine Development. J Infect Dis. 15;218(suppl_3):S141-S146; incorporated herein by reference). In some embodiments, the cholera antigen comprises the O-specific polysaccharide of V. cholerae, a CtxA antigen, or a CtxB antigen.

[0270]

[0259] In some embodiments, the immunogen comprises or consists of a polysaccharide antigen, e.g., a bacterial polysaccharide. In some embodiments, the immunogen is a polysaccharide antigen, e.g., a bacterial polysaccharide. Polysaccharides are major components on the surface of bacteria. Polysaccharide-encapsulated bacteria are the leading cause for several serious bacterial infection in childen, such as bacterial meningitis and pneumonia. The polysaccharide capsules of bacteria determine their virulence, and therefore targeting their capsidal polysaccharide can confer significant protection against bacteria infections.

[0271]

[0260] Bacterial polysaccharides are very heterogeneous within and between species, and they are also T-lymphocyte independent antigens. With a few exceptions, immunization with free polysaccharides generally stimulates short-lived B-cell responses and can even result in hyporesponsiveness to future vaccine doses. Recent studies suggest that polysaccharide conjugates may induce T-cell dependent response and stronger B-cell response, resulting in long-term immunity (see, e.g., Pollard et al (2009) Maintaining protection against invasive bacteria with protein-polysaccharide conjugate vaccines. Nat Rev Immunol 9, 213-220).

[0272]

[0261] Polysaccharide antigens are commonly known in the art. Teachings of known polysaccharide antigens, particularly those that have been developed to be polysaccharide vaccines, may be found at, e.g., Perera et al (2021) (Polysaccharide Vaccines: A Perspective on Non-Typhoidal Salmonella” Polysaccharides 2, no. 3: 691-714); and Aithal et al (2012) (PolysacDB: A Database of Microbial Polysaccharide Antigens and Their Antibodies. PLoS ONE 7(4): e34613), the entire contents of each of which are incorporated herein by reference.

[0273]

[0262] In some embodiments, the polysaccharide antigen is a cholera (e.g., Vibrio cholerae) antigen. In some embodiments, the polyssachride antigen is an O-specific polysaccharide of V. cholera.

[0274]

[0263] In some embodiments, the immunogen further comprises a pan human leukocyte antigen DR-binding epitope (PADRE) peptide (e.g., AKFVAAWTLKAAA, SEQ ID NO: 3), a T-helper epitope from tetanus toxoid (e.g., QYIKANSKFIGITEL, SEQ ID NO: 4), or a T- helper peptide from diphtheria toxoid (e.g., QSIALSSLMVAQAIP, SEQ ID NO: 5). The presence of these helper peptide has been shown to improve antibody immune response postvaccination in several studies (see, e.g., Rosa et al. “The pan HLA DR-binding epitope improves adjuvant-assisted immunization with a recombinant protein containing a malaria vaccine candidate” Immunol Lett. 2004; 92(3):259-68; Laubreton et al. “The fully synthetic MAG-Tn3 therapeutic vaccine containing the tetanus toxoid-derived TT830-844 universal epitope provides anti-tumor immunity” Cancer Immunol Immunother. 2016, 65(3):315-25; PMCID: PMC4779142.; Du et al “Diphtheria Toxoid-Derived T-Helper Epitope and a- galactosylceramide Synergistically Enhance the Immunogenicity of Glycopeptide Antigen” ACS Pharmacology & Translational Science 2024, 7(12): 3889-3901; the entire content of each are incorporated herein by reference).

[0275]

[0264] In another aspect, the present disclosure provides a kit comprising a vaccine as described herein. In some embodiments, the kit further comprises instructions for any of the methods described herein.

[0276] III. METHODS OF PREPARATION

[0277] Methods of Preparing the Immunogen

[0265] In some embodiments, the antigenic peptide or protein antigen described herein for use in the immunogen-loaded nanoemulsions s are made in transformed host cells using recombinant nucleic acid, e.g., DNA or RNA, techniques. To do so, a recombinant nucleic acid molecule coding for the antigenic peptide or protein antigen is prepared. Methods of preparing such nucleic acid molecules are well known in the art. For example, sequences coding for the antigenic peptides or protein antigens can be excised from a nucleic acid molecule using suitable restriction enzymes. Alternatively, the nucleic acid molecule can be synthesized using chemical synthesis techniques, such as the phosphoramidate method. A combination of these techniques can be used.

[0278]

[0266] The methods of making an antigenic peptide or protein antigen also include preparing a vector capable of expressing the antigenic peptide or protein antigen in an appropriate host. The vector comprises the nucleic acid molecule that codes for the peptide or protein antigen operatively linked to appropriate expression control sequences. Methods of affecting this operative linking, either before or after the nucleic acid molecule is inserted into the vector, are well known in the art. Expression control sequences include promoters, activators, enhancers, operators, ribosomal nuclease domains, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved with the control of transcription or translation. The resulting vector comprising the nucleic acid molecule encoding the peptide or protein antigen is used to transform an appropriate host. This transformation may be performed using methods well known in the art.

[0279]

[0267] Any of a large number of available and well-known host cells may be suitable for use in the methods disclosed herein. The selection of a particular host is dependent upon a number of factors recognized by the art. These include, for example, compatibility with the chosen expression vector, toxicity of the peptides encoded by the nucleic acid molecule, rate of transformation, ease of recovery of the peptides, expression characteristics, bio-safety and costs. A balance of these factors must be struck with the understanding that not all hosts may be equally effective for the expression of a particular nucleic acid sequence. Within these general guidelines, useful microbial hosts include bacteria (such as E. coli sp.y yeast (such as Saccharomyces .sp.) and other fungi, insects, plants, mammalian (including human) cells in culture, or other hosts known in the art.

[0280]

[0268] Next, the transformed host is cultured and purified. Host cells may be cultured under conventional fermentation conditions so that the desired compounds are expressed. Such fermentation conditions are well known in the art. Finally, the antigenic peptides or protein antigens are purified from the cells or culture medium by methods well known in the art.

[0281]

[0269] The antigenic peptides or protein antigens may also be prepared by synthetic methods. For example, solid phase synthesis techniques may be used. Suitable techniques are well known in the art, and include those described in Merrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.); Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis et al. (1985), Biochem. Inti. 10: 394-414; Stewart and Young (1969), Solid Phase Peptide Synthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976), The Proteins (3rd ed.) 2: 105- 253; and Erickson et al. (1976), The Proteins (3rd ed.) 2: 257-527, the entire contents of each of which are incorporated by reference herein. Solid phase synthesis is the preferred technique of making individual peptides since it is the most cost-effective method of making small peptides. Compounds that contain derivatized peptides or which contain non-peptide groups may be synthesized by well-known organic chemistry techniques.

[0282]

[0270] Other methods of nucleic acid expression and synthesis are generally known to one of ordinary skill in the relevant art.

[0283]

[0271] The nucleic acid molecules described above can be contained within a vector that is capable of directing their expression in, for example, a cell that has been transduced with the vector. Accordingly, expression vectors containing a nucleic acid molecule encoding a peptide or protein antigen and cells transfected with these vectors are among the embodiments provided herein.

[0284]

[0272] Vectors suitable for use include T7 -based vectors for use in bacteria (see, for example, Rosenberg et al., Gene 56: 125, 1987), the pMSXND expression vector for use in mammalian cells (Lee and Nathans, J. Biol. Chem. 263:3521, 1988), and baculovirus-derived vectors (for example the expression vector pBacPAKS from Clontech, Palo Alto, Calif.) for use in insect cells. The nucleic acid inserts, which encode the polypeptide of interest in such vectors, can be operably linked to a promoter, which is selected based on, for example, the cell type in which expression is sought. For example, a T7 promoter can be used in bacteria, a polyhedrin promoter can be used in insect cells, and a cytomegalovirus or metallothionein promoter can be used in mammalian cells. Also, in the case of higher eukaryotes, tissuespecific and cell type-specific promoters are widely available. These promoters are so named for their ability to direct expression of a nucleic acid molecule in a given tissue or cell type within the body. Skilled artisans are well aware of numerous promoters and other regulatory elements which can be used to direct expression of nucleic acids.

[0273] In addition to sequences that facilitate transcription of the inserted nucleic acid molecule, vectors can contain origins of replication, and other genes that encode a selectable marker. For example, the neomycin-resistance (neor) gene imparts G418 resistance to cells in which it is expressed, and thus permits phenotypic selection of the transfected cells. Those of skill in the art can readily determine whether a given regulatory element or selectable marker is suitable for use in a particular experimental context.

[0285]

[0274] Viral vectors that are suitable for use include, for example, retroviral, adenoviral, and adeno-associated vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.).

[0286]

[0275] Prokaryotic or eukaryotic cells that contain and express a nucleic acid molecule that encodes a peptide or protein antigen are also suitable for use. A cell is a transfected cell, i.e., a cell into which a nucleic acid molecule, for example a nucleic acid molecule encoding a peptide or protein antigen has been introduced by means of recombinant DNA techniques. The progeny of such a cell are also considered suitable for use in the methods disclosed herein.

[0287]

[0276] The precise components of the expression system are not critical. For example, a peptide or protein antigen can be produced in a prokaryotic host, such as the bacterium E. coli, or in a eukaryotic host, such as an insect cell (e.g., an Sf21 cell), or mammalian cells (e.g., COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, Va.). In selecting an expression system, it matters only that the components are compatible with one another. Artisans or ordinary skill are able to make such a determination. Furthermore, if guidance is required in selecting an expression system, skilled artisans may consult Ausubel et al. (Current Protocols in Molecular Biology, John Wiley and Sons, New York, N.Y., 1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985 Suppl. 1987).

[0288]

[0277] The expressed peptide or protein antigens can be purified from the expression system using routine biochemical procedures, and can be used, e.g., conjugated to an amphiphilic linker as described herein.

[0289] Methods of preparing the immunogen-loaded nanoemulsions

[0290]

[0278] In some aspects, the present disclosure provides methods for assembling the nanoemulsion of the vaccine of the present disclosure.

[0279] In some embodiments, to facilitate conjugation of the immunogen to the amphiphilic linker, the amphiphilic linker and immunogen each further comprise a reactive functional group, wherein the reactive functional groups can react with each other under appropriate conditions to form a linkage, such as a covalent bond.

[0291]

[0280] In some embodiments, a reactive functional group is attached to the hydrophilic group of the amphiphilic linker.

[0292]

[0281] In some embodiments, a nanoemulsion is first prepared with an amphiphilic linker comprising a reactive functional group (herein referred to as “functionalized amphiphilic linker”) tethered to the surfactant shell. The reactive functional group (e.g., azide group) is located on the hydrophilic group of the amphiphilic linker. For example, in the process of making nanoemulsions, the functionalized amphiphilic linker can be added to the mixture of oil phase, aqueous phase and surfactant(s) prior to high-speed mixing and sonication or high- pressure homogenization. When nanoemulsions are formed, the amphiphilic linker, having physical properties similar to the surfactant(s), settles in the layer of surfactant shell and aligns in the same manner as the surfactant(s), with the lipid end facing inward to the lipophilic (oil) core and the hydrophilic group facing outward to the aqueous phase. In this way, the reactive functional group located on the hydrophilic group is displayed on the surface of the nanoemulsions. Under appropriate conditions, when the nanoemulsions are mixed with an immunogen carrying a functional group that is reactive with the functional group of the functionalized amphiphilic linker (e.g., dibenzocyclooctyne (DBCO) group), the two functional groups react to form a linkage between the immunogen and amphiphilic linker, thereby conjugating the immunogen to the surface of the nanoemulsion.

[0293]

[0282] Pairs of reactive functional groups used for forming a linkage between two moieties (e.g., an immunogen and an amphiphilic linker) are well known in the art, and any such groups are applicable to prepare the nanoemulsion disclosed herein. In some embodiments, the immunogen is covalently linked to the amphiphilic linker using “click chemistry”, including, but not limited to, [3+2] cycloadditions, thiol-ene reaction, Diels- Alder reactions, and [4+1] cycloadditions between isonitriles (isocyanides) and tetrazines.

[0294]

[0283] In certain embodiments, an immunogen (e.g., antigen peptide or protein antigen) is covalently conjugated to an amphiphilic linker by reacting a free thiol group of a cysteine residue comprised in the antigen or protein antigen with a reactive maleimide group present in the amphiphilic linker. In some embodiments, the cysteine residue having a free thiol group is at or near the N-terminus of the antigenic peptide or protein antigen. In some embodiments, the cysteine residue having a free thiol group is at or near the C-terminus of the antigenic peptide or protein antigen.

[0295]

[0284] In some embodiments, an immunogen (e.g., antigen peptide or protein antigen) comprising a cysteine residue containing a free thiol group at or near the N-terminus or C- terminus is allowed to react with the malemide group comprised in maleimide-funcionalized PEG-lipid linker (e.g., cholesterol-PEG24-maleimide) to form a covalent bond, thereby non- covalently conjugating the immunogen to the nanoemulsion via the amphiphilic linker.

[0296]

[0285] In some embodiments, an immunogen (e.g., antigen peptide or protein antigen) comprising a dibenzocyclooctyne (DBCO) group is allowed to react with a reactive azide group of an azide-funcionalized PEG-lipid linker (e.g., cholesterol-PEG24-azide) to form a covalent bond, thereby non-covalently conjugating the immunogen to the nanoemulsion via the amphiphilic linker.

[0297]

[0286] The nanoemulsions of the invention can be purified and characterized using standard methods in the art.

[0298]

[0287] It is within the skill of the ordinarily skilled person in the art to adjust the composition of componenets of the vaccine (e.g., the amount of the functionalized amphiphilic linker) in order to adjust the amount of immunogen carried by the nanoemulsion. In some embodiments, the immunogen-loaded nanoemulsion comprises at least 1 immunogen molecule, at least 2 immunogen molecules, at least 3 immunogen molecules, at least 4 immunogen molecules, at least 5 immunogen molecules, at least 6 immunogen molecules, at least 7 immunogen molecules, at least 8 immunogen molecules, at least 9 immunogen molecules, at least 10 immunogen molecules, at least 15 immunogen molecules, at least 20 immunogen molecules, at least 25 immunogen molecules, or at least 30 immunogen molecules per nanoemulsion particle.

[0299]

[0288] In some embodiments, the immunogen-loaded nanoemulsion comprises between 1-50 immunogen molecules, between 1-40 immunogen molecules, between 1-30 immunogen molecules, between 1-25 immunogen molecules, between 1-20 immunogen molecules, between 1-15 immunogen molecules, between 1-10 immunogen molecules, or between 1-5 immunogen molecules. In some embodiments, the immunogen-loaded nanoemulsion comprises between 2-50 immunogen molecules, between 2-40 immunogen molecules, between 2-30 immunogen molecules, between 2-25 immunogen molecules, between 2-20 immunogen molecules, between 2-15 immunogen molecules, between 2-10 immunogen molecules, or between 2-5 immunogen molecules per nanoemulsion particle.

[0289] In some embodiments, the immunogen-loaded nanoemulsion comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 immunogen molecules per nanoemulsion particle.

[0300] IV. METHODS OF USE

[0301] Formulations

[0302]

[0290] The present disclosure provides compositions comprising vaccines containing the immunogen-loaded nanoemulsions disclosed herein. The compositions and vaccines are preferably for administration by intraperitoneal injection. The vaccines can be administered using bioerodible inserts and can be formulated in dosage forms appropriate for the route of administration.

[0303]

[0291] As further studies are conducted, information will emerge regarding appropriate dosage levels for treatment of various conditions in various subjects or patients, and the ordinary skilled worker, considering the therapeutic context, age, and general health of the recipient, will be able to ascertain proper dosing. The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment desired.

[0304]

[0292] The vaccine is formulated such that the nanoemulsions are stable and intact for intraperitoneal administration. Standard pharmaceutically acceptable carrier are available from any formulator.

[0305]

[0293] In some embodiments, the vaccine composition comprising the immunogen- loaded nanoemulsions futher comprises an adjuvant. In some embodiments, the adjuvant is mixed with the immunogen-loaded nanoemulsions shortly prior to administration to the subject.

[0306] Adjuvants

[0307]

[0294] A vaccine comprising the immunogen-loaded nanoemulsions can be administered alone, or in combination with an adjuvant. In some embodiments, the vaccine can be administered separately from the adjuvant. In some embodiments, the vaccine is formulated together with the adjuvant.

[0308]

[0295] The adjuvant may be, without limitation, alum (e.g., aluminum hydroxide, aluminum phosphate); saponins purified from the bark of the Q. saponaria tree such as QS21 (a glycolipid that elutes in the 21st peak with HPLC fractionation; Antigenics, Inc., Worcester, Mass.); poly[di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus Research Institute, USA); Flt3 ligand; Leishmania elongation factor (a purified Leishmania protein; Corixa Corporation, Seattle, Wash.); ISCOMS (immunostimulating complexes which contain mixed saponins, lipids and form virus-sized particles with pores that can hold antigen; CSL, Melbourne, Australia); Pam3Cys; SB-AS4 (SmithKline Beecham adjuvant system #4 which contains alum and MPL; SBB, Belgium); non-ionic block copolymers that form micelles such as CRL 1005 (these contain a linear chain of hydrophobic polyoxypropylene flanked by chains of polyoxyethylene, Vaxcel, Inc., Norcross, Ga.); and Montanide IMS (e.g., IMS 1312, water-based nanoparticles combined with a soluble immuno stimulant, Seppic).

[0309]

[0296] Adjuvants may be TLR ligands. Adjuvants that act through TLR3 include without limitation double- stranded RNA. Adjuvants that act through TLR4 include without limitation derivatives of lipopolysaccharides such as monophosphoryl lipid A (MPLA; Ribi ImmunoChem Research, Inc., Hamilton, Mont.) and muramyl dipeptide (MDP; Ribi) andthreonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland). Adjuvants that act through TLR5 include without limitation flagellin. Adjuvants that act through TLR7 and / or TLR8 include without limitation single- stranded RNA, oligoribonucleotides (ORN), synthetic low molecular weight compounds such as imidazoquinolinamines (e.g., imiquimod (R-837), resiquimod (R-848)). Adjuvants acting through TLR9 include without limitation DNA of viral or bacterial origin, or synthetic oligodeoxynucleotides (ODN), such as CpG ODN. Another adjuvant class is phosphorothioate containing molecules such as phosphorothioate nucleotide analogs and nucleic acids containing phosphorothioate backbone linkages.

[0310]

[0297] The adjuvant can also be oil emulsions (e.g., Freund's adjuvant); saponin formulations; virosomes and viral-like particles; bacterial and microbial derivatives; immunostimulatory oligonucleotides; ADP-ribosylating toxins and detoxified derivatives; alum; BCG; mineral-containing compositions (e.g., mineral salts, such as aluminium salts and calcium salts, hydroxides, phosphates, sulfates, etc.); bioadhesives and / or mucoadhesives; microparticles; liposomes; polyoxyethylene ether and polyoxyethylene ester formulations; polyphosphazene; muramyl peptides; imidazoquinolone compounds; and surface active substances (e.g. lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).

[0298] Adjuvants may also include immunomodulators such as cytokines, interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL- 12, etc.), interferons (e.g., interferon-. gamma.), macrophage colony stimulating factor, and tumor necrosis factor.

[0311]

[0299] In some embodiments, the adjuvant is a STING (STimulator of Interferon Genes) agonist. The STING signaling pathway in immune cells is a central mediator of innate immune response and when stimulated, induces expression of various interferons, cytokines and T cell recruitment factors that amplify and strengthen immune activity. Recent work has shown that STING agonists are effective adjuvants and efficiently elicit an immune response, described, for example in Dubensky, T., et al., Therapeutic Advances in Vaccines, Vol. 1(4): 131-143 (2013); and Hanson, M., et al., The Journal of Clinical Investigation, Vol. 125 (6): 2532-2546 (2015), the entire contents of each of which are hereby incorporated by reference.

[0312]

[0300] In some embodiments, a STING agonist is a cyclic dinucleotide. In certain embodiments, cyclic dinucleotides include, but are not limited to, cdAMP, cdGMP, cdIMP, c-AMP-GMP, c-AMP-IMP, and c-GMP-IMP, and analogs thereof including, but not limited to, phosphorothioate analogues. In some embodiments, suitable cyclic dinucleotides for use in the present disclosure are described in some detail in, e.g., US Patent Nos. 7,709,458 and 7,592,326; WO 2007 / 054279; US 2014 / 0205653; and Yan et al. Bioorg. Med. Chem Lett.

[0313] 18: 5631 (2008), each of which is hereby incorporated by reference. In some embodiments, the STING agonist is cyclic di-guanylate (c-di-GMP).

[0314]

[0301] In certain embodiments, a STING agonist is chemically synthesized. In certain embodiments, a STING agonist is an analog of a naturally occurring cyclic dinucleotide.

[0315] STING agonists, including analogs of cyclic dinucleotides, suitable for use in the disclosure are provided in US Patent Nos. 7,709,458 and 7,592,326; and US 2014 / 0205653.

[0316]

[0302] In some embodiments, the adjuvant is saponin monophosphoryl-lipid-A (MPLA) nanoparticle adjuvant (SMNP).

[0317]

[0303] In some embodiments, the adjuvant is polyinosinic-polycytidylic acid (poly-IC), or a derivative thereof e.g., poly-IC 12U (Ampligen) and / or poly-ICLC (Hiltonol)). Poly-IC is a synthetic dsRNA that can activate multiple elements of the host defense in a pattern that parallels that of a viral infection. When properly combined with an antigen, it can be utilized as a PAMP-adjuvant, resulting in modulation and optimization of the antigen- specific immune response.

[0318] Methods of Use of the Vaccines

[0304] In certain aspects, the present disclosure provides methods of vaccinating a subject, comprising administering, e.g., intraperitioneally, to the subject a vaccine comprising an immunogen-loaded nanoemulsion disclosed herein. The present disclosure also provides methods of immunizing a subject, comprising administering, e.g., intraperitioneally, to the subject a vaccine comprising an immunogen-loaded nanoemulsion disclosed herein. Administration of the vaccine, e.g., intraperitioneally, to the subject induces or enhances an immune response, e.g., humoral immune response or cell-mediated immune response in the gastrointestinal tract, in the subject. In some embodiments, administering, e.g., intraperitioneally, the vaccine to the subject also induces or enhances a mucosal immune response, e.g., mucosal antibody response in the gastrointestinal tract, in the subject. In some embodiments, administering, e.g., intraperitioneally, the vaccine induces a greater immune response, e.g., humoral immune response, and in particular, mucosal antibody response, than the immunogen (e.g., peptide antigen or protein antigen) alone or the immunogen administered together with the nanoemulsion (i.e., in the absence of an amphiphilic linker).

[0319]

[0305] In some embodiments, the method comprises inducing a humoral immune response. In some embodiments, the humoral immune response (e.g., antibody expression) is systemic. In some embodiments, the humoral immune response (e.g., antibody expression) is localized. In some embodiments, the humoral immune response (e.g., antibody expression) is at mucosal surfaces.

[0320]

[0306] In some embodiments, the method comprises, causes or results in the induction of production of an antibody that binds to the immunogen (e.g., peptide or protein antigen, or polysaccharide antigen) of the nanoemulsion vaccine. The antibody produced can be, e.g., an IgG antibody and / or IgA antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgA antibody. In some embodiments, the antibody is a neutralizing antibody. In some embodiments, the method comprises, causes or results in the induction of production of a neutralizing antibody against the pathogenic antigen (e.g., HIV, SARS-CoV2). In some embodiments, the method comprises, causes or results in the induction of sustained levels of a neutralizing antibody against the pathogenic antigen (e.g., HIV, SARS-CoV2). In some embodiments, the method comprises, causes or results in the induction of increased levels of IgG and / or IgA antibodies in serum and / or the gastrointestinal tract. In some embodiments, the method comprises inducing increased GC and / or follicular helper T cell (Tfh) responses in the mesenteric lymph nodes.

[0307] In some embodiments, the method comprises inducing a sustained level of antibody (e.g., IgG and / or IgA) titre in the serum, vaginal feces, and / or mucosa of the gastrointestinal tract of a subject for at least 10 weeks, 15 weeks, 20 weeks, 25 weeks 30 weeks, 35 weeks, 40 weeks, 45 weeks or 50 weeks. In some embodiments, the method comprises inducing a high level of antibody (e.g., IgA and / or IgG) titre in the serum, vaginal feces and / or mucosa of the gastrointestinal tract of a subject for at least 10 weeks, 15 weeks, 20 weeks, 25 weeks 30 weeks, 35 weeks, 40 weeks, 45 weeks or 50 weeks. In some embodiments, the antibody secreting cells (ASC) that produce the antibody are present in a subject at least 0.5 years, at least 1 year, at least 1.5 years, at least 2 years, at least 3 years, at least 4 years, or at least 5 years after administration of the vaccine.

[0321]

[0308] The present disclosure further provides methods of vaccinating and / or immunizing a subject comprising administering, e.g., intraperitioneally, to the subject the vaccine in an effective amount to deliver to and / or accumulate the immunogen-loaded nanoemulsion in mesenteric lymph nodes and thereby induce an immune response in the gastrointestinal tract. In a preferred embodiment, the method comprises peritoneal administration of the vaccine because the mesenteric lymph nodes are part of a lymphatic chain that drains the peritoneal cavity, and nanoparticles administered intraperitoneally (i.p.) have been shown to accumulate in mediastinal and mesenteric lymph nodes.

[0322]

[0309] In some embodiments, the present disclosure provides methods of vaccinating a subject comprising intraperitoneally administering the vaccine in an effective amount to the subject. In some embodiments, the present disclosure provides methods of immunizing a subject comprising intraperitoneally administering the vaccine in an effective amount to the subject. In some embodiments, after i.p. administration, the immunogen-loaded nanoemulsion traffics to the mesenteric lymph node, mediastinal lymph node, or both the mesenteric lymph node and mediastinal lymph node. In some embodiments, after i.p. adminsitration, the immunogen accumulates at the mesenteric lymph node, mediastinal lymph node, or both the mesenteric lymph node and mediastinal lymph node.

[0323]

[0310] In some embodiments, administration, e.g., intraperitioneally, of the vaccine or the composition elicits or enhances production of antibodies that bind to the immunogen in the gastrointestinal tract. In some embodiments, administration, e.g., intraperitioneally, of the vaccine or the composition elicits or enhances production of IgA antibodies that bind to the immunogen in the subject.

[0311] In some embodiments, the disclosure further provides a method of eliciting antiimmunogen IgA antibodies in a subject, comprising administering via intraperitoneal injection to the subject an effective amount of a vaccine described herein, thereby eliciting anti-immunogen IgA antibodies in the subject.

[0324]

[0312] In some embodiments, the disclosure further provides a method of eliciting a mucosal antibody response in the gastrointestinal tract of a subject, comprising administering via intraperitoneal injection to the subject an effective amount of a vaccine described herein, thereby eliciting a mucosal antibody response.

[0325]

[0313] In some embodiments, the nanoemulsion traffics to the mesenteric lymph node, mediastinal lymph node, or both the mesenteric lymph node and mediastinal lymph node. In some embodiments, the immunogen accumulates at the mesenteric lymph node, mediastinal lymph node, or both the mesenteric lymph node and mediastinal lymph node.

[0326]

[0314] In certain other aspects, the present disclosure provides methods of preventing or treating a disease (e.g., an infectious diseae), comprising admintering to a subject in need thereof a vaccine or composition provided herein. In certain other aspects, the present disclosure provides methods of inducing an immune response to prevent or reduce severity of an infectious disease.

[0327]

[0315] In some embodiments, the infectious disease is caused by a pathogen. In some embodiments, the pathogen can infect a subject through mucosal surfaces.

[0328]

[0316] Infectious disease that can benefit from the methods provided herein include, but are not limited to, HIV / AIDS, coronavirus disease 19 (CO VID-19), influenza, rotavirus infection (e.g., diarrhea), cytomegalovirus (CMV) infection, Epstein-Barr virus infection (e.g., mononucleosis), respiratory syncytial virus (RSV) infection, and cholera.

[0329]

[0317] Acquired immunodeficiency syndrome (AIDS) is a syndrome that is caused by human immunodeficiency virus (HIV). HIV is spread primarily by unprotected sex (including anal and vaginal sex), contaminated hypodermic needles or blood transfusions, and from mother to child during pregnancy, delivery, or breastfeeding. Following initial infection of HIV, an individual may not notice any symptoms, or may experience a brief period of influenza-like illness. Typically, this is followed by a prolonged incubation period with no symptoms. If the infection progresses, it interferes with the immune system, increasing the risk of developing common infections such as tuberculosis, as well as other opportunistic infections, and tumors which are rare in people who have normal immune function. These late symptoms of infection are referred to as acquired immunodeficiency syndrome (AIDS).

[0330]

[0318] Coronavirus disease 19 (COVID-19) is a respiratory disease caused by the SARS- CoV-2 virus, a member of a large family of viruses called coronaviruses. The virus is thought to spread from person to person through droplets released when an infected person coughs, sneezes, or talks. It may also be spread by touching a surface with the virus on it and then touching one’s mouth, nose, or eyes, though less common.

[0331]

[0319] Influenza (also known as “flu”) is an infection of the nose, throat and lungs caused by influenza virus. There are four types of influenza virus, termed influenza viruses A, B, C, and D. Aquatic birds are the primary source of Influenza A virus (IAV), which is also widespread in various mammals, including humans and pigs. Influenza B virus (IBV) and Influenza C virus (ICV) primarily infect humans, and Influenza D virus (IDV) is found in cattle and pigs. IAV and IBV circulate in humans and cause seasonal epidemics, and ICV causes a mild infection, primarily in children. In humans, influenza viruses are primarily transmitted through respiratory droplets produced from coughing and sneezing. Transmission through aerosols and intermediate objects and surfaces contaminated by the virus also occur.

[0332]

[0320] Rotarovirus infection commonly results in severe, watery diarrhea and vomiting in infants and young children, which could lead to hospitalization and death in children. People who are infected with rotavirus shed the virus in their stool, and rotarovirus spreads via fecal-oral transmission.

[0333]

[0321] Cytomegalovirus (CMV) infection is a common infection that infects people of all ages. Most people infected with CMV show no signs or symptoms, and the virus can be dormant (inactive) in various tissues for a long time. Various stimuli can reactivate the dormant CMV, resulting in virus growth which can sometimes cause disease. Serious infections typically develop only in infants infected before birth and in people with a weakened immune system. Infected people may shed CMV in their urine or saliva intermittently. The virus is also excreted in mucus in the cervix (the lower part of the uterus), semen, stool, and breast milk. Thus, the virus is spread through sexual and nonsexual contact.

[0334]

[0322] Epstein-Barr virus (EBV, also known as human herpesvirus 4) is the virus that infects B cells, with infection ranging from asymptomatic to infectious mononucleosis. EBV spreads most commonly through bodily fluids, especially saliva. However, EBV can also spread through blood and semen during sexual contact, blood transfusions, and organ transplantations.

[0323] Respiratory syncytial virus (RSV) is a respiratory virus that infects lungs and breathing passages. In adults and older, healthy children, RSV symptoms are mild and typically mimic the common cold. However, in young children, older adults, people with heart and lung disease, or anyone with a weak immune system, RSV infection can be severed. RSV is spread through contact with droplets from the nose and throat of infected people when they cough and sneeze. RSV can also spread through dried respiratory secretions on bedclothes and similar items.

[0335]

[0324] Cholera is an acute diarrheal illness caused by infection of the intestine with Vibrio cholerae bacteria. People can get sick when they swallow food or water contaminated with cholera bacteria. The infection is often mild or without symptoms, but can sometimes be severe and life-threatening.

[0336]

[0325] In certain aspects, the methods provided herein comprise inducing immunity to an infectious pathogen. Non-limiting examples of the infectious pathogen include a human immunodeficiency virus (HIV), a SARS-CoV-2 virus, an influenza virus, a rotavirus , a cytomegalovirus (CMV), an Epstein-Barr virus (EBV), a respiratory syncytial virus (RSV), and a cholera bacteria. Immunity against other common infectious pathogens can also be induced using the methods described herein.

[0337]

[0326] In some embodiments of the methods provided herein, the immune response that is induced in the subject comprises expression of an IgA antibody targeting the pathogen. In some embodiments, the immune response that is induced in the subject comprises expression of IgG antibodies targeting the pathogen. In some embodiments, the immune response that is induced in the subject comprises expression of both IgA and IgG antibodies targeting the pathogen. In some embodiments, the immune response that is induced in the subject comprises expression of neutralizing antibodies targeting the pathogen.

[0338]

[0327] In some embodiments, the subject is a mammal. In some embodiments, the subject is a non-human mammal, or a primate. In some embodiments, the subject is human.

[0339]

[0328] In some embodiments, the vaccine is administered repeatedly. In certain embodiments, an initial dose may be followed by administration of a second or a plurality of subsequent doses of the vaccine in an amount that can be approximately the same or less or more than that of the initial dose. In some embodiments, at least 2 doses, at least 3 doses, at least 4 doses, or at least 5 doses of the vaccine are administered to elicit an effective immune response (e.g., inducing an antibody-mediated immune response, inducing a cell-mediated immune response, and / or achieving a desired level of neutralizing antibodies).

[0329] In some embodiments, a subsequent dose of the vaccine is administered about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, a month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months, 9 months, or a year or more after administration of a previous dose.

[0340]

[0330] In some embodiments, doses of the vaccine are administered about 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, a month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months, 9 months, or a year apart.

[0341]

[0331] In some embodiments, the vaccine is administered every 2 weeks, every 4 weeks, every 6 weeks, every 8 weeks, every 10 weeks, every 12 weeks, or every 16 weeks.

[0342]

[0332] In some embodiments, a booster dose of the vaccine is administered one to several years (e.g., 2 years, 3 years, 5 years, 10 years, 15 years) after a previous dose.

[0343]

[0333] In some embodiments, a dose of the vaccine comprises about 1 to 500 pg, 5-300 pg, 20 to 500 pg, 50 to 450 pg, 75 to 400 pg, 100 to 300 pg, or 150 to 250 pg of immunogen. In some embodiments, a dose of the vaccine comprises about 1 pg , 2 pg, 3 pg, 4 pg, 5pg, 6 pg, 7 pg, 8 pg, 9 pg, 10 pg, 15 pg, 20 pg, 25 pg, 30 pg, 35 pg, 40 pg, 45 pg, 50 pg, 55 pg, 60 pg, 65 pg, 70 pg, 75 pg, 80 pg, 85 pg, 90 pg, 95 pg, 100 pg, 105 pg, 110 pg, 115 pg, 120 pg, 125 pg, 130 pg, 135 pg, 140 pg, 145 pg, 150 pg, 155 pg, 160 pg, 165 pg, 170 pg, 175 pg, 180 pg, 185 pg, 190 pg, 195 pg, 200 pg, 210 pg, 220 pg, 230 pg, 240 pg, 250 pg, 260 pg, 270 pg, 280 pg, 290 pg, or 300 pg of the immunogen.

[0344]

[0334] In some embodiments, the vaccine comprising the 3M-052 adjuvant is administered. In some embodiments, an amout of about 1 to 400 pg, 1 to 200 pg, 1 to 75 pg, 1 to 50 pg, 50 to 100 pg, 50 to 200 pg, 50 to 300 pg, 50 to 400 pg, 100 to 200 pg, 100 to 300 pg, 100 to 400 pg, 200 to 400 pg, or 300 to 400 pg of 3M-052 is administered in a dose of the vaccine. In some embodiments, about 1 pg , 2 pg, 3 pg, 4 pg, 5pg, 6 pg, 7 pg, 8 pg, 9 pg, 10 pg, 20 pg, 30 pg, 40 pg, 50 pg, 60 pg, 70 pg, 75 pg, 80 pg, 90 pg, 100 pg, 125 pg, 150 pg, 175 pg, 200 pg, 225 pg, 250 pg, 275 pg, 300 pg, 325 pg, 350 pg, 375 pg, 400 pg of 3M-052 is administered in a dose of the vaccine.

[0345]

[0335] In some embodiments, the vaccine is administered in combination with an cdGMP adjuvant. In some embodiments, an amout of about 5 to 50 pg, 50 to 150, or 100 to 400 pg of cdGMP is administered in combination with a dose of the vaccine. In some embodiments, about 5pg, 10 pg, 15 pg, 20 pg, 25pg, 30 pg, 40 pg, 50 pg, 60 pg, 70 pg, 80 pg, 90 pg, 100 pg, 125 pg, 150 pg, 175 pg, 200 pg, 225 pg, 250 pg, 275 pg, 300 pg, 325 pg, 350 pg, 375 pg, 400 pg of cdGMP is administered in combination with a dose of the vaccine. EXAMPLES

[0346]

[0336] This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, GenBank Accession and Gene numbers, and published patents and patent applications cited throughout the application are hereby incorporated by reference. Those skilled in the art will recognize that the invention may be practiced with variations on the disclosed structures, materials, compositions and methods, and such variations are regarded as within the ambit of the invention.

[0347]

[0337] Reference numbers in brackets “[ ]” herein refer to the corresponding literature listed in the attached Bibliography which forms a part of this Specification, and the literature is incorporated by reference herein.

[0348] Example 1: Summary

[0349]

[0338] Many viruses infect the mucosal and lymphoid tissues of the lower gastrointestinal tract.1 4For example, Human immunodeficiency virus (HIV) can be transmitted via the rectal mucosa and spreads to adjacent sites.5,6Gut-associated lymphoid tissues (GALT) are near this site of viral entry and are rich with CD4+T cells that HIV primarily infects.7 9Other viruses, such as SARS-CoV-2 and Rotavirus can directly infect the intestinal mucosa since intestinal epithelial cells express their target receptors.10,11In addition to viruses, a variety of bacteria and parasites also cause gastrointestinal infections.12 14

[0350]

[0339] To provide optimal protection from such pathogens, mucosal immunity, and particularly mucosal antibody responses, are highly desirable. Secretory IgA antibodies are optimized for mucosal defense, as they have greater stability than monomeric IgA and IgG in the gastrointestinal (GI) mucosa.15,16In addition, secretory IgA provides more effective virus neutralization and enhances trapping of antigen in mucus compared to monomeric IgA or IgG in the GI tract.17 20However, traditional routes of immunization, such as intramuscular injection, prime robust systemic immunity but typically fail to elicit sustained responses at mucosal portals of pathogen entry.21,22Gut mucosal immune responses are programmed in the GALT and mesenteric lymph nodes (mesLNs), but delivery of vaccines to these inductive sites is a major challenge. Antigen uptake directly from the gut lumen can directly enter Peyer’s patches or traffic in lymph to the mesenteric LNs, but orally- administered vaccines must survive the proteolytic environment of the GI tract, penetrate the mucus overlying the gut epithelium, and overcome the tolerogenic state favored for gut-derived antigens.23 26 Thus, of 5 oral vaccines licensed for use in the US, 4 are based on live- attenuated pathogens that have evolved to infect the GI tract23. However, use of live- attenuated vaccines is problematic for use against highly mutable pathogens such as HIV.

[0351]

[0340] Nanoparticles naturally clear from tissues by convection into lymphatics rather than entering the blood vasculature.27,28Notably, in addition to collecting lymph fluid from the gut, the mesenteric lymph nodes are part of a lymphatic chain that drains the peritoneal cavity29, and nanoparticles administered intraperitoneally (i.p.) have been shown to accumulate in mediastinal and mesenteric lymph nodes.30Although i.p. administration is not traditionally considered for vaccine administration, it is used in the clinic for administration of microencapsulated cell therapies31, chemotherapy32, and has been used as a route for gene therapy and cancer vaccines in the setting of ovarian cancer33 33. It is thus hypothesized that i.p. administration of particles carrying both antigen and adjuvant compounds could target vaccines to mesenteric LNs and provide a practical route to induce strong gut-associated mucosal immunity.

[0352]

[0341] To test this idea, clinically safe and effective nanoparticle vaccine adjuvants based on oil-in-water nanoemulsions (NEs) were further developed. These adjuvants are comprised of surfactant- stabilized biodegradable oils, and are used in multiple licensed vaccines including Fluad (MF59- adjuvanted influenza vaccine from Novartis) and Pandemrix (AS03- adjuvanted influenza vaccine from GSK).36 38Traditional NE adjuvants have been shown to not directly bind to co-formulated antigens37, and thus NEs that could carry an associated protein immunogen by linking antigens to an amphiphilic poly(ethylene glycol)-lipid localized at the surface of the nanoemulsion were designed. To further amplify the immunogenicity of these vaccines, the potent adjuvant TLR7 / 8 agonist 3M-052,39,40which is being used in multiple ongoing vaccine clinical trials, were encapsulated in the oil phase of the NE. It was found that these antigen / adjuvant-carrying nanoemulsions efficiently targeted mesLNs following i.p. but not s.c. immunization, and that persistence of antigen in these mucosa-draining LNs could be optimized by anchoring the antigen to a cholesterol tail that allowed exchange of the antigen from the NE to surrounding cells in the lymphoid tissue. Optimized NEs promoted robust GC responses in mesLNs and IgA class switching, leading to strong and sustained serum and mucosal antigen- specific IgA responses. These findings suggest that engineered nanoparticles can be used to efficiently prime gut mucosal immunity from a simple parenteral injection. Example 2: Oil-in-water nanoemulsions engineered as dual antigen / adjuvant carriers

[0353]

[0342] A squalene-based nanoemulsion (NE, consisting of squalene oil, span® 85 and Tween® 80)41,42was engineered as a carrier to co-deliver antigen and a potent TLR agonist to mucosa-draining lymphoid tissues. The TLR7 / 8 agonist 3M-052 was incorporated in the emulsion oil phase, and protein antigens were anchored to the surface of the emulsion droplets by reacting dibenzocyclooctyne (DBCO)-derivatized antigens with azide groups installed at the surface of the emulsion droplets by inclusion of one of two different azide- functionalized PEG-lipid surfactants in the NE: cholesterol-PEG24-azide or 1,2- distearoyl- sn-glycero-3-phosphoethanolamine-PEG24-azide (DSPE-PEG24-azide, FIG. 1A). A germline-targeting antigen termed engineered Outer Domain-GT8 (eOD-GT8) was selected as a testbed immunogen; this antigen was developed to prime B cells against the CD4 binding of HIV Env gp 1204345, and recently shown in a phase I clinical trial to successfully prime VRCOl-class memory B cells in humans.46Monomeric eOD-GT8 was fused at its C terminus with the pan human leukocyte antigen DR-binding epitope (PADRE) peptide47,48to provide extra T cell help, and a terminal free cysteine was introduced at the N-terminus to enable coupling to maleimide-containing molecules. This modified Cys-eOD-GT8 was coupled to DBC0-PEG12-maleimide to enable conjugation of the immunogen to the nanoemulsions via azide-DBCO cycloaddition (FIGs. 6A-6B).

[0354]

[0343] Although currently-licensed nanoemulsion adjuvants have mean particle sizes of 100-200 nm in diameter49,50, NEs with a smaller mean size to promote efficient lymphatic delivery was prepared based on a previously described protocol41. Azide-functionalized PEG-lipid surfactants and 3M-052 were dissolved in dichloromethane and added to a mixture of squalene oil, span® 85, and Tween® 80. After evaporating the organic solvent, citrate buffer was added to form nanoemulsions by self- assembly in a single step. Incorporation of 3M-052 into the NEs was confirmed by UV-vis spectroscopy and size exclusion chromatography (FIGs. 7A-7F). DBCO-modified eOD- GT8 was then conjugated to the NEs bearing cholesterol or DSPE-anchored PEG-azide groups to form eOD-GT8-NE conjugates (referred to hereafter as eOD-Chol-NE and eOD-DSPE-NE, respectively, FIG. 7G), which were separated from unreacted antigen by centrifugal filtration. Coupling of fluorescently- labeled eOD-GT8 antigen to fluorescent NEs followed by SEC analysis showed that the antigen co-migrated with the NE post coupling (FIG. IB). Dynamic light scattering (DLS) showed that Chol-NE and DSPE-NE exhibited a small shift to increased size following antigen coupling, consistent with the small size of the eOD antigen (FIG. 1C). Cryo-TEM measurements also showed a diameter for both antigen-conjugated NEs in the range of 10-15 nm (FIG. ID). The composition and properties of the NEs are summarized in Table 2. As a control, free eOD-GT8 protein was physically mixed with TLR7 a- loaded nanoemulsions without chemical conjugation (hereafter, eOD / NE).

[0355] Table 2: Compositions and Properties of Nanoemulsions

[0356] Example 3: Nanoemulsions target antigen to mucosa-draining lymph nodes following intraperitoneal injection

[0357]

[0344] The in vivo biodistribution behavior of antigen-NE conjugates following different routes of administration was first assessed. eOD-GT8 antigen was labeled with an Alexa Fluor 647 dye prior to NE conjugation, and then eOD-Chol-NE, eOD-DSPE-NE or eOD / NE formulations were administered to mice intravenously, subcutaneously at the tail-base, or intraperitoneally. Six hours after injection, popliteal, inguinal, mesenteric, posterior mediastinal, axillary and brachial lymph nodes were harvested and whole-tissue antigen uptake was quantified by in vivo imaging system (IVIS) fluorescence measurements (FIGs. 2A-2F). Following i.v. administration, very little antigen uptake was detected in all of the lymph nodes analyzed, for all 3 vaccine formulations (FIGs. 2A-2B). Subcutaneous administration led to substantial vaccine targeting to the immediate draining inguinal LNs for the two antigen-conjugated NE formulations, but no uptake in the gut-draining mesenteric LNs (FIGs. 2C-2D). By contrast, i.p. administration led to antigen accumulation in mesenteric and mediastinal lymph nodes, with NEs carrying cholesterol-anchored antigen providing a significantly increased mesLN uptake of ~5-fold over eOD-DSPE-NE or eOD / NE formulations (FIG. 2E-2F).

[0358]

[0345] Given the promising biodistribution seen for i.p. -administered nanoemulsions, a study was conducted to simultaneously characterize the kinetics of antigen and nanoemulsion accumulation and clearance from mesLNs. BODIPY-cholesteryl ester, a non-exchangeable lipophilic dye51,52, was incorporated to track the oil core of the nanoemulsions, while the eOD-GT8 antigen was Alexa-dye labeled as before. After i.p. administration of fluorophore- modified eOD-Chol-NE, eOD- DSPE-NE or eOD / NE, mesenteric lymph nodes were harvested at different time points and analyzed by IVIS. NE signal peaked for all 3 formulations at ~1 hr post administration, then decreased over time, in a manner indistinguishable for the 3 groups (FIGs. 2G-2H). By contrast, the antigen kinetics for each group were distinct. While eOD-GT8 mixed with NE showed peak accumulation at 1 hr followed by steady clearance, roughly mirroring the pattern of NE kinetics, the eOD-GT8 anchored to NE via cholesterol or DSPE lipids showed peak accumulation later, at ~3 hr (FIGs. 2I-2J). Notably, by 24 hrs, antigen was largely cleared from mesLNs for the eOD- DSPE-NE and eOD / NE formulations, while some antigen delivered by the eOD-Chol-NE still persisted at this timepoint (FIG. 2G). A control experiment was also conducted with eOD-GT8 conjugated to free cholesterol-PEG24-azide (i.e. without nanoemulsion). In this case, eOD-GT8 accumulation in the mesenteric lymph nodes was 2-3 times lower in comparison to eOD-Chol-NE (FIGs. 8A-8C). Thus, nanoemulsions carrying cholesterol- anchored eOD effectively delivered antigen to the mesenteric lymph nodes.

[0359]

[0346] Amphiphilic molecules can spontaneously exchange between organized lipid structures such as emulsions, lipoproteins, extracellular vesicles, and cell membranes, and the chemical structure of the amphiphile plays an important role in the exchange rate.53Given the data above showing more prolonged retention of cholesterol-anchored eOD-GT8 in mesLNs compared to the NE itself, it was hypothesized that the cholesterol-anchored antigen might be dissociating from nanoemulsions in mesenteric LNs and decorating cell membranes in the tissue, thereby promoting prolonged tissue retention. To explore this idea, eOD-GT8 and NE uptake by lymphocytes in vitro were first assessed. Fluorescent eOD-Chol-NE, eOD- DSPE-NE or eOD / NE were incubated with splenocytes at different molar concentrations of the antigen in the presence of serum for Ih at 37°C, followed by washing and flow cytometry analysis. Splenocytes showed minimal uptake of the nanoemulsion on this timeframe (FIGs. 3A-3B). Strikingly however, at higher antigen concentrations, cholesterol-anchored eOD- GT8 was transferred to more than 90% of the cells after incubation with eOD-Chol-NE, while only less than 2% of the cells took up DPSE-anchored eOD-GT8 or free antigen mixed with NE at the same concentrations (FIGs. 3A-3B). It was hypothesized that this transfer to cells could reflect either cell membrane insertion or endocytosis of the cholesterol-PEG- conjugate; to distinguish these possibilities dye-labeled eOD-GT8 was conjugated to NEs, incubated with splenocytes for 1 hr, washed, and then the cells were stained with the HIV broadly neutralizing antibody VRC01, which binds with high affinity to the eOD-GT8 antigen.43,54As shown in FIGs. 3C-3D, cholesterol-anchored eOD-GT8 associated with the cells was essentially all detectable by VRC01, with VRC01 staining proportional to the amount of eOD-GT8 uptake, suggesting predominantly cell surface plasma membrane insertion of the conjugate. This cell surface decoration was cell type-independent, as nearly the entire splenocyte population became surface labeled following eOD-Chol-NE incubation. By contrast, negligible amounts of DSPE-anchored eOD-GT8 or free antigen that transferred to the cells could be stained by VRC01 (FilGs. 3C-3D). Similar results were obtained when nanoemulsions or free eOD-GT8 were incubated with splenocytes in the absence of serum, suggesting serum proteins did not play a critical role in this cell transfer behavior (FIGs. 9A- 9B).

[0360]

[0347] Whether cell transfer of the cholesterol-anchored antigen also occurs in lymphoid tissues in vivo was next determined. The same formulations were injected i.p. and mesenteric lymph nodes were harvested 24h later to assess eOD-GT8-NE uptake by antigen presenting cells (FIGs. 10A-10C). In line with the in vitro experiments, eOD-GT8 showed substantially greater association with CDl lb'CDl lc+dendritic cells (DCs) (FIGs. 3E-3F), CDl lb+F4 / 80+macrophages (FIGs. 3G-3H), and B cells (FIGs. 31-3 J) when administered as eOD-Chol-NE compared to the other two vaccine formulations. Cells that were antigen+were generally negative for NE uptake, suggesting dissociation of the antigen from the NE in the lymph nodes (FIGs. 3E, 3G, 31). Altogether, these data suggest that the effective LN accumulation of cholesterol-anchored antigen reflects a dynamic process of NE targeting to mesLNs and subsequent transfer to lymph node-resident cell populations.

[0361] Example 4: Nanoemulsion targeting of antigen and adjuvant to mesLNs primes strong systemic and mucosal immune responses

[0348] To assess immune responses elicited by nanoemulsions, mice were immunized with eOD-Chol-NE, eOD-DSPE-NE or eOD / NE i.p, and mesenteric lymph nodes were harvested for flow cytometry analysis of germinal center (GC) responses 12 days later (FIGs. 11A- 11B). eOD-Chol-NE induced the highest frequency of both PD-1+CXCR5+T follicular helper (Tfh) cells and total GC B cells (FIGs. 4A-4D). Most strikingly, when GC B cells were stained with eOD-GT8 antigen tetramers, the antigen- specific GC B cells population primed by eOD-Chol-NE was ~20-fold greater than the other two formulations (FIGs. 4E- 4F). Moreover, a portion of these eOD-GT8 specific GC B cells were class switched to IgA, as desired for optimal mucosal immunity (FIGs. 4G-4H).

[0362]

[0349] In parallel, mice were immunized with the same formulations, boosted 4 weeks later, and Ig titers were measured longitudinally by ELISA. Unlike free antigen mixed with NE adjuvant, both antigen- conjugated nanoemulsions induced systemic IgG in all animals by 2 weeks post prime, and responses to eOD-Chol-NE and eOD-DSPE-NE remained ~ 100-fold higher than the eOD / NE condition post boost (FIGs. 41, FIGs. 12A-12D). eOD-Chol-NE induced the highest serum IgG titers after the prime, ~5 times higher than eOD-DSPE-NE and ~10 times higher than eOD / NE. Serum IgA was absent in all groups post prime, but following the boost, robust IgA titers were detected for both antigen-conjugated NEs, with the cholesterol-anchored NE eliciting ~ 10-fold higher titers than the eOD-DSPE-NE group (FIG. 4J). To assess mucosal antibody responses elicited in the gut, fecal Ig titers were also measured. Low fecal IgG titers were detectable after the boost for the antigen-conjugated NEs (FIG. 4K). By contrast, post boost, the antigen-coupled nanoemulsions elicited strong fecal IgA titers that were stable through at least 12 weeks, and similar to the serum data, eOD-Chol-NE elicited responses ~ 15-fold greater than eOD-DSPE-NE (FIG. 4L). As expected, control s.c. administration of eOD-Chol-NE induced high systemic antibody titers but no mucosal IgA (FIGs. 13A-13D). Notably, immunization using the eOD-Chol-NE formulation without encapsulated 3M-052 elicited much weaker GC responses and no IgA class switching in the mesLNs (FIGs. 14A-14C), and failed to prime systemic or mucosal IgA, indicating the importance of the co-delivered TLR agonist (FIGs. 14D-14G).

[0363]

[0350] eOD-GT8 is clinically relevant as a germline targeting antigen for HIV VRC01 -class broadly neutralizing antibodies in humans, but wild type mice cannot generate VRCOl-class neutralizing antibody responses. Thus, to test the capacity of mesLN-targeted vaccination to promote protective gut antibody responses, immunizations were carried out with a second antigen, an optimized mutant of the SARS-CoV-2 receptor binding domain termed RBDJ based on the Wuhan SARS-CoV-2 sequence.55,56Nanoemulsions were prepared with equivalent compositions as used for the eOD-GT8 immunogen (Table 2), and mice were primed and boosted with RBD-carrying NEs. Antibody titers were assessed in the serum and feces of the mice against the WT RBDJ as well as two different SARS-CoV-2 variant antigens: whole SI spike protein from the D614G variant and RBD from Omicron. Mirroring the findings with eOD-GT8, RBDJ-Chol-NE and RBDJ-DSPE-NE elicited serum IgG responses that were 2 orders of magnitude higher than free RBDJ mixed with nanoemulsions, and these sera also recognized the D614G and Omicron variants (FIGs. 5A, 5E, 51). RBDJ- Chol-NE also elicited strong serum and fecal IgA titers against all 3 antigens (FIGs. 5B, 5F, 5J, 5D, 5H, 5L). As with eOD-GT8, RBDJ mixed with NE was unable to prime any mucosal Ig responses. Although RBDJ-Chol-NE induced high serum and fecal IgA titers, IgA titers were not detected in the BAL fluid (BALF, FIGs. 15A-15B). However, high BALF IgG titers were measured for both RBDJ- DSPE-NE and RBDJ-Chol-NE immunizations.

[0364]

[0351] The protective capacity of these antibody responses was assessed by a pseudovirus neutralizing assay. RBDJ-Chol-NE immunization elicited a serum neutralizing titer ID50 (50% inhibitory dose) of ~104against SARS-CoV-2 D614G, -10 and -300 times higher than RBDJ-DSPE-NE and RBDJ / NE immunizations, respectively (FIG. 5M). Moreover, RBDJ-Chol-NE immunization elicited readily detectable fecal neutralizing ID50 titers, - 10-fold greater than RBDJ-DSPE-NE and RBDJ / NE immunizations (FIG. 5N). Altogether, these data suggest that targeting mesenteric LNs with nanoemulsion vaccines is an effective strategy to promote neutralizing antibody responses in the gut mucosa.

[0365] Example 5: Discussion

[0366]

[0352] Induction of mucosal antibody responses, especially IgA, in the gastrointestinal tract (GI) is desirable for optimal protection from a variety of pathogens that infect through the gut epithelium, but achieving robust gut immunity using non-live microbial vaccines has been challenging. Most commonly, vaccines aiming to promote gut immunity are administered by the oral route,23,57,58and antigens that penetrate the gut mucus are captured from the intestinal lumen by M cells in the small intestine and transferred to antigen-presenting cells in the Peyer’s patches. In addition, dendritic cells dispersed among epithelial cells of the gut can extend processes into the gut lumen to sample antigen59, and migrate to lymphoid follicles and mesenteric lymph nodes where B-cell class switching to IgA- producing cells is induced.60,61However, oral vaccination is limited by the efficiency of these antigen uptake processes and the tendency of gut antigen uptake to promote tolerance rather than immunity.23,24Here, the facts that mesLNs collect lymph not only from the gut but also from the peritoneal space, and targeted vaccines to this gut immunity inductive site via a simple i.p. injection using vaccine-loaded nanoemulsions were exploited. Formulation of vaccines in particulate carriers is a well-established approach to limit antigen / adjuvant dispersal into the bloodstream from tissues and instead favor lymphatic uptake, and to additionally promote vaccine capture at draining lymph nodes.27,28To favor the safety of this approach, nanoemulsions based on the biodegradable oil squalene used in several licensed vaccines and a TLR agonist that has been shown to be safe in ongoing clinical trials were employed.

[0367]

[0353] We conjugated antigen to these NE vaccines via PEG-lipids, motivated by the desire to mimic viral display of many copies of antigen at the particle surfaces. As the chemical structure of lipids is known to influence their tendency to exchange with serum proteins / membranes62,63, two different lipid “anchors”, DSPE and cholesterol, were tested. Unexpectedly, NEs carrying antigen linked via a cholesterol anchor were substantially more effective in achieving antigen accumulation in mesenteric LNs and drove much stronger GC and gut antibody responses than the less-exchangeable DSPE lipid tail. These enhanced vaccine responses correlated with an ability of the cholesterol-anchored antigen to transfer from the NE to lymphocytes in the mesenteric LNs, which is suspected to be favored due to the dense cellularity of lymph nodes compared to the i.p. space.

[0368]

[0354] Vaccine administration via Intramuscular injection (i.m.) is often the preferred route to administer vaccines due to the low reactogenicity of i.m.-dosed vaccines.64However, administering tetanus toxoid or measles virus vaccines via the intraperitoneal route has also been shown to be well- tolerated in humans.34,65Notably, the trial immunizing against tetanus toxoid using a traditional TT vaccine failed to elicit mucosal IgA in humans65, consistent with the preclinical findings that unformulated antigens are not efficiently delivered to the mucosa-draining lymphoid tissues. This route also used in the clinic for administration of cell therapies31, chemotherapy32, gene therapy, and cancer vaccines in the setting of ovarian cancer33 35. Thus, while more complex than a simple i.m. injection, the potential of i.p. vaccination to promote strong humoral immunity in the gut (and other mucosal tissues) is an important advantage for gut- tropic pathogens.

[0369]

[0355] In conclusion, this study demonstrates the potential of intraperitoneal injection of engineered squalene-based vaccines to elicit robust mucosal immunity in the gut. This approach holds promise for enhancing protection against infectious diseases in the lower gastrointestinal tract, particularly when oral vaccine delivery is not feasible or effective.

[0370] Example 6: Materials and methods

[0371]

[0356] Nanoemulsion-immunogen conjugate preparation. The TLR7 agonist 3M-052 (0.5 mg, Cayman Chemical) and 1 mg cholesterol-PEG24-azide or DSPE- PEG24-azide (BroadPharm) was dissolved in 100 pL dichloromethane and mixed with 10 p L of squalene oil (Sigma- Aldrich), 5 pL Span® 85 (Sigma- Aldrich) and 37.5 mg of TWEEN® 80 (Sigma- Aldrich). To prepare fluorescent nanoemulsions, additionally 0.25 mg of CholEsteryl BODIPY (ThermoFisher) dissolved in 100 pL dichloromethane was also added. The organic solvent was remvoed using a rotary evaporator, then 1 mL of 10 mM citrate buffer (pH 6.0, heated to 37 °C) was added to form nanoemulsions and sonicated for 1 minute in an ultrasonic bath (VWR B1500A-DTH / 50 W at 42 kHz). The solution was filtered with 0.1 pm syringe filters and characterized by dynamic light scattering (DLS) and TEM.

[0372]

[0357] HIV env gpl20 engineered outer domain-GT8 (eOD-GT8) and SARS-CoV-2 spike receptor binding domain (RBD) protein antigens with N-terminal cysteines (synthesis protocols and protein sequences previously reported56,66) at a concentration of 1 mg / mL and 0.5 mg / mL, respectively, were reduced with 4 molar equivalents of tris(2- carboxyethyl)phosphine (TCEP) for 15 minutes at 25°C in PBS. The reduced proteins (1 mg / mL) were then reacted with 10 equivalents DBCO-PEG12-maleimide (Sigma- Aldrich) in PBS for 18 hr at 25°C. Unreacted linker was removed from the samples by centrifugal filtration using 10 kDa molecular weight cutoff (MWCO) Amicon spin filters. Free protein controls were not modified DBCO-PEG12-maleimide to prevent conjugation to the nanoemulsions. To prepare fluorescent proteins, 10 equivalents of Alexa Fluor 647-NHS ester dye (ThermoFisher) was mixed with proteins and left on an orbital shaker for 18 hr at 25°C. Unconjugated dye was removed by centrifugal filtration using 10 kDa molecular weight cutoff (MWCO) Amicon spin filters.

[0373]

[0358] To form nanoemulsion-immunogen conjugates, ~55 pL of nanoemulsion was added to 350 pL PBS and centrifuged using 50 kDa molecular weight cutoff (MWCO) Amicon spin filters twice for buffer exchange into PBS. The resulting nanoemulsions and 80 pg of modified protein were mixed and reacted on an orbital shaker for 18 hr at 25°C. Unbound protein was removed by centrifugal filtration using 100 kDa molecular weight cutoff (MWCO) Amicon spin filters and characterized by size exclusion chromatography (SEC).

[0374]

[0359] In vivo biodistribution studies with IVIS. In vivo trafficking of the eOD-Chol- NE, eOD-DSPE-NE and eOD / NE was evaluated following intraperitoneal administration using an In Vivo Imaging System (IVIS) fluorescence imaging (Perkin Elmer). BALB / c mice were immunized with formulations containing 10 pg AF647-eOD-GT8, 5 pg CholEsteryl BODIPY™ 542 / 563 Cl l, 5 pg 3M-052 and 0.1 pg squalene oil. After 1 hour, 3 hours, 6 hours, 24 hours, or 72 hours post-immunization, mesenteric lymph nodes and other lymph nodes as indicated were harvested. AF647 and BODIPY fluorescence (radiant efficiency) was measured on freshly excised tissues.

[0375]

[0360] In-vitro membrane insertion in splenocytes. Nanoemulsion / immunogen uptake by cells was evaluated in vitro in murine splenocytes isolated from naive BALB / c mice (The Jackson Laboratory). Fluorescently labeled eOD-Chol-NE, eOD-DSPE-NE and eOD / NE formulations (eOD-GT8 modified with Alexa Fluor 647, nanoemulsions prepared with encapsulated CholEsteryl BODIPY™ FL C12 and 3M-052) were incubated with 2xl06cells per mL (4xl05cells per well in a 96- well plate) in cRPMI (RPMI-1640 + 5% fetal bovine serum (FBS) or in FBS-free RPMI medium at different dilutions: 3, 15, or 75 nM eOD-GT8. Following Ih incubation, cells were washed once with PBS, stained with Live / Dead Aqua (BioLegend) at 1:1000 in 100 pl PBS for 15 minutes at 25°C, washed once in flow cytometry buffer (PBS+2% BSA), then stained with the Env CD4 binding site-specific monoclonal antibody PE-VRC01 at 1 pg / 106cells in 100 pl flow cytometry buffer for 20 minutes at 25°C. Cells were then washed twice, fixed with 2% paraformaldehyde, and stored at 4°C until flow cytometry analysis on BD LSR II.

[0376]

[0361] eOD-GT8 / nanoemulsion cellular uptake studies in vivo. BALB / c mice were immunized with nanoemulsion formulations containing 10 pg AF647-eOD-GT8, 5 pg green fluorescent BODIPY™ FL C12 cholesteryl ester, 5 pg 3M-052 and 0.1 pg squalene oil. Twenty-four hours later, mesenteric lymph nodes were collected and single cell suspensions were prepared. Cells were washed with PBS and first stained with Zombie UV live / dead stain (BioLegend) at 1:750 in 100 pl PBS for 15 minutes at 25°C, then treated with antimouse CD16 / 32 Fc block (TruStain FcX, BioLegend) at 1:100 in 50 pl flow cytometry buffer for 10 minutes at 25 °C. To identify different cell populations with vaccine uptake, cells were stained with the following antibodies at a dilution of 1:100 in lOOpl flow cytometry buffer for 15 minutes at 25°C: anti-mouse CD3s PE-CF594 (clone 145-2C11; BD Biosciences), CD19 BUV395 (1D3; BD Biosciences), CDl lb BUV737 (MI / 70; BD Biosciences), CDl lc BV711 (N418, BioLegend), F4 / 80 PE (BM8; BioLegend), Ly6C BV785 (HK1.4, BioLegend), Ly6G BV421 (1A8, BioLegend). Cells were fixed with 2% paraformaldehyde and stored at 4°C until flow cytometry analysis. Counting beads (Invitrogen) were added prior to flow cytometry measurements. Cells were analyed on a BD LACS Symphony A3.

[0377]

[0362] Tfh cell response. BALB / c mice were immunized with nanoemulsion formulations containing 10 pg eOD-GT8, 5 pg 3M-052 and 0.1 pg squalene oil. After 12 days, mesenteric lymph nodes were collected, and single cell suspensions were prepared. Cells were washed with PBS and first stained with Zombie Aqua (Bio Legend) at 1:750 in 100 pl PBS for 10 minutes at 25°C, then treated with anti-mouse CD16 / 32 Pc block (TruStain PcX, BioLegend) at 1:100 in 100 pl flow cytometry buffer for 10 minutes at 25°C. Cells were stained with the following panel in 100 pl flow cytometry buffer for 20 minutes at 25°C: anti-mouse CD19 PITC at 1:200 (clone 1D3; BioLegend), CD4 AP647 at 1:200 (GK1.5; BioLegend), CD44 PE-Cy7 at 1:200 (IM7; BioLegend), PD-1 PE at 1:50 (RMP1- 14; BioLegend), and CXCR5 BV421 at 1:50 (L138D7; BioLegend). Counting beads (Invitrogen) were added prior to flow cytometry measurements. Cells were analyed on a BD LSR II.

[0378]

[0363] GC B cell response. BALB / c mice were immunized with nanoemulsion formulations containing 10 pg eOD-GT8, 5 pg 3M-052 and 0.1 pg squalene oil. After 12 days, mesenteric lymph nodes were collected, and single cell suspensions were prepared. Cells were washed with PBS and first stained with Zombie UV (BioLegend) at 1:750 in 100 pl PBS for 10 minutes at 25°C, then treated with anti-mouse CD16 / 32 Pc block (TruStain PcX, BioLegend) at 1:100 in 100 pl flow cytometry buffer for 10 minutes at 25 °C. Cells were stained with the following panel in lOOpl flow cytometry buffer for 20 minutes at 25°C: anti-mouse CD90.2 BV785 at 1:200 (clone 30-H12; BioLegend), CD19 BUV395 at 1:200 (1D3; BD Biosciences), CD38 PITC at 1:200 (90; BioLegend), GL7 PerCP-Cy5.5 at 1:150 (GL7; BioLegend), IgA AP647 at 1:100 (SouthernB iotech), eOD-tetramer BV605 at 1:100, and eOD-tetramer BV421 at 1:100 (tetramers formed by incubating 4 equivalents of biotinylated-eOD with 1 equivalent of streptavidin-BV605 or streptavidin-BV421

[0379] (Bio Legend) 18 hr at 4°C, prior to staining). Counting beads (Invitrogen) were added prior to flow cytometry measurements. Cells were analyed on a BD LACS Symphony A3.

[0364] Mouse sample collection. Fecal washes were collected from mouse fecal pellets (2 pellets of -0.75 cm each per mouse) combined with 200 pl PBS supplemented with Complete protease inhibitor cocktail (Roche, 11836153001) samples were vortexed, incubated overnight at 4°C, vortexed a second time, then centrifuged at 12,000xg for 5 minutes to collect supernatant. BALF was collected from 2x1 mL instillations of PBS supplemented with Complete protease inhibitor cocktail (Roche) in the lungs using a catheter through the trachea. Samples were centrifuged at 12,000xg for 5 min to collect the supernatant.

[0380]

[0365] ELISA analyses of murine antibody titers. To capture eOD-specific or RBD- specific antibodies from immunized mice, Costar Polystyrene High Binding 96-well plates (Coming) were coated directly with eOD antigen or RBD antigen at 2 pg / ml in PBS overnight at 4°C. Plates were then blocked with PBS + 2% BSA for 2 hours at 25°C. Mouse serum samples were diluted in blocking buffer (PBS + 2% BSA) starting at 1:50 followed by 4X serial dilutions and mucosal samples were diluted in block buffer starting at 1:50 followed by 3X serial dilutions. Samples were incubated in plates for 2 hours at 25°C. To determine antigen- specific IgG antibodies, goat anti-mouse IgG-horseradish peroxidase (HRP, BioRad, 1:5000) was added to the wells and incubated for 1 hour. To determine antigen-specific IgA antibodies, goat anti-mouse IgA- biotin (SouthernBiotech, 1:5000) was added and incubated for 1 hour followed by incubation with streptavidin-HRP (ThermoFisher Pierce High Sensitivity Streptavidin-HRP, 1:40,000) for 30 minutes.

[0381]

[0366] Plates were developed using 3,3',5,5'-Tetramethylbenzidine (TMB) substrate for 1 to 5 minutes and stopped with 2N sulfuric acid, and the resulting absorbance (A450 / A540) was measured on a plate reader. For all titer analyses, samples directly compared across groups were developed for the same amount of time. Endpoint cut-off titers are reported as inverse serum dilutions giving an HRP absorbance (A450 - A540) of 0.3 above background.

[0382]

[0367] Pseudovirus-based SARS-CoV-2 neutralization assay. The SARS-CoV-2 pseudoviruses (D614G variant) expressing GFP were purchased from GeneCopoeia. To determine the neutralization activity of mouse serum and fecal samples, HEK293T expressing human ACE2 (hACE2) (GeneCopoeia) were seeded in 96- well tissue culture plates at a density of 1 x 104cells per well overnight. Serum and fecal samples were first heat-inactivated at 56°C for 30 minutes. Serum samples were diluted in DMEM (+10% FBS and p / s) at 1:50 followed by 5X serial dilutions and fecal samples were diluted at 1:10 followed by 5X serial dilutions. Diluted samples were mixed with pseudovirus (-105 RLU / well) and incubated at 37°C for 1 hour before adding to HEK293T-hACE2 cells. 5mg / mL polybrene was also added to the wells. At 24h, the old medium was replaced with fresh complete medium. 48 hours after, cells were analyzed by flow cytometry. SARS- CoV- 2 neutralization titers (NT50) were defined as the sample dilution at which a 50% reduction in GFP positive cells was observed relative to the average of the virus control wells.

[0383]

[0368] Statistics. Statistics were analyzed using GraphPad Prism software. All graphs represent mean and standard deviations unless otherwise noted. Statistical comparison was performed using a one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for single-time point data and two-way ANOVA followed by Tukey’s post hoc test for multi-time point longitudinal data.

[0384]

[0369] Immunogen

[0385]

[0370] HIV eOD. eOD-GT8 gpl20 protein was synthesized as previously described. The eOD protein, with a free N-terminal cysteine (bold and underlined) and C-terminal PADRE universal helper T cell epitope (AKFVAAWTLKAAA), has the following sequence (with PADRE epitope italicized and underlined):

[0386] ETGCHHHHHHGGDTITLPCRPAPPPHCSSNITGLILTRQGGYSNDNTVIFRPSGGDWR DIARCQIAGTVVSTQLFLNGSLAEEEVVIRSEDWRDNAKSICVQLNTSVEINCTGAGH CNISRAKWNNTLKQIASKLREQYGNKTIIFKPSSGGDPEFVNHSFNCGGEFFYCDSTQ LFNSTW FNSTGSA FF VA A WTLKAAA (SEQ ID NO: 1)

[0387] SARS-CoV-2 RBD. For conjugation, the engineered RBD protein (‘RBD-E452K-F490W’) was genetically modified to include an N- terminal cysteine residue (bold and underlined).

[0388] SARS-CoV-2 RBD:

[0389] CITNECPFGEVFNATRFASVYAWNRKRISNCVADYSVEYNSASFSTFKCYGVSPTKE NDECFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKEPDDFTGCVIAWNSNNEDS KVGGNYNYKYREFRKSNEKPFERDISTEIYQAGSTPCNGVEGFNCYWPEQSYGFQPT NGVGYQPYRVVVESFEEEHAPATVCGPKKSTN (SEQ ID NO: 2)

[0390] EQUIVALENTS

[0391]

[0371] It is understood that the detailed examples and embodiments described herein are given by way of example for illustrative purposes only, and are in no way considered to be limiting to the invention. Various modifications or changes in light thereof will be suggested to persons skilled in the art and are included within the spirit and purview of this application and are considered within the scope of the appended claims. For example, the relative quantities of the ingredients may be varied to optimize the desired effects, additional ingredients may be added, and / or similar ingredients may be substituted for one or more of the ingredients described. Additional advantageous features and functionalities associated with the systems, methods, and processes of the present invention will be apparent from the appended claims. Moreover, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0392] BIBLIOGRAPHY

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Claims

IN THE CLAIMS:

1. A vaccine comprising: a) a nanoemulsion comprising a surfactant shell and a lipophilic core; and b) an immunogen non-covalently conjugated to the surface of the nanoemulsion by an amphiphilic linker.

2. The vaccine of claim 1, wherein the vaccine is capable of inducing a mucosal antibody response in the gastrointestinal tract.

3. The vaccine of claim 1 or 2, wherein the amphiphilic linker is capable of dissociating from the nanoemulsion to transfer the immunogen to a cell.

4. The vaccine of any one of claims 1-3, wherein the amphiphilic linker comprises a lipid covalently linked to a hydrophilic group.

5. The vaccine of claim 4, wherein the lipid of the amphiphilic linker is embedded in the surfactant shell of the nanoemulsion.

6. The vaccine of claim 4 or 5, wherein the immunogen is covalently linked to the hydrophilic group of the amphiphilic linker.

7. The vaccine of any one of claims 4-6, wherein the lipid of the amphiphilic linker is a monoacyl lipid or a diacyl lipid.

8. The vaccine of claim 7, wherein the monoacyl lipid or diacyl lipid has at least 8 carbons in the hydrocarbon tail(s).

9. The vaccine of claim 7, wherein the monoacyl lipid or diacyl lipid has between 8 and 18 carbons in the hydrocarbon tail(s).

10. The vaccine of any one of claims 4-9, wherein the lipid is l,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE).

11. The vaccine of any one of claims 4-9, wherein the lipid comprises a palmitoyl group.

12. The vaccine of any one of claims 4-6, wherein the lipid comprises cholesterol.

13. The vaccine of any one of claims 4-12, wherein the hydrophilic group comprises a polyethylene glycol (PEG), a polysaccharide, a polyvinyl alcohol, or a polypeptide.

14. The vaccine of claim 13, wherein the hydrophilic group is a PEG.

15. The vaccine of claim 14, wherein the PEG comprises 2 to 96 ethylene glycol subunits(PEG2-PEG96).

16. The vaccine of claim 15, wherein the PEG is PEG24.

17. The vaccine of any one of claims 1-16, wherein the amphiphilic linker comprises cholesterol-PEG24 or DSPE-PEG24.

18. The vaccine of any one of claims 1-17, wherein the nanoemulsion is an oil-in-water nanoemulsion.

19. The vaccine of any one of claims 1-18, wherein the lipophilic core comprises a biodegradable oil.

20. The vaccine of claim 19, wherein the biodegradable oil is an animal oil or plant oil.

21. The vaccine of claim 19 or 20, wherein the biodegradable oil is selected from the group consisting of squalene oil, castor oil and sesame oil.

22. The vaccine of any one of claims 1-21, wherein the surfactant shell comprises one or more surfactants selected from the group consisting of Span® 85, Span® 80, Tween® 80, and Tween® 20.

23. The vaccine of any one of claims 1-22, wherein the nanoemulsion is(i) smaller than 200 nm in diameter; or(ii) about 1-200 nm, about 1-100 nm, about 1-50 nm, about 1-30 nm, about 1-20 nm, about 5- 50 nm, about 5-30 nm, or about 5-20 nm in diameter.

24. The vaccine of any one of claims 1-23, wherein the immunogen is derived from a disease-causing organism selected from a bacteria, fungus, parasite, and virus.

25. The vaccine of any one of claims 1-24, wherein the immunogen comprises a peptide antigen or a protein antigen.

26. The vaccine of any one of claims 1-25, wherein the immunogen is selected from the group consisting of a human immunodeficiency virus (HIV) antigen, a SARS-CoV-2 antigen, an influenza antigen, a rotavirus antigen, a cytomegalovirus (CMV) antigen, an Epstein-Barr virus (EBV) antigen, a respiratory syncytial virus (RSV) antigen, and a cholera antigen.

27. The vaccine of claim 26, wherein the HIV antigen comprises HIV gpl20 engineered outer domain-germ line-targeting immunogen 8 (eOD-GT8),28. The vaccine of claim 26, wherein the SARS-CoV-2 antigen comprises an antigen from the receptor-binding domain (RBD) of SARS-CoV-2 spike protein.

29. The vaccine of any one of claims 1-28, wherein the immunogen further comprises a pan human leukocyte antigen DR-binding epitope (PADRE) peptide, a T-helper epitope from tetanus toxoid, or a T-helper peptide from diphtheria toxoid.

30. The vaccine of any one of claims 1-24, wherein the immunogen comprises a polysaccharide, optionally wherein the polysaccharide is a bacterial polysaccharide.

31. The vaccine of any one of claims 1-30, wherein the nanoemulsion further comprises an adjuvant in the lipophilic core.

32. The vaccine of claim 31, wherein the adjuvant is lipophilic.

33. The vaccine of claim 31 or 32, wherein the adjuvant comprises a TLR7 / 8 agonist, optionally wherein the TLR7 / 8 agonist comprises 3M-052.

34. The vaccine of claim 31 or 32, wherein the adjuvant comprises CpG-cholesterol or 3D(6-acyl)-PHAD™.

35. The vaccine of any one of claims 1-34, wherein intraperitoneal (i.p.) administration of the vaccine elicits or enhances production of antibodies that bind to the immunogen.

36. The vaccine of claim 35, wherein the antibodies comprise IgA antibodies, IgG antibodies, or IgA and IgG antibodies.

37. The vaccine of claim 35 or 36, wherein the antibodies are neutralizing antibodies.

38. A composition comprising the vaccine of any one of claims 1-37 and a pharmaceutically acceptable carrier.

39. The composition of claim 38, further comprising an adjuvant.

40. The composition of claim 39, wherein the adjuvant is polyinosinic-polycytidylic acid (poly-IC) or a STING agonist, optionally wherein the STING agonist comprises cyclic diguanylate (c-di-GMP).

41. A method of vaccinating a subject, comprising administering to the subject an effective amount of the vaccine of any one of claims 1-37 or the composition of any one of claims 38-40, thereby vaccinating the subject.

42. A method of immunizing a subject, comprising administering to the subject an effective amount of the vaccine of any one of claims 1-37 or the composition of any one of claims 38-40, thereby immunizing the subject.

43. The method of claim 41 or 42, wherein the vaccine or the composition is administered via intraperitoneal (i.p.) injection.

44. The method of any one of claims 41-43, wherein administration of the vaccine or the composition elicits or enhances production of antibodies that bind to the immunogen in the gastrointestinal tract.

45. The method of any one of claims 41-44, wherein administration of the vaccine or the composition elicits or enhances production of IgA antibodies that bind to the immunogen in the subject.

46. A method of eliciting anti-immunogen IgA antibodies in a subject, comprising administering via intraperitoneal (i.p.) injection to the subject an effective amount of the vaccine of any one of claims 1-37 or the composition of any one of claims 38-40, thereby eliciting anti-immunogen IgA antibodies in the subject.

47. A method of eliciting a mucosal antibody response in the gastrointestinal tract of a subject, comprising administering via intraperitoneal (i.p.) injection to the subject an effective amount of the vaccine of any one of claims 1-37 or the composition of any one of claims 38- 40, thereby eliciting a mucosal antibody response in the gastrointestinal tract of the subject.

48. The method of any one of claims 41-47, wherein the nanoemulsion traffics to the mesenteric lymph node, mediastinal lymph node, or both the mesenteric lymph node and mediastinal lymph node.

49. The method of anj one of claims 41- 48, wherein the immunogen accumulates at the mesenteric lymph node, mediastinal lymph node, or both the mesenteric lymph node and mediastinal lymph node.

50. The method of any one of claims 41-49, wherein the vaccine or the composition is administered in one or more doses.

51. The method of claim 50, wherein the vaccine or the composition is administered in at least 2 doses.

52. The method of claim 50 or 51, wherein doses of the vaccine are administered about 2,3, 4, 5, 6, 7, or 8 weeks apart.

53. The method of any one of claims 50-52, wherein each dose comprises about 5-300 pg of the immunogen.

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