Transmucosal amphiphile-protein conjugate vaccines

Abstract

Disclosed is a vaccine comprising an immunogen conjugated to an albumin-binding polymeric lipid tail, which is suitable for mucosal (e.g., intranasal) administration. Also disclosed is a method of using the vaccine to immunize a subject by mucosally (e.g., intranasally) administering an effective amount of the vaccine, alone or with an adjuvant. The invention provides, for example, a vaccine comprising an amphipathic conjugate, which comprises an immunogen operably linked to an albumin-binding lipid, which is suitable for mucosal administration to induce a humoral immune response.

Description

[Technical field]

[0001] STATEMENT REGARDING FEDERALLY FUNDED RESEARCH This invention was made with Government support under AI144462 and AI048240 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 316,919, filed March 4, 2022, the entire contents of which are expressly incorporated herein by reference.

[0003] Incorporation by Reference All documents cited or referenced in this specification, and all documents cited or referenced in the documents cited herein, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned in this specification or any document incorporated herein by reference, are hereby incorporated by reference and may be used in the practice of this invention. [Background technology]

[0004] Combating long-standing epidemics such as HIV and emerging threats such as SARS-CoV-2 requires immune strategies that can simultaneously induce systemic antibody responses and humoral immunity at the mucosal portal of entry [1–6]. Many pathogens, including HIV, SARS-CoV-2, influenza, rotavirus, and cholera, infect the host through mucosal surfaces and are therefore believed to require the engagement of both systemic and mucosal arms of the immune system using a combination of IgG and IgA antibodies for effective control and defense [1, 6, 7]. Secretory IgA (SIgA) is the primary humoral defense at mucosal tissue sites [4] and plays a particularly important role in providing protection through mechanisms such as immune exclusion, inhibition of transcytosis, and direct neutralization of viruses [8, 9]. The establishment of antigen-specific SIgA antibodies at mucosal surfaces provides a first-line defense that can help prevent infection and transmission

[10] . In the case of HIV, 90% of transmission occurs via the mucosal route, and 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 [11, 12]. Similarly, clinical studies of SARS-CoV-2 have shown that mucosal IgA exhibits potent neutralization and is strongly correlated with protection against a virus that primarily infects cells of the upper and lower respiratory tract mucosa [13, 14].

[0005] Conventional parenteral immunotherapy usually induces poor mucosal immunity. In contrast, vaccination at mucosal surfaces is known to be a highly effective strategy to initiate immune responses in mucosa-associated lymphoid tissues (MALT) and promote protective immunity in barrier tissues by programming mucosa-specific lymphocyte function and tissue homing at these sites [1, 3]. Priming of mucosal T and B lymphocytes occurs at MALT-inducing sites, such as nasal-associated lymphoid tissue (NALT) and gut-associated lymphoid tissue (GALT) [3, 15, 16]. Here, through the properties of the “mucosal immune circulatory homing pathway”, antigen priming can induce the expression of homing markers that direct the migration of activated antigen-specific T cells, B cells, and plasma cells to other local or distal mucosal effector sites [2, 3, 7, 17]. The location of antigen exposure determines which homing markers are expressed, dictating the homing destination and ultimate effector site. Usually, the strongest responses are elicited at the site of antigen exposure and the anatomically most adjacent mucosal tissues. For example, antigen-primed cells in the nasal-associated lymphoid tissue (NALT) acquire chemokine receptors (i.e., CCR10, α4β1) that can home to both the respiratory and genitourinary tracts, allowing intranasal immunization to establish humoral responses at both mucosal sites [2, 17].

[0006] Although well motivated by the biology of mucosal immunity, delivery of vaccine components across mucosal barriers remains a major challenge for mucosal vaccine development [1–3]. Vaccine uptake into the underlying mucosal immune compartment is hindered by multiple factors, including potential rapid antigen loss due to proteolytic degradation and acidic conditions at mucosal surfaces, high mucociliary clearance rates, and lack of diffusive uptake through tight junctions of epithelial monolayers [18–20]. Indeed, only a few mucosal vaccines are licensed, and all, except for the inactivated oral cholera vaccine, are based on live attenuated pathogens that naturally infect mucosal surfaces, such as oral polio vaccine (OPV) or intranasal influenza A / B vaccine (FluMist) [3, 21, 22]. However, live attenuated vaccines often face manufacturing challenges, reduced stability, and safety concerns. These challenges have been addressed in parenteral vaccines by focusing on safe, stable, and highly manufacturable recombinant protein or polysaccharide-based subunit vaccines, but subunit vaccines have historically demonstrated reduced immunogenicity and short-lived responses when applied to mucosal barriers, primarily due to delivery issues and poor uptake.[3] The development of technologies that overcome the barriers of mucosal delivery while meeting the safety and efficacy requirements of prophylactic vaccines remains an urgent unmet need. Summary of the Invention [Means for solving the problem]

[0007] This Summary introduces a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to limit the scope thereof.

[0008] In one aspect, the present disclosure provides a vaccine comprising an amphipathic conjugate, the amphipathic conjugate comprising an immunogen operably linked to an albumin-binding lipid, the vaccine being suitable for mucosal administration to induce a humoral immune response.

[0009] In some embodiments, the transmucosal administration is intranasal administration.

[0010] In some embodiments, the immunogen is a protein antigen having a molecular weight of about 10 kDa to about 500 kDa.

[0011] In some embodiments, the immunogen comprises a protein antigen 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.

[0012] In some embodiments, the immunogen comprises a monomeric or trimeric antigen.

[0013] In some embodiments, the immunogen comprises an antigenic peptide.

[0014] In some embodiments, the albumin-binding lipid is selected from the group consisting of cholesterol, monoacyl lipid, and diacyl lipid. In some embodiments, the albumin-binding lipid is a diacyl lipid. In some embodiments, the albumin-binding lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

[0015] In some embodiments, the immunogen is operably linked to the albumin-binding lipid via a first linker. In some embodiments, the first linker is selected from the group consisting of a hydrophilic polymer, a series of hydrophilic amino acids, a polysaccharide, an oligonucleotide, or a combination thereof. In some embodiments, the first linker comprises a polyethylene glycol (PEG) linker. In some embodiments, the first linker comprises 45-150 repeating units of a PEG monomer. In some embodiments, the first linker comprises a PEG2K linker.

[0016] In some embodiments, the vaccine further comprises a second linker, the second linker being located between the immunogen and the first linker, or between the albumin-binding lipid and the first linker. In some embodiments, the second linker comprises a PEG linker. In some embodiments, the second linker comprises 2-20 repeat units of a PEG monomer. In some embodiments, the second linker comprises 4 repeat units of a PEG monomer. In some embodiments, the second linker comprises a dibenzocyclooctyne (DBCO) group covalently bonded to the repeat unit of a PEG monomer.

[0017] In some embodiments, the immunogen comprises an HIV antigen. In some embodiments, the HIV antigen comprises HIV gp120 modified ectodomain germline targeted immunogen 8 (eOD-GT8).

[0018] In some embodiments, the immunogen comprises a SARS-CoV-2 antigen. In some embodiments, the SARS-CoV-2 antigen comprises an antigen derived from the receptor binding domain (RBD) of the SARS-CoV-2 spike protein.

[0019] In some embodiments, the vaccine further comprises an adjuvant, hi some embodiments, the adjuvant is selected from the group consisting of bis-(3'-5')-cyclic dimeric guanosine monophosphate (cdGMP) and saponin monophosphoryl lipid-A (MPLA) nanoparticle adjuvant (SMNP).

[0020] In some embodiments, mucosal administration of the vaccine induces or enhances the production of antibodies that bind to the immunogen. In some embodiments, the antibodies include IgA antibodies, IgG antibodies, or IgA and IgG antibodies. In some embodiments, the antibodies are neutralizing antibodies.

[0021] In another aspect, the disclosure provides a method of vaccinating a subject, the method comprising vaccinating the subject by mucosally administering to the subject an effective amount of a vaccine of the disclosure.

[0022] In yet another aspect, the present disclosure provides a method of immunizing a subject, comprising immunizing the subject by mucosally administering to the subject an effective amount of a vaccine of the present disclosure.

[0023] In some embodiments, the vaccine is administered to the subject intranasally.

[0024] In some embodiments, the vaccine is administered in two or more doses. In some embodiments, the doses of the vaccine are administered about 2, 4, 6, or 8 weeks apart. In some embodiments, the vaccine is administered at weeks 0, 8, 16, and 24. In some embodiments, the vaccine is administered in a dose of about 5 μg to about 300 μg. In some embodiments, the vaccine is administered in a dose of about 50 μg, 100 μg, or 150 μg.

[0025] In some embodiments, the vaccine is administered in combination with an adjuvant. In some embodiments, the adjuvant comprises SMNP. In some embodiments, the SMNP is administered at a dose of about 5 μg to about 500 μg. In some embodiments, the SMNP is administered at a dose of about 300 μg, 375 μg, or 450 μg. In some embodiments, the adjuvant comprises cdGMP. In some embodiments, the cdGMP is administered at a dose of about 25 μg to about 500 μg.

[0026] The following detailed description refers to the accompanying drawings which form a part of this application and which show, by way of illustration, specific exemplary implementations. Other implementations may be made without departing from the scope of the disclosure. [Brief description of the drawings]

[0027] [Figure 1A]1 shows the synthesis of albumin-binding amphiphile-protein immunogen conjugates. FIG. 2 is a schematic diagram of the amph-eOD structure. [Figure 1B] 1 shows the synthesis of albumin-binding amphiphile-protein immunogen conjugates. Dynamic light scattering analysis of eOD and amph-eOD. [Figure 1C] 1 shows the synthesis of albumin-binding amphiphile-protein immunogen conjugates. SEC profiles of eOD and amph-eOD. [Figure 1D] Synthesis of albumin-binding amphiphile-protein immunogen conjugates. AF647-eOD or AF647-amph-eOD proteins were incubated with albumin-functionalized agarose resin at 37° C., and the amount of each protein bound to the resin after 2 hours was quantified. Statistical significance was determined by unpaired t-test. [Figure 1E] Synthesis of albumin-binding amphiphile-protein immunogen conjugates. Fluorescent eOD or amph-eOD was incubated at various concentrations with mouse C57Bl / 6 spleen cells for 1 h at 37° C., followed by washing and staining with fluorescent VRC01 antibody: Representative flow cytometry plots of cell-bound eOD / amph-eOD and VRC01. [Figure 1F] Synthesis of albumin-binding amphiphile-protein immunogen conjugates. Fluorescent eOD or amph-eOD were incubated at various concentrations with mouse C57Bl / 6 spleen cells for 1 h at 37° C., then washed and stained with fluorescent VRC01 antibody: percentage of cells positive for eOD alone or double positive for eOD and VRC01, statistical significance determined by two-way ANOVA followed by Sidak's post-hoc test. [Figure 1G]Synthesis of albumin-binding amphiphile-protein immunogen conjugates. Fluorescent eOD or amph-eOD were incubated with mouse C57Bl / 6 spleen cells at various concentrations for 1 hour at 37°C, followed by washing and staining with fluorescent VRC01 antibody: Mean fluorescence intensity (MFI) of eOD and VRC01 as a function of eOD concentration, with statistically significant non-zero slopes determined by simple linear regression. All data represent the mean ± standard error. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 2A] We demonstrate that amph protein conjugates have improved persistence within the nasal mucosa and are transported across the mucosal surface. Ventral views of the upper palate and underside of the maxilla of a mouse showing the ROIs used to quantify IVIS signal in Figure 2B and Figure 2E (top) and sagittal views of the mouse skull and nasal cavity (bottom) showing the approximate locations of the corresponding coronal sections used for histology in Figure 2G-2H. [Figure 2B] Figure 1 shows that the amph protein conjugates have improved persistence in the nasal mucosa and are transported across mucosal surfaces. Representative IVIS images of fluorescent signal in the nasal cavity of BALB / c mice (n = 3 animals / group) over time after intranasal administration of 5 μg of AF647-eOD or AF647-amph-eOD mixed with 5 μg of saponin monophosphoryl lipid A (MPLA) nanoparticle adjuvant (SMNP). Regions of interest (ROIs) used for quantification of IVIS signal are marked with dotted white ellipses. [Figure 2C] Figure 2 shows that amph protein conjugates have improved persistence in the nasal mucosa and are transported across mucosal surfaces. Quantified IVIS signal from Figure 2B in the nasal cavity over time. Statistical significance was determined by unpaired t-test. Data shown from one representative of two independent experiments. [Figure 2D]Quantified IVIS signal area under the curve (AUC, total radiance x time) from Figure 2C shows that amph protein conjugates have improved persistence in the nasal mucosa and are transported across the mucosal surface. Statistical significance was determined by unpaired t-test. [Figure 2E] Figure 1 shows that amph protein conjugates enhance persistence in the nasal mucosa and are transported across mucosal surfaces.Representative IVIS images showing intranasal vaccine uptake and retention over time following intranasal administration of 5 μg AF647-eOD or AF647-amph-eOD mixed with 5 μg SMNP adjuvant to WT C57Bl / 6 and FcRn- / - mice (n=3 animals / group). [Figure 2F] Figure 2E shows that the amph protein conjugate has improved persistence in the nasal mucosa and is transported across the mucosal surface. Quantified IVIS signal in the nasal cavity of WT and FcRn− / − mice at 6 h. Statistical significance was determined by two-way ANOVA followed by Tukey's post hoc test. [Figure 2G] Figure 1. Representative histology images of vaccine in the nasal cavity of WT and FcRn- / - mice after 6 hours, showing that the amph protein conjugate has improved persistence in the nasal mucosa and is transported across mucosal surfaces. Images in (ii) are higher magnifications of the dashed area marked in (i). Scale bars represent (i) 1 mm and (ii) 500 μm. [Figure 2H] We show that amph protein conjugates have improved persistence in the nasal mucosa and are transported across mucosal surfaces. Representative histology images of vaccine in the nasal cavity of WT and FcRn- / - mice after 24 hours. Images in (ii) are higher magnification views of the dashed area indicated in (i). (iii) shows higher magnification sections stained with DAPI to identify the epithelial cell barrier. "e" stands for epithelium, "lp" for lamina propria, and "m" for mucus. Scale bars represent (i) 1 mm, (ii) 500 μm, and (iii) 100 μm. All data are shown as mean ± SEM. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 3A] Figure 1 shows that amph protein conjugates promote enhanced GC B cell and Tfh responses in the NALT in an FcRn-dependent manner. Groups of BALB / c mice (n=5 animals / group) were intranasally immunized with 10 μg AF647-amph-eOD or AF647-eOD mixed with 5 μg saponin adjuvant, and NALT tissue was isolated 1 or 4 days later for flow cytometric analysis of antigen uptake: (i) NALT tissue location and (ii) experimental timeline. [Figure 3B] We show that amph protein conjugates promote enhanced GC B cell and Tfh responses in the NALT in an FcRn-dependent manner. Groups of BALB / c mice (n=5 animals / group) were intranasally immunized with 10 μg AF647-amph-eOD or AF647-eOD mixed with 5 μg saponin adjuvant, and NALT tissues were isolated 1 or 4 days later for flow cytometry analysis of antigen uptake: Representative flow cytometry plots of eOD signal gating and mean fluorescence intensity in F4 / 80+ macrophages. [Figure 3C] Figure 1 shows that amph protein conjugates promote enhanced GC B cell and Tfh responses in NALT in an FcRn-dependent manner. Groups of BALB / c mice (n=5 animals / group) were intranasally immunized with 10 μg AF647-amph-eOD or AF647-eOD mixed with 5 μg saponin adjuvant, and NALT tissues were isolated 1 or 4 days later for flow cytometry analysis of antigen uptake: Representative flow cytometry plots of eOD signal gating and mean fluorescence intensity in B cells. [Figure 3D]Figure 1 shows that amph protein conjugates promote enhanced GC B cell and Tfh responses in NALT in an FcRn-dependent manner. Groups of BALB / c mice (n=5 animals / group) were intranasally immunized with 10 μg AF647-amph-eOD or AF647-eOD mixed with 5 μg saponin adjuvant, and NALT tissues were isolated 1 or 4 days later for flow cytometry analysis of antigen uptake: representative flow cytometry plots of eOD signal gating and mean fluorescence intensity in CD11c+ dendritic cells. Statistical significance was determined by unpaired t-test. [Figure 3E] Figure 1 shows that amph protein conjugates promote enhanced GC B cell and Tfh responses in the NALT in an FcRn-dependent manner. Groups of C57Bl / 6 (WT) or FcRn- / - mice (n=5 animals / group) were immunized with 5 μg eOD or amph-eOD mixed with 5 μg saponin adjuvant and GC / Tfh responses were analyzed by flow cytometry on day 12: Schematic diagram of experimental timeline. [Figure 3F] We show that amph protein conjugates promote enhanced GC B cell and Tfh responses in the NALT in an FcRn-dependent manner. Groups of C57Bl / 6 (WT) or FcRn- / - mice (n=5 animals / group) were immunized with 5 μg eOD or amph-eOD mixed with 5 μg saponin adjuvant and GC / Tfh responses were analyzed by flow cytometry on day 12: Representative flow cytometry gating and total GC B cell counts. [Figure 3G] We show that amph protein conjugates promote enhanced GC B cell and Tfh responses in the NALT in an FcRn-dependent manner. Groups of C57Bl / 6 (WT) or FcRn- / - mice (n=5 animals / group) were immunized with 5 μg eOD or amph-eOD mixed with 5 μg saponin adjuvant and analyzed GC / Tfh responses by flow cytometry on day 12: antigen-specific GC B cells. [Figure 3H]We show that amph protein conjugates promote enhanced GC B cell and Tfh responses in the NALT in an FcRn-dependent manner. Groups of C57Bl / 6 (WT) or FcRn- / - mice (n=5 animals / group) were immunized with 5 μg eOD or amph-eOD mixed with 5 μg saponin adjuvant and analyzed GC / Tfh responses by flow cytometry on day 12:Tfh cells. Data shown from one representative of two independent experiments. Statistical significance was determined by ordinary one-way ANOVA followed by Tukey's post-hoc test. All data are shown as mean ± SEM. (*p<0.05, **p<0.01, **p<0.001, ****p<0.0001). [Figure 4A] Figure 4 shows that amph protein conjugates induce enhanced systemic and mucosal immune responses after intranasal vaccination. BALB / c mice (n=5 animals / group) were immunized intranasally with 5 μg eOD or amph-eOD mixed with 25 μg cdGMP adjuvant and boosted 6 weeks later with the same formulations: Schematic diagram showing experimental timeline; (Figure 4B) IgG and IgA titers in serum, (Figure 4C) vaginal washes, and (Figure 4D) feces. [Figure 4B] We show that amph protein conjugates induce enhanced systemic and mucosal immune responses following intranasal vaccination. BALB / c mice (n=5 animals / group) were immunized intranasally with 5 μg eOD or amph-eOD mixed with 25 μg cdGMP adjuvant and boosted 6 weeks later with the same formulations. Statistical significance was determined by ordinary two-way ANOVA followed by Sidak's post-hoc test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 4C]Figure 1 shows that amph-protein conjugates induce enhanced systemic and mucosal immune responses following intranasal vaccination. BALB / c mice (n=5 animals / group) were immunized intranasally with 5 μg eOD or amph-eOD mixed with 25 μg cdGMP adjuvant and boosted 6 weeks later with the same formulations. Statistical significance was determined by ordinary two-way ANOVA followed by Sidak's post-hoc test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 4D] We show that amph protein conjugates induce enhanced systemic and mucosal immune responses following intranasal vaccination. BALB / c mice (n=5 animals / group) were immunized intranasally with 5 μg eOD or amph-eOD mixed with 25 μg cdGMP adjuvant and boosted 6 weeks later with the same formulations. Statistical significance was determined by ordinary two-way ANOVA followed by Sidak's post-hoc test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 4E] Figure 1 shows that amph-protein conjugates induce enhanced systemic and mucosal immune responses after intranasal vaccination. BALB / c mice (n=5 animals / group) were immunized intranasally with 5 μg eOD or amph-eOD mixed with 25 μg cdGMP adjuvant and boosted 6 weeks later with the same formulations: FRT and BM eOD-specific IgA antibody secreting cells assessed by ELISPOT 1 year after immunization. Data shown from one representative of two independent experiments. Statistical significance was determined by unpaired t-test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 4F]Figure 4 shows that amph-protein conjugates induce enhanced systemic and mucosal immune responses after intranasal vaccination. BALB / c mice (n=5 animals / group) were immunized with 5 μg eOD or amph-eOD mixed with 5 μg SMNP adjuvant and boosted 6 weeks later with the same formulations: schematic diagram showing experimental timeline. IgG and IgA titers in (Figure 4G) serum, (Figure 4H) vaginal washes, and (Figure 4I) feces. [Figure 4G] Figure 1 shows that amph protein conjugates induce enhanced systemic and mucosal immune responses following intranasal vaccination. BALB / c mice (n=5 animals / group) were immunized with 5 μg eOD or amph-eOD mixed with 5 μg SMNP adjuvant and boosted 6 weeks later with the same formulations. Statistical significance was determined by ordinary two-way ANOVA followed by Sidak's post-hoc test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 4H] Figure 1 shows that amph-protein conjugates induce enhanced systemic and mucosal immune responses following intranasal vaccination. BALB / c mice (n=5 animals / group) were immunized with 5 μg eOD or amph-eOD mixed with 5 μg SMNP adjuvant and boosted 6 weeks later with the same formulations. Statistical significance was determined by ordinary two-way ANOVA followed by Sidak's post-hoc test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 4I] Figure 1 shows that amph-protein conjugates induce enhanced systemic and mucosal immune responses following intranasal vaccination. BALB / c mice (n=5 animals / group) were immunized with 5 μg eOD or amph-eOD mixed with 5 μg SMNP adjuvant and boosted 6 weeks later with the same formulations. Statistical significance was determined by ordinary two-way ANOVA followed by Sidak's post-hoc test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 4J]Figure 1 shows that amph protein conjugates induce enhanced systemic and mucosal immune responses after intranasal vaccination. BALB / c mice (n=5 animals / group) were immunized with 5 μg eOD or amph-eOD mixed with 5 μg SMNP adjuvant and boosted 6 weeks later with the same formulations: FRT and BM eOD-specific IgA antibody secreting cells assessed by ELISPOT 35 weeks after immunization. Data shown from one representative of two independent experiments. Statistical significance was determined by unpaired t-test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). All data show mean ± SEM. [Figure 5A] FIG. 1 shows that amph-RBD conjugates induce enhanced systemic and mucosal neutralizing antibody responses against SARS-CoV-2 immunogens following intranasal vaccination. FIG. 1 shows a schematic diagram of the structure of amph-RBD. [Figure 5B] Figure 1 shows that amph-RBD conjugates induce enhanced systemic and mucosal neutralizing antibody responses against SARS-CoV-2 immunogens following intranasal vaccination. BALB / c mice (n=5 animals / group) were immunized intranasally with 5μg RBD or amph-RBD mixed with 5μg SMNP adjuvant and boosted 4 weeks later with the same formulations: Schematic showing the experimental timeline. [Figure 5C] We show that amph-RBD conjugates induce enhanced systemic and mucosal neutralizing antibody responses to SARS-CoV-2 immunogens following intranasal vaccination. BALB / c mice (n=5 animals / group) were immunized intranasally with 5 μg RBD or amph-RBD mixed with 5 μg SMNP adjuvant and boosted 4 weeks later with the same formulations. IgG titers in serum, vaginal washes, fecal washes, saliva, nasal washes, and bronchoalveolar lavage fluid (BALF) at week 6 are shown. Statistical significance was determined by two-way ANOVA followed by Sidak's post-hoc test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 5D]We show that amph-RBD conjugates induce enhanced systemic and mucosal neutralizing antibody responses to SARS-CoV-2 immunogens following intranasal vaccination. BALB / c mice (n=5 animals / group) were immunized intranasally with 5μg RBD or amph-RBD mixed with 5μg SMNP adjuvant and boosted 4 weeks later with the same formulations. IgA titers in serum, vaginal washes, fecal washes, saliva, nasal washes, and bronchoalveolar lavage fluid (BALF) at week 6 are shown. Statistical significance was determined by two-way ANOVA followed by Sidak's post-hoc test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 5E] We show that amph-RBD conjugates induce enhanced systemic and mucosal neutralizing antibody responses to SARS-CoV-2 immunogens following intranasal vaccination.BALB / c mice (n=5 animals / group) were immunized intranasally with 5μg RBD or amph-RBD mixed with 5μg SMNP adjuvant and boosted 4 weeks later with the same formulations: ACE2:RBD binding inhibition (IC50) of antibodies in serum and BALF at week 6. [Figure 5F] Figure 1 shows that amph-RBD conjugates induce enhanced systemic and mucosal neutralizing antibody responses to SARS-CoV-2 immunogens following intranasal vaccination. BALB / c mice (n=5 animals / group) were immunized intranasally with 5μg RBD or amph-RBD mixed with 5μg SMNP adjuvant and boosted 4 weeks later with the same formulations: pseudovirus neutralizing antibody (NAb) titers (NT50) in serum, nasal washes, and BALF at week 6. Dotted lines represent the limit of quantification. Data shown from one representative of two independent experiments. All data show mean ± SEM. [Figure 6A]Figure 1 shows that intranasal immunization with amph protein conjugates leads to improved humoral immune responses in non-human primates. Rhesus macaques (n=3 animals / group) were immunized intranasally with 100 μg AF647-eOD or AF647-amph-eOD mixed with 375 μg SMNP adjuvant. Fluorescent signal of vaccine immunogen in the nasal cavity was quantified 24 hours later by IVIS imaging. Statistical significance was determined by unpaired t-test. (BE) Rhesus macaques (n=6 animals / group) were immunized intranasally with 100 μg eOD or amph-eOD mixed with 375 μg SMNP adjuvant and boosted with the same formulations at weeks 8, 16, and 24. [Figure 6B] 1 shows that intranasal immunization with amph protein conjugates leads to improved humoral immune responses in non-human primates. [Figure 6C] Figure 1 shows that intranasal immunization with amph protein conjugates leads to improved humoral immune responses in non-human primates. Frequencies of antigen-specific IgM, IgG and IgA secreting plasmablasts in peripheral blood as determined by ELISPOT. Statistical significance was determined by two-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 6D] Figure 1 shows that intranasal immunization with amph protein conjugates leads to improved humoral immune responses in non-human primates. Serum IgG and IgA titers over time and IgG titers of individual animals at 6 weeks (middle panel, left: Amph-eOD, right: eOD). Statistical significance was determined by two-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 6E]Figure 1 shows that intranasal immunization with amph protein conjugates leads to improved humoral immune responses in non-human primates. IgG and IgA titers in nasal washes over time. Statistical significance was determined by two-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). All data are shown as mean ± SEM. [Figure 7A] 1 shows the synthesis of amphiphile-protein conjugates. Sequence of eOD protein with PADRE peptide underlined. [Figure 7B] 1 shows the synthesis of amphiphile-protein conjugates. FIG. 2 is a reaction scheme for preparing amph-eOD antigen conjugate. [Figure 8A] Insertion of amph protein conjugates into the cell membrane. AF647-labeled eOD or amph-eOD were incubated at various concentrations with mouse C57Bl / 6 spleen cells for 1 h at 37° C., washed and stained with rhodamine-labeled VRC01 antibody and assessed by flow cytometry to evaluate cell membrane insertion of eOD and amph-eOD: gating strategy to identify VRC01+ and AF647+ cells. [Figure 8B] Figure 1 shows insertion of amph protein conjugates into the cell membrane. AF647-labeled eOD or amph-eOD were incubated with mouse C57Bl / 6 spleen cells at various concentrations for 1 h at 37°C, washed and stained with rhodamine-labeled VRC01 antibody and assessed by flow cytometry to evaluate cell membrane insertion of eOD and amph-eOD: Representative flow cytometry plots of eOD / amph-eOD and VRC01 binding to cells at various concentrations of eOD. [Figure 8C]Insertion of amph protein conjugates into the cell membrane. AF647-labeled eOD or amph-eOD were incubated at various concentrations with mouse C57Bl / 6 spleen cells for 1 h at 37° C., washed and stained with rhodamine-labeled VRC01 antibody and assessed by flow cytometry to evaluate cell membrane insertion of eOD and amph-eOD: ELISA measurements are shown for human serum albumin binding to plate-bound human FcRn in the presence of various concentrations of DSPE-PEG2K-FITC. [Figure 9A] Figure 1 shows systemic distribution of amph protein conjugates in mice. BALB / c mice (n=3 animals / group) were immunized intranasally with 5 μg AF647-eOD or AF647-amph-eOD mixed with 5 μg SMNP adjuvant, and tissues were harvested 24 h later for IVIS analysis of AF647 fluorescent signal to assess systemic dissemination and distal lymphatic drainage of eOD and amph-eOD: representative IVIS images in the intestine, mesenteric lymph nodes (mLN), cervical lymph nodes (cLN), liver, and spleen after 24 h. [Figure 9B] Figure 1 shows systemic distribution of amph protein conjugates in mice. BALB / c mice (n=3 animals / group) were immunized intranasally with 5 μg AF647-eOD or AF647-amph-eOD mixed with 5 μg SMNP adjuvant, and tissues were harvested 24 hours later for IVIS analysis of AF647 fluorescent signal to assess systemic dissemination and distal lymphatic drainage of eOD and amph-eOD: quantified IVIS signal in gut, mesenteric lymph nodes (mLN), cervical lymph nodes (cLN), liver, and spleen after 24 hours. Data show mean ± SEM. [Figure 9C]Figure 1 shows the systemic distribution of amph protein conjugates in mice. BALB / c mice (n=3 animals / group) were immunized intranasally with 5 μg AF647-eOD or AF647-amph-eOD mixed with 5 μg SMNP adjuvant, and 24 h later tissues were harvested for IVIS analysis of AF647 fluorescent signal to assess systemic dissemination and distal lymphatic drainage of eOD and amph-eOD: ELISA analysis is shown for albumin concentration in nasal washes of wild-type (WT) and FcRn- / - mice (n=5 animals per group). Statistical comparisons were made using Welch's t-test. All data are presented as mean ± standard error of the mean (sem). ns is not significant. [Figure 10] Figure 1 shows uptake of amph protein in NALT cell populations in mice. Groups of BALB / c mice (n=5 animals / group) were intranasally immunized with 10 μg AF647-amph-eOD or AF647-eOD mixed with 5 μg SMNP adjuvant, and NALT tissue was isolated 1 or 4 days later for flow cytometric analysis of antigen uptake. Schematic showing gating strategy to identify uptake of AF647-labeled vaccine in NALT macrophages, B cells, and dendritic cells. MHC is major histocompatibility complex. [Figure 11A] Figure 1 shows GC B cell responses in mouse NALT after intranasal immunization with amph protein. Groups of C57Bl / 6 (WT) or FcRn- / - mice (n=5 animals / group) were immunized with 5 μg eOD or amph-eOD mixed with 5 μg SMNP adjuvant and GC responses were analyzed by flow cytometry on day 12. Gating strategy to identify GC B cells. [Figure 11B] Figure 1 shows GC B cell responses in mouse NALT after intranasal immunization with amph protein. Groups of C57Bl / 6 (WT) or FcRn- / - mice (n=5 animals / group) were immunized with 5 μg eOD or amph-eOD mixed with 5 μg SMNP adjuvant and GC responses were analyzed by flow cytometry on day 12. Representative FACS plots. [Figure 11C]Figure 1 shows GC B cell responses in mouse NALT after intranasal immunization with amph protein. Groups of C57Bl / 6 (WT) or FcRn- / - mice (n=5 animals / group) were immunized with 5 μg eOD or amph-eOD mixed with 5 μg SMNP adjuvant and GC responses were analyzed by flow cytometry on day 12: absolute cell counts showing CD38-GL7+ GC B total cells for all NALT samples including controls. [Figure 11D] Figure 1 shows GC B cell responses in mouse NALT after intranasal immunization with amph protein. Groups of C57Bl / 6 (WT) or FcRn- / - mice (n=5 animals / group) were immunized with 5 μg eOD or amph-eOD mixed with 5 μg SMNP adjuvant and GC responses were analyzed by flow cytometry on day 12. Representative FACS plots. [Figure 11E] Figure 1 shows GC B cell responses in mouse NALT after intranasal immunization with amph protein. Groups of C57Bl / 6 (WT) or FcRn- / - mice (n=5 animals / group) were immunized with 5 μg OD or amph-eOD mixed with 5 μg SMNP adjuvant and GC responses were analyzed by flow cytometry on day 12: absolute number of cells representing eOD tetramer+ GC B cells for all NALT samples including controls. Statistical significance was determined using one-way ANOVA followed by Tukey's post-hoc test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). All data show mean ± SEM. [Figure 12A] Figure 1 shows Tfh cell responses in mouse NALT after intranasal immunization with amph protein. Groups of C57Bl / 6 (WT) or FcRn- / - mice (n=5 animals / group) were immunized with 5 μg eOD or amph-eOD mixed with 5 μg SMNP adjuvant and Tfh responses were analyzed by flow cytometry on day 12. Gating strategy to identify Tfh cells. [Figure 12B]Figure 1 shows Tfh cell responses in mouse NALT after intranasal immunization with amph protein. Groups of C57Bl / 6 (WT) or FcRn- / - mice (n=5 animals / group) were immunized with 5 μg eOD or amph-eOD mixed with 5 μg SMNP adjuvant and Tfh responses were analyzed by flow cytometry on day 12. Representative FACS plots showing activated ICOS+CD4+CD44+ T cells in all NALT samples including controls. [Figure 12C] Figure 1 shows Tfh cell responses in mouse NALT after intranasal immunization with amph protein. Groups of C57Bl / 6 (WT) or FcRn- / - mice (n=5 animals / group) were immunized with 5 μg eOD or amph-eOD mixed with 5 μg SMNP adjuvant and Tfh responses were analyzed by flow cytometry on day 12. Absolute cell counts represent activated ICOS+CD4+CD44+ T cells for all NALT samples including controls. [Figure 12D] Figure 1 shows Tfh cell responses in mouse NALT after intranasal immunization with amph protein. Groups of C57Bl / 6 (WT) or FcRn- / - mice (n=5 animals / group) were immunized with 5μg eOD or amph-eOD mixed with 5μg SMNP adjuvant and Tfh responses were analyzed by flow cytometry on day 12. Representative FACS plot showing PD-1+CXCR5+Tfh cells in all NALT samples including controls. [Figure 12E] Figure 1 shows Tfh cell responses in mouse NALT after intranasal immunization with amph protein. Groups of C57Bl / 6 (WT) or FcRn- / - mice (n=5 animals / group) were immunized with 5μg eOD or amph-eOD mixed with 5μg SMNP adjuvant and Tfh responses were analyzed by flow cytometry on day 12. Absolute cell counts represent PD-1+CXCR5+Tfh cells for all NALT samples including controls. Statistical significance was determined using one-way ANOVA followed by Tukey's post-hoc test (*p<0.05, **p<0.01). All data show mean ± SEM. [Figure 13A]Control parenteral immunization with amph-protein conjugates induces little mucosal antibody response compared to intranasal immunization. BALB / c mice (n=5 animals per group) were immunized in the scruff of the neck with 5 μg amph-eOD mixed with 25 μg cdGMP adjuvant by intranasal (in) or subcutaneous (sc) injection and boosted 6 weeks later with the same formulation (arrows). IgG and IgA titers were measured in serum. [Figure 13B] Control parenteral immunization with amph-protein conjugates induces little mucosal antibody response compared to intranasal immunization. BALB / c mice (n=5 animals per group) were immunized in the scruff of the neck with 5 μg amph-eOD mixed with 25 μg cdGMP adjuvant by intranasal (in) or subcutaneous (sc) injection and boosted 6 weeks later with the same formulation (arrows). IgG and IgA titers were measured in vaginal washes. [Figure 13C] Figure 1 shows that control parenteral immunization with amph-protein conjugates induces little mucosal antibody response compared to intranasal immunization. BALB / c mice (n=5 animals per group) were immunized in the scruff of the neck with 5 μg amph-eOD mixed with 25 μg cdGMP adjuvant by intranasal (in) or subcutaneous (sc) injection and boosted 6 weeks later with the same formulation (arrows). IgG and IgA titers were measured in feces. Statistical significance comparing in and sc groups was determined by unpaired t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. All data are presented as mean ± SEM. [Figure 14A]Figure 1 shows that long-lived antigen-specific IgG and IgA plasma cells were established in mice after intranasal immunization with amph protein without induction of anti-PEG antibodies. BALB / c mice (n=3 animals / group) were immunized intranasally with 5 μg amph-eOD mixed with 25 μg cdGMP adjuvant and boosted 6 weeks later with the same formulation. Female reproductive tract (FRT) and bone marrow (BM) eOD-specific IgG and IgA antibody secreting cells were assessed by ELISPOT 20 weeks after immunization: (A) representative well images and (B) quantified number of antibody-secreting plasma cells per 500,000 cells. All data show mean ± SEM. [Figure 14B] Figure 1 shows that long-lived antigen-specific IgG and IgA plasma cells were established in mice after intranasal immunization with amph protein without induction of anti-PEG antibodies. BALB / c mice (n=3 animals / group) were immunized intranasally with 5 μg amph-eOD mixed with 25 μg cdGMP adjuvant and boosted 6 weeks later with the same formulation. Female reproductive tract (FRT) and bone marrow (BM) eOD-specific IgG and IgA antibody secreting cells were assessed by ELISPOT 20 weeks after immunization: (A) representative well images and (B) quantified number of antibody-secreting plasma cells per 500,000 cells. All data show mean ± SEM. [Figure 14C]Figure 4 shows that long-lived antigen-specific IgG and IgA plasma cells were established in mice after intranasal immunization with amph protein without induction of anti-PEG antibodies. BALB / c mice (n=3 animals / group) were immunized intranasally with 5 μg amph-eOD mixed with 25 μg cdGMP adjuvant and boosted 6 weeks later with the same formulation. Female reproductive tract (FRT) and bone marrow (BM) eOD-specific IgG and IgA antibody secreting cells were assessed by ELISPOT at 20 weeks post-immunization: (A) representative well images, and (B) quantified number of antibody-secreting plasma cells per 500,000 cells. All data show mean ± SEM. Serum samples from mice immunized with saponin (taken at week 11) or cdGMP adjuvant (taken at week 12) as in Figure 4A and F were analyzed by ELISA for anti-PEG IgG and compared to a reference anti-PEG IgG standard. [Figure 15A] Synthesis and characterization of amph-RBD. Gel comparison of RBD and cys-RBD. [Figure 15B] Synthesis and characterization of amph-RBD. Results of an antigenic ELISA comparing binding of RBD and cys-RBD to monoclonal antibody CR3022 and angiotensin-converting enzyme 2 (ACE2)-Fc. [Figure 15C] Synthesis and characterization of amph-RBD. Dynamic light scattering analysis of RBD and amph-RBD. Shown as value-weighted % frequency. Dh denotes hydrodynamic diameter. [Figure 15D] Synthesis and characterization of amph-RBD. Size-exclusion chromatography (SEC) profiles of RBD and amph-RBD. [Figure 15E] 5A-5C show the synthesis and characterization of amph-RBD. FIG. 5D shows the raw absorbance curves of ACE2 binding inhibition in week 6 serum used to determine the IC50 values ​​shown in FIG. [Figure 15F] Synthesis and characterization of amph-RBD. Raw absorbance curves of ACE2 binding inhibition in bronchoalveolar lavage fluid (BALF) used to determine IC50 values ​​shown in Figure 5E. All data shown are mean ± SEM. [Figure 16A] Figure 16 shows that intranasal immunization with amph protein conjugates leads to improved humoral immune responses in non-human primates. Rhesus macaques (n=6 animals / group) were intranasally immunized with 100 μg eOD or amph-eOD mixed with 375 μg SMNP adjuvant and boosted with the same formulations (arrows in Figures 16C and 16D) at weeks 8, 16 and 24. The percentage of antigen-specific IgM, IgG and IgA secreting plasmablasts in peripheral blood (total % eOD) as determined by ELISPOT is shown. Statistical significance was determined using multiple unpaired t-tests. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 16B] Figure 16 shows that intranasal immunization with amph protein conjugates leads to improved humoral immune responses in non-human primates. Rhesus macaques (n=6 animals / group) were intranasally immunized with 100 μg eOD or amph-eOD mixed with 375 μg SMNP adjuvant and boosted with the same formulations (arrows in Figures 16C and 16D) at weeks 8, 16, and 24. Total frequencies of IgM, IgG, and IgA secreting plasmablasts. Statistical significance was determined using multiple unpaired t-tests. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 16C] Figure 16 shows that intranasal immunization with amph protein conjugates leads to improved humoral immune responses in non-human primates. Rhesus macaques (n=6 animals / group) were intranasally immunized with 100 μg eOD or amph-eOD mixed with 375 μg SMNP adjuvant and boosted with the same formulations (arrows in Figures 16C and 16D) at weeks 8, 16, and 24. Vaginal IgG and IgA titers over time. Statistical significance was determined using a two-way ANOVA comparing eOD to amph-eOD across all time points. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 16D]Figure 16 shows that intranasal immunization with amph protein conjugates leads to improved humoral immune responses in non-human primates. Rhesus macaques (n=6 animals / group) were immunized intranasally with 100 μg eOD or amph-eOD mixed with 375 μg SMNP adjuvant and boosted with the same formulations (arrows in Figures 16C and 16D) at weeks 8, 16, and 24. Rectal IgG and IgA titers over time. Statistical significance was determined using a two-way ANOVA comparing eOD to amph-eOD across all time points. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). All data represent mean ± SEM. [Figure 17A] Synthesis of amphiphile MD39 conjugates is shown. Reduced MD39 trimeric protein with a terminal cysteine ​​(cys-MD39) was first reacted with the linker DBCO-PEG4-maleimide to form the intermediate product DBCO-PEG4-MD39. [Figure 17B] The synthesis of amphiphile MD39 conjugates is shown. DBCO-PEG4-MD39 is then reacted with DSPE-PEG2K-azide in a click chemistry reaction to form the final product amph-MD39, which can exist as an amph-MD39 monomeric conjugate or an amph-MD39 trimer conjugate. [Figure 18]Characterization of amph-MD39 by UV-Vis spectrophotometry. MD39 protein with a terminal cysteine ​​(cys-MD39, spectrum shown as solid grey line) was first reacted with the linker DBCO-PEG4-maleimide to form the intermediate product DBCO-PEG4-MD39 ("amph pre click", spectrum shown as solid black line), identified by the presence of a DBCO peak at 309 nm. DBCO-PEG4-MD39 was then reacted with DSPE-PEG2K-azide in a click chemistry reaction to form the final product amph-MD39 ("amph post click", spectrum shown as dotted line). The absence of a DBCO peak at 309 nm in the final product provided evidence that the reaction had proceeded to completion. MD39 protein was identified and quantified using the peak at 280 nm. The concentration of MD39 in the amph-MD39 product was quantified using the peak at 280 nm corrected for background lipid absorbance from 310 to 500 nm. [Figure 19A] Figure 1 shows that amph-MD39 trimer conjugates induced enhanced systemic and mucosal immune responses following intranasal immunization. BALB / c mice (n=5 animals per group) were immunized intranasally with 5 μg MD39 or amph-MD39 mixed with 5 μg saponin MPLA nanoparticle (SMNP) adjuvant and boosted with the same formulations at weeks 6 and 12. [Figure 19B] Figure 1 shows that amph-MD39 trimer conjugates induced enhanced systemic and mucosal immune responses after intranasal immunization. Antigen-specific serum IgG and vaginal mucosal IgA titers were measured by ELISA against MD39. Red arrows indicate vaccination. Statistical significance was determined by ordinary two-way ANOVA followed by Sidak's post-hoc test comparing MD39 with amph-MD39 at each time point. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. All data represent mean ± standard deviation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] overview Humoral immune (antibody) responses are desired both systemically and locally at mucosal surfaces to combat infectious pathogens that infect the host through mucosal transmission.

[0029] The present invention is based on the surprising discovery that vaccines containing large protein antigens conjugated to lipid tails (amphiphilic conjugates) can elicit humoral immune responses against antigens such as HIV and SARS-CoV-2 after mucosal (e.g., intranasal) administration significantly more effectively than free protein antigens. Amphiphilic conjugates containing protein antigens surprisingly showed enhanced persistence and uptake across mucosa compared to unmodified antigens, leading to a large increase in germinal center (GC) and follicular helper T cell (Tfh) responses in the nasal associated lymphoid tissue (NALT). Intranasal (in) immunization with amphiphilic conjugates also surprisingly resulted in high levels of IgG and IgA in serum, upper and lower respiratory tract mucosa, and distal urogenital mucosal sites, including the induction of substantial neutralizing antibody responses in mice. Furthermore, intranasal immunization with amphiphilic conjugates enhanced vaccine uptake in the nasal cavity and enhanced IgG and IgA responses compared to soluble protein immunization in non-human primates.

[0030] Thus, the present disclosure provides vaccines suitable for mucosal administration and methods of use thereof to induce an immune response or immunity (including, for example, a humoral antibody response) against an infectious pathogen.

[0031] definition Terms used in the claims and specification are defined as follows, unless otherwise specified.

[0032] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0033] As used herein, "about" will be understood by one of ordinary skill in the art and will vary to some extent depending on the context in which it is used. If there are uses of the term that are not clear to persons of ordinary skill in the art given the context in which it is used, "about" will mean up to plus or minus 10% of the particular value.

[0034] As used herein, the term "adjuvant" refers to a compound that enhances or otherwise alters or modifies the resulting immune response with a particular immunogen or antigen. Modification of the immune response includes enhancing or broadening the specificity of either or both the antibody immune response and the cellular immune response. Modification of the immune response may also mean decreasing or suppressing a particular antigen-specific immune response. In certain embodiments, the adjuvant is a cyclic dinucleotide. In some embodiments, the adjuvant is an immunostimulatory oligonucleotide as described herein. In some embodiments, the adjuvant is administered before, simultaneously with, or after administration of the amphiphilic conjugate or a composition comprising the conjugate. In some embodiments, the adjuvant is co-formulated in the same composition as the amphiphilic conjugate.

[0035] "Amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid (i.e., an α carbon attached to a hydrogen, a carboxyl group, an amino group, and an R group) (e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium). 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 refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function similarly to a naturally occurring amino acid.

[0036] Amino acids may 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, may be referred to by their commonly accepted single-letter codes.

[0037] An "amino acid substitution" refers to the replacement of at least one existing amino acid residue in a given amino acid sequence (the amino acid sequence of the 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 given amino acid sequence. An insertion typically consists of the insertion of one or two amino acid residues, although larger "peptide insertions" of the invention can involve the insertion of, for example, about 3 to about 5, or even up to about 10, 15, or 20 amino acid residues. The inserted residue(s) can 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 given amino acid sequence.

[0038] As used herein, "amphiphile" or "amphiphilic" refers to a conjugate that includes a hydrophilic head group and a hydrophobic tail, thereby forming an amphiphilic conjugate. In some embodiments, the amphiphilic conjugate includes an immunogen, such as, for example, a protein antigen, and one or more hydrophobic lipid tails. In some embodiments, the amphiphilic conjugate further includes a polymer (e.g., polyethylene glycol), which is attached to one or more lipids and / or the immunogen.

[0039] The term "ameliorate" refers to any therapeutically beneficial outcome in the treatment of a condition such as cancer, including prevention, reduction in severity or progression, remission, or cure.

[0040] As used herein, the term "antibody" refers to an immunoglobulin molecule that contains four polypeptide chains, two heavy chains (HC) and two light chains (LC), interconnected by disulfide bonds. Antibodies are composed of two structural regions: a variable fragment (Fab), which mediates antigen binding, and a constant fragment (Fc), which mediates downstream effector functions.

[0041] 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 have a heavy chain known as a gamma chain; IgM has a μ chain; IgA has an alpha chain; IgE has an epsilon chain; and IgD has a delta chain. Variations in the heavy chain polypeptides allow each immunoglobulin class to function in different types of immune responses and at different stages of the body's defense. The amino acid sequences that cause these functional differences are located primarily within the Fc domain. IgG (immunoglobulin IgG) is expressed on the surface of mature B cells and is also the most widespread Ig in serum and the extravascular space. There are four subtypes of IgG: IgG1, IgG2, IgG3, and IgG4. IgA (immunoglobulin A) plays a vital role in gastrointestinal, respiratory, and genitourinary mucosal homeostasis, and in this role functions as the primary antibody of immunity. There are two subtypes of IgA: IgA1 and IgA2.

[0042] Immunoglobulin class switching, also known as isotype switching, is a biological mechanism that alters the production of immunoglobulins by B cells from one type to another. Class switching occurs rapidly after activation of mature naive B cells, resulting in a switch from expression of IgM and IgD to expression of IgG, IgE, or IgA. This switch improves the ability of antibodies to induce a humoral immune response and eliminate pathogens.

[0043] As used herein, the term "antigen" or "immunogen" refers to a molecule that induces an immune response when administered to a vertebrate, particularly a mammal.

[0044] The terms "antigenic peptide" or "peptide antigen," as used interchangeably herein, refer to a peptide that induces an immune response, e.g., a cell-mediated immune response, when administered to a vertebrate, particularly a mammal.

[0045] As used herein, the term "antigenic protein" or "protein antigen" refers to a protein that induces an immune response, e.g., a humoral antibody-mediated immune response, when administered to a vertebrate, particularly a mammal.

[0046] The term "antigen-presenting cell" or "APC" refers to a cell that presents foreign antigens on its surface in complex with MHC. T cells recognize this complex using the T cell receptor (TCR). Examples of APCs include, but are not limited to, dendritic cells (DCs), peripheral blood mononuclear cells (PBMCs), monocytes (such as THP-1), B lymphoblastoid cells (such as C1R.A2, 1518B-LCL), and monocyte-derived dendritic cells (DCs). Some APCs internalize antigens by either phagocytosis or receptor-mediated endocytosis.

[0047] The term "B cell" refers to a type of lymphocyte responsible for mediating the production of antigen-specific immunoglobulins (Ig), commonly known as antibodies, against invading pathogens.

[0048] As used herein, "CG oligodeoxynucleotide (CG ODN)," also referred to as "CpG ODN," is a short, single-stranded synthetic DNA molecule that contains a cytosine nucleotide (C) followed by a guanine nucleotide (G). In certain embodiments, the immunostimulatory oligonucleotide is a CG ODN.

[0049] A polypeptide or amino acid sequence "derived from" a specified polypeptide or protein refers to the origin of the polypeptide. Preferably, a polypeptide or amino acid sequence derived from a particular sequence is that sequence or a portion thereof, the portion consisting of at least 10 to 20 amino acids, preferably at least 20 to 30 amino acids, more preferably at least 30 to 50 amino acids, having an amino acid sequence essentially identical to that portion, or an amino acid sequence recognizable by a person skilled in the art as having that origin within the sequence.

[0050] A polypeptide derived from another peptide may have one or more mutations relative to the starting polypeptide, for example, one or more amino acid residues replaced by another amino acid residue, or one or more amino acid residues inserted or deleted.

[0051] Polypeptides can include non-naturally occurring amino acid sequences. Such variants will necessarily have less than 100% sequence identity or similarity with the starting molecule. In preferred embodiments, the variants have about 75% to less than 100% amino acid sequence identity or similarity, more preferably about 80% to less than 100%, more preferably about 85% to less than 100%, more preferably about 90% to less than 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and most preferably about 95% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide, for example over the length of the variant molecule.

[0052] In one embodiment, there is one amino acid difference between the 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 with the starting amino acid residues (i.e., the same residues) after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.

[0053] As used herein, the term antigen "cross-presentation" refers to the presentation of exogenous protein antigens to T cells via MHC class I and class II molecules on APCs.

[0054] As used herein, the term "cytotoxic T lymphocyte (CTL) response" refers to an immune response induced by cytotoxic T cells. CTL responses are primarily mediated by CD8+ T cells.

[0055] As used herein, the term "effective amount" or "effective dose" is defined as an amount sufficient to achieve or at least partially achieve a desired effect, such as inducing or enhancing an immune response to an immunogen, or providing immunity. The term "therapeutically effective amount" or "therapeutically effective dose" is defined as an amount effective for alleviating symptoms of a disease. Prevention can be considered treatment, and thus a therapeutically effective amount can be a "prophylactically effective amount."

[0056] As used herein, the term "effector cell" or "effector immune cell" refers to a cell that is involved in an immune response, e.g., promoting an immune effector response. In some embodiments, an immune effector cell specifically recognizes an antigen. Examples of immune effector cells include, but are not limited to, natural killer (NK) cells, B cells, monocytes, macrophages, and T cells (e.g., cytotoxic T lymphocytes (CTLs)). In some embodiments, an effector cell is a T cell.

[0057] As used herein, the term "humoral immune response" refers to an immune response mediated by antibody molecules secreted by B cells. The presence of an antigen triggers the activation and differentiation of B cells into antibody-secreting plasma cells, usually involving helper T cells (CD4+ T cells).

[0058] As used herein, the term "immune effector function" or "immune effector response" refers to the function or response of immune effector cells that promote an immune response against a target.

[0059] As used herein, an "immune cell" is a cell of hematopoietic origin 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).

[0060] As used herein, an "immunostimulatory oligonucleotide" is an oligonucleotide that is capable of stimulating (eg, inducing or enhancing) an immune response.

[0061] 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., passive or adaptive) to a particular antigen. The term "inducing" as used in reference to the induction of CDC or ADCC refers to the stimulation of a specific direct cell killing mechanism.

[0062] As used herein, a subject "in need of prophylaxis," "in need of treatment," "in need of immunization," or "in need of" refers to a subject who, as determined by a competent medical practitioner (e.g., a physician, nurse or clinical nurse in the case of a human, or a veterinarian in the case of a non-human mammal), would reasonably benefit from a given treatment (such as treatment with a composition comprising an amphipathic conjugate for immunization against an immunogen).

[0063] As used herein, "intranasal administration" refers to a route of transmucosal drug administration in which a drug (e.g., a vaccine) is instilled through the nose and passes through or across the nasal epithelium to enter the underlying cells / tissues. In embodiments, intranasal administration provides for local, systemic, or both local and systemic delivery of a drug.

[0064] The term "in vivo" refers to a process that takes place in a living organism.

[0065] As used herein, the terms "linked," "operably linked," "fused," or "fusion" are used interchangeably. These terms refer to the joining together of three or more elements, components, or domains by suitable means, including chemical conjugation or recombinant DNA technology. Methods of chemical conjugation (e.g., the use of heterobifunctional crosslinkers or the use of "click" chemistry) are known in the art, as are methods of recombinant DNA technology.

[0066] The term "lipid" refers to a biomolecule that is soluble in a non-polar solvent and insoluble in water. Lipids are often described as hydrophobic or amphipathic molecules that can form structures such as vesicles or membranes in an aqueous environment. Lipids include fatty acids, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids (including cholesterol), prenol lipids, glycolipids, and polyketides. In some embodiments, lipids suitable for the amphipathic conjugates of the present disclosure bind to human serum albumin under physiological conditions. In some embodiments, lipids suitable for the amphipathic conjugates of the present disclosure insert into cell membranes under physiological conditions. In some embodiments, the lipid binds to albumin and inserts into cell membranes under physiological conditions. In some embodiments, the lipid is a diacyl lipid. In some embodiments, the diacyl lipid contains more than 12 carbons. In some embodiments, the diacyl lipid contains at least 13, at least 14, at least 15, at least 16, at least 17, or at least 18 carbons.

[0067] As used herein, "neutralizing antibody" refers to an antibody that not only binds to a pathogen (e.g., virus, bacteria), but also binds to prevent infection. For example, neutralizing antibodies can prevent a virus from entering a host cell by blocking the interaction of the viral capsid protein with a receptor on the host cell. Only a small subset of antibodies that bind to a pathogen can neutralize. After infection, it may take some time for a subject to produce highly effective neutralizing antibodies, but these can persist and protect against future encounters with the agent.

[0068] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids containing known analogs 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 not only the sequence explicitly indicated, but also conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences. 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). In the case of arginine and leucine, the second base modification can also be conservative. The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0069] In some embodiments, the peptides of the invention are encoded by nucleotide sequences that may be useful for many applications, including cloning, gene therapy, protein expression and purification, introduction of mutations, DNA vaccination of a host in need thereof, antibody generation, e.g., for passive immunization, PCR, primer and probe generation, etc.

[0070] 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, but are not limited to, intravenous, intranasal, intraocular, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid, and intrasternal injection and infusion.

[0071] As generally used herein, "pharmacologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues, organs, and / or body fluids of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0072] As used herein, the term "physiological conditions" refers to the in vivo conditions of a subject. In some embodiments, physiological conditions refer to a neutral pH (e.g., a pH between 6 and 8).

[0073] "Polypeptide," "peptide," and "protein" refer to a polymer of amino acid residues. These terms apply to naturally occurring amino acid polymers and to non-naturally occurring amino acid polymers, including those in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid.

[0074] As used herein, "protein" refers to a molecule that contains or consists of more than 50 amino acids. As used herein, "peptide" refers to a molecule that consists of 2 to 50 amino acids. "Oligopeptide" refers to a molecule that consists of 2 to about 20 amino acids.

[0075] As used herein, a "small molecule" is a molecule having a molecular weight of less than about 500 Daltons.

[0076] 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, ovines, fowl, amphibians, or reptiles.

[0077] The term "sufficient amount" or "an amount sufficient for" means an amount sufficient to produce a desired effect, eg, an amount sufficient to immunize a subject against an immunogen.

[0078] 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 T helper cells (also known as TH cells or CD4+ T cells) and subtypes (including TH1, TH2, TH3, TH17, TH9 and THC 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 (central memory T cells (TCM cells), effector memory T cells (TEM and TEMRA cells), and resident memory T cells). T cells include, but are not limited to, T cells that are specific for the immune system, including T cells, regulatory T cells (also known as Treg cells or suppressor T cells) and subtypes (including CD4+FOXP3+Treg cells, CD4+FOXP3-Treg cells, Tr1 cells, Th3 cells, and Treg17 cells), natural killer T cells (also known as NKT cells), mucosal-associated invariant T cells (MAIT), and gamma delta T cells (γδ T cells), including Vγ9 / Vδ2 T cells. Any one or more of the above or unmentioned T cells may be the target cell type for the methods of use of the present invention.

[0079] As used herein, "T cell activation" or "activation of T cells" refers to the cellular process in which mature T cells expressing antigen-specific T cell receptors on their surface recognize their cognate antigen and respond by entering the cell cycle, secreting cytokines or lytic enzymes, and gaining the ability to initiate or execute cell-based effector functions. T cell activation requires at least two signals for full activation. The first occurs after engagement of the T cell antigen-specific receptor (TCR) by the antigen major histocompatibility complex (MHC), and the second occurs by subsequent engagement of costimulatory molecules (e.g., CD28). These signals are transmitted to the nucleus and cause T cell clonal expansion, upregulation of cell surface activation markers, differentiation into effector cells, induction of cytotoxicity or cytokine secretion, induction of apoptosis, or a combination thereof.

[0080] 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.

[0081] The term "T cell cytotoxicity" includes any immune response mediated by activation of CD8+ T cells. Exemplary immune responses include cytokine production, proliferation of CD8+ T cells, granzyme or perforin production, and clearance of infectious agents.

[0082] As used herein, "mucosal administration" refers to a route of drug administration in which a drug (e.g., a vaccine) passes through or across the mucosal epithelium to enter the underlying tissue. In some embodiments, a drug administered transmucosally enters the systemic circulation. In some embodiments, transmucosal administration provides for local delivery of a drug. In some embodiments, transmucosal administration provides for both local and systemic delivery of a drug.

[0083] As used herein, the terms "treat," "treating," and "treatment" refer to therapeutic or prophylactic measures as described herein. The method of "treatment" employs administration of a vaccine or amphipathic conjugate of the present disclosure to a subject in need of such treatment, e.g., a subject at risk of infection with an immunogen. In some embodiments, the amphipathic conjugate is administered to a subject in need of an enhanced immune response against a particular antigen to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of a disorder or a recurrent disorder, or a subject who may eventually develop such a disorder.

[0084] As used herein, "vaccine" refers to a composition comprising an amphiphilic conjugate as described herein, in a form that can be administered (e.g., mucosally or intranasally) to a subject, and capable of inducing a protective immune response. In embodiments, the protective immune response is sufficient to induce immunity and / or prevent and / or ameliorate an infection or disease, and / or reduce at least one symptom of an infection or disease, and / or enhance the effectiveness of another dose of the amphiphilic conjugate. When introduced into a host, the vaccine elicits an immune response, including, but not limited to, the production of 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.

[0085] Amphiphilic conjugates In some aspects, the present disclosure provides a vaccine comprising an immunogen (e.g., a peptide or protein antigen) operably linked to an albumin-binding lipid, the vaccine being suitable for mucosal administration (e.g., intranasal administration) to induce an immune response (e.g., a cell-mediated immune response or a humoral antibody-mediated immune response).

[0086] Amphiphile vaccine technology has been previously developed in which adjuvants or antigenic peptides are attached to a lipophilic polymer tail to promote localization of the vaccine to lymph nodes (Liu et al. (2014) Nature 507:519-522). Such amph peptides can also insert into cell membranes (see, e.g., Liu et al. (2011) Angewandte Chemie-Intl. Ed. 50:7052-7055). However, previous work on amphiphile vaccines has focused on amphiphile conjugates that contain relatively low molecular weight peptide antigens that target T cell immunity and are introduced systemically into a subject by intravenous or subcutaneous injection. The present disclosure provides amphiphile conjugates that are suitable for mucosal delivery (e.g., intranasal administration) and contain immunogens such as peptide or protein antigens that stimulate protective immune responses locally and / or systemically.

[0087] A variety of amphiphilic conjugate structures are provided in which a lipophilic albumin binding moiety, or "lipid tail" (e.g., DSPE), is linked (e.g., covalently attached) to an immunogen, such as a peptide or protein antigen, via a linker (e.g., a PEG linker). Without being bound by theory, it is believed that the amphiphilic conjugates included in the vaccines of the present disclosure use albumin as a non-covalent chaperone to cross mucosal surfaces through the interaction of albumin with the neonatal Fc receptor (FcRn) expressed by mucosal epithelial cells. Increased uptake of the amphiphilic conjugates through mucus and epithelial linings enhances the immune response in local tissues, such as lymphoid tissues.

[0088] In some embodiments, the amphipathic conjugate included in the vaccine of the present disclosure is a lipid conjugate described in US 2013 / 0295129, the entire contents of which are incorporated herein by reference. In some embodiments, the amphipathic conjugate comprises a hydrophobic tail that inserts into a cell membrane.

[0089] In some embodiments, the hydrophobic tail enhances the association of the conjugate to a cell surface (e.g., an epithelial cell surface). In some embodiments, the hydrophobic tail allows the amphiphilic conjugate to anchor to a cell membrane and retain the amphiphilic conjugate in local tissue (e.g., a mucosal epithelium). In some embodiments, the hydrophobic tail allows the amphiphilic conjugate to anchor to a cell membrane and retain the amphiphilic conjugate in tissue near the site of administration. In some embodiments, the hydrophobic tail allows the amphiphilic conjugate to anchor to a cell membrane and reduce systemic circulation of the conjugate. In some embodiments, the hydrophobic tail allows the amphiphilic conjugate to anchor to a cell membrane and reduce distribution of the conjugate to distant tissues.

[0090] In some embodiments, the amphiphilic conjugates of the present disclosure include albumin-binding lipids that allow the conjugates to efficiently cross mucosal epithelia with albumin in vivo. In some embodiments, the amphiphilic conjugates include albumin-binding lipids that include a hydrophobic tail that inserts into cell membranes and allows the conjugates to efficiently cross mucosal epithelia with albumin in vivo. In some embodiments, the amphiphilic conjugates bind to endogenous albumin, thereby allowing the uptake of the conjugate with albumin by neonatal Fc receptors (FcRn), targeting the conjugate to local lymphoid tissues where it accumulates. In some embodiments, the amphiphilic conjugates include immunogens, such as antigenic peptides or protein antigens, to induce or enhance a protective immune response.

[0091] In some embodiments, the amphiphilic conjugate is efficiently targeted to lymph nodes or local lymphatic tissues. In some embodiments, the lymph node targeting conjugate comprises a highly lipophilic albumin binding domain (e.g., albumin binding lipid), and a cargo such as an immunogen (e.g., an antigenic peptide or protein antigen). In some embodiments, the lymph node targeting conjugate comprises three domains: a highly lipophilic albumin binding domain (e.g., albumin binding lipid), a cargo such as an immunogen (e.g., an antigenic peptide or protein antigen), and a linker (e.g., a polar block linker) that promotes the solubility of the conjugate. Thus, in certain embodiments, the general structure of the amphiphilic conjugate is LPC, where "L" is an albumin binding lipid, "P" is a polar block linker, and "C" is a cargo such as an immunogen (e.g., an antigenic peptide or protein antigen). In some embodiments, the cargo itself can also function as a polar block domain, and a separate polar block domain is not required. Thus, in certain embodiments, the conjugate has only two domains: the albumin-binding lipid and a cargo, such as an immunogen (eg, an antigenic peptide or protein antigen).

[0092] In some embodiments, the amphiphilic conjugate is administered or formulated with an adjuvant.

[0093] (i) Lipids In some embodiments, the lipid component of the amphipathic conjugates of the present disclosure comprises a hydrophobic tail. In some embodiments, the hydrophobic tail is inserted into or capable of inserting into a cell membrane. In some embodiments, the lipid is linear, branched, or cyclic. In some embodiments, the lipid is greater than 12 carbons in length. In some embodiments, the lipid is 13 carbons in length. In some embodiments, the lipid is 14 carbons in length. In some embodiments, the lipid is 15 carbons in length. In some embodiments, the lipid is 16 carbons in length. In some embodiments, the lipid is 17 carbons in length. In some embodiments, the lipid is 18 carbons in length. In some embodiments, the lipid is 19 carbons in length. In some embodiments, the lipid is 20 carbons in length. In some embodiments, the lipid is 21 carbons in length. In some embodiments, the lipid is 22 carbons in length. In some embodiments, the lipid is 23 carbons in length. In some embodiments, the lipid is 24 carbons in length. In some embodiments, the lipid is 25 carbons in length. In some embodiments, the lipid is 26 carbons in length. In some embodiments, the lipid is 27 carbons in length. In some embodiments, the lipid is 28 carbons in length. In some embodiments, the lipid is 29 carbons in length. In some embodiments, the lipid is 30 carbons in length. In some embodiments, the lipid is at least 17-18 carbons in length, but may be shorter if it exhibits good albumin binding and adequate targeting to lymph nodes.

[0094] In certain embodiments, the activity of the amphiphilic conjugate depends in part on the ability of the conjugate to target lymph nodes. In certain embodiments, the activity of the amphiphilic conjugate depends in part on the ability of the conjugate to bind to albumin, for example, in the blood, tissue, lymph, or mucosal epithelium of a subject. In some embodiments, the activity of the amphiphilic conjugate depends in part on the ability of the conjugate to bind to albumin and be transported across a mucosal barrier via the interaction of albumin with FcRn expressed by mucosal epithelial cells. Thus, the amphiphilic conjugate of the present disclosure typically comprises a lipid capable of binding to albumin. In a preferred embodiment, the amphiphilic conjugate comprises a lipid capable of binding to albumin under physiological conditions.

[0095] A lipid suitable for targeting lymph nodes and / or transporting a conjugate across mucosal epithelium can be selected based on the ability of the lipid or lipid conjugate comprising the lipid to bind to albumin. Suitable methods for testing the ability of a lipid or lipid conjugate to bind to albumin are known in the art. For example, in certain embodiments, a plurality of lipid conjugates are allowed to spontaneously form micelles in aqueous solution. The micelles are incubated with albumin or a solution comprising albumin, such as fetal bovine serum (FBS). The sample can be analyzed, for example, by ELISA, size exclusion chromatography, or other methods, to determine whether binding has occurred. A lipid conjugate can be selected as a lymph node targeting conjugate if, in the presence of albumin or a solution comprising albumin, such as fetal bovine serum (FBS), the micelles dissociate and the lipid conjugate binds to albumin as described above.

[0096] Examples of preferred lipids for use in lymph node-targeting lipid conjugates include, but are not limited to, fatty acids having an aliphatic tail of 8-30 carbons, including, but not limited to, linear unsaturated and saturated fatty acids, branched saturated and unsaturated fatty acids, and fatty acid 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 comprises at least one lipid tail containing 8-30, 12-30, 15-25, or 16-20 carbons.

[0097] In certain embodiments, the lipid is a diacyl lipid or a bilateral lipid. In some embodiments, the tail of the diacyl lipid comprises about 8 to about 30 carbons and may be saturated, unsaturated, or a combination thereof. In some embodiments, the diacyl lipid is saturated. In some embodiments, the diacyl lipid is saturated and each tail comprises about 8 to about 30 carbons. In some embodiments, the diacyl lipid is saturated and each tail comprises 12 carbons. In some embodiments, the diacyl lipid is saturated and each tail comprises 13 carbons. In some embodiments, the diacyl lipid is saturated and each tail comprises 14 carbons. In some embodiments, the diacyl lipid is saturated and each tail comprises 15 carbons. In some embodiments, the diacyl lipid is saturated and each tail comprises 16 carbons. In some embodiments, the diacyl lipid is saturated and each tail comprises 17 carbons. In some embodiments, the diacyl lipid is saturated and each tail comprises 18 carbons. In some embodiments, the diacyl lipids are saturated and each tail comprises 19 carbons. In some embodiments, the diacyl lipids are saturated and each tail comprises 20 carbons. In some embodiments, the diacyl lipids are saturated and each tail comprises 21 carbons. In some embodiments, the diacyl lipids are saturated and each tail comprises 22 carbons. In some embodiments, the diacyl lipids are saturated and each tail comprises 23 carbons. In some embodiments, the diacyl lipids are saturated and each tail comprises 24 carbons. In some embodiments, the diacyl lipids are saturated and each tail comprises 25 carbons. In some embodiments, the diacyl lipids are saturated and each tail comprises 26 carbons. In some embodiments, the diacyl lipids are saturated and each tail comprises 27 carbons. In some embodiments, the diacyl lipids are saturated and each tail comprises 28 carbons. In some embodiments, the diacyl lipids are saturated and each tail comprises 29 carbons. In some embodiments, the diacyl lipids are saturated and each tail contains 30 carbons. The tails can be attached to the head group via an ester bond, an amide bond, a thioester bond, or a combination thereof.In some embodiments, the diacyl lipid is a phospholipid, a glycolipid, a sphingolipid, or a combination thereof.

[0098] In some embodiments, the lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). In some embodiments, the diacyl lipid is synthesized as described in U.S. Patent No. 9,107,904, the entire contents of which are incorporated herein by reference. In some embodiments, the diacyl lipid is synthesized as follows: [ka]

[0099] Preferably, the amphiphilic conjugate comprises a lipid that is 8 or more carbon units in length. It is believed that increasing the number of lipid units reduces the lipid's insertion into the plasma membrane of cells, while still allowing the lipid conjugate to freely bind albumin and migrate through mucosal epithelia and / or lymph nodes. For example, in some embodiments, the lipid may be a diacyl lipid composed of two C18 hydrocarbon tails. In some embodiments, the lipid used in the preparation of the amphiphilic conjugate is not a single chain hydrocarbon (e.g., C18).

[0100] (ii) Cargo In some aspects, the cargo of the amphipathic conjugates provided herein is an immunogen. In some embodiments, the immunogen is a peptide antigen (also referred to herein as an antigenic peptide), a protein antigen, 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 a peptide antigen or a protein antigen. In some embodiments, the immunogen comprises or consists of a polysaccharide antigen.

[0101] In some embodiments, the immunogen is an antigenic peptide. As used herein, an "antigenic peptide" has fewer than 50 amino acids and contains at least one amino acid sequence sufficient to elicit an immune response, such as a cell-mediated immune response.

[0102] In some embodiments, the immunogen is not an antigenic peptide. In some embodiments, the immunogen does not elicit a cell-mediated immune response.

[0103] For many types of infectious diseases transmitted via mucosal routes, such as HIV, SARS-CoV-2, and influenza, larger protein antigens that more closely resemble the native proteins of the infectious agent are believed to be significantly more effective at inducing an immune response by vaccination than small peptides. Thus, in some embodiments, the immunogen is a protein antigen. As used herein, a "protein antigen" includes at least 50 or more amino acids and includes at least one amino acid sequence sufficient to elicit an immune response, e.g., a humoral antibody-mediated immune response.

[0104] 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 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.

[0105] In some embodiments, the protein antigen is more than 50 amino acids, more than 51 amino acids, more than 52 amino acids, more than 53 amino acids, more than 54 amino acids, more than 55 amino acids, more than 56 amino acids, more than 57 amino acids, more than 58 amino acids, more than 59 amino acids, more than 60 amino acids, more than 75 amino acids, more than 100 amino acids, more than 125 amino acids, more than 150 amino acids, more than 175 amino acids, more than 200 amino acids, more than 250 amino acids, more than 300 amino acids, more than 350 amino acids, more than 400 amino acids, more than 5 ... amino acids, more than 450 amino acids, more than 500 amino acids, more than 550 amino acids, more than 600 amino acids, more than 650 amino acids, more than 700 amino acids, more than 750 amino acids, more than 800 amino acids, more than 850 amino acids, more than 900 amino acids, more than 950 amino acids, more than 1000 amino acids, more than 1050 amino acids, more than 1100 amino acids, more than 1150 amino acids, more than 1200 amino acids, more than 1250 amino acids, more than 1300 amino acids, more than 1350 amino acids, more than 1400 amino acids, 14 More than 50 amino acids, more than 1500 amino acids, more than 1550 amino acids, more than 1600 amino acids, more than 1650 amino acids, more than 1700 amino acids, more than 1750 amino acids, more than 1800 amino acids, more than 1850 amino acids, more than 1900 amino acids, more than 1950 amino acids, more than 2000 amino acids, more than 2050 amino acids, more than 2100 amino acids, more than 2150 amino acids, more than 2000 amino acids, more than 2300 amino acids, more than 2400 amino acids, more than 2500 amino acids, more than 2600 amino acids The amino acid sequence may comprise more than 2700 amino acids, more than 2800 amino acids, more than 2900 amino acids, more than 3000 amino acids, more than 3100 amino acids, more than 3200 amino acids, more than 3300 amino acids, more than 3400 amino acids, more than 3500 amino acids, more than 3600 amino acids, more than 3700 amino acids, more than 3800 amino acids, more than 3900 amino acids, more than 4000 amino acids, more than 4100 amino acids, more than 4200 amino acids, more than 4300 amino acids, more than 4400 amino acids, or more than 4500 amino acids.

[0106] 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 contains 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 about 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, 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.

[0107] In some embodiments, the protein antigen has a molecular weight (MW) of about 10 kDa to about 500 kDa. In some embodiments, the protein antigen has a molecular 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, or about 10 kDa to about 50 kDa. In some embodiments, the protein antigen has a MW of 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 50 kDa to about 500 kDa, about 50 kDa to about 450 kDa, about 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 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 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 150 kDa to about 500 kDa, about 150 kDa to about 450 kDa, about 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, or 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.

[0108] In some embodiments, the protein antigen is a monomeric antigen (ie, a single antigenic polypeptide chain).

[0109] In some embodiments, the protein antigen is a multimeric antigen, e.g., a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, or decamer. In some embodiments, the protein antigen is a dimeric antigen. In some embodiments, the protein antigen is a trimeric antigen. In some embodiments, the multimeric antigen comprises identical monomeric subunits, i.e., a repeat sequence of the same antigen, such as two repeat sequences of the same antigen (i.e., a homodimeric antigen) or three repeat sequences of the same antigen (i.e., a homotrimer). In some embodiments, the multimeric antigen comprises different monomeric subunits, i.e., different protein antigen sequences from the same pathogen, such as two different sequences from the same pathogen (i.e., a heterodimeric antigen) or three different sequences from the same pathogen (i.e., a heterotrimeric antigen). In some embodiments, two or more of the protein antigen sequences of the monomeric subunits of the multimeric antigen are each derived from a different pathogen.

[0110] In some embodiments, the protein antigen can be derived from a virus, a bacterium, a parasite, a plant, a protozoan, or a fungus.

[0111] Suitable antigenic peptide or protein antigens are widely known in the art and are available from commercial, governmental, and scientific sources. The antigen may be a purified or partially purified polypeptide derived from a viral or bacterial source. The antigen may be a recombinant polypeptide produced by expressing DNA encoding the polypeptide antigen in a heterologous expression system.

[0112] In some embodiments, the antigenic peptide or protein antigen is selected from the group consisting of Arenaviridae, Arteriviridae, Astroviridae, Baculoviridae, Badnavirus, Barnaviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Capillovirus, Carlavirus, Calimovirus, Circoviridae, Closterovirus, Comoviridae, Coronaviridae (e.g., coronaviruses such as Severe Acute Respiratory Syndrome (SARS) virus), Corticoviridae, Cystoviridae, Deltavirus, Dianthovirus, Enamovirus, Filoviridae (e.g., Marburg virus, Ebola virus (e.g., Zaire, Reston, Cote d'Ivoire, or Sudan strains)), Flaviviridae (e.g., Hepatitis C virus, Dengue virus type 1, Dengue virus type 2, Dengue virus type 3, and Dengue virus type 4), Hepadnaviridae, Herpesviridae (e.g., human herpesvirus types 1, 3, 4, and 5), and / or other viruses. and 6, and Cytomegalovirus), Hypoviridae, Iridoviridae, Leviviridae, Lipotryxviridae, Microviridae, Orthomyxoviridae (e.g., influenza viruses types A, B, and C), Papovaviridae, Paramyxoviridae (e.g., measles, mumps, and human respiratory syncytial virus), Parvoviridae, Picornaviridae (e.g., poliovirus, rhinovirus, hepatovirus, and aphthovirus), Poxviridae (e.g., vaccinia virus and smallpox virus), Reoviridae (e.g., rotavirus), Retroviridae (e.g., lentiviruses such as human immunodeficiency virus (HIV) 1 and HIV 2), Rhabdoviridae (e.g., rabies virus, measles virus, respiratory syncytial virus, etc.), Togaviridae (e.g., 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.

[0113] Viral antigens may be derived from specific strains of papillomavirus, herpesviruses, such as herpes simplex types 1 and 2, hepatitis virus, e.g., hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis delta D virus (HDV), hepatitis E virus (HEV), and hepatitis G virus (HGV), hepatitis viruses, such as tick-borne encephalitis virus, parainfluenza, varicella zoster, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, coxsackievirus, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, and lymphocytic choriomeningitis.

[0114] In some embodiments, the antigenic peptide or protein antigen is selected from the group consisting of Actinomycetes, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Helicobacter, Haemophilus, Haemophilus influenzae type B (HIB), Hyphomicrobium, Legionella, Leptospiraceae, Listeria monocytogenes, and the like. The bacterial strain may be derived from bacteria including, but not limited to, bacteria from any of the following families: Neisseria meningitidis A, B and C, Methanobacterium, Micrococcus, Mycobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Procloron, Proteus, Pseudomonas, Hodospirillum, Rickettsia, Salmonella, Shigella, Spirillum, Spirochete, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus, Treponema, Vibrio, and Yersinia.

[0115] In some embodiments, antigenic peptide or protein antigens may be derived from parasites including, but not limited to, parasites from any of the following families: Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroidea, Rickettsia rocky mountain spotted fever, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydia psittacosis, Chlamydia trachomatis, Plasmodium falciparum, Trypanosoma brucei, Amoebiasis histolytica, Toxoplasmosis, Trichomonas vaginalis, and Schistosoma mansoni. These include sporozoite antigens, Plasmodium antigens, such as all or a portion of circumsporozoite proteins, sporozoite surface proteins, liver stage antigens, apical membrane associated proteins, or merozoite surface proteins.

[0116] 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.

[0117] 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.

[0118] In some embodiments, the amino acid sequence of the antigenic peptide or protein antigen can be the amino acid sequence of a naturally occurring antigen. In some embodiments, the antigenic peptide or protein antigen can be a sequence that has been modified from the amino acid sequence of the naturally occurring antigen. These modifications can serve to enhance antigenicity or improve the production of amphipathic conjugates.

[0119] Human immunodeficiency virus (HIV) antigens are widely known in the art. Non-limiting examples of HIV antigens can 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.

[0120] 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 gp120 antigen or a gp140 antigen. In some embodiments, the HIV Env antigen is a gp120 antigen. In some embodiments, the HIV Env antigen is a gp120 modified ectodomain germline targeted immunogen 8 (eOD-GT8). In some embodiments, the eOD-GT8 gp120 antigen comprises or consists of the amino acid sequence of SEQ ID NO:1.

[0121] In some embodiments, the HIV envelope protein antigen is a native-like trimeric antigen (of repeating monomers) that mimics the structure of the virion-associated spike (e.g., HIV MD39 SOSIP). In some embodiments, the monomers that make up the HIV envelope protein antigen trimeric MD39 SOSIP comprise or consist of the amino acid sequence of SEQ ID NO:3.

[0122] SARS-CoV-2 antigens are widely known in the art. Examples of SARS-CoV-2 antigens used in existing vaccine technologies, as well as antigens under test and experimentation, can 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. USA118, 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.

[0123] In some embodiments, the SARS-CoV-2 antigen comprises a SARS-CoV-2 spike protein (also known as an "S protein"), or an antigenic fragment of the spike protein. In some embodiments, the SARS-CoV-2 antigen comprises an antigen from the S1 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 from the S2 subunit of the spike protein.

[0124] 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.

[0125] Influenza antigens are widely known in the art and can 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 gene-based 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.

[0126] Rotavirus antigens are generally known in the art. Teachings regarding antigens known to induce the development of antibodies against rotavirus, particularly neutralizing antibodies, can be found, for example, in US7311918B2, US6,16431, and Dennehy (2008) (Rotavirus vaccines: an overview. Clin Microbiol Rev. 21(1):198-208), which are incorporated herein by reference. In some embodiments, the rotavirus antigen comprises a VP4 antigen, a VP6 antigen, or a VP7 antigen.

[0127] Cytomegalovirus (CMV) antigens are generally known in the art. Teachings regarding CMV antigens can be found, for example, in Nelson et al (2018) (A new era in cytomegalovirus vaccinology: Considerations for reasonable design of next-generation vaccines to prevent congenital cytomegalovirus infection. npj Vaccines 3, 38), which is incorporated herein by reference. Neutralizing antibodies targeting CMV proteins gB, gH, and UL128-131A have been found following natural infection. In some embodiments, the CMV antigen comprises a gB antigen, a gH antigen, or a UL128-131A antigen.

[0128] Currently, no vaccine against Epstein-Barr Virus (EBV) has been successfully developed, but EBV antigens that induce antibody production, particularly neutralizing antibody production, are widely known in the art. Teachings regarding known EBV antigens can be found in Cui et al (2021) (Epstein Barr Virus: Development of Vaccines and Immune Cell Therapy for EBV-Associated Diseases. Front. Immunol., Vol 12), which is incorporated herein by reference. In some embodiments, the EBV antigen comprises a gp350 antigen, a gH antigen, a gL antigen, or a gB antigen.

[0129] Most recent attempts to generate a respiratory syncytial virus (RSV) vaccine have been based on the F protein of RSV because it mediates viral entry into host cells and anti-F antibodies have been shown to reduce severe RSV disease in high-risk infants. Other proteins that have been shown to be capable of eliciting neutralizing antibodies include the N protein and the M2-1 protein. Teachings regarding known RSV antigens can be found, for example, in 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 Immunol. 35:30-8), which are incorporated herein by reference. In some embodiments, the RSV antigen comprises an F protein antigen, an N protein antigen, or an M2-1 protein antigen.

[0130] Research on the immune response to cholera (e.g., Vibrio cholerae) infection has focused primarily on antibodies. Antibody responses have been found against the O-specific polysaccharide of Vibrio cholerae and 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 Vibrio cholerae, the CtxA antigen, or the CtxB antigen.

[0131] In some embodiments, the immunogen is a polysaccharide antigen. Polysaccharides are the major components of the bacterial surface. Polysaccharide-encapsulated bacteria are the main cause of several serious bacterial infections in children, such as bacterial meningitis and pneumonia. Because the polysaccharide capsule of bacteria determines its virulence, targeting capsid polysaccharides can provide significant protection against bacterial infections.

[0132] Bacterial polysaccharides are highly heterogeneous within and between species and are also T-lymphocyte-independent antigens. With some exceptions, immunization with free polysaccharides generally stimulates short-lived B-cell responses and may even cause hyporesponsiveness to future vaccine challenges. Recent studies suggest that polysaccharide conjugates may induce T-cell-dependent responses and stronger B-cell responses, resulting in long-term immunity (see, for example, Pollard et al (2009) Maintaining protection against invasive bacteria with protein-polyhydrate conjugate vaccines. Nat Rev Immunol 9, 213-220).

[0133] Polysaccharide antigens are generally known in the art. Teachings of known polysaccharide antigens, particularly those being developed as polysaccharide vaccines, can be found, for example, in Perera et al. (2021) (Polyhydrate Vaccines: A Perspective on Non-Typhoidal Salmonella Polysaccharides 2, no. 3: 691-714); and Aithal et al. (2012) (PolysacDB: A Database of Microbial Polysaccharides Antigens and Their Antibodies. PLoS ONE 7(4): e34613), the entire contents of each of which are incorporated herein by reference.

[0134] In some embodiments, the polysaccharide antigen is a cholera (e.g., Vibrio cholerae) antigen. In some embodiments, the polysaccharide antigen is the O-specific polysaccharide of Vibrio cholerae.

[0135] (iii) Linker In various aspects of the present disclosure, the lipid, e.g., the albumin-binding lipid, and the cargo, e.g., the immunogen, are connected by a linker molecule. In some embodiments, the linker is covalently attached to the lipid, the cargo, or both the lipid and the cargo. In embodiments, the linker is disposed between each of the lipid and the cargo, and is covalently attached to each of the lipid and the cargo.

[0136] Depending on the amino acid sequence, some amino acid-based immunogens can be essentially insoluble, therefore, in certain embodiments, a polar blocking linker is included as a linker between the cargo and the lipid to increase the solubility of the amphiphilic conjugate.

[0137] In some embodiments, the polar block linker enables the amphipathic conjugate to bind to albumin. In some embodiments, the polar block linker enhances the ability of the amphipathic conjugate to bind to albumin. In some embodiments, the polar block linker enhances the solubility of the conjugate without interfering with its ability to bind to albumin.

[0138] In some embodiments, the polar blocking linker modulates (eg, decreases or enhances) the ability of the lipid to insert into the plasma membrane of cells, such as cells adjacent to the mucosa to which it is administered.

[0139] One of skill in the art will recognize that the length and composition of the linker can be adjusted based on the lipid and cargo selected. Additional non-limiting examples of linkers applicable to the amphiphilic conjugates of the present disclosure can be found in WO 2019 / 060425, the entire contents of which are incorporated herein by reference.

[0140] In some embodiments, suitable polar blocks include, but are not limited to, oligonucleotides as described below, hydrophilic polymers including, but not limited to, poly(ethylene glycol) (molecular weight: 500 Da to 20,000 Da), polyacrylamide (molecular weight: 500 Da to 20,000 Da), polyacrylic acid, a series 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.

[0141] In some embodiments, the polar block provides solubility to the entire lipid conjugate based on the molecular weight of the polar block, whether it is an individual component or the cargo itself. For example, in some embodiments, a polar block having a molecular weight of 2,000 Da is sufficient to make the lipid conjugate soluble for albumin binding. In some embodiments, the polar block has a molecular weight of about 300 to about 20,000 Da. In some embodiments, the polar block has a molecular weight of about 1,000 to about 15,000 Da. In some embodiments, the polar block has a molecular weight of about 1,500 to about 10,000 Da. In some embodiments, the polar block has a molecular weight of about 2,000 to about 5,000 Da. In some embodiments, the polar block has a molecular weight of about 1,000 to about 2,500 Da. In some embodiments, the polar block has a molecular weight of about 1,000 to about 3,000 Da. In some embodiments, the polar block has a molecular weight of about 1,000 to about 3,500 Da. In some embodiments, the polar block has a molecular weight of about 1,000 to about 4,000 Da. In some embodiments, the polar block has a molecular weight of about 1,000 to about 5,000 Da. In some embodiments, the polar block has a molecular weight of about 5,000 to about 10,000 Da. In some embodiments, the polar block has a molecular weight of about 15,000 to about 20,000 Da.

[0142] In some embodiments, the hydrophobic lipid and the linker / cargo are covalently bonded. In some embodiments, the covalent bond is a non-cleavable bond or a cleavable bond. In some embodiments, the non-cleavable bond comprises an amide bond or a phosphate bond, and the cleavable bond comprises a disulfide bond, an acid cleavable bond, an ester bond, an anhydride bond, a biodegradable bond, or an enzyme cleavable bond.

[0143] Ethylene glycol (EG): In certain embodiments, the linker (first and / or second linker) comprises one or more ethylene glycol (EG) units, more preferably two or more EG units (i.e., polyethylene glycol (PEG)). For example, in certain embodiments, the amphiphilic conjugate comprises a cargo (i.e., a peptide or protein antigen) and a hydrophobic lipid (e.g., an albumin-binding lipid) linked by a polyethylene glycol (PEG) molecule or a derivative or analog thereof.

[0144] In some embodiments, amphiphilic conjugates suitable for use in the methods disclosed herein comprise an immunogen, such as an antigenic peptide or protein antigen, covalently attached to PEG, which is in turn covalently attached to a hydrophobic lipid, such as an albumin-binding lipid. The exact number of EG units depends on the lipid and cargo.

[0145] In some embodiments, the linker (e.g., the first linker) comprises a PEG molecule (e.g., the first PEG molecule) or other similarly soluble polymer. The PEG molecule (e.g., the first PEG molecule) can be (PEG) n where n represents the number of repeating PEG monomers (i.e., EG units). In some embodiments, the number of repeating PEG monomers (n) in the PEG molecule may be about 1 to about 150, about 1 to about 125, about 1 to about 100, about 1 to about 50, about 50 to about 100, or about 100 to about 150. In some embodiments, the number of repeating PEG monomers (n) in the PEG molecule may be about 10 to about 90, about 20 to about 80, about 30 to about 70, or about 40 to about 60 monomers. In certain embodiments, the number of repeating PEG monomers (n) in the PEG molecule may be about 45 to about 150. In certain embodiments, the number of repeating PEG monomers in the PEG monomer (e.g., the first linker) is about 45 to 55 monomers. For example, in certain embodiments, the number of repeating PEG monomers in the PEG linker (e.g., the first linker) is about 48 monomers.

[0146] In some embodiments, the PEG linker (e.g., the first linker) or PEG molecule has a molecular weight of about 300 to 20,000 daltons. In some embodiments, the PEG linker or PEG molecule has a molecular weight of about 1,000 daltons. In some embodiments, the PEG linker or PEG molecule has a molecular weight of about 1,500 daltons. In some embodiments, the PEG linker or PEG molecule has a molecular weight of about 2,000 daltons. In some embodiments, the PEG linker or PEG molecule has a molecular weight of about 2,500 daltons. In some embodiments, the PEG linker or PEG molecule has a molecular weight of about 3,500 daltons. In some embodiments, the PEG linker or PEG molecule has a molecular weight of about 4,000 daltons. In some embodiments, the PEG linker or PEG molecule has a molecular weight of about 5,000 daltons. In some embodiments, the PEG linker or PEG molecule has a molecular weight of about 6,000 daltons. In some embodiments, the PEG linker or PEG molecule has a molecular weight of about 7,000 daltons. In some embodiments, the PEG linker or PEG molecule has a molecular weight of about 8,000 daltons. In some embodiments, the PEG linker or PEG molecule has a molecular weight of about 9,000 daltons. In some embodiments, the PEG linker or PEG molecule has a molecular weight of about 10,000 daltons. In some embodiments, the PEG linker or PEG molecule has a molecular weight of about 11,000 daltons. In some embodiments, the PEG linker or PEG molecule has a molecular weight of about 12,000 daltons. In some embodiments, the PEG linker or PEG molecule has a molecular weight of about 13,000 daltons. In some embodiments, the PEG linker or PEG molecule has a molecular weight of about 14,000 daltons. In some embodiments, the PEG linker or PEG molecule has a molecular weight of about 15,000 daltons. In some embodiments, the PEG linker or PEG molecule has a molecular weight of about 16,000 Daltons, hi some embodiments, the PEG linker or PEG molecule has a molecular weight of about 17,000 Daltons.In some embodiments, the PEG linker or PEG molecule has a molecular weight of about 18,000 daltons. In some embodiments, the PEG linker or PEG molecule has a molecular weight of about 19,000 daltons. In some embodiments, the PEG linker or PEG molecule has a molecular weight of about 20,000 daltons.

[0147] Second linker: In some embodiments, the cargo of the amphiphilic conjugate is a large protein antigen (e.g., a dimeric or trimeric antigen), and a longer linker is required to avoid steric hindrance of the large protein antigen in the amphiphilic conjugate. In such a case, in some embodiments, the second linker is bonded to the first linker to form a linker suitable for the large protein antigen, and the first linker is any linker described above. The second linker and the first linker are bonded to each other directly or indirectly (e.g., bonded via click chemistry) and are disposed between the lipid and the cargo.

[0148] In some embodiments, the second linker is disposed between the first linker and the cargo, connecting the first linker and the cargo. In some embodiments, the second linker is disposed between the first linker and the lipid, connecting the first linker and the lipid.

[0149] In some embodiments, the second linker comprises a PEG molecule, e.g., a second PEG molecule (e.g., a second repeat unit of a PEG monomer). In some embodiments, the PEG molecule of the second linker is the same as the PEG molecule in the first linker. In some embodiments, the PEG molecule of the second linker is different from the PEG molecule in the first linker. In some embodiments, the number of repeating PEG monomers (m) in the second linker can be 1 to 20 monomers. In some embodiments, the number of repeating PEG monomers (m) in the second linker can be 2 to 18, 5 to 15, or 8 to 12 monomers. In some embodiments, the number of repeating PEG monomers (m) in the second linker is 4 monomers.

[0150] In some embodiments, the second linker comprises a dibenzocyclooctyne (DBCO) group (or equivalent functional group) linked to a PEG molecule, e.g., a second PEG molecule (e.g., a second repeat unit of a PEG monomer). In some embodiments, the second linker comprises a dibenzocyclooctyne-(PEG) m (or DBCO-(PEG) m ), where m represents the number of repeating PEG monomers. In some embodiments, the number of repeating PEG monomers (m) in the second linker can be 1 to 20 monomers. In some embodiments, the number of repeating PEG monomers (m) in the second linker can be 2 to 18, 5 to 15, or 8 to 12 monomers. In some embodiments, the number of repeating PEG monomers (m) in the second linker is 4 monomers.

[0151] In some embodiments, the second linker further comprises a maleimide group. In some embodiments, the second linker is DBCO-(PEG). m In certain embodiments, the second linker comprises DBCO-(PEG)4-maleimide, the structure of which is shown below: [ka]

[0152] A representative schematic of the structure of a non-limiting example of an amphiphilic conjugate comprising a second linker as DBCO-(PEG)4-maleimide is shown below: [ka]

[0153] A non-limiting example of an amphiphilic conjugate comprising DBCO-(PEG)4-maleimide (DSPE-PEG2K-DBCO-PEG4-MD39) is shown in FIG. 17B.

[0154] Oligonucleotide linkers. In certain embodiments, the linker is an oligonucleotide. Non-limiting examples of oligonucleotide linkers applicable to the amphiphilic conjugates of the present disclosure can be found in WO 2019 / 060425, the entire contents of which are incorporated herein by reference. The linker can have any sequence, for example, the sequence of the oligonucleotide can be a random sequence or a sequence specifically selected for its molecular or biochemical properties (e.g., high polarity). In certain embodiments, the polar block linker comprises one or more consecutive series of adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analogs thereof. In certain embodiments, the polar block linker consists of a series of consecutive adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analogs thereof.

[0155] In certain embodiments, the linker is one or more guanines, for example, 1-10 guanines. It has been discovered that altering the number of guanines between the cargo, such as a CpG oligonucleotide, and the lipid tail can control the stability of the micelles in the presence of serum proteins. Thus, the number of guanines in the linker can be selected based on the desired affinity of the conjugates of the present disclosure for serum proteins, such as albumin. It has previously been shown that when the cargo in the amphiphilic conjugate is a CpG immunostimulatory oligonucleotide and the lipid tail is a diacyl lipid, the number of guanines affects the ability of micelles formed in aqueous solution to dissociate in the presence of serum: 20% of the unstabilized micelles (lipo-G0T10-CG) were intact, while the remaining 80% were disrupted and bound to FBS components. In the presence of guanines, the percentage of intact micelles increased from 36% (lipo-G2T8-CG) to 73% (lipo-G4T6-CG), eventually reaching 90% (lipo-G6T4-CG). Increasing the number of guanines to 8 (lipo-G8T2-CG) and 10 (lipo-G10T0-CG) did not further improve micelle stability. Thus, in certain embodiments, linkers in conjugates suitable for use in the methods disclosed herein can include 0, 1, or 2 guanines.

[0156] Methods for preparing amphiphilic conjugates (i) Method for preparing antigen peptide or antigen protein In some embodiments, the antigenic peptide or protein antigen described herein for use in the amphiphilic conjugate is produced in a transformed host cell using recombinant nucleic acid, e.g., DNA or RNA technology. To do so, a recombinant nucleic acid molecule encoding the antigenic peptide or protein antigen is prepared. Methods for preparing such nucleic acid molecules are well known in the art. For example, the sequence encoding the antigenic peptide or protein antigen can be excised from the nucleic acid molecule using an appropriate restriction enzyme. Alternatively, the nucleic acid molecule can be synthesized using chemical synthesis techniques such as the phosphoramidate method. These techniques can be used in combination.

[0157] The method of producing an antigenic peptide or protein antigen also includes preparing a vector capable of expressing the antigenic peptide or protein antigen in a suitable host. The vector comprises a nucleic acid molecule encoding the peptide or protein antigen operably linked to a suitable expression control sequence. Methods for affecting this operably linkage, 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 in the control of transcription or translation. The resulting vector containing the nucleic acid molecule encoding the peptide or protein antigen is used to transform a suitable host. This transformation can be carried out using methods well known in the art.

[0158] Any of the numerous available and well-known host cells may be suitable for use in the methods disclosed herein. The selection of a particular host depends on many factors recognized in the art. These include, for example, compatibility with the selected expression vector, toxicity of the peptide encoded by the nucleic acid molecule, transformation rate, ease of recovery of the peptide, expression characteristics, biological safety, and cost. These factors must be balanced, with the understanding that not all hosts are equally effective in expressing a particular nucleic acid sequence. Within these general guidelines, useful microbial hosts include bacteria (such as E. coli), yeast (such as Saccharomyces) and other fungi, insects, plants, mammalian (including human) cells in culture, or other hosts known in the art.

[0159] The transformed host is then cultured and purified. The host cells may be cultured under conventional fermentation conditions such that the desired compound is expressed. Such fermentation conditions are well known in the art. Finally, the antigenic peptide or protein antigen is purified from the cells or medium by methods well known in the art.

[0160] Antigenic peptide or protein antigens can also be prepared by synthetic methods, for example solid phase synthetic techniques can 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. Intl. 10:394-414; Stewart and Young (1969), Solid Phase Peptide Synthesis; US 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 herein by reference. Solid phase synthesis is the preferred technique for producing individual peptides because it is the most cost-effective method for producing small peptides. Compounds containing derivatized peptides or compounds containing non-peptide groups can be synthesized by well-known organic chemistry techniques.

[0161] Other methods of nucleic acid expression and synthesis are generally known to those of skill in the relevant art.

[0162] The nucleic acid molecules described above can be included within vectors that can, for example, direct their expression in cells transduced with the vector. Thus, expression vectors that contain nucleic acid molecules encoding peptide or protein antigens, and cells transfected with these vectors, are among the embodiments provided herein.

[0163] Vectors suitable for use include T7-based vectors for use in bacteria (see, e.g., Rosenberg et al., Gene 56:125, 1987), pMSXND expression vector for use in mammalian cells (Lee and Nathans, J. Biol. Chem. 263:3521, 1988), and baculovirus-derived vectors for use in insect cells (e.g., expression vector pBacPAKS from Clontech, Palo Alto, Calif.). The nucleic acid insert encoding the polypeptide of interest in such vectors can be operably linked to a promoter selected, for example, based on the cell type in which expression is desired. For example, the T7 promoter can be used in bacteria, the polyhedrin promoter in insect cells, and the cytomegalovirus or metallothionein promoters in mammalian cells. Also, for higher eukaryotes, tissue-specific and cell type-specific promoters are widely available. These promoters are named for their ability to direct the expression of a nucleic acid molecule in a particular tissue or cell type in the body. Those skilled in the art are familiar with the numerous promoters and other regulatory elements that can be used to direct the expression of a nucleic acid.

[0164] In addition to sequences that facilitate transcription of the inserted nucleic acid molecule, vectors can also contain origins of replication and other genes that code for selection markers. For example, the neomycin resistance (neor) gene confers G418 resistance to cells in which it is expressed, thus allowing phenotypic selection of transfected cells. Those skilled in the art can readily determine whether a given regulatory element or selection marker is suitable for use in a particular experimental situation.

[0165] Viral vectors suitable for use include, for example, retrovirus, adenovirus, and adeno-associated virus vectors, herpes viruses, simian virus 40 (SV40), and bovine papilloma virus vectors (see, e.g., Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, NY).

[0166] Prokaryotic or eukaryotic cells that contain and express a nucleic acid molecule encoding a peptide or protein antigen are also suitable for use. The cell is a transfected cell, i.e., a cell into which a nucleic acid molecule, e.g., a nucleic acid molecule encoding a peptide or protein antigen, has been introduced by recombinant DNA techniques. The progeny of such cells are also considered suitable for use in the methods disclosed herein.

[0167] The exact components of the expression system are not critical. For example, peptide or protein antigens can be produced in prokaryotic hosts such as E. coli, or in eukaryotic hosts such as insect cells (e.g., Sf21 cells), 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). When selecting an expression system, it is only important that the components are compatible with each other. A craftsman or person skilled in the art can make such a judgment. Furthermore, if guidance is needed in selecting an expression system, the skilled artisan can refer to Ausubel et al. (Current Protocols in Molecular Biology, John Wiley and Sons, New York, NY, 1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985 Suppl. 1987).

[0168] The expressed peptide or protein antigens can be purified from the expression system using routine biochemical procedures and can be used, for example, conjugated to an albumin-binding lipid via a linker, as described herein.

[0169] (ii) Methods for preparing amphiphilic conjugates In some embodiments, the present disclosure provides methods of assembling amphiphilic conjugates.

[0170] In certain embodiments, the cargo immunogen (e.g., an antigenic peptide or protein antigen) is covalently attached to the linker by reacting a free thiol group of a cysteine ​​residue contained in the antigen or protein antigen with a reactive maleimide group present in the linker. In some embodiments, the cysteine ​​residue having the free thiol group is at or near the N-terminus of the antigenic peptide or protein antigen. In some embodiments, the cysteine ​​residue having the free thiol group is at or near the N-terminus of the antigenic peptide or protein antigen.

[0171] In some embodiments, a cargo immunogen (e.g., an antigenic peptide or protein antigen) that includes a cysteine ​​residue containing a free thiol group at or near the N-terminus can be reacted with a maleimide group contained in a lipid-PEG linker-maleimide molecule (e.g., DSPE-PEG2K-maleimide) to form a covalent bond to form an amphipathic conjugate (e.g., DSPE-PEG2K-protein antigen, see FIG. 1A).

[0172] In some embodiments, a cargo immunogen (e.g., an antigenic peptide or protein antigen) that includes a cysteine ​​residue containing a free thiol group at or near the N-terminus is linked to a second linker (e.g., DBCO-(PEG) such as DBCO-(PEG)4-maleimide). m -maleimide) to produce an intermediate product (e.g., DBCO-(PEG) mThe DBCO group of the intermediate can then be reacted with a reactive azide group of a lipid-PEG linker-azide molecule (e.g., DSPE-PEG-2K-azide) to form a covalent bond to form an amphiphilic conjugate (e.g., DSPE-PEG2K-DBCO-PEG4-protein antigen, see Figures 17A and 17B).

[0173] The amphiphilic conjugates of the invention can be purified and characterized using standard methods in the art.

[0174] How the vaccine is used In certain aspects, the present disclosure provides a method of vaccinating a subject, comprising administering a vaccine comprising an amphiphilic conjugate disclosed herein to the subject mucosally (e.g., intranasally). The present disclosure also provides a method of immunizing a subject, comprising administering a vaccine comprising an amphiphilic conjugate disclosed herein to the subject mucosally (e.g., intranasally). Mucosally (e.g., intranasally) administering the vaccine to the subject induces or enhances an immune response, such as, for example, a humoral immune response or a cell-mediated immune response, in the subject. In some embodiments, mucosally (e.g., intranasally) administering the vaccine induces a greater immune response, such as, for example, a humoral immune response or a cell-mediated immune response, than a peptide or protein antigen alone.

[0175] In some embodiments, the method includes 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 local. In some embodiments, the humoral immune response (e.g., antibody expression) occurs at a mucosal surface.

[0176] In some embodiments, the method includes inducing production of an antibody that binds to the peptide or protein antigen of the amphipathic conjugate. The antibody produced can be an IgG antibody or an 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 includes inducing production of a neutralizing antibody against a pathogenic antigen (e.g., HIV, SARS-CoV2). In some embodiments, the method includes inducing a sustained level of a neutralizing antibody against a pathogenic antigen (e.g., HIV, SARS-CoV2). In some embodiments, the method includes inducing an increase in the level of IgG and / or IgA antibodies in any one or more of the serum, the upper and / or lower respiratory tract mucosa, or the urogenital mucosa. In some embodiments, the method includes inducing an increase in GC and / or follicular helper T cell (Tfh) responses in the NALT.

[0177] In some embodiments, the method includes inducing sustained levels of antibody (e.g., IgA and / or IgA) titers in the serum, vagina, and / or feces of the 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 includes inducing high levels of antibody (e.g., IgA and / or IgG) titers in the serum, vagina, and / or feces of the 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, antibody-secreting cells (ASCs) that produce antibodies are present in the subject for 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. In some embodiments, the ASC cells are detected in the female reproductive tract (FRT) and / or bone marrow (BM).

[0178] (i) Preparation The present disclosure provides a vaccine comprising the amphiphilic conjugate disclosed herein. The vaccine is administered by a mucosal (e.g., nasal, vaginal, rectal, or sublingual) route. The vaccine can be administered using a bioerodible insert and can be formulated in a dosage form suitable for each administration route.

[0179] As further research progresses, information will become available regarding appropriate dosage levels for the treatment of various conditions in various subjects or patients, and those skilled in the art will be able to ascertain appropriate dosage levels, taking into account the treatment situation, age, and general health of the recipient. The selected dosage depends on the desired therapeutic effect, the route of administration, and the desired duration of treatment.

[0180] Formulations for administration to mucosa can be spray-dried drug particles that can be incorporated into tablets, gels, capsules, suspensions or emulsions. Standard pharmaceutical excipients are available from any formulations supplier.

[0181] In some embodiments, the vaccine comprising the amphiphilic conjugate further comprises an adjuvant.

[0182] (ii) Adjuvant Vaccines comprising amphipathic conjugates 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 with an adjuvant.

[0183] Adjuvants include, but are not limited to, alum (e.g., aluminum hydroxide, aluminum phosphate), saponins purified from the bark of Q. saponaria such as QS21 (a glycolipid eluting in the 21st peak in HPLC fractionation, Antigenics, Inc., Worcester, MA), poly[di(carboxylatophenoxy)phosphazene (PCPP polymer, Virus Research Institute, USA), Flt3 ligand, Leishmania elongation factor (a purified Leishmania protein, Corixa Corporation, Seattle, WA), ISCOMS (mixed saponin, lipid-containing immunostimulatory conjugates that form virus-sized particles with pores capable of retaining antigens, CSL, Melbourne, Australia), Pam3Cys, SB-AS4 (SmithKline Beecham Adjuvant System #4 containing alum and MPL, SBB, Belgium), CRL The excipients can be micelle-forming non-ionic block copolymers such as 1005 (which contain linear chains of hydrophobic polyoxypropylene flanked by chains of polyoxyethylene, Vaxcel, Inc., Norcross, GA), and Montanide IMS (e.g., IMS 1312, a water-based nanoparticle combined with Seppic, a soluble immunostimulant).

[0184] The adjuvant may be a TLR ligand. Adjuvants acting through TLR3 include, but are not limited to, double-stranded RNA. Adjuvants acting through TLR4 include, but are not limited to, monophosphoryl lipid A (MPLA, Ribi ImmunoChem Research, Inc., Hamilton, Montana) and muramyl dipeptide (MDP; Ribi), threonyl-muramyl dipeptide (t-MDP; Ribi), OM-174 (glucosamine disaccharide related to lipid A, OM Pharma SA, Meylin, Switzerland). Adjuvants acting through TLR5 include, but are not limited to, flagellin. Adjuvants acting through TLR7 and / or TLR8 include, but are not limited to, single-stranded RNA, oligoribonucleotides (ORN), synthetic low molecular weight compounds such as imidazoquinoline amines (e.g., imiquimod (R-837), resiquimod (R-848)). Adjuvants that act through TLR9 include, but are not limited to, DNA of viral or bacterial origin, or synthetic oligodeoxynucleotides (ODNs), such as CpG ODNs. Another class of adjuvants are phosphorothioate-containing molecules, such as phosphorothioate nucleotide analogs and nucleic acids that contain phosphorothioate backbone bonds.

[0185] Adjuvants can also be oil emulsions (e.g., Freund's adjuvant), saponin preparations, virosomes and virus-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 aluminum salts, calcium salts, hydroxides, phosphates, sulfates, etc.), bioadhesives and / or mucoadhesives, microparticles, liposomes, polyoxyethylene ether and polyoxyethylene ester preparations, polyphosphazenes, muramyl peptides, imidazoquinolone compounds, and surfactants (e.g., lysolecithin, Pluronic® polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).

[0186] Adjuvants can 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-γ, etc.), macrophage colony-stimulating factor, and tumor necrosis factor.

[0187] 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 the innate immune response, and when stimulated, it induces the expression of various interferons, cytokines, and T cell recruiting factors that amplify and enhance immune activity. Recent studies have shown that STING agonists are effective adjuvants and efficiently induce immune responses, as 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 incorporated herein by reference.

[0188] In some embodiments, the STING agonist is a cyclic dinucleotide.In certain embodiments, the cyclic dinucleotide includes, but is 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 analogs.In some embodiments, the cyclic dinucleotide suitable for use in the present disclosure is described in some detail in, for example, U.S. Patent Nos. 7,709,458 and 7,592,326, WO 2007 / 054279, U.S. Patent No. 2014 / 0205653, and Yan et al. Bioorg.Med.Chem Lett.18:5631 (2008), each of which is incorporated herein by reference in its entirety.

[0189] In certain embodiments, STING agonist is chemically synthesized.In certain embodiments, STING agonist is a naturally occurring cyclic dinucleotide analogue.STING agonist including cyclic dinucleotide analogue suitable for use in the present disclosure is described in U.S. Patent No. 7,709,458 and U.S. Patent No. 7,592,326 and U.S. Patent Application Publication No. 2014 / 0205653.

[0190] In some embodiments, the adjuvant is a saponin monophosphoryl lipid A (MPLA) nanoparticle adjuvant (SMNP). In some embodiments, the adjuvant is cdGMP.

[0191] (iii) Transmucosal Administration The present disclosure provides a method of vaccinating and / or immunizing a subject, comprising administering an effective amount of a vaccine to the subject mucosally (e.g., intranasally). Mucosal administration includes nasal, oral (sublingual), intratracheal, vaginal and rectal routes. Mucosal administration may be preferred over parenteral routes of administration (e.g., subcutaneous, intramuscular, intravenous, and intrathecal) because it is non-invasive, does not require a trained medical professional to administer, and can be self-administered by the subject.

[0192] For example, in some embodiments where the immunogen is a peptide antigen, transmucosal administration does not include intratracheal administration.

[0193] In some embodiments, the present disclosure provides a method of vaccinating a subject, comprising intranasally administering to the subject an effective amount of a vaccine.In some embodiments, the present disclosure provides a method of immunizing a subject, comprising intranasally administering to the subject an effective amount of a vaccine.

[0194] 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 a human.

[0195] In some embodiments, the vaccine is administered repeatedly. In certain embodiments, a first dose may be followed by a second or subsequent multiple doses of the vaccine, with doses similar to, or less than, the first dose. In some embodiments, at least two, at least three, at least four, or at least five doses of the vaccine are administered to induce an effective immune response (e.g., to induce an antibody-mediated immune response, to induce a cell-mediated immune response, and / or to achieve a desired level of neutralizing antibodies).

[0196] In some embodiments, a subsequent dose of the vaccine is administered about 1 week, 2 weeks, 3 weeks, 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months, 9 months, or more than 1 year after the previous dose.

[0197] 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.

[0198] In some embodiments, a booster dose of the vaccine is administered one to several years (eg, 2, 3, 5, 10, 15 years) after the previous dose.

[0199] In some embodiments, the vaccine dose comprises about 1-500 μg, 20-500 μg, 50-450 μg, 75-400 μg, 100-300 μg, or 150-250 μg of the amphipathic conjugate. In some embodiments, the vaccine dose comprises about 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, 100 μg, 105 μg, 110 μg, 115 μg, 120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg, 200 μg, 210 μg, 220 μg, 230 μg, 240 μg, 250 μg, 260 μg, 270 μg, 280 μg, 290 μg, 300 μg, 310 μg, 320 μg, 330 μg, 340 μg, 350 μg, 360 μg, 370 μg, 380 μg, 390 μg, 400 μg, 400 μg, 400 μg, 400 μg, 400 μg, 400 μg, 40

[0036] In one embodiment, the compound comprises 100 μg, 125 μg, 130 μg, 135 μg, 140 μg, 145 μg, 150 μg, 155 μg, 160 μg, 165 μg, 170 μg, 175 μg, 180 μg, 185 μg, 190 μg, 195 μg, 200 μg, 210 μg, 220 μg, 230 μg, 240 μg, 250 μg, 260 μg, 270 μg, 280 μg, 290 μg, or 300 μg of amphiphilic conjugate.

[0200] In some embodiments, the vaccine is administered in combination with an SMNP adjuvant. In some embodiments, an amount of about 1-400 μg, 1-50 μg, 50-100 μg, 50-200 μg, 50-300 μg, 50-400 μg, 100-200 μg, 100-300 μg, 100-400 μg, 200-400 μg, or 300-400 μg of SMNP is administered in combination with a dose of vaccine. In some embodiments, an amount of about 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 125 μg, 150 μg, 175 μg, 200 μg, 225 μg, 250 μg, 275 μg, 300 μg, 325 μg, 350 μg, 375 μg, 400 μg of SMNP is administered in combination with a dose of vaccine.

[0201] In some embodiments, the vaccine is administered in combination with a cdGMP adjuvant. In some embodiments, about 5-50 μg, 50-150, or 100-400 μg of cdGMP is administered in combination with a dose of vaccine. In some embodiments, about 5 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 125 μg, 150 μg, 175 μg, 200 μg, 225 μg, 250 μg, 275 μg, 300 μg, 325 μg, 350 μg, 375 μg, or 400 μg of cdGMP is administered in combination with a dose of vaccine.

[0202] (iv) Target infectious disease In certain aspects, the methods provided herein include inducing an immune response to prevent or reduce the severity of an infectious disease.

[0203] In some embodiments, the infectious disease is caused by a pathogen, hi some embodiments, the pathogen is capable of infecting the subject through a mucosal surface.

[0204] Infectious diseases that may benefit from the methods provided herein include, but are not limited to, HIV / AIDS, coronavirus disease 19 (COVID-19), influenza, rotavirus infection (e.g., diarrhea), cytomegalovirus (CMV) infection, Epstein-Barr virus infection (e.g., mononucleosis), respiratory syncytial virus (RSV) infection, and cholera.

[0205] Acquired immune deficiency syndrome (AIDS) is a syndrome caused by the human immunodeficiency virus (HIV). HIV is spread primarily through unprotected sexual intercourse (including anal and vaginal intercourse), contaminated needles and blood transfusions, and from mothers to children during pregnancy, childbirth, and breastfeeding. After an initial infection with HIV, a person may not experience any symptoms at all or may experience a short period of flu-like illness. This is usually followed by a long, symptom-free incubation period. As the infection progresses, the immune system is hindered, increasing the risk of developing common infections such as tuberculosis, as well as other opportunistic infections and tumors that are rare in people with normal immune function. These later stages of infection are called acquired immune deficiency syndrome (AIDS).

[0206] Coronavirus disease 19 (COVID-19) is a respiratory illness 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 expelled when an infected person coughs, sneezes, or talks. It can also, less commonly, be spread by touching a surface that has the virus on it and then touching your mouth, nose, or eyes.

[0207] Influenza (also called "the flu") is an infection of the nose, throat, and lungs caused by influenza viruses. There are four types of influenza viruses, called influenza viruses A, B, C, and D. Waterfowl are the main source of infection for influenza A viruses (IAV), which are also widely distributed in humans and various mammals, including pigs. Influenza B viruses (IBV) and influenza C viruses (ICV) mainly infect humans, while influenza D viruses (IDV) are found in cattle and pigs. IAV and IBV circulate in humans and cause seasonal epidemics, while ICV causes mild infections, mainly in children. In humans, influenza viruses are transmitted primarily through respiratory droplets produced by coughing and sneezing. Transmission also occurs through aerosols and intermediate objects, as well as surfaces contaminated by the virus.

[0208] Rotavirus infection typically causes severe watery diarrhea and vomiting in infants and young children, which can lead to hospitalization and death in children. Humans infected with rotavirus shed the virus in their stool, and rotavirus is spread by fecal-oral transmission.

[0209] Cytomegalovirus (CMV) infection is a common infection that affects people of all ages. Most people infected with CMV do not show signs or symptoms, and the virus can remain dormant (inactive) for long periods of time in various tissues. Various stimuli can reactivate dormant CMV, allowing the virus to multiply and, in some cases, cause disease. Severe infection usually occurs only in infants infected before birth and in people with weakened immune systems. Infected people may shed CMV intermittently in urine or saliva. The virus is also shed in mucus from the cervix (lower part of the uterus), semen, stool, and breast milk. Thus, the virus spreads through sexual and non-sexual contact.

[0210] Epstein-Barr virus (EBV, also known as human herpesvirus 4) is a virus that infects B cells, causing a spectrum of infections ranging from asymptomatic to infectious mononucleosis. EBV is most commonly spread through bodily fluids, especially saliva. However, EBV can also be spread through blood and semen during sexual contact, blood transfusions, and organ transplants.

[0211] Respiratory syncytial virus (RSV) is a respiratory virus that infects the lungs and respiratory tract. In adults and older healthy children, symptoms of RSV are mild and usually similar to a cold. However, in young children, the elderly, and people with heart and lung disease or weakened immune systems, RSV infection can become severe. RSV spreads through contact with droplets from the nose and throat when an infected person coughs and sneezes. RSV can also spread through dried respiratory secretions on bedding and similar objects.

[0212] Cholera is an acute diarrheal disease caused by infection of the intestine with the bacterium Vibrio cholerae. People can become ill if they swallow food or water contaminated with the bacteria. Infection is often mild or asymptomatic, but can be severe and life-threatening.

[0213] In certain embodiments, the methods provided herein include inducing immunity against infectious pathogens. Non-limiting examples of infectious pathogens include human immunodeficiency virus (HIV), SARS-CoV-2 virus, influenza virus, rotavirus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), respiratory syncytial virus (RSV), and Vibrio cholerae. Immunity against other common infectious pathogens can also be induced using the methods described herein.

[0214] In some embodiments of the methods provided herein, the immune response induced in the subject comprises expression of IgA antibodies that target the pathogen. In some embodiments, the immune response induced in the subject comprises expression of IgG antibodies that target the pathogen. In some embodiments, the immune response induced in the subject comprises expression of both IgA and IgG antibodies that target the pathogen. In some embodiments, the immune response induced in the subject comprises expression of neutralizing antibodies that target the pathogen. EXAMPLES

[0215] The present invention is further illustrated by the following examples, which should not be construed as limiting. The contents of all references, GenBank accessions and gene numbers, and published patents and patent applications cited throughout this application are hereby incorporated by reference. Those skilled in the art will recognize that the invention can be practiced with modifications of the disclosed structures, materials, compositions and methods, and that such modifications are considered to be within the scope of the invention.

[0216] Reference numbers within brackets "[ ]" herein refer to the corresponding documents listed in the accompanying references which form part of this specification and which are hereby incorporated by reference.

[0217] Example 1: Overview Vaccine platforms have previously been developed that use endogenous albumin as a chaperone to enhance lymph node transport of peptide antigens or molecular adjuvants after parenteral injection. One of the main functions of albumin in vivo is to act as a fatty acid transporter, as albumin has seven distinct lipid-binding pockets [23, 24]. By conjugating peptide or Toll-like receptor agonist adjuvants to amphiphilic albumin-binding lipid tails (forming "amph vaccines"), important changes in the pharmacokinetic behavior of these vaccine components can be achieved: first, after injection, the lipid tails of the amph vaccines bind to endogenous albumin present in the interstitial fluid at the injection site, efficiently redirecting the conjugates to lymphatic vessels and draining lymph nodes following the convective pathway of albumin (unmodified peptides would disperse in the blood and be rapidly diluted and degraded)

[25] . Second, upon reaching the dense cellular microenvironment of lymph nodes, the lipid tails of amph peptides insert into cell membranes, promoting long-term antigen retention in the draining lymphoid tissues [26, 27]. These changes in the pharmacokinetics of amph peptides compared to soluble peptide vaccines translate into robust enhancement of systemic T cell responses and antitumor immunity after parenteral immunization [25, 28, 29].

[0218] In addition to constitutive transport from blood to tissues and lymph, albumin is transported bidirectionally across mucosal barriers via interactions with the neonatal Fc receptor (FcRn) expressed by mucosal epithelial cells. FcRn has emerged as a “mucosal gateway” to improve drug uptake across the mucosal epithelium of nasopharyngeal, pulmonary, and gastrointestinal tissues [7, 30–32]. It is widely expressed on mucosal epithelial cells in adult animals and humans and plays a key role in recycling IgG and albumin through bidirectional transcytosis of both molecules [33–35]. Albumin-bound amph-vaccines are capable of FcRn-mediated uptake through mucosae, e.g., the nasal mucosa, allowing higher levels of antigen to reach the NALT. Furthermore, membrane binding of amph immunogens may prolong antigen availability in nasal and NALT tissues to promote local immune priming while avoiding systemic diffusion of antigen from the site of action of locally co-administered mucosal adjuvants. It was hypothesized that these two effects may combine to promote enhanced mucosal and systemic immunity.

[0219] Given that the majority of licensed vaccines are believed to act through the induction of protective antibody responses [36,37], in the examples provided herein, large protein immunogen amphiphilic conjugates designed to elicit humoral immune responses in the context of HIV and SARS-CoV-2 were prepared and tested. As described below, the amphiphilic conjugates showed improved persistence and uptake through the nasal mucosa compared to the unmodified antigen, and significantly increased GC and follicular helper T cell (Tfh) responses in the NALT. Intranasal immunization with the amphiphilic conjugates elicited high levels of IgG and IgA in serum, upper and lower respiratory tract mucosa, and distal urogenital mucosal sites, including the induction of substantial neutralizing antibody responses against the SARS-Cov-2 RBD immunogen. Furthermore, immunization with the amphiphilic conjugates enhanced vaccine uptake in the nasal cavity of non-human primates and enhanced IgG and IgA responses compared to immunization with soluble protein. Taken together, the data presented in the Examples herein demonstrate that the vaccines of the present disclosure enhance both mucosal and systemic immunity induced by intranasal immunization.

[0220] Example 2: Synthesis of protein antigen-amphiphile conjugates with albumin binding and membrane insertion properties. To assess whether the addition of an albumin-binding moiety to a subunit protein vaccine antigen alters antigen uptake through the nasal mucosa, we first synthesized a conjugate of an HIV Env protein immunogen bound to a poly(ethylene glycol) (PEG)-DSPE amphiphile. This PEG lipid has an equilibrium K DIt was previously demonstrated to bind albumin at approximately 125 nM

[25] . As a test antigen for this concept, Env immunogen eOD-GT8 (gp120 modified ectodomain germline-targeted immunogen 8, hereafter eOD), a germline-targeted antigen of approximately 25 kDa that was recently shown to successfully prime antibody responses broadly neutralizing VRC01 class HIV in phase I clinical trials [38-41], was selected. eOD was fused at its C-terminus with the PADRE universal helper epitope and introduced a terminal free cysteine ​​at its N-terminus, allowing it to be conjugated with maleimide-functionalized PEG2K-DSPE to form a thioether bond (Figures 7A and 7B). The resulting amph-eOD (Figure 1A) formed micelles with a diameter of approximately 30 nm in aqueous solution (Figure 1B), facilitating purification from unreacted eOD (approximately 5 nm) by size-exclusion chromatography (SEC) (Figure 1C).

[0221] Conjugation of small peptide antigens with PEG-DSPE has previously been shown to confer the conjugate the ability to bind albumin and also to interact with cell membranes, altering their transport behavior in vivo [25, 26]. To assess whether amphiphile tails could similarly alter the behavior of much larger protein immunogens, fluorescently labeled amph-eOD was first incubated with albumin-functionalized agarose resin at 37 °C for 2 h, followed by separation of the resin to measure the protein remaining in solution. Sixty percent of the added amph-eOD bound to the albumin resin, compared with less than 5% of unmodified eOD (Figure 1D). Next, the interaction of amph-eOD with lymphocytes was evaluated. Titrated concentrations of Alexa dye-labeled eOD or amph-eOD were added to mouse spleen cells in 10% serum at 37 °C, followed by extracellular staining at 4 °C with fluorescently labeled VRC01 monoclonal antibody to detect eOD coating the cell surface. Flow cytometry analysis revealed that both eOD and amph-eOD showed binding to spleen cells within 1 h, but amph-eOD showed more than 15-fold higher uptake levels (Figures 1E-1G, 8A-8B). Furthermore, the majority of cell-associated amph-eOD was localized to the cell surface as revealed by VRC01 staining (Figures 1E-1G). The percentage of eOD+VRC01+ double positive cells increased proportionally with amph-eOD but not with eOD concentration (Figures 1F-1G). Thus, surprisingly, amph protein conjugates were found to exhibit similar albumin binding and membrane insertion properties as previously studied amph peptide conjugates, and we hypothesized that this may alter antigen trafficking and persistence in vivo.

[0222] Example 3: Amphiphile modification enhances uptake and retention of eOD antigen in the nasal cavity following intranasal immunization of mice Albumin is transported bidirectionally through respiratory mucosal surfaces via interactions with the neonatal Fc receptor (FcRn) [31, 42, 43]. Amph protein immunogens may show enhanced uptake through nasal epithelia by using albumin as a non-covalent chaperone. To test this idea, we first assessed whether conjugation of DSPE-PEG to albumin inhibits its interaction with FcRn using an enzyme-linked immunosorbent assay (ELISA) that measures albumin binding to plate-bound FcRn. Incubation with fluorescein isothiocyanate-labeled DSPE-PEG at concentrations up to 1 μM on albumin showed no inhibition of albumin binding to FcRn (Figure 8C).

[0223] Next, we investigated the transport of fluorescent amph-eOD vaccine in the nasal cavity of mice over time after intranasal administration. Total vaccine uptake in the nasal cavity was quantified by an in vivo imaging system (IVIS) measuring the fluorescent signal in a predefined region of interest (ROI) in the mouse nose over time (Figure 2A, (i)), and further characterized by histological images of cross-sections of the nasal cavity (Figure 2A, (ii)). First, BALB / c mice were intranasally immunized with Alexa fluorescent-labeled eOD or amph-eOD mixed with saponin adjuvant. The upper jaw was removed from the mouse nose, and the signal in the ventral side of the nasal cavity was quantified by IVIS over an 11-day period (Figure 2B). Amph-eOD showed significant accumulation and persistence in the nasal cavity over 72 hours, with the vaccine still detectable at days 7 and 11 post-immunization (Figure 2B-C). In contrast, free eOD showed some initial signal (less than 40% of amph-eOD) at 24 h, which quickly decreased to background. Vaccine exposure, assessed as the area under the curve (AUC) of nasal fluorescence signal over time, was approximately 5.7-fold greater for amph-eOD than for eOD (Figure 2D). Furthermore, amph-eOD did not diffuse to reach systemic compartments or distal lymphoid tissues, as only minimal vaccine accumulation was observed by IVIS in the spleen, liver, intestine, cervical LNs, or mesenteric LNs at 24 h (Figures 9A-9B).

[0224] Without wishing to be bound by theory, the enhanced persistence of amphiphile vaccines in the nasal cavity may be mediated by a combination of (1) the lipid tails facilitating association with the epithelial cell surface and (2) the amphiphiles binding to albumin in the mucus layer, facilitating FcRn-mediated transcytosis to the underlying nasal submucosa. Notably, IVIS imaging revealed that FcRn-mediated transcytosis was significantly greater in nasal patients compared to wild-type (WT) animals. - / - We found that amph-eOD administered with intranasal adjuvant was rapidly cleared in mice, and the persistence of amph-eOD in FcRn-deficient animals was similar to that of unmodified eOD in WT mice (Figures 2E-2F).

[0225] To determine whether enhanced antigen persistence correlated with actual uptake into nasal tissue, histological sections from the midpoint of the nasal cavity were imaged (Figure 2A, (ii)). Confocal imaging immediately revealed qualitative differences in vaccine accumulation and uptake in the nasal cavity at the 6-hour time point (Figure 2G). eOD was only sparsely observed on the epithelial cell surface ("e") and instead appeared to be primarily confined to the upper mucus layer ("m") lining the airways (Figure 2G, (ii) right panel). In contrast, in WT mice, amph-eOD accumulated primarily on the epithelial surface overlying the lamina propria ("lp") and was concentrated in the respiratory nasal turbinates. Amph-eOD also downregulated FcRn - / - The amph-eOD showed obvious accumulation on the epithelial surface of mice (Figure 2G, (i, ii)), which was attributed to the ability of the amphiphilic tail to insert into the cell membrane. At 24 hours after administration, eOD was nearly undetectable in the nasal cavity, whereas amph-eOD increased the cellular ... - / - In WT mice, amph-eOD still accumulated at the epithelial surface (Figure 2H, (i, ii) left and middle panels). However, high-magnification imaging using DAPI staining to delineate the epithelium and underlying submucosa revealed distinct pockets of amph-eOD uptake in the lamina propria of WT mice. This submucosal accumulation was due to the FcRn - / -It was absent in mice (Figure 2H, (iii) right panel). These data suggest that binding of eOD to epithelial cells is facilitated by the DSPE lipid tail, but transport across the epithelial barrier is highly dependent on FcRn.

[0226] Example 4: Induction of superior germinal center and Tfh cell responses in the NALT in an FcRn-dependent manner by intranasal amph-gp120. It was hypothesized that enhanced vaccine retention in the nasal cavity and increased uptake through the nasal epithelium would result in greater antigen delivery to the NALT, located beneath the nasal cavity and behind the soft palate (Figure 3A, (i)), thereby initiating a stronger local GC response. Therefore, the accumulation and persistence of fluorescent eOD or amph-eOD in the NALT over time was investigated by flow cytometry after intranasal immunization (Figure 3A, (ii)). + Amph-eOD accumulation in macrophages and B cells significantly exceeded that of eOD both 1 and 4 days after immunization (Figures 3B-C, Figure 10). + MHCII + Uptake in dendritic cells was also greater for amph-eOD compared to eOD 1 day after immunization (Figure 3D, Figure 10). These findings indicate that amph-eOD reaches the NALT and is taken up by important antigen-presenting cell (APC) populations to a greater extent than unmodified eOD. To determine the impact of enhanced antigen delivery to nasal lymphoid tissues at the early stage of the adaptive immune response to eOD, germinal center (GC) B cell and follicular helper T (Tfh) cell responses in the NALT were assessed 12 days after in immunization with eOD and saponin adjuvant (Figure 3E). Amph-eOD induced a greater GC response in the NALT of WT mice, in terms of both total GC B cells (4.8-fold) and eOD-bound antigen-specific GC B cells (6.8-fold), compared to soluble eOD immunization (Figure 3F-G, Figure 11). Strikingly, FcRn - / -These amplified responses were entirely FcRn dependent, as amph-eOD immunization in animals elicited responses comparable to eOD in WT mice (Figure 3F-G, Figure ​(Figure11B-E). These trends were mirrored in NALT follicular helper T cell (Tfh) responses, with eOD and FcRn in WT mice. - / - Compared with both amph-eOD and amph-eOD in WT mice (Figure 3H and Figures 12A-E), amph-eOD induced a greater Tfh response than eOD ​​(p<0.01) and FcRn in WT mice. - / - T cells (ICOS) compared with amph-eOD in mice (p<0.05). + CD4 + CD44 + Thus, amph conjugate immunization induced greater overall activation of mucosal GC and T cell responses in an FcRn-dependent manner.

[0227] Example 5: Intranasal amph-eOD induces potent systemic and mucosal antibody responses in mice. Output antibody responses elicited by intranasal immunization with amphiphiles or soluble proteins were evaluated both systemically and at distal mucosal sites relevant for HIV infection, such as the rectum and urogenital mucosa. First, studies were performed combining eOD with the cyclic dinucleotide, cyclic dimeric guanosine monophosphate (cdGMP) (Figure 4A). Cyclic dinucleotides (CDNs) activate the innate immune sensor stimulator of interferon genes (STING) and have previously been reported to be effective mucosal vaccine adjuvants in mice [44–46]. Intranasal immunization with amph-eOD and cdGMP induced very high serum IgG and IgA responses, with endpoint antigen-specific serum IgG titers of approximately 10 6 , and IgA titers are around 10 3 ~10 4and sustained for 35 weeks (Figure 4B). Amph vaccination increased IgG responses by more than 2 logs over unmodified eOD and stimulated strong serum IgA responses that were completely absent after soluble protein immunization. Notably, amph-eOD also induced significant and sustained mucosal IgG and IgA responses in the vaginal tract (Figure 4C) and rectal mucosa (Figure 4D), whereas soluble eOD immunization also elicited only weak to undetectable responses. Intranasal and parenteral (subcutaneous) vaccination with amph-eOD were also directly compared. Subcutaneous immunization with amph-eOD induced strong systemic IgG titers in the blood but failed to induce mucosal responses (Figures 13A-13C).

[0228] Cohorts of mice were then euthanized at various time points and the female reproductive tract (FRT) and bone marrow (BM) were isolated and analyzed via antibody secreting cell (ASC) ELISPOT to identify long-lived plasma cells. Amph-eOD immunization resulted in high levels of both eOD-specific IgA and IgG plasma cells in the FRT and BM 20 weeks after immunization (Figures 14A-14B). Even more strikingly, more than a year after immunization, mice immunized with amph-eOD retained a significant population of eOD-specific IgA plasma cells present in the FRT and BM, whereas mice immunized with eOD showed few ASCs in either niche (Figure 4E).

[0229] CDNs are in clinical trials as immune stimulants for cancer treatment, but have not yet been used in human vaccines. Therefore, a similar study was next performed using an ISCOM-like saponin adjuvant called SMNP, which has a nanoparticle structure and composition similar to the matrix M adjuvant in advanced clinical trials of a SARS-CoV-2 vaccine by Novavax

[48] (Figure 4F). Similar to CDNs, ISCOM-based adjuvants have been shown to be effective intranasal adjuvants in preclinical studies [49, 50]. Similar to the results of cdGMP, intranasal immunization with amph-eOD and SMNP increased immunization rates by approximately 10% each. 6 and about 104 Amph-eOD / SMNP immunization also induced strong and long-lasting mucosal IgG and IgA responses in the vaginal tract (Fig. 4H) and rectal mucosa (Fig. 4I), with post-boost amph-eOD titers consistently ∼10 higher than eOD ​​titers in the vaginal mucosa. 3 2-fold higher in mice immunized with amph-eOD and 10- to 100-fold higher in fecal samples. After 35 weeks, analysis of FRT and BM by ASC ELISPOT again demonstrated a significant increase in the number of eOD-specific IgA plasma cells in the FRT (P<0.05) and BM (P<0.1) of mice immunized with amph-eOD compared to eOD (Figure 4J). Strikingly, with both cdGMP and SMNP adjuvants, the population of IgA plasma cells established in the female reproductive tract was equal to or greater than that in the bone marrow (Figures 4E-4J).

[0230] Taken together, these studies demonstrate that intranasal immunization with amph-conjugated antigens can promote potent and long-lasting systemic and mucosal antigen-specific humoral immunity in mice using multiple adjuvants.

[0231] Recently, clinical studies of SARS-CoV-2 mRNA vaccines have raised concerns about the possibility of antibody responses to PEG included in the vaccine formulation, which may induce allergic reactions in human volunteers. Therefore, serum samples from the above studies using saponin or cdGMP adjuvants were analyzed for the presence of anti-PEG IgG. Despite the use of a strong adjuvant, the anti-PEG response induced by amph-eOD was only slightly above background (Figure 14C).

[0232] Example 6: Induction of high levels of neutralizing antibodies against SARS-CoV-2 in the respiratory mucosa by amph vaccination. eOD is a germline-targeted immunogen designed to initiate priming of human B cells with the ability to produce broadly neutralizing antibodies similar to the CD4 binding site bnAb VRC01 [38–41], however, this immunogen fails to induce neutralizing antibody responses in wild-type mice due to genetic differences in the CDR3 region of mouse and human antibodies. Furthermore, responses elicited in the local respiratory mucosa by intranasal immunization are not relevant for protection from HIV. These considerations motivated us to test the utility of amph conjugation in the context of a vaccine for SARS-CoV-2, as WT mice readily produce neutralizing antibodies against this virus and nAb responses in the nasal and respiratory tract are highly relevant for protection [51–53]. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein was selected as the target antigen to incorporate into the amphiphile platform, as it is the target of most human neutralizing antibodies

[54] . Soluble RBD proteins are known to be poorly immunogenic [55, 56]. Therefore, we tested whether amphiphilic conjugation of the RBD could enhance its immunogenicity and simultaneously promote protective antibody responses in the systemic and respiratory mucosa. To this end, a recently developed modified artificial RBD immunogen was employed, which was expressed in Pichia pastoris and is expressed at much higher levels than the wild-type RBD sequence and shows substantially higher stability

[57] . Modification of the RBD immunogen with an N-terminal cysteine ​​did not affect its production, stability, or antigenic profile (Figures 15A and 15B), and maleimide-functionalized PEG-RBD immunogens were also modified to enhance its immunogenicity (Figures 15B). 2K This allowed the conjugation of the protein with -DSPE (Figure 5A). Similar to amph-eOD, the conjugated amph-RBD formed micelles with diameters of approximately 35 nm in aqueous solution, facilitating purification from unreacted RBD (approximately 5 nm) by SEC (Figures 15C-15D).

[0233] To assess the immunogenicity of amph-RBD, BALB / c mice were immunized intranasally with amph-RBD or RBD in combination with SMNP adjuvant at weeks 0 and 4. At week 6, serum and mucosal samples were collected and measured for RBD-specific IgG / A titers and pseudovirus neutralization (Figure 5B). As shown in Figures 5C-5D, amph-RBD dramatically outperformed soluble RBD in eliciting antigen-specific serum and mucosal IgG and IgA responses. Serum Ig levels were three orders of magnitude higher with amph-RBD than with RBD, and importantly, amph-RBD elicited strong IgG and IgA responses in nasal and bronchial lavage fluid (BALF), whereas soluble RBD immunization elicited weak or no responses (Figures 5C-5D). ACE2-RBD binding inhibition assays revealed IC50 for blockade of ACE2 binding by RBD of ~25,000 in serum and ~300 in BALF of amph-RBD immunized mice (Figure 5E, Figures 15E-15F). Finally, analysis of SARS-CoV-2 pseudovirus neutralization revealed serum nAb titers of ~30,000 and mean nasal and BAL nAb titers of ~500 and ~200, respectively (Figure 5F). In contrast, intranasal immunization with soluble RBD did not elicit detectable neutralizing responses in any compartment (Figure 5F). Thus, intranasal inoculation of amph-RBD vaccine dramatically enhances the induction of neutralizing antibody responses at the mucosal entry site of the SARS-CoV-2 virus.

[0234] Example 7: Amph conjugate vaccines demonstrate enhanced immunogenicity in non-human primates. Although the systemic and mucosal antibody responses elicited by the amph conjugate vaccine in mice were compelling, many of the vaccine techniques effective in small animals have not been successfully applied to larger animals and humans. Therefore, we next evaluated whether amph conjugates are also effective in non-human primates (NHPs) using eOD immunogens. Transport of amphiphile vaccines and soluble proteins after intranasal immunization was first evaluated in rhesus macaques. Alexafluor-labeled amph-eOD or soluble eOD was administered intranasally with SMNP adjuvant, and 24 hours later, nasal tissues including tonsils, adenoids, cervical LNs, axillary LNs, and nasal turbinates were harvested and the fluorescent signal from the labeled immunogen was evaluated by IVIS imaging. Similar to the observations in mice, amph-eOD was detected at significantly higher levels than eOD ​​in nasal tissues (Figure 6A). Negligible signal was detected in cervical LNs or axillary LNs (data not shown).

[0235] To evaluate vaccine immunogenicity, NHPs were immunized intranasally with amph-eOD or eOD combined with SMNP at weeks 0, 8, 16, and 24 (Figure 6B). PBMCs were harvested 5 days after each immunization to measure plasma blast responses by antibody-secreting cell (ASC) ELISPOT. Amph-eOD induced significantly higher eOD-specific IgM, IgG, and IgA plasma blast responses, quantified as total antigen-specific plasmablasts or percentage of total plasmablasts, after the second and third boost (Figure 6C, Figures 16A-16B). In serum, intranasal immunization with amph-eOD seroconverted all animals after a single dose, whereas serum IgG titers primed by soluble eOD remained close to baseline until the first boost was administered (Figure 6D). Antigen-specific serum IgG and IgA titers were consistently about 10-fold higher in NHPs immunized with amph-eOD compared to eOD, even after repeated boosts (Figure 6D). In the nasal mucosa, IgG and IgA were about 1 log higher in NHPs immunized with amph-eOD compared to eOD at weeks 18 and 26, and were maintained after boosts (Figure 6E). Unlike the findings in mice, amph-eOD induced sporadic vaginal and rectal IgG and IgA responses, whereas overall vaginal IgG, vaginal IgA, and rectal IgG from amph-eOD were significantly greater than eOD ​​(p<0.01, p<0.05, and p<0.0001, respectively), and these responses were not consistently maintained throughout the study (Figures 16C-16D). Taken together, these data in the available animal model that most closely resembles humans suggest that intranasal immunization with amph conjugates is a promising strategy to enhance both systemic and mucosal immunity to subunit vaccines.

[0236] Example 8: Synthesis of amphiphilic conjugates with HIV trimeric antigen and intranasal immunization of mice The best immunogen candidates for eliciting broadly neutralizing antibodies against HIV are native-like trimers such as MD39 SOSIP. Therefore, motivated by the promising results with amph-eOD, amphiphile conjugates with a much larger protein antigen, the HIV MD39 SOSIP trimer, were synthesized. A longer linker was used to conjugate this larger trimeric protein to avoid steric hindrance during incorporation into the amphiphile platform.

[0237] Synthesis and purification of amph-MD39: HIV MD39 SOSIP trimers with a C-terminal cysteine ​​(≥1 mg / ml) were first reduced with 10 molar equivalents of tris(2-carboxyethyl)phosphine (TCEP) for 15 min at 25 °C. TCEP was removed by centrifugal filtration using a 10 kDa molecular weight cut-off (MWCO) Amicon spin filter while the protein was washed three times with phosphate-buffered saline (PBS). The protein (1–5 mg / ml) was then reacted with 5 molar equivalents of DBCO-PEG4-maleimide (dibenzocyclooctyne-PEG4-maleimide, MW 674.74 Da) (Sigma) in PBS for 18 h at 4 °C. Unreacted maleimide-PEG4-DBCO was then removed using a 10 kDa MWCO Amicon spin filter, and the product was analyzed for the presence of the DBCO peak at 309 nm by UV-Vis spectrophotometer (Nanodrop One, Thermo Fisher Scientific). MD39-DBCO was then mixed (≥1 mg / ml) with 5 molar equivalents of dry DSPE-PEG2K-azide (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azide(polyethylene glycol)-2000], molecular weight 2816.519 Da) (Avanti Polar Lipids) in PBS and vortexed intermittently for 2 h at 25 °C, followed by gentle mixing for 18 h at 4 °C. The product was then measured again by UV-Vis, which indicated the absence of a DBCO peak at 309 nm, confirming that the reaction had proceeded to completion. MD39 concentration was determined by the protein peak at 280 nm and corrected for background lipid absorbance from 310 to 500 nm. The protein amphiphiles were purified by affinity chromatography using azide-functionalized agarose beads in a gravity column eluted with PBS to separate unreacted MD39-DBCO amph-MD39. Bound protein amphiphiles were quantified by UV-Vis.

[0238] Mouse immunization and blood collection: Immunization studies were performed using age-matched 8-10 week old female BALB / cJ mice (strain 000651) purchased from Jackson Laboratory.

[0239] BALB / c mice were immunized intranasally by administering the vaccine to anesthetized mice in supine position with 20 μl of phosphate-buffered saline (PBS, 10 μl per nostril, 30–60 s interval between nostrils). Animals were primed on day 0 and boosted with 5 μg of MD39 (soluble MD39 or amph-MD39) combined with 5 μg of saponin monophosphoryl lipid A (MPLA) nanoparticle (SMNP) adjuvant on days 42 and 84. For long-term immune monitoring, blood and mucosal samples were collected every 2 or 3 weeks for ELISA. Blood was collected by buccal or retroorbital bleeding, serum was isolated using serum separator tubes, and supernatants were collected by centrifugation at 10,000 g for 5 min. Vaginal mucosal fluid was collected from anesthetized mice by vaginal lavage using 75 μl of sterile PBS (3 × 25 μl drops, 3–5 aspirations each) combined with 5 μl of 25X protease inhibitor (EDTA-free SIGMAFAST protease inhibitor cocktail tablets, Sigma-Aldrich). The fluid was centrifuged at 12,000 × g for 10 min at 4 °C, and the supernatant was collected.

[0240] Results: The inclusion of a second linker was effective for the efficient synthesis of amphiphile-MD39 trimer conjugates. MD39 bearing a C-terminal cysteine ​​was first reacted with DBCO-PEG4-maleimide linker to form intermediate product DBCO-PEG4-MD39, which was then subjected to click chemistry reaction with functionalized lipid DSPE-PEG2K-azide to form final product amph-MD39 (Figure 17A-17B). The intermediate product DBCO-PEG4-MD39 was clearly identified by UV-Vis spectrophotometer by the coexistence of MD39 peak at 280 nm and DBCO peak at 309 nm, and the post-click amph-MD39 product was identified by the absence of MD39 peak at 280 nm and DBCO peak at 309 nm, indicating the reaction was complete (Figure 18).

[0241] Intranasal immunization with amph-MD39 induced significantly higher serum and mucosal antigen-specific antibody responses compared to soluble MD39 protein (FIG. 19B). Amph-MD39 induced significantly higher serum IgG at weeks 7 (p<0.0001), 9 (p<0.0001), and 11 (p<0.01) and significantly higher vaginal mucosal IgA at weeks 9 (p<0.05), 11 (p<0.001), and 22 (p<0.01) after priming compared to soluble MD39. These results indicate that intranasal immunization with amphiphile conjugates of larger proteins such as MD39 trimers (approximately 10-fold larger than eOD ​​or RBD monomers) can induce potent antibody responses in both serum and urogenital mucosa.

[0242] This immunization strategy can be used to elicit broadly neutralizing antibodies against HIV at clinically relevant sites of infection, such as the urogenital mucosa, by combining amphiphile conjugates with natural trimeric immunogens known to elicit broadly neutralizing antibodies to enhance transmucosal uptake.

[0243] Example 9: Discussion It has previously been demonstrated that linking peptide antigens to amphiphilic lipid tails promotes albumin-mediated transport into lymphatics after parenteral injection, thereby enhancing antigen-specific T cell responses important for cancer immunity [25, 27, 29]. Here, it was surprisingly found that this strategy can be used for much larger protein immunogens associated with humoral immunity, and that by exploiting another natural transport mechanism of endogenous albumin, its ability to be transported across mucosal epithelia by the neonatal Fc receptor (FcRn), “albumin hitchhiking” can be applied to significantly enhance intranasal delivery of immunogens [31, 42]. Amph proteins were shown to persist for extended periods in nasal tissues after intranasal administration in both mice and non-human primates. In mice, it was demonstrated that this persistence is associated with increased transport across mucosal barriers and increased uptake into the NALT, a secondary lymphoid organ located dorsal to the soft palate beneath the nasal cavity in rodents, and similar to Waldeyer's ring in primates and humans

[16] . In mice, the NALT is composed of localized aggregates, whereas in primates, Waldeyer's rings are more abundantly composed of tonsils and adenoids [15, 58]. Importantly, the NALT, tonsils, and adenoids all serve as important sites for the initiation and coordination of local mucosal antigen-specific immune responses [3, 59, 60]. A significant increase in germinal center B cell and Tfh cell responses was observed in the NALT after intranasal immunization with amph-conjugated immunogens when compared to free protein. This increase in antigen delivery and local immune priming correlated with a significant enhancement of systemic IgG and IgA responses, as well as mucosal antibody responses, in both mice and nonhuman primates. Amph modification of protein immunogens allowed us to induce strong serum IgG responses in conjunction with strong mucosal IgA responses by intranasal immunization. This is of great interest, as many infectious diseases, such as SARS-CoV-2, influenza, rotavirus, and cholera, are thought to require a combination of mucosal IgA and serum IgG antibodies for optimal protection [1-7]. Therefore, the ability to activate both systemic IgG and mucosal IgA may be of value in a variety of vaccines.

[0244] Amph proteins overcome a major obstacle to mucosal vaccine development: the delivery of underlying antigens across mucus and epithelial barriers to the mucosal immune compartment [18, 19]. In addition to an efficient mucociliary clearance mechanism, mucosal surfaces are lined with an epithelial monolayer formed by tight junctions between cells, which prevent the uptake of macromolecules by diffusion

[61] . Thus, transport of molecules through the nasal epithelium is thought to be limited to active transport of small soluble proteins by goblet cells [22, 62] and transport of larger inert particles by differentiated microfold cells (M cells). Similar to Peyer's patches in the gut, M cells are also found inside the nasal cavity, present in both the nasal turbinate epithelium and the follicle-associated epithelium lining the NALT, where they sit atop the subepithelial dome (SED) of organized mucosal lymphoid tissue and function as "antigen delivery cells" [16, 63, 64]. Here, M cells acquire antigens from the nasal cavity, transcytose it through the submucosal epithelium, and then pass the antigen to underlying DCs, macrophages, B cells, and other APCs within the SED. After intranasal administration, a significant amount of amph-eOD was observed to be concentrated in the nasal turbinates, allowing M cell capture and transcytosis to serve as another mechanism for intranasal amph-eOD uptake [62, 65]. However, FcRn-expressing columnar epithelial cells are much more abundant than M cells in the respiratory mucosa

[62] . This, combined with data showing that amph-eOD uptake and immune responses are clearly dependent on FcRn, indicates that FcRn-mediated transcytosis is a more efficient route of antigen delivery in the nasal mucosa. Albumin-bound amph antigens transcytosed by airway epithelial cells can be released at the basolateral surface and subsequently taken up by the underlying APCs. Interestingly, APCs such as macrophages, DCs, and B cells, where the highest amph-eOD uptake was observed, also express high levels of FcRn

[66] .

[0245] FcRn is recognized to play a role in recycling and half-life extension of IgG and albumin and has received increasing attention as a means to alter drug delivery and pharmacokinetics [30, 31, 67]. To date, the focus has been primarily on developing engineered therapeutic monoclonal antibodies with altered FcRn binding affinity (i.e., Fc fusions), or drug-albumin fusions, to take advantage of FcRn-mediated recycling in the blood and extend serum half-life by increasing overall molecular weight and decreasing renal clearance rate. More recently, the FcRn transcytosis pathway has been investigated for non-invasive protein delivery via FcRn-mediated transcytosis [43, 68–70]. For example, Pridgen et al. compared FcRn-targeted versus non-targeted nanoparticles as a means of oral delivery of encapsulated insulin through the intestinal mucosa in mice and observed approximately 10-fold higher uptake across the intestinal epithelium

[71] , while Bern et al. found that engineered albumin-protein fusions with improved FcRn binding showed enhanced uptake across the nasal epithelium and extended serum half-life in mice

[43] .

[0246] More directly relevant to the present work, Roopenian and Zhu demonstrated that fusions of protein antigens with antibody Fc domains could enhance intranasal vaccination against HSV-2

[72] and HIV gag

[73] . These antigen-Fc fusions enhanced systemic antibody and T cell responses to intranasal immunization, as well as mucosal antibodies in BALF and vaginal fluid, but to our knowledge, the efficacy of this approach has not been evaluated in large animal models. An important difference between approaches that exploit FcRn interactions alone and the amphivaccine approach studied here is that Fc or albumin fusions administered to airway surfaces are not only delivered to local mucosal lymphoid tissues, but also reach the systemic circulation and subsequently exhibit the circulation time in blood seen for antibodies / albumin. This motivated the use of Fc and albumin fusions for the delivery of systemic therapeutics such as erythropoietin [69, 70]. However, such a wide distribution is problematic for vaccines: vaccine adjuvants are designed to provide a very local inflammatory trigger to avoid systemic toxicity, but if antigens coadministered with these adjuvants do not remain local, competing tolerogenic responses may occur in non-inflamed distal lymphoid tissues such as lymph nodes and spleen

[74] . In contrast, the lipid tails of amphiphile conjugates promote cell membrane interactions and prevent systemic diffusion of these conjugates. Here, we observed a local stimulation of immune responses after intranasal administration of amph proteins, which activated responses in NALT but did not reach even the nearby draining cervical LNs and did not accumulate in tissues such as the spleen, liver, and intestine, indicating negligible systemic distribution.

[0247] The development of amph-RBD COVID vaccines demonstrated the ability of this amph protein vaccine platform to induce functional neutralizing antibody responses at mucosal sites of respiratory pathogen entry. Clinical studies have shown that mucosal IgA strongly correlates with protection against SARS-CoV-2 [6, 13, 14], but so far, most COVID vaccines have not focused on targeting mucosal tissues, and few vaccines have been shown to induce functional neutralization at mucosal sites [53, 75, 76]. Amph-RBD immunization induced significant IgG and IgA antibody responses, including nAbs, in both the serum and upper and lower respiratory tract mucosa of mice. Thus, intranasal amph-RBD vaccination is a promising approach to induce mucosal protection against COVID. Furthermore, needle-free mucosal vaccination offers practical advantages over parenteral vaccination when mass vaccination is required, such as in the current global COVID-19 pandemic, namely ease of administration, administration without the need for medically trained personnel, improved compliance, and avoiding the risk of spreading blood-borne infections due to needle contamination, all of which leads to improved vaccination coverage

[77] .

[0248] A limitation of these studies is the inherent challenge of immunological differences between animal models and humans. In mice, immunization with amph proteins not only induced strong local mucosal Ig responses, but also stimulated long-lived, high-titer IgG and IgA at distal mucosal sites in the vagina and rectum, accompanied by the generation of resident antibody-secreting cells. In contrast, intranasal immunization with amph proteins in nonhuman primates induced enhanced systemic and nasal IgG and IgA responses compared with soluble protein administration, but did not sustain distal mucosal responses in the vaginal tract and rectum. However, such “common mucosal immunity” has been reported in small studies in macaques [1, 78–81] and humans. For example, intranasal immunization with cholera toxin B (CTB), a potent mucosal adjuvant, led volunteers to demonstrate antibody responses in urine or vaginal secretions [82, 83]. Although CTB has not been advanced as an intranasal adjuvant due to the risk of causing Bell's palsy

[82] , these data suggest that with the appropriate adjuvant it can elicit distal mucosal responses in humans. Despite this limitation, the robust systemic and local mucosal antibody priming observed in NHPs after intranasal amph protein administration combined with the saponin adjuvant SMNP, an adjuvant currently in GMP development for first-in-human clinical trials, suggests that this approach may be valuable for human vaccines.

[0249] Collectively, these results indicate that delivery of antigens across mucosal epithelia using amphiphile-protein vaccines represents a promising strategy for promoting mucosal immunity against HIV, SARS-CoV-2, and other infectious diseases.

[0250] Example 10: Materials and Methods Study Design. The primary objective of this study was to evaluate the effect of modification of protein antigens with amphiphilic PEG-lipid tails on systemic and mucosal immune responses elicited by intranasal vaccination in small and large animal models and to define the mechanism of action underlying the action of these modified immunogens. Mice and non-human primates were immunized with clinically relevant subunit protein immunogens combined with saponin or alternative adjuvants, and early local responses (antigen uptake, T cell priming, and germinal center induction) as well as subsequent events (serum and mucosal antibodies, plasma blasts, plasma cells) responses were evaluated over time. Mechanistic studies utilized fluorescently labeled proteins and gene knockout mouse models that allow immunogen trafficking in tissues to dissect key pathways of the immune response.

[0251] Immunogen synthesis and characterization HIV eOD. eOD-GT8 gp120 protein was synthesized as previously described [84, 85]. eOD protein with a free N-terminal cysteine ​​and a C-terminal PADRE universal helper T cell epitope (AKFVAAWTLKAAA) was expressed in HEK cells and purified on a nickel affinity column followed by size exclusion chromatography on a Superdex75 10 / 300 column (GE Healthcare). eOD gp120 monomer (PADRE epitope is italicized and underlined): MW 21.787 kDa HHHHHHGGDTITLPCRPAPPPHCSSNITGLILTRQGGYSNDNTVIFRPSGGDWRDIARCQIAGTVVSTQLFLNGSLAEEEVVIRSEDWRDNAKSICVQLNTSVEINCTGAGHCNISRAKWNNTLKQIASKLREQYGNKTIIFKPSSGGDPEFVNHSFNCGGEFFYCDSTQLFNSTWFNSTGS AKFVAAWTLKAAA (SEQ ID NO:1) SARS-CoV-2 RBD. The modified RBD protein ("RBD-L452K-F490W") was produced in Komagataella phaffii (Pichia pastoris). The strain was grown in 200 mL flask cultures and the secreted protein was purified as described above

[57] . The RBD was genetically modified to contain an N-terminal cysteine ​​residue for amphiphile binding. SARS-CoV-2 RBD monomer: MW 22.684kDa CITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYKYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYWPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTN (SEQ ID NO: 2)

[0252] HIV MD39 SOSIP: MD39 SOSIP is an HIV native-like trimeric antigen (JM Steichen et al., Science. 366 (2019), the entire contents of which are incorporated herein by reference). The molecular weight (MW) of the MD39 SOSIP trimer is approximately 217.018 kDa. Monomer sequence of MD39: (monomer MW is 72.339 kDa) (SEQ ID NO:3)

[0253] Conjugation and labeling of amphiphiles eOD and RBD protein antigens bearing N-terminal cysteines (≥1 mg / ml) were first reduced with 10 molar equivalents of tris(2-carboxyethyl)phosphine (TCEP) for 15 min at 25°C. TCEP was removed by centrifugal filtration using a 10 kDa MWCO Amicon spin filter while the proteins were washed three times with PBS. Proteins (1–5 mg / ml) were then reacted with 4 equivalents of dry DSPE-PEG2K-maleimide (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000]) (Avanti Polar Lipids) in PBS for 2 h at 25°C with intermittent vortexing, followed by gentle mixing for 18 h at 4°C. Protein amphiphiles were purified by size-exclusion chromatography (SEC) using a Sepharose CL6B (Sigma-Aldrich) gravity column eluted with PBS. The bound protein amphiphilic micelle and unbound protein peaks were detected by tryptophan fluorescence (excitation: 280 nm / em: 340 nm). Peak micelle fractions were pooled, concentrated by centrifugal filtration using 10 kDa MWCO Amicon spin filters, and quantified by UV-Vis spectrophotometry (Nanodrop One, Thermo Scientific). Particle size was characterized by dynamic light scattering (Zetasizer Nano, Malvern).

[0254] Labeled eOD protein and protein amphiphiles were prepared using AF647 NHS ester (ThermoFisher Scientific) by reacting the fluorescent dye with eOD or amph-eOD (≥1 mg / ml) in 0.1 M sodium bicarbonate buffer for 1 h at 25 °C, according to the manufacturer's instructions. VRC01 was synthesized as previously described

[86] . Labeled VRC01 was prepared by reacting the fluorescent dye with human VRC01 (≥1 mg / ml) in PBS for 1 h at 25 °C, using Pierce NHS Rhodamine (ThermoFisher Scientific) according to the manufacturer's instructions. Labeled proteins were purified by centrifugal filtration using 10 kDa Amicon spin filters, and the degree of labeling (DOL) was characterized by UV-Vis spectrophotometry and confirmed to be ≥1.0.

[0255] Adjuvants. The STING agonist adjuvant bis(3'-5')-cyclic dimeric guanosine monophosphate (cdGMP) was purchased from InvivoGen. Saponin MPLA nanoparticle adjuvant (SMNP) was synthesized as previously described

[87] .

[0256] Albumin binding: affinity chromatography. Albumin binding of the conjugates was assessed using albumin-immobilized agarose affinity chromatography as previously described

[25] . Pierce NHS-activated agarose resin (ThermoFisher Scientific) was functionalized with albumin by adding 26.4 mg of BSA in 4.4 ml of PBS directly to 330 mg of agarose according to the manufacturer's instructions. The resin reaction was mixed for 1 h at 25 °C and then overnight at 4 °C before being quenched with 1 M Tris-HCl (pH 8.0) followed by extensive washing with PBS. AF647-labeled eOD or amph-eOD was then applied to the albumin-functionalized resin (0.3 μM final concentration in 2 ml column volume) and incubated at 37 °C for 2 h with end-over-end mixing. The eluate was collected after centrifugation of the column at 1000 × g for 2 min. The amount of protein or amphiphile conjugate retained on the column was determined by measuring the AF647 fluorescence (640 / 670 nm) of the eluate relative to the starting sample in a fluorescence plate reader and normalizing to the DOL.

[0257] Membrane insertion into splenocytes. Insertion of amphiphiles into cell membranes was evaluated in vitro in mouse splenocytes isolated from naive mice. Single cell suspensions were incubated at 5x106 cells / ml (1x106 cells / well in 96-well plates) in cRPMI (RPMI-1640+10% FBS+1% penicillin / streptomycin) containing 25, 100 or 250 nM AF647-eOD or AF647-amph-eOD for 1 h at 37°C. Cells were washed once with PBS and stained with Live / Dead Aqua (Invitrogen) 1:1000 in 100 μl PBS for 15 min at 25°C, washed once with FACS buffer (PBS+1% BSA) and then stained with Rhodamine-VRC01 at 1.0 μg / 106 cells in 100 μl FACS buffer for 30 min at 4°C. Cells were then washed twice, fixed with 2% paraformaldehyde, and stored at 4°C until flow cytometry analysis on a BD LSR Fortessa.

[0258] Measurement of albumin-neonatal Fc receptor (FcRn) binding. To measure FcRn binding, 96-well enzyme-linked immunosorbent assay (ELISA) plates (Corning, #3690) were coated with 5.0 μg / ml streptavidin in phosphate-buffered saline (PBS), incubated at 25° C. for 4 hours, and blocked. Plates were washed with 1% casein in PBS (G-biosciences, 786-194) for 18 hours at 4° C., then washed three times with PBS+0.05% Tween® 20 (pH 5.5). Biotinylated human FcRn (ACRO Biosystems, FCMH82W4) was added at 5 μg / ml in 1% casein in PBS (pH 5.5) and incubated at 25° C. for 2 hours before washing. Human albumin (Sigma, A3782, serial dilutions 5–0 μg / ml) was preincubated with fluorescein isothiocyanate (FITC)-labeled 1,2-distearoyl sn-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol)-2000] (DSPE-PEG2K-FITC, Creative PEGworks, PLS-9927, serial dilutions 10–0 μM) in 1% casein in PBS (pH 5.5) for 2 h at 25 °C, then added to the FcRn-coated plates and incubated for another 2 h at 25 °C. Goat anti-human albumin antibody, horseradish peroxidase (HRP)-conjugated (Bethyl Laboratories, A80-129P), diluted 1:3000 in 1% casein in PBS, was added and incubated for 30 min at 25 °C. After washing the plate three times, tetramethylbenzidine (TMB) substrate (Thermo Fisher Scientific, 34028) was added, followed by 2N H2SO4 as a stop solution. The absorbance was measured at 450 nm.

[0259] Mouse studies Animal strains. All procedures were in accordance with local, state, and federal regulations and approved by the Massachusetts Institute of Technology Institutional Animal Care and Use Committee (IACUC). Immunologic studies were performed using age-matched 8-10 week-old female BALB / cJ mice (strain 000651), C57BL / 6J mice (strain 000664), or FcRn- / - mice on a C57BL / 6J background (strain 003982) purchased from The Jackson Laboratory.

[0260] Trafficking in IVIS. In vivo transport of AF647-labeled amph-eOD and eOD was assessed after intranasal administration using an IVIS fluorescent imaging system (Perkin Elmer). To eliminate background autofluorescence in the gut, mice were fed an alfalfa-free diet (AIN-93M, Bio-Serv) from 3 days prior to immunization and throughout the study period. BALB / c mice were intranasally immunized with 5 μg of AF647-amph-eOD or AF647-eOD combined with 5 μg of SMNP and compared to naive controls. Intranasal immunizations were administered by instillation of 20 μl of PBS (10 μl per nostril, 30–60 s nostril interval) into anesthetized mice in the supine position. After administration, mice were kept anesthetized for a minimum of 5 min in the supine position to allow uptake and prevent drainage. At 24 h, 48 h, 72 h, 7 days, and 11 days after immunization, the nasal cavity (from the nose tip to the mandible), cervical lymph nodes, intestine, mesenteric lymph nodes, liver, and spleen were excised and AF647 fluorescence (radiant efficiency) was measured by IVIS. The nasal cavity was imaged by removing the head from the mouse body and then dissecting the mandible from the nose and discarding it. Images were collected of the inferior ventral surface of the upper palate (Figure 2A, (i)).

[0261] To evaluate the FcRn dependency of amphiphile transport in the nasal mucosa, FcRn- / - mice were immunized intranasally with 5 μg AF647-amph-eOD combined with 5 μg SMNP and compared to WT mice (C57BL / 6J) immunized intranasally with 5 μg AF647-amph-eOD or AF647-eOD combined with 5 μg SMNP. At 6, 24, and 72 hours post-immunization, nasal cavities were isolated as described above and AF647 fluorescence (radiant efficiency) was measured by IVIS.

[0262] Histology and fluorescence microscopy of the nasal epithelium. Nasal samples from FcRn- / - and C57BL / 6 mice were processed histologically by FFPE (formalin-fixed paraffin embedding) as follows: samples were fixed in 10% neutral buffered formalin (NBF) for 24 h at 25 °C, then transferred to 70% ethanol and stored at 4 °C. Fixed samples were decalcified in 10% EDTA disodium salt dihydrate (Sigma) at pH 7.4 for 10 days at 4 °C, changing the EDTA solution every 3 days. Decalcified tissues were embedded in paraffin and sliced ​​into approximately 5 μm coronal sections using a microtome, starting at 1 mm from the nostril and proceeding to a depth of 7.5 mm at 500 μm step intervals throughout the nasal cavity. Sections located 1.5–3 mm from the nostril were identified as the main sites of vaccine deposition for detailed imaging (Figure 2A, (ii)). Slices were mounted on glass slides and stained with DAPI using Vectashield HardSet Antifade Mounting Medium (Vector Laboratories) and then imaged using a Leica SP8 laser scanning confocal microscope equipped with a 25x water objective or a 63x oil objective. Images were processed with ImageJ.

[0263] ELISA for albumin quantification. To assay albumin concentrations in the nasal mucosa, nasal washes were collected from C57BL / 6 or FcRn mice as described above. Albumin concentrations in nasal secretions were measured using a commercially available mouse albumin ELISA kit (Abcam, catalog no. ab207620) according to the manufacturer's instructions.

[0264] Flow cytometric analysis of NALT uptake. BALB / c mice were immunized intranasally with 10 μg AF647-eOD or AF647-amph-eOD in combination with 5 μg SMNP. One and four days later, mice were euthanized and the upper palate was excised to isolate NALT

[88] and processed into a single cell suspension as follows: the upper palate was enzymatically and mechanically digested in 1 ml RPMI-1640 containing 0.8 mg / ml collagenase / dispase (Roche) and 0.1 mg / ml DNase (Roche) by first cutting into <1 mm chunks using fine-tip spring scissors and then crushing in a 1.5 ml biomasher tube (Kimble). After 15 min incubation at 37° C. with shaking, the supernatant was removed and added to 10 ml FACS buffer (PBS+1% BSA) at 4° C., and the remaining tissue was subjected to a second round of digestion in 1 ml fresh enzyme mix for another 15 min at 37° C., after which the supernatant was removed and added again to cold FACS buffer, which was centrifuged at 500×g for 5 min to pellet the cells, washed once with FACS buffer, passed through a 70 μm filter, and finally centrifuged and resuspended in FACS buffer in a Vbottom plate for antibody staining.

[0265] Cells were washed with PBS and first stained with Live / Dead Near-IR (Invitrogen) at 1:500 in 100 μl PBS for 15 min at 25°C, and then treated with anti-mouse CD16 / 32 Fc block (TruStain FcX, BioLegend) at 1:100 in 50 μl FACS buffer for 10 min at 4°C. To identify the different cell populations that had taken up the vaccine, cells were stained for 30 min at 4°C with the following antibodies diluted 1:100 in 50 μl of FACS buffer: anti-mouse CD3ε APC-Cy7 (clone 145-2C11, BioLegend), B220 PerCP-Cy5.5 (RA3-6B2, BioLegend), CD45 BUV737 (30-F11, BD Biosciences), MHCII BV605 (M5 / 114.15.2, BioLegend), CD11b BV421 (M1 / 70, BioLegend), CD11c BV510 (N418, BioLegend), F4 / 80 BV711 (BM8, BioLegend), CD103 PE (2E7, BioLegend), CD8α. BV786 (53-6.7, BD Biosciences), and CD169 PE-Cy7 (3D6.112, BioLegend). Cells were fixed with 2% paraformaldehyde and stored at 4°C until flow cytometry analysis. Counting beads (Invitrogen) were added before running on the BD LSR Fortessa.

[0266] Flow cytometry analysis of NALT GC B cell and Tfh cell responses. FcRn- / - and C57BL / 6 mice were immunized intranasally with 5 μg eOD or amph-eOD in combination with 5 μg SMNP. After 12 days, mice were euthanized and NALT were isolated and processed as described above. Cells were washed with PBS and first stained with 1:500 Live / Dead Aqua (Invitrogen) in 100 μl PBS for 15 min at 25°C, then treated with 1:100 anti-mouse CD16 / 32 Fc block (TruStain FcX, BioLegend) in 50 μl FACS buffer for 10 min at 4°C. To identify eOD-specific GC B cells, half of the cells from each NALT sample were stained in 50 μl of FACS buffer for 30 min at 4 °C with the following panel: anti-mouse CD3ε BV711 (clone 145-2C11, BioLegend) at 1:200, B220 PE-Cy7 (RA3-6B2, BioLegend) at 1:200, CD38 FITC (90, BioLegend) at 1:200, GL7 PerCP-Cy5.5 (GL7, BioLegend) at 1:150, eOD-tetramer PE at 1:100, and eOD-tetramer BV421 at 1:50. Fluorescently labeled eOD tetramers were prepared by first reacting eOD with maleimide-PEG2-biotin (ThermoFisher) according to the manufacturer's instructions and then conjugating 5 molar equivalents of biotinylated eOD with 1 equivalent of streptavidin-PE or streptavidin-BV421 (BioLegend) for 30 min at 25°C.To identify Tfh cells, half of the cells from each NALT sample were stained for 30 min at 4 °C with the following antibodies in 50 μl FACS buffer: anti-mouse B220 BV510 (clone RA3-6B2, BioLegend) at 1:200, CD4 BV711 (GK1.5, BioLegend) at 1:200, CD44 PE-Cy7 (IM7, BioLegend) at 1:200, ICOS PE (7E.17G9, BioLegend) at 1:100, PD-1 BV650 (J43, BD Biosciences) at 1:50, and CXCR5-biotin (2G8, BD Biosciences) at 1:50, followed by streptavidin-BV421 (BioLegend) at 1:100.

[0267] Mouse immunization and sample collection. BALB / c mice were immunized intranasally as described above. Mice were primed on day 0 and boosted on days 28 or 42 with a 5 μg dose of eOD or RBD combined with 25 μg cdGMP or 5 μg SMNP adjuvant, as indicated.

[0268] For long-term immune monitoring, blood and mucosal samples were collected every 2 or 3 weeks for ELISA or PVNT antibody analysis, as indicated. Blood was collected by buccal or retroorbital bleed, serum was isolated using serum separator tubes, and supernatants were collected by centrifugation at 10,000 x g for 5 min. Vaginal mucosal fluid was collected from anesthetized mice by vaginal lavage using 75 μl of sterile PBS (3 × 25 μl drops, 3–5 aspirations each) combined with 5 μl of 25X protease inhibitor (EDTA-free SIGMAFAST protease inhibitor cocktail tablets, Sigma). Fluids were centrifuged at 12,000 x g for 10 min at 4 °C to collect supernatants. Fecal washes were collected from mouse fecal pellets (four pellets of approximately 0.75 cm per mouse) combined with 300 μl of 1X protease inhibitor, samples were vortexed and incubated at 4 °C for 1 h, vortexed a second time, and then centrifuged at 13,000 x g at 4 °C for 15 min to collect the supernatant. Saliva washes were collected by injecting 30 μl of sterile PBS between the mouse cheek and the gum line (aspirated 3-5 times), repeated on both sides, and then mixed with 10 μl of 2X protease inhibitor. All fluid samples were stored in aliquots at -80 °C for future analysis.

[0269] After euthanasia, bone marrow (BM) and female reproductive tract (FRT) tissues were harvested to assess immune memory and resident plasma cell responses in the vaginal mucosa.

[0270] FRTs were isolated from the vaginal opening to the ovaries, cut into 1-3 mm chunks using fine-tipped spring scissors, and digested with 2 ml / sample of RPMI-1640 containing 2 mg / ml collagenase D (Roche), 0.6 U / ml dispase II (StemCell Technologies), and 0.2 mg / ml DNase I (Roche) for 30 min at 37°C with shaking. Samples were then centrifuged at 500xg for 5 min to pellet the tissue and cells, the supernatant discarded, and resuspended in 2 ml of fresh digestion medium and incubated for an additional 30 min at 37°C with shaking. Digestion was quenched by adding an equal volume of RPMI-1640 containing 10% FBS and 1% penicillin / streptomycin. This solution and remaining tissue were further mechanically digested by passing through a 70 μm cell strainer using the plunger end of a 1 ml syringe, followed by centrifugation at 500 × g for 5 min and resuspension in 5 ml of ACK lysis buffer for 5 min at 4 °C to lyse residual RBCs. An equal volume of cRPMI was added to quench the ACK, and the samples were then centrifuged at 500 × g for 5 min, rinsed once with cRPMI, passed a second time through a 70 μm filter, centrifuged a final time, and resuspended in cRPMI for counting and further analysis (ELISPOT, flow cytometry).

[0271] In the RBD study, nasal washes and bronchoalveolar lavage fluid (BALF) were collected to assess mucosal antibody responses present in the upper and lower respiratory tract. Nasal washes were collected from 2x15μl instillations of PBS, one into each nostril (3-5x aspirates), combined with 10μl of 2X protease inhibitor. BALF was collected by instilling 2x1ml of sterile PBS into the lungs using a 24Gx catheter through the trachea. Both fluid samples were centrifuged at 12,000xg for 10 minutes at 4°C to collect the supernatants and stored at -80°C.

[0272] ELISA analysis of mouse antibody titers. Anti-eOD and anti-RBD IgG and IgA binding titers were measured by ELISA in mouse sera and mucosal samples (vaginal washes, fecal washes, saliva, nasal washes, and BALF). To capture eOD-specific antibodies from immunized mice, MAXIsorp (ThermoFisher) 96-well plates were directly coated with 2 μg / ml eOD antigen in PBS overnight at 4°C. To capture RBD-specific antibodies, Costar polystyrene high-binding 96-well plates (Corning) were directly coated with 2 μg / ml RBD antigen in PBS overnight at 4°C. Plates were then blocked with PBS+2% BSA for 2 h at 25°C. Mouse sera were diluted from 1:100 or 1:200 in blocking buffer (PBS+2% BSA) and mucosal samples were diluted from 1:10 in blocking buffer followed by 4-fold serial dilutions. For the eOD ELISA, 5 μg / ml of VRC01 was used as a positive control. For the RBD ELISA, 5 μg / ml of mAb CR3022 or Fc fusion protein ACE2-Fc were used as positive controls. Samples were incubated in the plates for 2 h at 25 °C and then detected with 1:5000 goat anti-mouse IgG-HRP (BioRad) or 1:2000 goat anti-mouse IgA-HRP (Invitrogen) in blocking buffer for 1 h. Plates were developed using TMB substrate for 1–20 min, stopped with 2 N sulfuric acid, and the resulting absorbance (A 450 / A 540 ) was measured on a plate reader. For all titer analyses, samples that were directly compared between groups were developed over the same time period. The cutoff titer was determined based on background HRP absorbance (A 450 ~A 540 ) is reported as the reciprocal dilution at which the concentration becomes 0.2 (RBD) or 0.1 (eOD).

[0273] ELISPOT analysis of mouse plasma cells. IgG and IgA plasma cells were analyzed in BM and FRT tissues from 35 or 52 weeks after priming as indicated, using PVDF-MSIP filter plates (0.45 μm high protein binding Immobilon-P membrane filter plates, Millipore) and the Mouse IgG / A ELISpot-BASIC kit (Mabtech). To quantify eOD antigen-specific IgG and IgA plasma cells, filter plates were coated with 10 μg / ml eOD in 100 μl of sterile PBS, incubated overnight at 4° C., and cells were seeded at 500,000 and 250,000 cells / well in 100 μl of cRPMI. To quantify the total amount of IgG and IgA plasma cells, filter plates were coated with 15 μg / ml anti-IgG (purified goat anti-mouse IgG capture antibody, Mabtech) or anti-IgA (monoclonal antibody MT45A, Mabtech) in 100 μl sterile PBS, incubated overnight at 4°C, and cells were seeded at 100,000 and 50,000 cells / well in 100 μl cRPMI. Plates were then incubated at 37°C for 18-20 h, spot detection was performed according to the manufacturer's instructions, and plates were read on a CTL ImmunoSpot Analyzer.

[0274] Parental control immunization of mice. To compare intranasal immunization with parenteral controls, BALB / c mice were immunized intranasally or subcutaneously in the scruff of the neck with 5 μg amph-eOD in combination with 25 μg cdGMP. Mice were primed on day 0 and boosted on day 42. Blood, vaginal, and fecal samples were collected at regular intervals as described above.

[0275] Anti-PEG antibody ELISA. Antibody responses to PEG contained in amph-protein conjugates were assayed by ELISA. Briefly, MaxiSorp ELISA plates were coated with 1 μg / mL streptavidin in PBS for 4 h at 25°C, blocked with PBS+2% bovine serum albumin (BSA) overnight at 4°C, and then washed three times with wash buffer (PBS with 0.2% Tween 20). Biotin-PEG-OH (Creative PEGWorks, cat.#PJK-1946) was added to the plates in blocking buffer (1 μg / mL) and incubated for 2 h at 25°C. After washing the plates three times with wash buffer, mouse serum samples and mouse anti-PEG IgG standard antibody (AffinityImmuno kit cat.#EL-141-PEG-mIGG, starting at 1 μg / ml followed by two-fold serial dilutions) were added and incubated for 2 h before washing. Anti-mouse IgG-HRP diluted 1:5000 in blocking buffer was used as the detection antibody. Samples were incubated for 1 h at 25°C, then washed and TMB substrate was added, followed by 2N H2SO4 as a stop solution. Absorbance was measured at 450 nm.

[0276] ACE2:RBD binding inhibition assay. Functional antibody inhibition of ACE2:RBD binding was measured in mouse serum and BALF as a preliminary indicator of neutralizing antibodies using a SARS-CoV-2 Surrogate Virus Neutralization Test Kit (Genscript) according to the manufacturer's instructions. Mouse serum was diluted from 1:10 and BALF was diluted 1:2, followed by 4-fold serial dilutions. Inhibition (IC50) was defined as the sample dilution at which a 50% reduction in ACE2:RBD binding was observed compared to the negative control (no inhibition).

[0277] Pseudovirus-based SARS-CoV-2 neutralization assay. SARS-CoV-2 pseudoviruses expressing a luciferase reporter gene were generated with a similar approach as described above [89, 90]. Briefly, the packaging plasmid psPAX2 (AIDS Resource and Reagent Program), the luciferase reporter plasmid pLenti-CMV Puro-Luc (Addgene), and the spike protein expressing pcDNA3.1-SARS CoV-2 SΔCT were co-transfected into HEK293T cells by Lipofectamine 2000 (ThermoFisher). The pseudovirus-containing supernatant was collected 48 h after transfection and purified by centrifugation and filtration through a 0.45 μm filter. To determine the neutralization activity of mouse serum and mucosal samples, HEK293ThACE2 cells were cultured at 1.75 × 10 4 Cells were seeded overnight in 96-well tissue culture plates at a density of 1000 cells / well. Samples (serum, saliva, nasal wash, vaginal wash, fecal wash, and BALF) were first heat-inactivated at 56°C for 30 min. 3-fold serial dilutions of heat-inactivated serum or mucosal samples were then prepared and mixed with 50 μL of pseudovirus. The mixtures were incubated at 37°C for 1 h before being added to HEK293T-hACE2 cells. After 48 h postinfection, cells were lysed with a Steady-Glo luciferase assay (Promega) according to the manufacturer's instructions. SARS-CoV-2 neutralization titers (NT50) were defined as the sample dilution at which a 50% reduction in relative light units (RLU) was observed compared to the average of virus control wells.

[0278] NHP research Animals. Six female Indian rhesus macaques (Macaca mulatta) were assigned to the IVIS trafficking study (n = 3 animals per group). Twelve 3-4 year old female Indian rhesus macaques were assigned to the longitudinal immune study (n = 6 animals per group). Macaques were distributed to be comparable in age, weight, and MHC genotype between groups. Animals were bred and maintained at the New Iberia Research Center (NIRC) at the University of Louisiana at Lafayette in accordance with the rules and regulations of the Guide for the Care and Use of Laboratory Animals. The entire study (protocol 8789-08) was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Louisiana at Lafayette. All animals were negative for SIV, simian T-cell leukemia virus, and simian retrovirus. Animals were also grouped by MHC, excluding animals expressing the Mamu B*008 or B*017 alleles, and distributing animals expressing the Mamu A*001 allele evenly between groups.

[0279] Trafficking in IVIS. In vivo transport of AF647-labeled amph-eOD and eOD was assessed after intranasal administration using an IVIS fluorescent imaging system (Perkin Elmer). Macaques were intranasally immunized with 100 μg of AF647-amph-eOD or AF647-eOD mixed with 375 μg of SMNPs by dropping 200 μl per nostril (total of 400 μl per animal) directly into each nostril. After administration, animals remained in supine position under anesthesia for 10 min to allow for vaccine uptake and prevent shedding. After 24 h, nasal tissues including tonsils, adenoids, cervical LNs, axillary LNs, and nasal turbinates were harvested and fixed in 4% paraformaldehyde for 5 days, then transferred to PBS + 0.1% PFA + 0.05% sodium azide and stored at 4°C before evaluation by IVIS.

[0280] Vaccination studies and sample collection. Animals were immunized intranasally with 100 μg amph-eOD or eOD mixed with 375 μg SMNP at weeks 0, 8, 16, and 24 as described above. For longitudinal immune monitoring, peripheral blood mononuclear cells (PBMCs) were collected by venipuncture from the femoral vein, then separated with Ficoll and stored frozen, except for those freshly used for plasma blast ELISPOT assays. Serum samples were stored at −80°C until ELISA analysis. Mucosal samples were collected using Merocel sponges, processed as previously described

[91] , and stored at −80°C until analysis.

[0281] ELISA analysis of NHP antibody titers. To measure eOD-specific antibody titers, MAXIsorp 96-well plates (ThermoFisher) were coated with 2 μg / mL gp120 eOD monomer in PBS. Serum samples were diluted 1:50 and mucosal washes 1:10 in 2% BSA blocking buffer, followed by 4-fold serial dilutions. 5 μg / ml hVRC01 was included as a positive control. Samples were incubated for 2 h at room temperature and then detected with 1:5000 goat anti-human IgG-HRP (Jackson ImmunoResearch) or 1:2000 goat anti-human IgA-HRP (ThermoFisher Scientific). Cutoff titers were determined based on background HRP absorbance (A 450 ~A 540 ) is reported as the reciprocal dilution at which the titer becomes 0.2 (IgA) or 0.1 (IgG).

[0282] ELISPOT analysis of NHP plasma cells. Total and antigen-specific plasma blast responses in peripheral blood were measured by ELISPOT assay as described above

[92] . Briefly, 96-well Multiscreen HTS filter plates (Millipore) were coated overnight at 4°C with 100 μl / well of goat anti-monkey IgG, IgM, or IgA antibodies (Rockland) at 5 μg / ml, or HIV eOD-gp120 at 1 μg / ml, respectively. Plates were washed with PBS-0.05% Tween 20 (PBS-T) and blocked for 2 h at 37°C with complete medium. Freshly isolated cells were plated in duplicate at serial 3-fold dilutions and incubated overnight at 37°C in a 5% CO2 incubator. Plates were washed with PBS-T and incubated for 1 h at 37°C with biotin-conjugated anti-monkey IgG, IgM, or IgA antibodies (Rockland) diluted 1:1,000. After washing, the plates were incubated with 1:1,000 diluted horseradish peroxidase (HRP)-conjugated streptavidin (Vector labs) for 2 hours at room temperature and developed using an AEC substrate kit (BD Biosciences). To stop the reaction, the plates were washed extensively with water and then air-dried. Spots were imaged and counted using an Immunospot ELISPOT Analyzer (Cellular Technology Limited). The number of spots specific for each Ig isotype was reported as the number of total antibody-producing cells or antigen-specific antibody-producing cells per million PBMCs.

[0283] statistical analysis Statistics were analyzed using GraphPad Prism software. When comparing more than two groups, one-way or two-way ANOVA was performed with α = 0.05, followed by Tukey or Sidak's post-hoc test as indicated. For comparing two groups, unpaired two-tailed t-tests were performed with α = 0.05. Statistical significance in amphiphilic membrane insertion experiments was determined using simple linear regression assessing the dependence of AF647 or VRC01 MFI on eOD ​​concentration to determine significant non-zero slopes. ACE2:RBD binding inhibition (IC50) was determined using sigmoidal 4PL non-linear regression. All graphs represent the mean ± standard error of the mean (sem) unless otherwise noted. Statistical significance is marked as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0284] Equivalent It is understood that the detailed examples and embodiments described herein are given by way of example for illustrative purposes only and are not to be considered as limiting the present invention in any way. Various modifications or other changes in light of these will be suggested to those skilled in the art and are within the spirit and scope of the present application and are considered to be within the scope of the appended claims. For example, the relative amounts of the components may be changed to optimize the desired effect, additional components may be added, and / or similar components may be substituted for one or more of the components described. Additional advantageous features and functionality 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 routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

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Claims

1. A vaccine comprising an amphipathic conjugate, wherein the amphipathic conjugate comprises an immunogen operably linked to an albumin-binding lipid, and the vaccine is suitable for transmucosal administration to induce a humoral immune response.

2. The vaccine according to claim 1, wherein the transmucosal administration is intranasal administration.

3. The vaccine according to claim 1, wherein the immunogen is a protein antigen having a molecular weight of about 10 kDa to about 500 kDa.

4. The vaccine according to claim 1, wherein the immunogen comprises a protein antigen selected from the group consisting of human immunodeficiency virus (HIV) antigen, SARS-CoV-2 antigen, influenza antigen, rotavirus antigen, cytomegalovirus (CMV) antigen, Epstein-Barr virus (EBV) antigen, respiratory syncytial virus (RSV) antigen, and cholera antigen.

5. The vaccine according to claim 1, wherein the immunogen comprises a monomeric antigen or a trimeric antigen.

6. The vaccine according to claim 1, wherein the immunogen comprises an antigen peptide.

7. The vaccine according to claim 1, wherein the albumin-binding lipid is selected from the group consisting of cholesterol, monoacyl lipids, and diacyl lipids.

8. The vaccine according to claim 7, wherein the albumin-binding lipid is a diacyl lipid.

9. The vaccine according to claim 7, wherein the albumin-binding lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

10. The vaccine according to claim 1, wherein the immunogen is operably linked to the albumin-binding lipid via a first linker.

11. The vaccine according to claim 10, wherein the first linker is selected from the group consisting of a hydrophilic polymer, a series of hydrophilic amino acids, polysaccharides, oligonucleotides, or a combination thereof.

12. The vaccine according to claim 11, wherein the first linker comprises a polyethylene glycol (PEG) linker.

13. The vaccine according to claim 12, wherein the first linker comprises 45 to 150 repeating units of PEG monomers.

14. The vaccine according to claim 10, wherein the first linker comprises a PEG2K linker.

15. The vaccine according to claim 10, further comprising a second linker, the second linker being located between the immunogen and the first linker, or between the albumin-binding lipid and the first linker.

16. The vaccine according to claim 15, wherein the second linker comprises a PEG linker.

17. The vaccine according to claim 16, wherein the second linker comprises 2 to 20 repeating units of PEG monomers.

18. The vaccine according to claim 17, wherein the second linker comprises a repeating unit of four PEG monomers.

19. The vaccine according to claim 16, wherein the second linker comprises a dibenzocyclooctin (DBCO) group covalently bonded to the repeating unit of the PEG monomer.

20. The vaccine according to claim 1, wherein the immunogen comprises an HIV antigen.

21. The vaccine according to claim 20, wherein the HIV antigen comprises HIV gp120 modified outer domain germline target immunogen 8 (eOD-GT8).

22. The vaccine according to claim 1, wherein the immunogen comprises the SARS-CoV-2 antigen.

23. The vaccine according to claim 22, wherein the SARS-CoV-2 antigen comprises an antigen derived from the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein.

24. The vaccine according to claim 1, further comprising an adjuvant.

25. The vaccine according to claim 24, wherein the adjuvant is selected from the group consisting of bis-(3'-5')-cyclic dimer guanosine monophosphate (cdGMP) and saponin monophosphoryllipid-A (MPLA) nanoparticle adjuvant (SMNP).

26. The vaccine according to claim 1, wherein transmucosal administration of the vaccine induces or enhances the production of antibodies that bind to the immunogen.

27. The vaccine according to claim 26, wherein the antibody comprises an IgA antibody, an IgG antibody, or both an IgA and an IgG antibody.

28. The vaccine according to claim 26, wherein the antibody is a neutralizing antibody.

29. A composition for administering vaccine to a target, wherein the composition comprises the vaccine described in claim 1, and is administered to the target via the mucous membrane.

30. A composition for immunizing a target, wherein the composition comprises the vaccine described in claim 1, and is administered to the target via mucosal administration.

31. The composition according to claim 29 or 30, characterized in that it is administered intranasally to the subject.

32. The composition according to claim 29 or 30, characterized in that it is administered in two or more doses.

33. The composition according to claim 32, characterized in that the dose of the composition is administered at intervals of about 2, 4, 6, or 8 weeks.

34. The composition according to claim 32, characterized in that it is administered at weeks 0, 8, 16, and 24.

35. The composition according to claim 29 or 30, characterized in that the vaccine is administered in a dose of about 5 μg to about 300 μg.

36. The composition according to claim 29 or 30, characterized in that the vaccine is administered in a dose of approximately 50 μg, 100 μg, or 150 μg.

37. The composition according to claim 29 or 30, characterized in that it is administered in combination with an adjuvant.

38. The composition according to claim 37, wherein the adjuvant comprises SMNP.

39. The composition according to claim 38, characterized in that the SMNP is administered in a dose of about 5 μg to about 500 μg.

40. The composition according to claim 39, characterized in that the SMNP is administered in a dose of approximately 300 μg, 375 μg, or 450 μg.

41. The composition according to claim 40, wherein the adjuvant comprises cdGMP.

42. The composition according to claim 41, characterized in that the cdGMP is administered in a dose of about 25 μg to about 500 μg.

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