Covalently modified antigens for improved immune response and / or stability

Abstract

Described herein are covalently modified polypeptide antigens with improved immunogenicity and / or stability, as well as compositions, cells, and methods related to the polypeptide antigens. The polypeptide antigens are covalently conjugated to one or more steroid acid moieties to improve their stability and / or to elicit improved cellular immunity or improved cellular and humoral immunity against the antigens after administration to a subject. The steroid acids include bile acids and bile acid analogs that enhance endosomal trapping of cargo by enhancing the enzymatic cleavage of endosomal membrane- ensheathed sphingomyelin to ceramide and / or endosomal escape. The steroid acid moieties can be preconjugated to a peptide, and the steroid acid-peptide moiety is subsequently conjugated to the polypeptide antigen. The peptide can comprise one or more domains that confer additional functionality to the modified polypeptide antigen.

Description

[0001] The present specification relates to covalently modified antigens to enhance or alter their immunogenicity and / or stability. More specifically, the present specification relates to polypeptide antigens covalently conjugated to one or more steroid acid moieties for improved cellular immunity and / or improved thermostability. BACKGROUND

[0002] While subunit vaccines based on polypeptide antigens are generally considered the safest vaccines, such antigens can not elicit a strong enough immune response to provide protective and long-lasting immunity. Furthermore, while the use of mRNA-based vaccines in response to the COVID-19 pandemic has garnered much attention, their relatively poor stability and stringent cold storage requirements are obstacles to their deployment on a global scale. Thus, methods to improve the immunogenicity, efficacy, and stability of polypeptide antigen-based vaccines would be highly desirable. SUMMARY

[0003] In a first aspect, described herein is a method of improving the immunogenicity and / or stability of a polypeptide antigen, the method comprising providing a polypeptide antigen to be modified, and covalently conjugating the polypeptide antigen to one or more steroid acid moieties to produce a modified polypeptide antigen. In some embodiments, the modified polypeptide antigen is conjugated to a sufficient number of steroid acid moieties to increase endosomal escape of the modified polypeptide antigen relative to the polypeptide antigen lacking the modification upon intracellular delivery, wherein the modified polypeptide antigen triggers an improved adaptive immune response to the polypeptide antigen upon administration to a subject as compared to the corresponding unmodified polypeptide antigen. In some embodiments, the modified polypeptide antigen is conjugated to a sufficient number of steroid acid moieties such that the modified polypeptide antigen exhibits greater stability than the polypeptide antigen prior to conjugation.

[0004] In a further aspect, described herein is a population of cells (e.g., in vitro or ex vivo) comprising a modified polypeptide antigen as described herein, or an immunogenic composition comprising: a modified polypeptide antigen and / or population of cells as described herein; and a pharmaceutically acceptable excipient and / or adjuvant.

[0005] In another aspect, described herein is a method for triggering an enhanced adaptive immune response in a subject against an unmodified polypeptide antigen of interest, the method comprising administering to the subject an immunogenic composition as described herein.

[0006] In another aspect, described herein is a method for treating or preventing a disease or disorder that can be treated by vaccination and / or immunotherapy, the method comprising administering to a subject an immunogenic composition as described herein.

[0007] In another aspect, described herein is a method for vaccinating a subject against an infectious disease, the method comprising administering to the subject an immunogenic composition described herein, wherein the polypeptide antigen comprises an antigenic fragment of a pathogen (e.g., virus, bacteria, fungus) that causes the infectious disease.

[0008] In another aspect, described herein is a method for treating or preventing a disease or disorder that can be treated by vaccination and / or immunotherapy, the method comprising administering to a subject an immunogenic composition as described herein.

[0009] In another aspect, described herein is a method for treating a cancer in a subject, the method comprising administering to the subject an immunogenic composition as described herein.

[0010] In another aspect, described herein is a modified polypeptide antigen as described herein for use in generating an immune response in a subject or for use in the manufacture of an immunogenic composition for generating an immune response in a subject.

[0011] In another aspect, described herein is a method for preparing a polypeptide antigen, the method comprising conjugating an unmodified polypeptide antigen to a sufficient number of steroid acid moieties to produce a modified polypeptide antigen that exhibits a higher stability (e.g., thermal stability) than the stability of the polypeptide antigen prior to conjugation.

[0012] General Definitions

[0013] Headings and other identifiers, e.g., (a), (b), (i), (ii), etc., are presented merely for convenience and do not necessarily limit the scope of the steps or elements to which they are presented.

[0014] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0015] As used in the specification and claims, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, e.g., "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0016] As used herein, the expression "consisting essentially of" or "consists essentially of" refers to those elements required for a given embodiment. The expression allows for the presence of additional elements that do not materially affect the basic and novel characteristic or functional characteristic of the embodiments of the application. In the context of the modified polypeptide antigens described herein, the expression "consisting essentially of" or "consists essentially of" refers to the elements required for improving the immunogenicity of the polypeptide antigen (e.g., by improving antigen presentation by professional antigen presenting cells) as compared to the unmodified antigen. For greater clarity, the expression does not exclude the possibility of other additional non-essential ingredients (e.g., excipients, fillers, stabilizers or inert components) that do not materially alter the function or ability of the steroid acid-peptide moiety to improve the immunogenicity of the polypeptide antigen.

[0017] The term "about" is used to indicate that a value includes the standard deviation of the error in the apparatus or method used to determine the value. In general, the term "about" is intended to encompass a possible variation of ±10% from the value. Accordingly, variations of ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, and ±10% from a given value are included within the scope of the term "about" as used herein. Unless otherwise stated, the use of the term "about" preceding a range applies to both ends of the range.

[0018] Other objects, advantages and features of the present specification will become more apparent upon reading the following non- restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0019] In the drawings:

[0020] Figure 1 shows the biochemical characterization of the ChAcNLS antigen formulation. Figure 1Ais a schematic representation of the covalent conjugation of a given antigen to the ChAcNLS moiety. Figure 1B Representative Coomassie blue staining showing unmodified OVA (lane 1), or ChAcNLS conjugated to OVA at a molar ratio of 25x (lane 2) or 50x (lane 3). Figure 1C The amino acid sequence of chicken OVA is shown. Lysine residues predicted to be accessible for ChAcNLS conjugation (>50%) are highlighted in black. Three lysine residues predicted to be weakly accessible are underlined. Figure 1D The ribbon structure of the OVA protein with lysine residues predicted to be highly (blue), moderately (green) or poorly (yellow) accessible is shown. Figure 1E Representative Western blot showing unmodified OVA (lane 1), ChAcNLS-OVA at a ratio of 25x (lane 2) and ChAcNLS-OVA at a ratio of 50x (lane 3). Figure 1F Intrinsic tryptophan fluorescence (ITF) analysis of nOVA or ChAcNLS-OVA (cOVA) at various ChAcNLS:OVA ratios in response to heat stress is shown. Figure 1G and Figure 1H The effect of various cOVA variants on the antigen-presenting efficacy of DCs is shown. Figure 1G Representative schematic of the different variants tested in Figure 1H Representative schematic of the different variants tested in Figure 1H Quantification of the response using the SIINFEKL-specific B3Z cell line co-cultured with DCs treated with different variants is shown. For this figure, n=5 / group, ***p<0.001 when compared to the nOVA group.

[0021] Figure 2 shows the results of the antigen cross-presentation assay. Figure 2A Antigen classical (MHC-II) and cross-presentation (MHC-I) assays for the evaluation of OVA-responsive OT-I (CD8) and OT-II (CD4) T cells are shown schematically. Figure 2B IFN-g produced by OT-I derived CD8 T cells incubated with DCs and either naked OVA (nOVA) or ChAcNLS-OVA (cOVA) is shown. Figure 2B IL-2 levels produced by OT-II derived CD4 T cells incubated with DCs and either naked OVA (nOVA) or ChAcNLS-OVA (cOVA) is shown. Figure 2D IFN-g production of the experiment of Figure 2C IFN-g production of the experiment of Figure 2E OVA-DQ TMResults of representative flow cytometry experiments for ChAcNLS-OVA-DQ (gray peak) versus cOVA-DQ (red peak) treatment. Figure 2F Quantification of the mean fluorescence intensity (MFI) of the OVA-DQ / ChAcNLS-OVA-DQ signal is shown. For this experiment, n=5 / group, ***p<0.001. Figure 2E Quantification of the mean fluorescence intensity (MFI) of the OVA-DQ / ChAcNLS-OVA-DQ signal is shown. For this experiment, n=5 / group, ***p<0.001. Figure 2G Representative experiment of Gal3-GFP expressing DC2.4 cells treated with nOVA (upper icon) versus cOVA (lower icon) is shown. White arrows point to some damaged endosomes.

[0022] Figure 3 shows syngeneic prophylactic vaccination against T cell lymphoma. Figure 3A is a schematic representation of the timeline for prophylactic vaccination using OVA protein. Figures 3B-3C Evaluation of tumor growth volume ( Figure 3B ) and survival rate ( Figure 3C ) of animals challenged with EG.7 tumors after prophylactic vaccination with DCs pulsed with naked OVA (nOVA; green) or ChAcNLS-OVA (cOVA; red) is shown. Non-immune mice injected with EG.7 are shown as "Ctl" (black). Figure 3D Antibody titers of vaccinated animals quantified by ELISA are shown. Figure 3E Quantification of central (T cm ) and effector (T eff ) CD4 T cells from mice immunized with nOVA / cOVA pulsed DCs derived from vaccinated animals from this study is shown. Figure 3F Quantification of central (T cm ) and effector (T eff ) CD8 T cells from mice immunized with nOVA / cOVA pulsed DCs derived from vaccinated animals from this study is shown. Figure 3G Luminex TM analysis of cytokine / chemokine production in response to in vitro restimulation of T cells isolated from vaccinated animals from this study. Cytokines / chemokines with the highest fold change are boxed. For Figure 3B , Figure 3C , Figure 3D and Figure 3E , n=10 / group, ***P<0.001.

[0023] Figure 4 shows immune evaluation after direct injection of OVA protein. Figure 4A is a schematic representation of the timeline for prophylactic vaccination using OVA protein with or without vaccine adjuvant.Figure 4B shows mean tumor measurements in animals immunized with naked OVA (green; nOVA) (1 pg), ChAcNLS-OVA (red; cOVA) (1 pg), cOVA (1 pg) with AddaS03 TM adjuvant (blue), and cOVA (1 pg) with AddaVax TM shows mean tumor measurements in animals immunized with cOVA (1 pg) with AddaS03 Figure 4C shows Figure 4A shows survival results for the experiments indicated. Figure 4D shows quantification of antibody titers from Figure 4A shows quantification of antibody titers from

[0024] Figure 5 shows therapeutic vaccination against T-cell lymphoma. Figure 5A is a schematic of the timeline for therapeutic vaccination. Figures 5B-5C shows tumor growth volume (A) and survival (B) of animals challenged with EG.7 tumors after syngeneic therapeutic vaccination with DCs pulsed with naked OVA (“nOVA”) or ChAcNLS-OVA (“cOVA”) alone, with anti-PD-1 (“aPD-1”), or without anti-PD-1. Non-immune mice injected with EG.7 are shown as “Ctl”. Figure 5B ) and survival (B) of animals challenged with EG.7 tumors after syngeneic therapeutic vaccination with DCs pulsed with ChAcNLS-OVA (“cOVA”) with or without anti-PD-1 at different cell numbers (3K, purple; 30K, blue; 100K, green; 300K, red) and with anti-PD-1 (black dashed line). Non-immune mice injected with EG.7 are shown as “Ctl”. Figure 5C ) and survival (B) of animals challenged with EG.7 tumors after syngeneic therapeutic vaccination with DCs pulsed with ChAcNLS-OVA (“cOVA”) with or without anti-PD-1 at different cell numbers (3K, purple; 30K, blue; 100K, green; 300K, red) and with anti-PD-1 (black dashed line). Non-immune mice injected with EG.7 are shown as “Ctl”. Figures 5D-5E Figure 5D Figure 5E Figure 6 shows tumor lysate-based therapeutic vaccination against T-cell lymphoma. is a schematic of the timeline for allogeneic therapeutic vaccination.

[0025] Figure 6A is a schematic of the timeline for allogeneic therapeutic vaccination. Figures 6B-6C ​​Evaluation of tumor growth volume (A) and survival rate (B) of animals challenged with EL4 tumors after immunization with BALB / c-derived allogeneic DCs pulsed with anti-PD-1 (black dotted line; "aPD-1"), EL4 lysate or EL4-ChAcNLS lysate ("clysate") with (EL4 lysate, purple; EL4-ChAcNLS lysate, red) or without (EL4 lysate, green; EL4-ChAcNLS lysate, blue) anti-PD-1 treatment (n=10 / group). Non-immunized mice injected with EG.7 are shown as "Ctl" (black). Figure 6B ) and survival rate ( Figure 6C ) of animals challenged with EL4 tumors after immunization with BALB / c-derived allogeneic DCs pulsed with anti-PD-1 (black dotted line; "aPD-1"), EL4 lysate or EL4-ChAcNLS lysate ("clysate") with (EL4 lysate, purple; EL4-ChAcNLS lysate, red) or without (EL4 lysate, green; EL4-ChAcNLS lysate, blue) anti-PD-1 treatment (n=10 / group). Non-immunized mice injected with EG.7 are shown as "Ctl" (black). Figure 6D A schematic representation of the experimental design of the tumor infiltrating lymphocyte (TIL) study is shown. Figure 6E Analysis of various immune cells in tumors of all groups shown in Figure 6B and Figure 6C is shown. Figure 6F Evaluation of CD8 / Treg ratio in tumors depicted in Figure 6B and Figure 6C is shown. For Figure 6B and Figure 6C , n=10 / group. For Figure 6E - Figure 6G, n=5 / group, 772*P<0.05, **P<0.01 and ***P<0.001.

[0026] Figure 7 shows the SARS-CoV-2 spike protein used for ChAcNLS-spike-CoV-2 formulations. Figure 7A A schematic representation of the ribbon structure of the SARS-CoV-2 spike protein (strain with D614G mutation) with lysine residues predicted to be highly (blue), moderately (green) or less (yellow) accessible is shown. Figure 7B The amino acid sequence of the SARS-CoV-2 spike protein is shown. Lysine residues predicted to be accessible for ChAcNLS conjugation (>50%) are highlighted in black. Lysine residues predicted to be weakly accessible are underlined.

[0027] Figure 8 shows the evaluation of the immunogenicity of ChAcNLS-spike-CoV-2 vaccines using different CoV-2 spike protein domains. Figure 8A Antibody titers of mice vaccinated with full-length "naked" spike-CoV-2 (unconjugated; nSpike-CoV-2; black bars) or ChAcNLS-spike-CoV-2 ("cSpike-CoV-2"; grey bars) in the presence of AddaS03 or AddaVax adjuvants are shown. Mice were given an additional booster injection at 17 weeks. IgG antibody titers were measured by ELISA.Figure 8B Different isotype titers from the study of Figure 8A Figure 8C Antibody titers in mice vaccinated with the S1-RBD portion of the CoV-2 spike protein, "naked" S1-RBD-CoV-2 (unconjugated; nS1-RBD-CoV-2; black bars) or ChAcNLS-S1-RBD-CoV-2 ("cS1-RBD-CoV-2"; grey bars) in the presence of AddaS03 or AddaVax adjuvant or injected alone are shown. IgG antibody titers were measured by ELISA. Figure 8D Antibody titers in mice vaccinated with the S2 portion of the CoV-2 spike protein, "naked" S2-CoV-2 (unconjugated; nS2-CoV-2; black bars) or ChAcNLS-S2-CoV-2 ("cS2-CoV-2"; grey bars) in the presence of AddaS03 or AddaVax adjuvant or injected alone at week 18 are shown. IgG antibody titers were measured by ELISA. Figure 8E Results of in vitro infection neutralization assays to assess the neutralizing capacity of the produced antibodies are shown. As NT 50 Titers show that antibodies isolated from mice immunized with cSpike-CoV-2 were more efficient in inhibiting viral infection of HEK cells compared to nSpike-CoV-2. For this figure, n=5 / group, *P<0.05, **P<0.01 and ***P<0.001.

[0028] Figure 9 shows the cytokine profile analysis by Luminex TM performed after in vitro T cell restimulation. Figure 9A Cytokine profile analysis using T cells derived from mice vaccinated with nSpike-CoV-2 containing two different adjuvants is shown. Figure 9B Cytokine profile analysis using T cells derived from mice vaccinated with cSpike-CoV-2 containing two different adjuvants is shown.

[0029] Figure 10 shows the evaluation of the immunogenicity of the vaccine in rabbits. Figure 10A A schematic representation of the experimental design is shown. In this experiment, three doses of cSpike-CoV-2 were tested. Figure 10B Antibody titers assessed for each 2 weeks collected serum are shown, n=3 / group, ***P<0.001.

[0030] Figure 11 shows the evaluation of the therapeutic efficacy of cSpike-CoV-2 in a challenge model. Figure 11A A schematic representation of the experimental design for the vaccine efficacy study in hamsters is shown.​Figure 11B This demonstrates a response to FDA-approved (GMP grade) MONTANIDE TM Antibody titers of cSpike-CoV-2 vaccines with ISA 720 VG adjuvant or AddaS03 mixture. Vaccines were tested using excess ratios of ChAcNLS with spike-CoV-2 protein (10X and 50X).

[0031] Figure 12 shows the evaluation of the cross-reactivity of the generated antibodies against various SARS-CoV-2 variants. Figure 12A The diagram shows the SARS-CoV-2 spike mutant used in this study and different mutations in the RBD domain. Figure 12B Antibody titers against different RBD domains are shown in serum from mice vaccinated with cSpike-CoV-2, with or without adjuvant. Figure 12C It shows the basis Figure 12B The percentage of cross-reactivity of the data shown with all test variants. Figure 12D The figure shows the neutralization levels obtained for various viral variants. The data shown in the figure are presented with n = 5 / group and *P < 0.05, **P < 0.01 and ***P < 0.001.

[0032] Figure 13 shows the Eurocine TM Evaluation of the immunogenicity of a cSpike-CoV-2 vaccine using an Indian (IN)CoV-2 spike protein variant (cSpike-CoV-2-IN) in the presence of adjuvant. Figure 13A A schematic diagram of the protocol for cSpike-CoV-2-IN vaccination is shown. Figure 13B and Figure 13C The IgG and IgA titers in the serum of mice vaccinated with cSpike-CoV-2-IN are shown separately compared to the saline control. For Figure 13C The samples collected in week 5 were analyzed. Figure 13D and Figure 13E The analysis of IgG and IgA titers in bronchoalveolar lavage fluid (BALF) of mice vaccinated with cSpike-CoV-2-IN at week 6 is shown. For this study, *P<0.05 and **P<0.01. Two-way ANOVA was applied to Figure B. One-way ANOVA (Bonferroni test) was performed for Figures C, D, and E.

[0033] Figure 14 shows a cytokine / chemokine analysis of the cSpike-CoV-2-IN vaccine using an Indian (IN)CoV-2 spike protein variant. The cytokine (Cokine) analysis is shown three days after in vitro spleen cell restimulation using the recombinant spike protein.Figure 14A ) and chemokines ( Figure 14B ) in response to Luminex TM Analysis. Depicted cytokines or chemokines with significant fluctuations compared to control (ctl; saline) animals are highlighted in gray. Units shown are pg / mL.

[0034] Figure 15 Cross-reactivity of serum-derived IgG from mice vaccinated with cSpike-CoV-2-IN using the Indian (IN) CoV-2 spike protein variant is shown with various CoV-2 spike protein variants. The original strain D614G spike protein was used as a comparison with the remaining variants. For this assay, n=5 / group, *P<0.05 against the original SARS-COV2 strain.

[0035] Figures 16A-16D Representative SDS-PAGE gels showing different bile acid-NLS-OVA conjugate preparations using 10X or 50X excess molar ratios of bile acid-NLS reactants to OVA antigen.

[0036] Figure 17 Effect of OVA antigen (0.1 mg / mL) conjugated with different types of bile acid-NLS moieties on antigen presentation to dendritic cells is shown. For this experiment, BMDCs were used as antigen presenting cells in a B3Z reporter system. Controls tested included no antigen (“PBS”) and antigen alone (i.e., unconjugated) (“OVA alone”; 5 mg / mL). OD 570 Levels represent OVA presentation levels, and the dashed line represents the signal obtained with the original ChAcNLS conjugated to OVA (“CA-SV40”). Bile acids: cholic acid (CA); glycodeoxycholic acid (GDCA); glycochenodeoxycholic acid (GCDCA): chenodeoxycholic acid (CDCA); ursodeoxycholic acid (UDCA); glyco ursodeoxycholic acid (GUDCA); deoxycholic acid (DCA); glycocholic acid (GCA); and lithocholic acid (LCA).

[0037] Figure 18 Effect of OVA antigen (0.1 mg / mL) conjugated with different types of bile acid-NLS moieties on antigen presentation to B cells is shown. For this experiment, isolated B cells were used as antigen presenting cells in a B3Z reporter system. Controls tested included no antigen (“PBS”) and antigen alone (i.e., unconjugated) (“OVA alone”; 5 mg / mL). OD 570 Levels represent OVA presentation levels, and the dashed line represents the signal obtained with the original ChAcNLS conjugated to OVA (“CA-SV40”). Bile acids: cholic acid (CA); glycodeoxycholic acid (GDCA);

[0038] Glycochenodeoxycholic acid (GCDCA): chenodeoxycholic acid (CDCA); urso- deoxycholic acid (UDCA);

[0039] Glycochenodeoxycholic acid (GCDCA): chenodeoxycholic acid (CDCA); urso- deoxycholic acid (UDCA);

[0040] Sequence Listing

[0041] The instant application contains a Sequence Listing in computer readable form created on November 1, 2021. The computer readable form is incorporated herein by reference.

[0042] SEQ ID NO: Description 1 ChAcNLS 2 Chicken egg white ovalbumin (OVA) 3 SARS-CoV-2 Spike glycoprotein (strain D614G) (NCBI Ref: 6XR8A) 4 SARS-CoV Spike glycoprotein (Uniprot P59594) 5 OVA OT-I (CD8) peptide 6 OVA OT-II (CD4) peptide 7 NLS from SV-40 large T antigen 8 GWG-SV40 NLS 9 hnRNP A1 M9 NLS 10 hnRNP D NLS 11 hnRNP M NLS 12 PQBP-1 NLS 13 NLS2 - RG domain RPS17 14 NLS1 RPS17 15 NLS2 RPS17 16 NLS3 RPS17 17 cMyc NLS 18 HuR NLS 19 Tus NLS 20 Nucleoplasmin NLS DETAILED DESCRIPTION

[0043] Described herein are compositions, cells, and methods relating to improving or altering adaptive immune responses to a polypeptide antigen and / or improving the stability of a polypeptide antigen. In some aspects, the present application stems from the demonstration herein that conjugation of a polypeptide antigen to a steroid acid moiety triggers improved cellular immunity, or improved cellular and humoral immunity, against the antigen. In some aspects, the present application stems from the demonstration herein that conjugation of a polypeptide antigen to a steroid acid moiety improves the stability of the polypeptide antigen (e.g., against heat stress). In some embodiments, the polypeptide antigens described herein can be covalently conjugated to a steroid acid moiety or to a steroid acid-peptide moiety via a functionalization linker. Advantageously, when a polypeptide antigen is conjugated to a steroid acid-peptide moiety, the peptide can be designed to comprise one or more domains that confer desired functionality (e.g., protein transduction and / or subcellular targeting) to the modified polypeptide antigen, which can further enhance immunogenicity.

[0044] In a first aspect, described herein is a method for improving the immunogenicity of a polypeptide antigen. The method generally comprises selecting / providing a suitable polypeptide antigen to be modified, and covalently conjugating the polypeptide antigen to a steroid acid moiety to produce a modified polypeptide antigen. In some embodiments, the modified polypeptide antigen is conjugated to a number of steroid acid moieties sufficient to increase the cellular and / or humoral immune response against the polypeptide antigen (e.g., as compared to the corresponding unmodified polypeptide antigen) upon administration to a subject. In some embodiments, the modified polypeptide antigen is conjugated to a number of steroid acid moieties sufficient to increase the endocytosis and / or endosomal escape of the modified polypeptide antigen upon intracellular delivery (e.g., as compared to the corresponding unmodified polypeptide antigen). In some embodiments, the modified polypeptide antigen triggers an improved adaptive immune response (e.g., an improved cellular and / or humoral immune response) against the polypeptide antigen upon administration to a subject as compared to the corresponding unmodified polypeptide antigen.

[0045] Polypeptide antigens are typically captured by antigen presenting cells (e.g., dendritic cells), but are initially entrapped in endosomes. Maturation of endosomes to lysosomes results in a decrease in pH and activation of proteolytic enzymes that mediate non-specific antigen degradation. Thus, some antigenic fragments that are produced can then pass through endosomal pores to the cytosol, where further antigen degradation occurs by proteasome machinery prior to MHC class I presentation. While this process occurs naturally, the resulting antigen fragments that ultimately exit the endosome can be small and / or damaged, rendering them unsuitable for proteasome degradation, thereby hindering their MHC class I presentation and thus cell-mediated immunity based on presentation. Without being bound by theory, the increased endosomal escape of the modified polypeptide antigens described herein can enable the antigens (or larger antigen fragments) to reach the cytosol in a more native conformation. Thus, proteasome degradation of these more native antigens can produce a greater number of immunogenic peptides and / or stable peptides presented at the surface of antigen presenting cells via MHC class I, thereby eliciting potent T cell activation.

[0046] As used herein, "polypeptide antigen" refers to an immunogenic peptide-linked chain of amino acids of any length, but typically at least 8, 9, 10, 11, or 12 amino acids long. For greater clarity, the polypeptide antigens referred to herein do not include antigen-binding antibodies or fragments thereof. As used herein, "protein antigen" refers to a polypeptide antigen of at least 50 amino acid residues in length, while "peptide antigen" refers to a polypeptide antigen of less than 50 amino acid residues in length. For greater clarity, the polypeptides, proteins, and peptides described herein can or can not contain any type of modification (e.g., chemical modification or post-translational modification, such as acetylation, phosphorylation, glycosylation, sulfation, sumoylation, prenylation, ubiquitination, etc.) or incorporation of one or more synthetic or unnatural amino acids to the extent that the modification or synthetic or unnatural amino acid does not destroy the antigenicity of the polypeptide antigen or the desired functionality of the peptide (or a domain contained therein).

[0047] In some embodiments, the modified polypeptide antigens described herein can be conjugated to a sufficient number of steroid acid moieties such that the modified polypeptide antigen exhibits greater stability (e.g., thermal stability) than the stability of the polypeptide antigen prior to conjugation (Example 2 and Figure 1G and Figure 1H ).

[0048] In some embodiments, the polypeptide antigens described herein can be protein antigens. In some embodiments, protein antigens can advantageously comprise a plurality of available functional groups that can be conjugated to a steroid acid or steroid acid-peptide moiety. In contrast, peptide antigens can not comprise a sufficient number of functional groups for steroid acid conjugation. Furthermore, steroid acid-peptide antigen conjugates can undesirably self-assemble into rod-like nanoparticles, as reported in Azuar et al., 2019, in which hydrophobic steroid acid groups from different modified peptide antigens aggregate and sequester inside, preventing their ability to interact with membranes and mediate endosomal escape. Insufficient endosomal escape can not negatively impact MHC class II presentation, and thus can be beneficial for humoral immunity, but not for cellular immunity (Azuar et al., 2019).

[0049] In some embodiments, the protein antigens described herein can comprise (or can be engineered to comprise) 1 to 50, 2 to 50, 5 to 50, or 10 to 50 functional groups (e.g., lysine and / or cysteine residues; or any other group) that can be used for conjugation to a steroid acid or steroid acid-peptide moiety described herein. In some embodiments, the polypeptide antigen can be a protein antigen that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 amino acids in length. In some embodiments, the polypeptide antigen can be a protein antigen that has a molecular weight of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 kDa. In some embodiments, the polypeptide antigens described herein can comprise one or more MHC class I epitopes and / or MHC class II epitopes.

[0050] In some embodiments, the polypeptide antigens described herein can be or can include a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a neoantigen, a viral antigen, a bacterial antigen, a fungal antigen, an antigen associated with a disease or condition that can be treated by vaccination and / or immunotherapy; or any antigenic fragment thereof. In some embodiments, the polypeptide antigens described herein can be or can include a spike protein from SARS-CoV-2 (SEQ ID NO: 3) or SARS-CoV (SEQ ID NO: 4) or an antigenic variant or antigenic fragment thereof. In some embodiments, the TAA, TSA, and / or neoantigen can be a single nucleotide variant antigen, a frameshift mutation antigen, a splice variant antigen, a gene fusion antigen, an endogenous retroelement antigen, or another class of antigen such as a human leukocyte antigen (HLA)-somatic mutation-derived antigen or a post-translational TSA (Smith et al., 2019). In some embodiments, the TSA can be a cancer antigen of viral origin such as from human papillomavirus (HPV), cytomegalovirus, or Epstein-Barr virus (EBV). In some embodiments, the TAA can be or can include a cancer-testis antigen, HER2, PSA, TRP-1, TRP-2, EpCAM, GPC3, CEA, MUC1, MAGE-A1, NY-ESO-1, SSX-2, mesothelin (MSLN), or EGFR (Patel et al., 2017; Tagliamonte et al., 2014). In some embodiments, the polypeptide antigens described herein can be or can include a cell lysate or other material derived from a tumor such as tumor-derived exosomes.

[0051] In some embodiments, the polypeptide antigens can be conjugated to a steroid acid moiety that enhances endocytosis and / or endosomal escape of the internalized cargo. Without being bound by theory, steroid acids (e.g., bile acids and bile acid analogs) have been demonstrated to be exploited / adopted by viruses to facilitate their infection of host cells, such as by increasing their endocytic uptake and / or endosomal escape to enable access to the cytosol (Shivanna et al., 2014; Shivanna et al., 2015; Murakami et al., 2020). For example, bile acids have been demonstrated to trigger the enzymatic acid sphingomyelinase (ASM) to cleave sphingomyelin into ceramide on the inner leaflet of the endosome. The increased amount of ceramide destabilizes the membrane and promotes endosomal escape. In some embodiments, steroid acids suitable for conjugation to the polypeptide antigens described herein include those that trigger the accumulation of ceramide on the inner leaflet of the endosome, thereby destabilizing the endosomal membrane and promoting endosomal escape following intracellular delivery of the modified polypeptide antigen. In some embodiments, steroid acids suitable for conjugation to the polypeptide antigens described herein include those that trigger increased acid sphingomyelinase (ASM)-mediated cleavage of sphingomyelin to form ceramide.

[0052] In some embodiments, the steroid acid suitable for conjugation to the polypeptide antigens described herein comprises or consists of a bile acid (e.g., a primary bile acid or a secondary bile acid). In some embodiments, the steroid acid can be or comprise cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), glycochenodeoxycholic acid (GDCA), glycocholic acid (GCA), taurocholic acid (TCA), glycodeoxycholic acid (CDCA), glycochenodeoxycholic acid (GCDCA), taurodeoxycholic acid (TDCA), glycolithocholic acid (GLCA), tauroolithocholic acid (TLCA), taurohyodeoxycholic acid (THDCA), taurochenodeoxycholic acid (TCDCA), ursodeoxycholic acid (UCA), tauro ursodeoxycholic acid (TUDCA), ursodeoxycholic acid (UDCA), or glyco ursodeoxycholic acid (GUDCA), or any analog thereof that induces endocytosis; triggers accumulation of ceramide on the inner leaflet of the endosome; triggers increased acid sphingomyelinase (ASM)-mediated sphingomyelin cleavage to ceramide; and / or has a higher hydrophobicity than cholic acid.

[0053] Hydrophobic bile acids such as GCDCA, TCA, GCA, and CA, but not hydrophilic bile acids such as UDCA, have been shown to increase GII.3 human norovirus infection and replication in host enterocytes by enhancing endosomal uptake and endosomal escape via ASM-mediated accumulation of ceramide on the apical membrane (Murakami et al., 2020). In some embodiments, the steroid acid suitable for conjugation to the polypeptide antigens described herein comprises or consists of a bile acid or bile acid analog that is more hydrophobic than cholic acid. In some embodiments, the steroid acid suitable for conjugation to the polypeptide antigens described herein comprises or consists of a bile acid or bile acid analog that is more hydrophobic than cholic acid (e.g., CDCA, DCA, LCA, TCA, TDCA, TCDCA, GCA, GDCA, or GCDCA; Hanafi et al., 2018).

[0054] In some embodiments, the average number of steroid acid moieties per modified polypeptide antigen can vary, e.g., based on the type of steroid acid selected and / or the type of polypeptide antigen selected (e.g., amino acid length, structure, number of available functional groups). In some embodiments, a polypeptide can be reacted with a molar excess of a steroid acid or steroid acid-peptide moiety to maximize the number of conjugated steroid acid moieties. In some embodiments, a polypeptide can be reacted with a limiting amount of a steroid acid or steroid acid-peptide moiety to control or limit the number of conjugated steroid acid moieties. In some embodiments, each modified polypeptide antigen molecule can be conjugated to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 steroid acid moieties. In some embodiments, the modified polypeptide antigen molecule can be conjugated to a steroid acid (or steroid acid-peptide) moiety at a solvent accessible amine (e.g., primary amine) and / or thiol of the polypeptide antigen. In some embodiments, the modified polypeptide antigen molecule can be conjugated to a steroid acid (or steroid acid-peptide) moiety at any other chemical group or functional group present on or engineered into the polypeptide antigen. It will be appreciated that the maximum number of steroid acid moieties included in a modified polypeptide antigen described herein is less than or equal to the number of available functional groups on the polypeptide antigen (or functionalized polypeptide antigen) available for conjugation. In some embodiments, the polypeptide antigen (and / or steroid acid or steroid acid-peptide moiety) can be pre-functionalized, e.g., with a bi-, tri-, or multi-functional linker group, prior to the reaction that conjugates the polypeptide antigen to the steroid acid or steroid acid-peptide moiety.

[0055] In some embodiments, a steroid acid described herein can be included in a steroid acid-peptide moiety. In some embodiments, a steroid acid can be pre-conjugated to a peptide, e.g., at a free N-terminal amino group of the peptide or at some other functional group within the peptide. In some embodiments, the polypeptide antigen can then be conjugated to the steroid acid-peptide moiety via the peptide, such as at the N-terminal or C-terminal residue of the peptide.

[0056] In some embodiments, the peptide can be a non-immunogenic peptide. In some embodiments, the peptide can be a water-soluble peptide, where conjugation of the peptide to the steroid acid increases the water-solubility of the steroid acid-peptide moiety compared to the steroid acid moiety alone. In some embodiments, the peptide can be a cationic peptide (e.g., that facilitates interaction with plasma and / or endosomal membranes).

[0057] In some embodiments, the peptide can comprise one or more domains that confer additional functionality to the modified polypeptide antigen. As used herein, a "domain" generally refers to a portion of a protein that has a particular functionality. Some domains retain their functionality when separated from the rest of the protein, and thus can be used in a modular fashion. The modular nature of many protein domains can provide flexibility in their placement within the peptides described herein. However, some domains can perform better when engineered at certain positions in the peptide (e.g., N-terminal or C-terminal regions, or in between). The position of the domain within its endogenous protein can be an indication of where the domain should be engineered within the peptide.

[0058] In some embodiments, the peptide can comprise a protein transduction domain (PTD) that stimulates endocytosis, endosome formation, or intracellular delivery in a non-cell specific manner. In some embodiments, the peptide can comprise a subcellular targeting signal that facilitates targeting of the modified polypeptide antigen to a particular subcellular compartment. In some embodiments, the peptide can comprise a nuclear localization signal (NLS) that targets the modified polypeptide antigen to the nucleus. Interestingly, while targeting the cytosolic compartment would be expected to be advantageous given that proteasome-mediated MHC class I peptide epitope processing occurs in the cytosol, the results shown herein surprisingly demonstrate that a modified polypeptide antigen comprising a nuclear localization signal triggers a significant increase in antigen immunogenicity. In some embodiments, the nuclear localization signal described herein can comprise or be derived from an NLS from the SV-40 large T antigen (e.g., PKKKRKV; SEQ ID NO: 7) or from other classical NLSs. In some embodiments, the nuclear localization signal described herein can comprise or be derived from a non-classical NLS (e.g., the acidic M9 domain in hnRNP Al protein; the sequence KIPIK in the yeast transcriptional repressor Matα2; PY-NLS; ribosomal NLS; or the composite signal of U snRNP). In some embodiments, the nuclear localization signal described herein comprises or consists essentially of the amino acid sequence of any of SEQ ID NOs: 1 or 7-20, or any portion thereof. In some embodiments, the nuclear localization signal described herein comprises or consists essentially of a nuclear localization signal that is or consists essentially of: an SV40 NLS (e.g., comprising in SEQ ID NO: 1 or 7), a GWG-SV40 NLS (e.g., comprising in SEQ ID NO: 8), a hnRNP Al M9 NLS (e.g., comprising in SEQ ID NO: 9), a hnRNP D NLS (e.g., comprising in SEQ ID NO: 10), a hnRNP M NLS (e.g., comprising in SEQ ID NO: 11), a PQBP-l NLS (e.g., comprising in SEQ ID NO: 12), a NLS2-RG domain RPS 17 (e.g., comprising in SEQ ID NO: 13), a NLS1 RPS 17 (e.g., comprising in SEQ ID NO: 14), a NLS2 RPS 17 (e.g., comprising in SEQ ID NO: 15), a NLS3 RPS 17 (e.g., comprising in SEQ ID NO: 16), a cMyc NLS (e.g., comprising in SEQ ID NO: 17), a HuR NLS (e.g., comprising in SEQ ID NO: 18), a Tus NLS (e.g., comprising in SEQ ID NO: 19), or a nucleoplasmin NLS (e.g., comprising in SEQ ID NO: 20).In some cases, the above-referenced SEQ ID NOs include an N-terminal cysteine residue (e.g., the thiol group of an N-terminal cysteine residue) for facilitating conjugation to a polypeptide antigen. Thus, in some embodiments, the NLS sequences referred to herein can not include the N-terminal cysteine residue included in any of SEQ ID NOs: 1 and 7-20. In some embodiments, other functional groups (e.g., carboxyl groups, synthetic amino acids, etc.) that are added or inserted (e.g., toward the N-terminal to C-terminal portion of the peptides described herein) to facilitate conjugation of the steroid acid-peptide to a given polypeptide antigen are also contemplated.

[0059] In some embodiments, the nuclear localization signal described herein can comprise the general consensus sequence: (i) K(K / R)X(K / R); (ii) (K / R)(K / R)X 10-12 (K / R) 3 / 5 wherein (K / R) 3 / 5 represents three lysine or arginine residues out of five consecutive amino acids; (iii) KRX 10-12 KRRK; (iv) KRX 10-12 K(K / R)(K / R); or (v) KRX 10-12 K(K / R)X(K / R), where X is any amino acid (Sun et al., 2016).

[0060] In some embodiments, the modified polypeptide antigens described herein can exhibit increased cytosolic delivery compared to the corresponding unmodified polypeptide antigen. In some embodiments, the modified polypeptide antigens described herein can exhibit increased total cellular delivery of the modified polypeptide antigen compared to the corresponding unmodified polypeptide antigen. In some embodiments, the modified polypeptide antigens described herein can exhibit enhanced cellular immunity to the polypeptide antigen compared to the corresponding unmodified polypeptide antigen. In some embodiments, the modified polypeptide antigens described herein exhibit increased IFN-g production by CD8+ T cells upon exposure to the polypeptide antigen compared to the corresponding unmodified polypeptide antigen. In some embodiments, the modified polypeptide antigens described herein exhibit enhanced humoral immunity to the polypeptide antigen compared to the corresponding unmodified polypeptide antigen. In some embodiments, the modified polypeptide antigens described herein trigger an increase in the repertoire (or biological diversity) of antibody species against the polypeptide antigen compared to the corresponding unmodified polypeptide antigen (e.g., including antibodies against weakly immunogenic epitopes).

[0061] In some aspects, described herein is a population of cells (e.g., in vitro or ex vivo) comprising or treated with a modified polypeptide antigen described herein. In some embodiments, the population of cells described herein can comprise immune cells (e.g., T cells), antigen presenting cells (e.g., dendritic cells, macrophages, engineered antigen presenting cells), cells expressing MHC class I, cells expressing MHC class II, or any combination thereof.

[0062] In some aspects, described herein is an immunogenic composition comprising: a modified polypeptide antigen described herein or produced by a method described herein, or a population of cells described herein, or any combination thereof, and a pharmaceutically acceptable excipient and / or adjuvant (e.g., a vaccine adjuvant suitable for human or animal use). In some embodiments, the adjuvant can be an emulsion adjuvant, such as an oil-in-water emulsion adjuvant (e.g., a squalene-based oil-in-water emulsion adjuvant). In some embodiments, the immunogenic composition described herein can be a therapeutic or prophylactic vaccine (e.g., an anti-cancer vaccine, an anti-viral vaccine, or an anti-bacterial vaccine). In some embodiments, the modified polypeptide antigen described herein can allow for a reduction in the amount of antigen and / or antigen presenting cells formulated in the immunogenic composition (e.g., vaccine) needed to generate an immune response, as compared to the amount when using the corresponding unmodified polypeptide antigen lacking a steroid acid conjugation.

[0063] In some aspects, described herein is a method for triggering an enhanced adaptive immune response against a target polypeptide antigen in a subject, the method comprising administering to the subject an immunogenic composition as described herein.

[0064] In another aspect, described herein is a method for treating or preventing a disease or disorder that can be treated by vaccination and / or immunotherapy, the method comprising administering to a subject an immunogenic composition as described herein.

[0065] In some aspects, described herein is a method for treating a cancer in a subject, the method comprising administering to a subject in need thereof an immunogenic composition as described herein. In some embodiments, the method can be combined with immune checkpoint inhibitor therapy or other anti-cancer treatment.

[0066] In some aspects, described herein is a modified polypeptide antigen as defined herein for use in generating an immune response in a subject. In some aspects, described herein is a modified polypeptide antigen as defined herein for use in the manufacture of an immunogenic composition (e.g., a vaccine or an immunotherapy) for generating an immune response in an individual. In some aspects, described herein is the use of a modified polypeptide antigen as defined herein, a modified polypeptide antigen produced by a method described herein, a population of cells described herein, or an immunogenic composition described herein for generating an immune response in a subject. In some aspects, described herein is the use of a modified polypeptide antigen as defined herein, a modified polypeptide antigen produced by a method described herein, a population of cells described herein, or an immunogenic composition described herein for the manufacture of a medicament (e.g., a vaccine or an immunotherapeutic agent) for generating an immune response in a subject. In some embodiments, the immune response comprises enhanced cellular immunity to the polypeptide antigen, increased IFN-g production by CD8+ T cells upon exposure to the polypeptide antigen, enhanced humoral immunity to the polypeptide antigen, or any combination thereof, as compared to that generated from a corresponding unmodified polypeptide antigen.

[0067] In some aspects, described herein is a method for preparing a polypeptide antigen, the method comprising conjugating an unmodified polypeptide antigen to a sufficient number of steroid acid moieties to produce a modified polypeptide antigen that exhibits greater stability (e.g., thermal stability) than the polypeptide antigen prior to conjugation. In some embodiments, the number of steroid acid moieties conjugated to the polypeptide antigen is sufficient to increase endosomal escape of the modified polypeptide antigen following intracellular delivery relative to the absence of said modified polypeptide antigen. In embodiments, the modified polypeptide antigen is a modified polypeptide antigen as defined herein.

[0068] Clause

[0069] In various aspects, described herein are one or more of the following clauses:

[0070] 1. A method of improving immunogenicity of a polypeptide antigen, the method comprising providing a polypeptide antigen to be modified, and covalently conjugating the polypeptide antigen to one or more steroid acid moieties to produce a modified polypeptide antigen conjugated to a sufficient number of steroid acid moieties to increase endosomal escape of the modified polypeptide antigen following intracellular delivery relative to the absence of said modified polypeptide antigen, wherein the modified polypeptide antigen triggers an improved adaptive immune response to the polypeptide antigen following administration to a subject as compared to a corresponding unmodified polypeptide antigen.

[0071] 2. The method of clause 1, wherein the modified polypeptide antigen is conjugated to a sufficient number of steroid acid moieties such that the modified polypeptide antigen exhibits a higher stability (e.g., thermal stability) than the polypeptide antigen prior to conjugation.

[0072] 3. The method of clause 1 or clause 2, wherein the polypeptide antigen is a protein antigen and / or has a molecular weight of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 kDa.

[0073] 4. The method of any one of clauses 1-3, wherein the polypeptide antigen comprises one or more MHC class I epitopes and / or MHC class II epitopes.

[0074] 5. The method of any one of clauses 1-4, wherein the polypeptide antigen is or comprises a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a neoantigen, a viral antigen, a bacterial antigen, a fungal antigen, an antigen associated with a disease or condition that can be treated by vaccination and / or immunotherapy; or any antigenic fragment thereof.

[0075] 6. The method of any one of clauses 1-5, wherein the polypeptide antigen is or comprises a coronavirus antigen (e.g., a SARS-CoV-2 spike protein (SEQ ID NO: 3) or a SARS-CoV spike protein (SEQ ID NO: 4) or an antigenic fragment thereof; or a cancer antigen such as a single nucleotide variant antigen, a mutation frameshift antigen, a splice variant antigen, a gene fusion antigen, an endogenous retroelement antigen, or another class of antigen such as a human leukocyte antigen (HLA)-somatic mutation-derived antigen or a post-translational TSA, a viral-derived cancer antigen (e.g., from human papillomavirus (HPV), cytomegalovirus, or Epstein-Barr virus (EBV)), a cancer-testis antigen, HER2, PSA, TRP-1, TRP-2, EpCAM, GPC3, CEA, MUC1, MAGE-A1, NY-ESO-1, SSX-2, mesothelin (MSLN), EGFR, a cell lysate, or other material derived from a tumor (e.g., tumor-derived exosomes).

[0076] 7. The method of any one of clauses 1-6, wherein the steroid acid triggers accumulation of ceramide on the inner leaflet of the endosome, thereby destabilizing the endosomal membrane and facilitating endosomal escape of the polypeptide antigen following intracellular delivery.

[0077] 8. The method of any one of clauses 1-7, wherein the steroid acid initiates increased acid sphingomyelinase (ASM)-mediated sphingomyelin cleavage to ceramide.

[0078] 9. The method of any one of clauses 1-8, wherein the steroid acid is a bile acid.

[0079] 10. The method of any one of clauses 1-9, wherein the steroid acid is a primary bile acid or a secondary bile acid.

[0080] 11. The method of any one of clauses 1-10, wherein the steroid acid is or comprises: (a) a bile acid that is cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), glycochenodeoxycholic acid (GDCA), glycocholic acid (GCA), taurocholic acid (TCA), glycodeoxycholic acid (CDCA), glycochenodeoxycholic acid (GCDCA), taurodeoxycholic acid (TDCA), glycolithocholic acid (GLCA), tauroolithocholic acid (TLCA), taurohyodeoxycholic acid (THDCA), taurochenodeoxycholic acid (TCDCA), ursodeoxycholic acid (UCA), tauro-ursodeoxycholic acid (TUDCA), ursodeoxycholic acid (UDCA), or glyco-ursodeoxycholic acid (GUDCA); (b) an analog of the bile acid of (a) that: induces endocytosis; triggers accumulation of ceramide on the inner leaflet of the endosome; triggers increased acid sphingomyelinase (ASM)-mediated sphingomyelin cleavage to ceramide; and / or has a higher hydrophobicity than cholic acid; (c) a bile acid or bile acid analog that is more hydrophobic than cholic acid (e.g., CDCA, DCA, LCA, TCA, TDCA, TCDCA, GCA, GDCA, or GCDCA); or (d) any combination of (a)-(c).

[0081] 12. The method of any one of clauses 1-11, wherein each modified polypeptide antigen molecule is conjugated to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 steroid acid moieties.

[0082] 13. The method of any one of clauses 1-12, wherein the modified polypeptide antigen molecule is conjugated to the steroid acid at a solvent-accessible amine (e.g., a primary amine) and / or thiol of the polypeptide antigen.

[0083] 14. The method of any one of clauses 1-13, wherein the modified polypeptide antigen molecule is conjugated to the steroid acid via a linker (e.g., a bifunctional linker, a trifunctional linker, or a multifunctional linker).

[0084] 15. The method of any one of clauses 1-14, wherein the steroid acid is comprised in a steroid acid-peptide conjugate and the polypeptide antigen is conjugated to the steroid acid- peptide conjugate (e.g., via the peptide, such as at an N-terminal or C-terminal residue).

[0085] 16. The method of clause 15, wherein the peptide: (i) comprises a protein transduction domain that stimulates endocytosis and / or endosome formation; (ii) comprises a subcellular targeting signal; (iii) is a cationic peptide (e.g., a non-cell-penetrating cationic peptide); (iv) is a non-immunogenic peptide; or (v) any combination of (i)-(iv).

[0086] 17. The method of clause 16, wherein the subcellular targeting signal is a nuclear localization signal, such as a classical NLS (e.g., an NLS from SV-40 large T antigen (e.g., PKKKRKV; SEQ ID NO: 7) or other classical NLS) or a non-classical NLS (e.g., acidic M9 domain in hnRNPA1 protein; sequence KIPIK in yeast transcriptional repressor Mata2; PY-NLS; ribosomal NLS; and composite signal of UsnRNP).

[0087] 18. The method of clause 16 or clause 17, wherein the nuclear localization signal is: an SV40 NLS (e.g., comprised in SEQ ID NO: 1 or 7), a GWG-SV40 NLS (e.g., comprised in SEQ ID NO: 8), an hnRNPA1 M9 NLS (e.g., comprised in SEQ ID NO: 9), an hnRNP D NLS (e.g., comprised in SEQ ID NO: 10), an hnRNP M NLS (e.g., comprised in SEQ ID NO: 11), a PQBP-1 NLS (e.g., comprised in SEQ ID NO: 12), an NLS2-RG domain RPS17 (e.g., comprised in SEQ ID NO: 13), an NLS1 RPS17 (e.g., comprised in SEQ ID NO: 14), an NLS2 RPS17 (e.g., comprised in SEQ ID NO: 15), an NLS3 RPS17 (e.g., comprised in SEQ ID NO: 16), a cMyc NLS (e.g., comprised in SEQ ID NO: 17), a HuR NLS (e.g., comprised in SEQ ID NO: 18), a Tus NLS (e.g., comprised in SEQ ID NO: 19), or a nucleoplasmin NLS (e.g., comprised in SEQ ID NO: 20); or a variant of an NLS having nuclear localization activity, the NLS comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 7-20.

[0088] 19. The method of any one of clauses 1-18, wherein the modified polypeptide antigen triggers: (i) increased cytosolic delivery of the modified polypeptide antigen compared to a corresponding unmodified polypeptide antigen; (ii) increased total cellular delivery of the modified polypeptide antigen compared to a corresponding unmodified polypeptide antigen; (iii) enhanced cellular immunity against the polypeptide antigen compared to a corresponding unmodified polypeptide antigen; (iv) increased IFN-g production by CD8+ T cells following exposure to the polypeptide antigen compared to a corresponding unmodified polypeptide antigen; (v) enhanced humoral immunity against the polypeptide antigen compared to a corresponding unmodified polypeptide antigen; (vi) increased number of antibody species against the polypeptide antigen compared to a corresponding unmodified polypeptide antigen; or (vii) any combination of (i)-(vi).

[0089] 20. A population of cells (e.g., in vivo or ex vivo), comprising the modified polypeptide antigen produced by the method of any one of clauses 1-19 or a modified polypeptide antigen of any one of clauses 1-19.

[0090] 21. The cell population of clause 20, comprising immune cells (e.g., T cells), antigen presenting cells (e.g., dendritic cells, macrophages, engineered antigen presenting cells), cells expressing MHC class I, cells expressing MHC class II, or any combination thereof.

[0091] 22. An immunogenic composition comprising: the modified polypeptide antigen produced by the method of any one of clauses 1 to 19, the modified polypeptide antigen of any one of clauses 1 to 19, the cell population of clause 20 or clause 21, or any combination thereof; and a pharmaceutically acceptable excipient and / or adjuvant (e.g., an emulsion adjuvant, an oil-in-water emulsion adjuvant, or a squalene-based oil-in-water emulsion adjuvant).

[0092] 23. The immunogenic composition of clause 22, which is a therapeutic vaccine or a prophylactic vaccine (e.g., an anti-cancer vaccine, an anti-viral vaccine, or an anti-bacterial vaccine).

[0093] 24. A method for triggering an enhanced adaptive immune response in a subject against an unmodified target polypeptide antigen, the method comprising administering to the subject the immunogenic composition of clause 22 or clause 23.

[0094] 25. A method for vaccinating a subject against an infectious disease, the method comprising administering to the subject the immunogenic composition of clause 22 or clause 23, wherein the polypeptide antigen comprises an antigenic fragment of a pathogen (e.g., a virus, a bacterium, a fungus) that causes the infectious disease.

[0095] 26. A method for treating cancer in a subject, the method comprising administering to the subject the immunogenic composition of clause 22 or clause 23.

[0096] 27. The method of clause 26, wherein the method is combined with immune checkpoint inhibitor therapy.

[0097] 28. The modified polypeptide antigen of any one of clauses 1 to 19, or produced by the method of any one of clauses 1 to 19, for use in generating an immune response in a subject or for use in the manufacture of an immunogenic composition for generating an immune response in a subject.

[0098] 29. Use of a modified polypeptide antigen according to any one of Clauses 1 to 19, the modified polypeptide antigen produced by the method according to any one of Clauses 1 to 19, the population of cells according to Clause 20 or Clause 21, or the immunogenic composition according to Clause 22 or Clause 23, for producing an immune response in a subject or for the manufacture of a medicament (e.g. a vaccine) for producing an immune response in a subject.

[0099] 30. The modified polypeptide antigen for use according to Clause 28 or the use according to Clause 29, wherein the immune response comprises enhanced cellular immunity to the polypeptide antigen, increased IFN-g production by CD8+ T cells following exposure to the polypeptide antigen, enhanced humoral immunity to the polypeptide antigen, or any combination thereof, compared to that produced from a corresponding unmodified polypeptide antigen.

[0100] 31. A method for preparing a polypeptide antigen, the method comprising conjugating an unmodified polypeptide antigen to a sufficient number of steroid acid moieties to produce a modified polypeptide antigen that exhibits greater stability (e.g. thermal stability) than the polypeptide antigen prior to conjugation.

[0101] 32. The method according to Clause 31, wherein the number of steroid acid moieties conjugated to the polypeptide antigen is sufficient to increase endosomal escape of the modified polypeptide antigen following intracellular delivery relative to the unmodified polypeptide antigen.

[0102] 33. The method according to Clause 31 or Clause 32, wherein the modified polypeptide antigen is as defined in any one of Clauses 3 to 19.

[0103] 34. A method of improving the immunogenicity of a polypeptide antigen, the method comprising providing a polypeptide antigen to be modified, and covalently conjugating the polypeptide antigen to one or more bile acid-peptide moieties to produce a modified polypeptide antigen that is conjugated to a sufficient number of bile acid-peptide moieties to trigger an improved adaptive immune response to the polypeptide antigen following administration to a subject compared to a corresponding unmodified polypeptide antigen, wherein the peptide comprised in the bile acid-peptide moiety comprises a nuclear localization signal (NLS).

[0104] 35. The method according to Clause 34, wherein the modified polypeptide antigen is conjugated to a sufficient number of bile acid-peptide moieties to increase antigen presentation of the modified polypeptide antigen following intracellular delivery relative to a corresponding unmodified polypeptide antigen.

[0105] 36. The method of clause 34 or clause 35, wherein the modified polypeptide antigen is conjugated to a sufficient number of bile acid-peptide moieties such that the modified polypeptide antigen exhibits higher thermostability relative to a corresponding unmodified polypeptide antigen.

[0106] 37. The method of any one of clauses 34-36, wherein covalent conjugation of the polypeptide antigen to one or more bile acid-peptide moieties is performed by reacting the polypeptide antigen with a molar excess of the bile acid-peptide moieties.

[0107] 38. The method of clause 37, wherein the polypeptide antigen is reacted with a 2-fold to 100-fold molar excess of the bile acid-peptide moieties.

[0108] 39. The method of clause 37, wherein the polypeptide antigen is reacted with a 2-fold to 50-fold molar excess of the bile acid-peptide moieties.

[0109] 40. The method of clause 37, wherein the polypeptide antigen is reacted with a 5-fold to 25-fold molar excess of the bile acid-peptide moieties.

[0110] 41. The method of any one of clauses 34-40, wherein the average number of bile acid- peptide moieties conjugated per modified polypeptide antigen is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50; or between about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 and n, where n is the total number of accessible sites on the polypeptide antigen available for conjugation.

[0111] 42. The method of any one of clauses 34-41, wherein the bile acid is cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), glycochenodeoxycholic acid (GDCA), glycocholic acid (GCA), taurocholic acid (TCA), glycodeoxycholic acid (CDCA), glycochenodeoxycholic acid (GCDCA), taurodeoxycholic acid (TDCA), glycolithocholic acid (GLCA), tauroolithocholic acid (TLCA), taurohyodeoxycholic acid (THDCA), taurochenodeoxycholic acid (TCDCA), ursodeoxycholic acid (UCA), tauroursodeoxycholic acid (TUDCA), ursodeoxycholic acid (UDCA), or glyco ursodeoxycholic acid (GUDCA).

[0112] 43. The method of any one of clauses 34-42, wherein the bile acid is an analog of CA, CDCA, DCA, LCA, GDCA, GCA, TCA, CDCA, GCDCA, TDCA, GLCA, TLCA, THDCA, TCDCA, UCA, TUDCA, UDCA, or GUDCA, wherein the analog: induces endocytosis; triggers accumulation of ceramide on the inner leaflet of the endosome; or triggers increased acid sphingomyelinase (ASM)-mediated sphingomyelin cleavage to ceramide.

[0113] 44. The method of any one of clauses 34-43, wherein the nuclear localization signal is: SV40 NLS (SEQ ID NO: 1 or 7), GWG-SV40 NLS (SEQ ID NO: 8), hnRNPA1 M9 NLS (SEQ ID NO: 9), hnRNP D NLS (SEQ ID NO: 10), hnRNP M NLS (SEQ ID NO: 11), PQBP-1 NLS (SEQ ID NO: 12), NLS2-RG domain RPS17 (SEQ ID NO: 13), NLS1 RPS17 (SEQ ID NO: 14), NLS2 RPS17 (SEQ ID NO: 15), NLS3 RPS17 (SEQ ID NO: 16), cMyc NLS (SEQ ID NO: 17), HuR NLS (SEQ ID NO: 18), Tus NLS (SEQ ID NO: 19), or nucleoplasmin NLS (SEQ ID NO: 20).

[0114] 45. The method of any one of clauses 34-44, wherein the nuclear localization signal is a variant of an NLS having nuclear localization activity, the NLS comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 7-20.

[0115] 46. The method of any one of clauses 34-45, wherein the polypeptide antigen is conjugated to the one or more bile acid-peptide moieties via a linker.

[0116] 47. The method of clause 46, wherein the linker is a bifunctional linker, a trifunctional linker, or a multifunctional linker.

[0117] 48. The method of any one of clauses 34-47, wherein the modified polypeptide antigen molecule is conjugated to the one or more bile acid-peptide moieties via a solvent-accessible functional group of the polypeptide antigen.

[0118] 49. The method of any one of clauses 34-48, wherein the polypeptide antigen is or comprises a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a tumor-derived cell lysate, a tumor-derived exosome, a neoantigen, a viral antigen, a bacterial antigen, a fungal antigen, or another antigen associated with a disease or condition that can be treated by vaccination and / or immunotherapy.

[0119] 50. The method of any one of clauses 34-49, wherein the polypeptide antigen is or comprises a SARS-CoV spike protein or an antigenic fragment thereof.

[0120] 51. An immunogenic composition comprising the modified polypeptide antigen produced by the method of any one of clauses 34-50 or a population of cells comprising the modified polypeptide antigen produced by the method of any one of clauses 34-50, and a pharmaceutically acceptable excipient and / or adjuvant.

[0121] 52. The immunogenic composition of clause 51, wherein the population of cells comprises dendritic cells, B cells, T cells, macrophages, engineered antigen presenting cells, cells expressing MHC class I, cells expressing MHC class II, or any combination thereof.

[0122] 53. A method for triggering an enhanced adaptive immune response against an unmodified target polypeptide antigen in a subject, the method comprising administering to the subject the immunogenic composition of clause 52.

[0123] Example

[0124] Example 1: General Materials and Methods

[0125] Animals and Ethics

[0126] BALB / c mice, six to eight weeks of age, were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and C57BL / 6 mice of similar age were purchased from Charles River (Montreal, QC, Canada). Littermate mice were bred and housed in a pathogen-free environment at the animal facility of the Institute for Research in Immunology and Cancer (IRIC). Animal protocols were approved by the Animal Protection Committee of the University of Montreal.

[0127] Cell lines and reagents

[0128] All cell culture media and reagents were purchased from Wisent Bioproducts (St-Bruno, QC, Canada) unless otherwise stated. All flow cytometry antibodies were purchased from BD Biosciences (San Jose, CA, USA) unless otherwise stated. Albumin from chicken egg white (ovalbumin; OVA), LPS, and Nunc MaxiSorp TM plates were purchased from Sigma-Aldrich (St-Louis, MI, USA). OVA-DQ TM was purchased from ThermoFisher (Waltham, MA, USA). SIINFEKL peptide was synthesized by Genscript (Piscataway, NJ, USA). Bradford reagent was purchased from Bio-Rad (Hercules, CA, USA). All cytokine ELISAs were purchased from R&D Systems (Minneapolis, MN, USA) unless otherwise stated. Recombinant GM-CSF was purchased from Peprotech (Rocky Hill, NJ, USA). CD8 and CD4 T cell isolation kits were purchased from StemCell Technologies (Vancouver, BC, Canada). PD-1 antibody (clone RMP1-14) for in vivo studies was purchased from BioXCell (West Lebanon, NH, USA).

[0129] Generation of bone marrow-derived DCs

[0130] RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 50 U / mL penicillin-streptomycin, 2 mM L-glutamine, 10 mM HEPES, 1% MEM non-essential amino acids, 1 mM sodium pyruvate, 0.5 mM dithiothreitol, 50 ng / mL recombinant mouse GM-CSF, and 1 ng / mL LPS from E. coli O111 were used to culture BMDCs. TM 1640 supplemented with 10% fetal bovine serum (FBS), 50 U / mL penicillin-streptomycin, 2 mM L-glutamine, 10 mM HEPES, 1% MEM non-essential amino acids, 1 mM sodium pyruvate, 0.5 mM dithiothreitol, 50 ng / mL recombinant mouse GM-CSF, and 1 ng / mL LPS from E. coli O111 were used to culture BMDCs. b 1640 supplemented with 10% fetal bovine serum (FBS), 50 U / mL penicillin-streptomycin, 2 mM L-glutamine, 10 mM HEPES, 1% MEM non-essential amino acids, 1 mM sodium pyruvate, 0.5 mM dithiothreitol, 50 ng / mL recombinant mouse GM-CSF, and 1 ng / mL LPS from E. coli O111 were used to culture BMDCs.

[0131] Modeling of accessible lysines in protein antigens

[0132] RCSB PDB and Swiss-Model Expasy TM Free access software models the 3D structure of the antigen. The accessible amino acids representing lysine residues are identified and highlighted according to their accessibility (blue: high; green: medium, and yellow: poor).

[0133] Cancer cell lysate preparation

[0134] To prepare cancer cell lysates, cultured EL4 cells were collected by centrifugation at 1500 rpm for 5 min, followed by two washing steps with PBS to remove traces of FBS. Cells were then subjected to 5 rounds of freezing and thawing in liquid nitrogen / boiling water, respectively. To remove large particles, the lysate was chopped using a G26 needle, passed through a 70 pm cell strainer, and then filtered through a 0.45 pm filter. The obtained lysate was then quantified using Bradford reagent, aliquoted and stored at -80°C until use.

[0135] Generation of ChAcNLS-antigen formulations

[0136] ChAcNLS was synthesized as previously described in Beaudolin et al., 2016, unless otherwise specified. OVA, OVA-DQ or cancer cell lysates were solubilized at 1-10 mg / mL in sterile PBS containing or not other formulation components but no amine (NH3) or thiol (SH) groups. SM(PEG)4 crosslinker was added to the reaction using different molar excess ratios (5x, 10x, 25x, 50x) for 1 h. Free SM(PEG)4 crosslinker was removed by Centricon TM filtration and Sephadex TM Free SM(PEG)4 crosslinker was removed by filtration and Sephadex column. ChAcNLS was added at the same molar excess ratio and incubated for 1 h to obtain different amounts of ChAcNLS moieties per antigen attachment. Unless otherwise specified, the cOVA conjugates tested in the examples were produced using a 50x molar excess ratio. Free unattached ChAcNLS was removed by centricon filtration and Sephadex column. ChAcNLS-modified antigens were concentrated in sterile PBS to obtain a final concentration of 5-10 mg / mL determined by UV absorbance.

[0137] To evaluate ChAcNLS loading, 10 pg of OVA or ChAcNLS-OVA conjugate were separated on 12% polyacrylamide gels under reducing conditions and stained with Coomassie Brilliant Blue R-250 TM(Bio-Rad, Mississauga, ON, Canada). Migration distance in the gel relative to the leading front of blue dye (Rf) was measured and the number of ChAcNLS moieties introduced into OVA was classified as low, medium and high ChAcNLS load by reference to a log plot of molecular weight vs. 1 / Rf of Kaleidoscope pre-stained standards (Bio-Rad) run under identical conditions. In addition, Western blot analysis against OVA was performed to confirm the Coomassie results.

[0138] Biochemical characterization of ChAcNLS-OVA

[0139] A series of tests were performed by Charles River (Wilmington, MA, USA) including: 1) differential scanning calorimetry or dynamic light scattering, 2) circular dichroism (CD) far- and near-UV spectroscopic scans and Fourier transform infrared spectroscopy (FTIR), 3) size exclusion chromatography coupled with multi-angle laser light scattering, 4) intrinsic tryptophan fluorescence (ITF), 5) peptide mapping (reference standard characterization by LC-MS / MS), and 6) determination of intact and subunit molecular weights via LC-MS to characterize the ChAcNLS-OVA modified antigen.

[0140] Generation of bile acid-NLS moieties

[0141] Unless otherwise specified, the synthesis of cholic acid-NLS moieties was similar to that of cholic acid-NLS (ChAcNLS) as previously described in Beaudolin et al., 2016. For example, for CA-SV40NLS, cholic acid was conjugated to the free amino group of the N-terminal cysteine residue of a 13-mer peptide (CGYGPKKKRKVGG; SEQ ID NO: 1) comprising a nuclear localization signal from the SV40 large T antigen (SEQ ID NO: 7) flanked by linker amino acids.

[0142] Evaluation of intrinsic tryptophan fluorescence (ITF)

[0143] Chirascan 4000 (Applied Photophysics, Leatherhead, Surrey, UK) was used to measure the CD spectra of ChAcNLS-OVA at 0.1 mg / mL in PBS at 25 °C. The spectra were recorded from 190 to 340 nm at a scan rate of 50 nm / min and a bandwidth of 1 nm. TMA Q100 circular dichroism (CD) spectrometer was used for intrinsic tryptophan fluorescence (ITF) analysis and dry-body temperature incubation using a VWR digital heating block (Radnor, PA). A Chirscan Q100 autosampler rack cooling system was used for all 4°C incubations. Data were analyzed using MATLAB software (Natick, MA). Briefly, samples were removed from storage at -20°C and allowed to equilibrate to room temperature. Samples were then diluted in PBS from a stock concentration ranging from 4 to 5 mg / mL to 0.8 mg / mL. Diluted samples were then analyzed for ITF after 10 minutes of heat stress without exposure to heat stress (native) or by dry-body incubation. One aliquot of each diluted sample was incubated at 4°C, a second aliquot was incubated at 37°C, and a third aliquot was incubated at 80°C. BSA diluted to 0.8 mg / mL was mixed with the samples under each of the above thermal conditions. All samples were allowed to equilibrate to room temperature after incubation. ITF analysis was performed by excitation at 280 nm with an emission scan range of 200-600 nm, a bandwidth of 1.0 nm, a time per point of 1 second, and a step size of 0.5, repeated in triplicate 8 times. Triplicate spectra were blank subtracted, averaged, and converted from units of mdeg to relative fluorescence intensity using MATLAB software. Diluted BSA solutions were measured as controls before and after the sample sequence.

[0144] DC2.4 transfection and evaluation of damaged endosomes by microscopy

[0145] For this assay, 15 x 10 3 DC2.4 cells were seeded onto sterile coverslips in 24-well plates. Two days after transfection of DC2.4 cells with eGFP-hGal3 mammalian expression vector, 0.1 mg / mL of nOVA or cOVA was added to the cells, which were then incubated at 37°C for 3 hours. Cells were then washed twice to remove excess protein and then fixed onto the coverslips. Coverslips were observed by fluorescence microscopy (Nikon, Eclipse Ti2-U) and results were analyzed using Image J TM software. TM

[0146] Phenotypic evaluation of generated BMDCs by flow cytometry

[0147] To assess expression of cell surface markers, BMDCs were incubated with various antibodies diluted according to the manufacturer’s instructions using staining buffer (PBS containing 2% FBS) for 30 minutes at 4°C in the dark. After extensive washing with staining buffer, cells were resuspended in 400 μL of staining buffer. Cells were analyzed by BD FACSDiva TM on a CANTO II TM ​Samples were acquired and then analyzed using FlowJo TM v10 analysis.

[0148] Monitoring of antigen processing

[0149] To assess OVA processing, cells were incubated with 10 pg / mL OVA-DQ (with or without ChAcNLS modification) at 37°C. After 30 min, cells were washed and regular culture medium was added. At the end of the indicated incubation time, cells were collected and washed with cold PBS containing 2% FBS. Cells were analyzed by flow cytometry to monitor fluorescence.

[0150] Antigen presentation assay

[0151] To assess antigen cross-presentation, cells were seeded at 25 x 10 3 cells / well in 24-well plates (Corning; Massachusetts, United States) and then pulsed with different concentrations of antigen for 3 h. At the end of the pulse period, cells were washed to remove excess antigen and co-cultured with 10 6 mL CD4 or CD8 T cells purified from the spleen of OT-II or OT-I mice, respectively, using a T cell isolation kit according to the manufacturer's protocol. After 72 h, supernatants were collected and used to quantify cytokine production by commercial enzyme-linked immunosorbent assay (ELISA).

[0152] For the B3Z assay, 5 x 10 4 DCs were first pulsed with the selected protein or cOVA variant for 3 h, followed by washing, before adding 5 x 10 4 B3Z cells. Cells were incubated for 17-19 h, before lysis and further incubation with chlorophenol red-beta-D-galactopyranoside (CPRG) solution at 37°C for 4-6 h. The optical density signal was detected using a Synergy H1 TM microplate reader (Biotek, Winooski, VT, United States).

[0153] Quantification of antibody titers by ELISA

[0154] Nunc MaxiSorp TMPlates were coated overnight with 1 pg of OVA diluted in coating buffer. The next day, plates were washed and then blocked with 3% non-fat milk for 1 hour at room temperature. Following this step, plates were washed before addition of diluted sera (two-fold dilutions were prepared). After 2 hours of incubation, plates were washed before addition of 1 : 1000 diluted HRP-linked anti-mouse IgG secondary antibody. After two hours, plates were washed and then incubated with HRP for 10-20 minutes at room temperature. After HRP quenching, signal was detected using Synergy TM H1 microplate reader (Biotek; Winooski, VT, United States).

[0155] Immunization and tumor challenge

[0156] For prophylactic vaccination, female C57BL / 6 mice (n=10 / group) were injected subcutaneously (SC) with OVA / OVA-ChAcNLS (1 pg / dose) on days 0 and 14, 10 4个 BMDC or tumor lysates (0.1 mg / mL). Two weeks after the second vaccination, mice were challenged subcutaneously (SC) with 5x10 5 EG.7 or EL4 cells and tumor growth was evaluated over time. To assess antigen-specific CD8 T cell activation, splenocytes isolated from immunized mice were first stimulated in vitro with 1 pg / mL OVA, and supernatants were collected three days later to assess cytokine / chemokine production by Luminex TM

[0157] For therapeutic vaccination, female C57BL / 6 mice (n=10 / group) received a SC injection of 5x10 5 EL4 or EG.7 cells on day 0. Five days later (appearance of palpable tumors of ~40-60 mm 3 , mice were injected SC with 3x10 4 BMDC pulsed with OVA- / OVA-ChAcNLS or tumor lysate- / ChAcNLS lysates (two injections; 1 week apart). Control animals received only 5x10 5 tumor cells. Tumor growth was followed thereafter in treated animals. For therapeutic vaccination in combination with immune checkpoint inhibitors (e.g. aPD-1), mice received a SC injection of 200 pg / dose of antibody or its isotype every 2 days for a total of 6 doses over two weeks. Similar procedures were performed in BALB / c mice for allogeneic vaccination.

[0158] Analysis of tumor infiltrating immune cells

[0159] ​After tumor resection, the tumor mass was first weighed, and then cut into small pieces using surgical scissors in 4-5 ml of master mix. This master mix contained 2 mg / ml collagenase D, 2 mg / ml collagenase IV, and 100 μg / mL DNase IV mixed in DMEM supplemented with 5% FBS. The mixture was then stirred in a cell culture incubator at 37°C. After incubation for 30 minutes, 10 ml of DMEM was added to neutralize the enzymatic reaction. The digested solution was filtered through a 70 μm cell filter, and all remaining fragments at the top of the filter were crushed with a plunger. 1-2 ml of DMEM was then added to wash the filter. The collected cells were then centrifuged at 1200 rpm (4°C) for 5 minutes, treated with erythrocyte lysis buffer for 1 minute, and then resuspended in 3-4 ml of DMEM supplemented with 5% FBS. After washing the cells, the pellet was resuspended in DMEM supplemented with 5% FBS, and then cell staining was performed for flow cytometry analysis.

[0160] Antigen presentation assay using the B3Z reporter system

[0161] Various bile acid-NLS conjugates were screened using the B3Z reporter system. The B3Z cell line is a T-cell hybridoma specific to the H2-Kb-SIINFEKL complex. The LacZ reporter gene (controlled by the NFAT promoter) is expressed once activated via its TCR. In short, 1.5 × 10⁻⁶ cells... 5 5 × 10⁵ BMDCs or isolated B cells treated with ovalbumin (OVA)-bile acid-NLS conjugate 4 B3Z cells were co-cultured overnight at 37°C and 5% CO2. The next day, all cells were washed twice with PBS (pH 7.4), and the cell pellet was lysed by adding 100 μL of lysis buffer in PBS containing 0.15 mM chlorophenol red-β-D-galactopyranoside (CPRG) substrate (Calbiochem, La Jolla, CA), 0.125% NP40 (EMD Sciences, La Jolla, CA), 9 mM MgCl2 (Aldrich, USA), and 100 mM 2-mercaptoethanol. After incubation at 37°C for 5 h or 24 h, absorbance was obtained at 570 nm, with 636 nm as the reference wavelength. For these experiments, the bile acid-NLS-OVA conjugate was resuspended at 0.1 mg / mL in PBS (pH 7.3) (prepared using a 10× molar ratio of bile acid-NLS fraction:OVA), and OVA alone was resuspended at 5 mg / mL.

[0162] Statistical analysis

[0163] P values were calculated using one-way ANOVA. Results are expressed as the mean with S.D. error bars, and statistical significance is indicated by asterisks: *P < 0.05, **P < 0.01, ***P < 0.001.

[0164] Example 2: Biochemical characterization of ChAcNLS antigen formulations

[0165] The steroid acid-peptide conjugate ChAcNLS was synthesized as described in Example 1. Briefly, cholic acid was conjugated to the free amino group of the N-terminal cysteine residue of a 13-mer peptide. This peptide (CGYG PKKKRKV GG; SEQ ID NO: 1) contains a nuclear localization signal from the SV40 large T antigen (underlined) flanked by linker amino acids. Multiple ChAcNLS moieties were then conjugated to the epsilon-amino groups of accessible lysine residues of the prototypic polypeptide antigen OVA (SEQ ID NO: 2; Figure 1C ). Figure 1A A schematic representation of the covalent attachment of a given antigen to ChAcNLS moieties is shown in FIG. 1. ChAcNLS-OVA was then biochemically characterized as described in Example 1, and the conjugation was confirmed by Coomassie blue staining ( Figure 1B ) and Western blotting ( Figure 1E ). Biochemical characterization revealed that ChAcNLS-OVA conjugated at a 25x ratio ( Figure 1B , lane 2) was on average conjugated to about four ChAcNLS moieties per OVA, corresponding to an increase in molecular weight (MW) of about 8.6 kDa. ChAcNLS-OVA conjugated at a 50x ratio ( Figure 1B , lane 3) was on average conjugated to about eight ChAcNLS moieties per OVA, corresponding to a MW of about 19.2 kDa. Figure 1D A ribbon structure of the OVA protein with lysine residues predicted to be highly (blue), moderately (green), or poorly (yellow) accessible lysine residues is shown in FIG. 2.

[0166] In addition, to assess the overall stability of ChAcNLS-OVA (cOVA), ITF analysis was performed to measure its unfolding after heat stress. In this assay, a shift in peak position or change in intensity is indicative of unfolding, as polypeptide residues can become exposed to solvent and undergo a change in orientation ( Figure 1F ). When different cOVA ratios were assayed under native or heat- variable conditions, nOVA underwent complete denaturation at 80°C, while a partial decrease in peak intensity was observed for 50x COVA ( Figure 1F ). No changes in ITF spectral measurements were observed for the other cOVA samples, indicating that conjugation to ChAcNLS moieties greatly increased antigen stability.

[0167] An antigen presentation assay using the SIINFEKL-specific B3Z cell line was then performed to compare the different OVA conjugates. As shown in Figure 1G different conjugates tested included: a cholic acid-NLS moiety ("ChAcNLS"); OVA conjugated to a cholic acid moiety without an NLS peptide ("ChAc-OVA"); OVA conjugated to a cholic acid-NLS moiety via a PEG4 bifunctional linker ("ChAcNLS-PEG4-OVA" or "cOVA"); and OVA conjugated to a cholic acid-NLS moiety via a PEG6 bifunctional linker ("ChAcNLS-PEG6-OVA"). As shown in Figure 1H conjugated to a cholic acid moiety without an NLS peptide ("ChAc-OVA") did not result in improved antigen presentation compared to naked OVA ("nOVA"). Strikingly, OVA conjugated to a 50x molar excess of ChAcNLS moiety via a PEG4 bifunctional linker ("cOVA (50x)") exhibited the same level of antigen presentation as the SIINFEKL positive control peptide ("SIINFEKL"). Interestingly, equivalent levels of antigen presentation were obtained by reducing the molar excess of ChAcNLS moiety to 5x, lOx, and 25x ["cOVA (ChAcNLS-PEG4-OVA)"], but this elevated antigen presentation was lost at a 2x molar excess of ChAcNLS moiety. For OVA conjugated to a 2x to 25x molar excess of ChAcNLS moiety via a PEG6 bifunctional linker ("ChAcNLS-PEG6-OVA"), comparable levels of antigen presentation to the SIINFEKL positive control were also observed. Finally, OVA conjugated to a 5x and lOx molar excess of ChAcNLS moiety via a much longer PEG 24 bifunctional linker ("ChAcNLS-PEG 24 -OVA") observed higher levels of antigen presentation than nOVA but lower than the SIINFEKL positive control peptide, but the increase in antigen presentation over nOVA was lost at 2x and 25x molar excess (data not shown).

[0168] Example 3: In vitro cross-presentation of ChAcNLS-OVA

[0169] To generate BMDCs, femurs and tibias of female C57BL / 6 or BALB / c mice were flushed to collect total nucleated cells. Cells were then plated with recombinant GM-CSF (10 ng / mL) for 8 days and replaced every 2 days. LPS was added on day 9 to trigger DC maturation, followed by antigen pulsing. Maturation of BMDCs was confirmed by flow cytometry. No T cells, B cells, or NK cells were detected on day 9, and over 80% of BMDCs expressed CD11c+, CD80+, CD86+, and I-Ab+. BMDCs were then incubated with different concentrations of naked OVA (nOVA) or ChAcNLS-OVA (cOVA), with the addition of CD4+ T cells from OT-II transgenic mice or CD8+ T cells from OT-I transgenic mice.

[0170] Figure 2A is a schematic showing the set-up for assessing antigen cross-presentation of OVA peptides (i.e., SIINFEKL [OT-I peptide for CD8+ T cells; SEQ ID NO: 5] or ISQAVHAAHAEINEAGR [OT-II peptide for CD4+ T cells; SEQ ID NO: 6]) for OVA-responsive OT-I (CD8) and OTII (CD4) T cell activation.

[0171] Figure 2B shows the amount of IFN-g produced using OT-I derived CD8 T cells, which is a measure of cross-presentation activity. Strikingly, CD8+ T cells incubated with BMDCs and cOVA produced significantly more IFN-g than when incubated with naked antigen (nOVA). Figure 2C and Figure 2D shows the amount of IL-2 and IFN-g produced using OT-II derived CD4 T cells, which is a measure of classical MHC class II cross-presentation activity. Strikingly, CD4+ T cells incubated with BMDCs and cOVA produced significantly more IFN-g than when incubated with naked antigen (nOVA). From these observations, intracellular processing of captured OVA was then monitored. To do this, ChAcNLS was cross-linked to OVA-DQ prior to pulsing of ex vivo generated primary bone marrow-derived DCs. While a difference in increase of signal density at 3 hours post-DC pulsing could be delineated between the two antigen conditions, signal density was significantly higher in DCs treated with OVA-DQ linked to ChAcNLS at 6 hours post-pulsing compared to nOVA Figure 2E and Figure 2F ). Interestingly, no difference in signal intensity was detected between 3 hours or 6 hours of nOVA pulsing, suggesting signal saturation Figure 2E). However, these observations were related to antigen presentation assays using primary DCs co-cultured with OT-I (CD8) Figure 2B ) or OT-II (CD4) T cells Figure 2C and Figure 2D ).

[0172] To determine whether cOVA enhances endosomal to cytosol escape, the galectin-3 (Gal3) expression system was used as a marker of damaged endomembranes. More specifically, Gal3 exhibits a high affinity for P-galactoside conjugates that are normally present on the cell surface, in the lumen of the Golgi and endocytic compartments. Thus, when expressed under normal conditions, Gal3 is uniformly distributed throughout the cytoplasm. In contrast, induction of endosomal membrane rupture brings Gal3 in close proximity to and binds luminal glycoproteins. We therefore transiently transfected the DC2.4 cell line with a construct to express Gal3 in the form of a fusion with enhanced green fluorescent protein (eGFP-Gal3) to evaluate its distribution pattern. As expected, after treatment of DC2.4 cells expressing eGFP-Gal3 with nOVA, the GFP signal was widely distributed throughout the cytosol Figure 2G (top panel). In contrast, treatment of DC2.4 with cOVA pulse induced the appearance of several dots, clearly indicating signal relocation to damaged endosomes Figure 2G (bottom panel).

[0173] Example 4: In vivo anti-cancer activity

[0174] To determine the effectiveness of ChAcNLS-modified OVA as a prophylactic vaccine, mice were vaccinated with cOVA as a cell-based vaccine or as a stand-alone vaccine. For the cell-based vaccine, BMDCs pulsed with nOVA or cOVA were injected subcutaneously into mice, followed by implantation of EG.7 lymphoma cells, followed by challenge. The immunization protocol is depicted in Figure 3A .

[0175] Remarkably, mice vaccinated with BMDCs pulsed with cOVA did not show any tumor growth and had a 100% survival rate, while control (non-vaccinated) and mice vaccinated with BMDCs pulsed with nOVA developed large tumors and were more prone to die Figure 3B and Figure 3C . Moreover, mice vaccinated with BMDCs pulsed with cOVA developed higher antibody titers Figure 3D . In addition, in the cOVA-DC group, the levels of CD4 effector (CD44hiCD62Llo) and CD8 central (CD44hiCD62Lhi) and effector memory T cells were significantly higher Figure 3E and Figure 3F). Finally, Luminex analysis of cytokine / chemokine profiles from T cells derived from in vitro restimulations TM Analysis revealed an increase in IFN-γ levels in the coVa group compared to nOVA-injected mice Figure 3G ). Similar data were observed for macrophage inflammatory proteins (MIP)-1 β and MIP-2, two strong chemotactic proteins for monocytes / macrophages, NK cells and neutrophils, and interleukins (IL)-6 and IL-10, two cytokines known to support B cell differentiation and antibody production Figure 3G . Altogether, the improved immune responses observed in animals vaccinated with cOVA-pulsed DCs are consistent with the resistance to multiple EG.7 re-challenges and the long-term survival benefit they acquired.

[0176] In a similar immunization protocol, mice were vaccinated with coVa or nOVA alone (non-BMDC-pulsed) prior to implantation of EG.7 lymphoma cells Figure 4A . As shown in Figure 4B , mice vaccinated with cOVA developed smaller tumors, had a significantly increased survival Figure 4C ) and antibody responses Figure 4D . The use of two squalene-based water-in-oil emulsion adjuvants (AddaS03 TM or AddaVax TM ) for vaccination improved the efficacy of the immune responses, with AddaVax triggering superior effects in cOVA-vaccinated mice.

[0177] Example 5: In vivo therapeutic vaccination against T cell lymphoma Figure 3A Figure 3B Figure 3C Figure 3D Figure 3E Figure 3F Figure 3G Figure 3G Figure 4A Figure 4B Figure 4C Figure 4D Example 5: In vivo therapeutic vaccination against T cell lymphoma

[0178] To determine the effectiveness of bile acid-conjugated polypeptide antigens as therapeutic vaccines, mice were first implanted with EG.7 lymphoma cells and then immunized with nOVA- or cOVA-pulsed BMDCs in the presence or absence of the immune checkpoint inhibitor / anti-cancer agent anti-PD-1 antibody. The immunization protocol is shown in Figure 5A .

[0179] Mice immunized with cOVA-pulsed BMDCs had significantly smaller tumors Figure 5B ) and increased survival Figure 5C). Remarkably, mice treated with the combination therapy of anti-PD-1 Ab and cOVA-pulsed BMDCs showed synergistic effects in treating T cell lymphoma in mice, as shown by the reduction in tumor volume and the increase in survival rate. This synergistic effect was directly related to the number of immunized cOVA-pulsed BMDCs in mice Figure 5D and 5E ).

[0180] Finally, ChAcNLS was covalently linked to EL4 T cell lymphoma lysates to determine the effect of an antigen-specific therapeutic vaccine. Mice engrafted with EL4 T cell lymphoma cells were immunized with BMDCs pulsed with EL4 lysates alone or ChAcNLS-EL4 lysates in the presence or absence of anti-PD-1 antibody Figure 6A ). Similar to cOVA, mice immunized with ChAcNLS-EL4 lysate-pulsed BMDCs had significantly smaller tumors and increased survival rate compared to mice immunized with EL4 lysate-pulsed BMDCs alone or with anti-PD-1 Ab, regardless of the presence of anti-PD-1 Ab Figure 6B and Figure 6C ). Notably, a synergistic effect was seen with the combination of ChAcNLS-EL4 lysate-pulsed BMDCs and anti-PD1 Ab therapy, as tumor growth in mice plateaued around 36 days and mice had a 70% survival rate at the end of the study (54 days). These observations were further supported by tumor infiltrating lymphocyte (TIL) analysis Figure 6E ), which revealed an enhanced recruitment of CD8, NK, and CD11c immune effector cells in DCs pulsed with ChAcNLS-EL4 lysate ("cLysate") and PD-1 Figure 6E ). In sharp contrast, the levels of regulatory CD4 T cells (Tregs) were greatly reduced in the same group Figure 6E ), supporting the notion that combining DCs pulsed with cLysate and PD-1 favors inflammation by tilting the balance against suppressive Treg infiltration in favor of CD8 T cells Figure 6F ). Altogether, these findings indicate that "off-the-shelf allogeneic DCs" prepared with ChAcNLS-EL4 lysate formulation can be effectively used as a universal vaccine to trigger potent anti-tumor responses.

[0181] Example 6: In vivo therapeutic vaccination against SARS-CoV-2

[0182] To determine the effectiveness of bile acid-conjugated polypeptide antigens as therapeutic vaccines for microbial infections, particularly viral infections such as SARS-CoV-2 infection, a vaccine consisting of ChAcNLS covalently linked to the SARS-CoV-2 spike protein was constructed, similar to the construction of the cOVA vaccine described in Example 1, Example 2, and Example 4.

[0183] Figure 7A and Figure 7B A schematic showing the SARS-CoV-2 spike protein for the ChAcNLS-spike-CoV-2 formulation, and ribbon structure of the SARS-CoV-2 spike protein (strain D614G) with lysine residues predicted to be highly (blue), moderately (green), or less (yellow) accessible lysine residues, similar to OVA. As better described by the amino acid sequence of the SARS-CoV-2 spike protein (SEQ ID NO: 3; Figure 7B ) more than 50% of the lysine residues are predicted to be accessible (highlighted in black) for ChAcNLS conjugation.

[0184] Mice were vaccinated with full-length “naked” spike-CoV-2 (unconjugated; nSpike-CoV-2; black bars) or with ChAcNLS-spike-CoV-2 (“cSpike-CoV-2”; grey bars) in the presence of AddaS03 or AddaVax adjuvant Figure 8A ). In the presence of either adjuvant, elevated IgG titers against the spike protein were observed in mice vaccinated with cSpike-CoV-2 compared to unconjugated nSpike-CoV-2. These IgG antibodies were predominantly of the IgG1 isotype, however, significant levels of IgG2a and IgG2b were observed Figure 8B ).

[0185] To evaluate the immunogenicity of different domains of the SARS-CoV-2 spike protein, mice were also vaccinated with unconjugated vaccine and conjugated vaccine containing either the S1-RBD or S2 portion. Titers from mice vaccinated with the S1-RBD and S2 portion of the CoV-2 spike protein were significantly elevated in the presence of AddaS03 or AddaVax adjuvant, as shown in Figure 8C and Figure 8D .

[0186] To evaluate whether anti-spike IgG antibodies from vaccinated mice had neutralizing activity, an in vitro infectivity neutralization assay was developed using spike 1-pseudotyped virus-like particles and HEK cells. As shown in Figure 8EAs shown, sera from mice inoculated with cSpike-CoV-2 were more effective in inhibiting viral infection of HEK cells and thus had stronger neutralizing activity as shown by the NT50 titers compared to nSpike-CoV-2.

[0187] Vaccines consisting of spike protein conjugated to ChAcNLS were shown to be effective in generating strong humoral responses. To determine if the same vaccines were effective in generating cellular responses, cytokine profiling after in vitro T cell restimulation was evaluated in mice inoculated with nSpike-CoV-2 or cSpike-CoV-2 in the presence of two different adjuvants. Results are shown in Figure 9A and Figure 9B In mice inoculated with cSpike-CoV-2, a strong IFN-g response was observed with either adjuvant compared to inoculation with nSpike-CoV-2, indicating a strong and consistent Th1 response needed to control viral infection.

[0188] To further evaluate the immunogenicity of the SARS-CoV-2 vaccine, rabbits and hamsters were inoculated with different doses of cSpike-CoV-2 in the presence of different adjuvants. Figure 10 shows a schematic of the vaccine inoculation design of rabbits with cSpike-CoV-2 Figure 10A and IgG titers at different time points and doses Figure 10B . Figure 11 shows the evaluation of therapeutic efficacy of cSpike-CoV-2 in a challenge model. Figure 11 shows a schematic of the experimental design of vaccine efficacy studies in hamsters Figure 11A and IgG titers in response to MONTANIDE® ISA 720 VG adjuvant or AddaS03 mixed with FDA approved (GMP grade) MONTANIDE® ISA 720 VG adjuvant or AddaS03 TM IgG antibody titers of cSpike CoV-2 vaccines in response to MONTANIDE® ISA 720 VG adjuvant or AddaS03 Figure 11B Here, the vaccines were tested using an excess molar ratio of ChAcNLS to spike-CoV-2 protein (10X and 50X). Hamsters were challenged intranasally with SARS-CoV-2 delta variant prior to the third dose. In general, all doses of vaccines were well tolerated by rabbits and hamsters and generated strong humoral responses.

[0189] Finally, to evaluate if inoculation with cSpike-CoV-2 was protective against infection with different SARS-CoV-2 variants, sera from vaccinated mice were tested for cross-reactivity to spike proteins from California, Brazil, South Africa, UK, India and delta strain, which have specific mutations in the RBD relative to the “wild type” strain D614G Figure 12A and Figure 12B and Figure 12CAs shown, sera from mice immunized with cSpike-CoV-2 had significant cross-reactivity with the spike protein from each of the SARS-CoV-2 variants tested. Moreover, the antibodies from the sera of vaccinated mice were protective and had strong neutralizing activity against the UK, Brazil, South Africa, delta, and California SARS-CoV-2 strains, as shown in in vitro neutralization assays Figure 12D

[0190] Overall, these findings indicate that ChAcNLS-CoV-2-spike protein formulations can be effective as universal vaccines that trigger potent antiviral responses.

[0191] Example 7: In vivo therapeutic vaccination against different SARS-CoV-2 variants

[0192] To determine if SARS-CoV-2 vaccines using spike proteins derived from different variants are effective using the same formulation, the cSpike-CoV-2-IN vaccine was produced using spike proteins from the Indian variant.

[0193] Mice were vaccinated with different doses of the cSpike-CoV-2-IN vaccine or with saline (control), and elevated IgG titers in sera and BALF were observed at different time points (Figure 13). Moreover, strong cellular responses were also observed by detecting different cytokine and chemokine levels in T cells from mice vaccinated with cSpike-CoV-2-IN (Figure 14).

[0194] Finally, sera from mice vaccinated with the cSpike-CoV-2-IN vaccine had cross-reactivity with the spike protein from all the different SARS-CoV-2 variants tested Figure 15

[0195] Overall, these findings indicate that ChAcNLS can be adapted to spike proteins from different SARS-CoV-2 variants to formulate effective vaccines that trigger broad, protective, and potent antiviral responses.

[0196] Example 8: Enhanced antigen presentation of antigens conjugated to different bile acid-NLS conjugates

[0197] Different bile acid-NLS conjugates were produced conjugated to OVA and their ability to enhance OVA antigen presentation by DCs or B cells was evaluated in a B3Z reporter assay. As shown in the SDS-PAGE in Figure 16, bile acid-NLS-OVA conjugates were produced with a 10x or 50x molar excess ratio of bile acid-NLS to OVA, but not with a 100x molar excess ratio of bile acid-NLS to OVA, as shown by the lack of a band corresponding to the conjugate in the SDS-PAGE. Figure 17 (dendritic cells) and Figure 18 ​​The results for the (B cell) antigen presentation assays are shown only for conjugates produced at a 10× molar excess ratio. Based on the relative migration distance (Rf) calculated from SDS-PAGE results (e.g., Figure 16): a 10× molar excess of bile acid-NLS reactant resulted in approximately 1–4 bile acid-NLS moles per OVA molecule; a 25× molar excess of bile acid-NLS reactant resulted in approximately 5–9 bile acid-NLS moles per OVA molecule; and a 50× molar excess of bile acid-NLS reactant resulted in approximately 12–20 bile acid-NLS moles per OVA molecule.

[0198] Dendritic cells as antigen presenting cells

[0199] like Figure 17 As shown, all the different bile acid-NLS conjugates tested enhanced BMDC presentation of OVA at levels similar to or higher than those of CA-SV40 (i.e., ChAcNLS; in Figure 17 (shown as a dashed line in the middle). Notably, Figure 17 The increased antigen presentation observed in all bile acid-NLS conjugates tested was superior to that observed in the "OVA alone" control, despite the fact that a 50-fold concentration of OVA antigen (5 mg / mL) was used compared to the OVA conjugate (0.1 mg / mL). Interestingly, several bile acid-NLS conjugates such as CDCA-SV40, UDCA-SV40, GDCA-SV40, GDCA-NLS2-RPS17, and LCA-NLS2-RPS17 significantly enhanced OVA presentation compared to CA-SV40. Figure 17 In addition, a negative control, in which OVA was conjugated with the SV40 peptide alone (without bile acids) at a more comparable concentration of 0.1 mg / mL, was used. Figure 17 The bile acid-NLS conjugate "SV40-CA" produces results similar to the negative control "PBS". The main difference between the bile acid-NLS conjugate "SV40-CA" and the conjugate "CA-SV40" lies in the arrangement of the bile acid groups. Specifically, in the "SV40-CA" conjugate, the bile acid groups are conjugated to the C-terminus of the SV40NLS peptide via a lysine residue added to the C-terminus. Figure 17 As shown, both the “SV40-CA” and “CA-SV40” conjugates produce similar B3Z responses, indicating that the arrangement of the bile acid groups relative to the peptide moiety does not affect the immunostimulatory effect of the bile acid-NLS moiety.

[0200] B cells as antigen presenting cells

[0201] like Figure 18As shown, most of the different bile acid-NLS conjugates tested produced equivalent or enhanced B cell antigen presentation compared to a fifty-fold dose of OVA alone (5 mg / mL) control or compared to CA-SV40 (i.e., ChAcNLS). Interestingly, several bile acid-NLS conjugates such as DCA-SV40, UDCA-SV40, LCA-SV40, GDCA-GWG-SV40, GDCA-PQBP1, CA-hnRNP M, CA-NLS2-RPS17, GDCA-hnRNPA1 M9, CA-cMyc, GDCA-SV40, GUDCA-PQBP1, and CDCA-NLS3-RPS17 significantly enhanced OVA presentation compared to CA-SV40.

[0202] Overall, these findings support the versatility of multiple bile acid-NLS conjugates to improve the immunogenicity of a given polypeptide antigen (e.g., resulting from enhanced antigen presentation), potentially enabling the use of lower doses of polypeptide antigens, which can be the most expensive component of manufacturing a subunit vaccine.

[0203] References

[0204] Anding AL, Baehrecke EH. Cleaning House: Selective Autophagy of Organelles. Dev Cell 2017; 41(l): 10-22.

[0205] Anguille S, Smits EL, Lion E, et al. Clinical use of dendritic cells for cancer therapy. Lancet Oncol 2014; 15(7): e257-67.

[0206] Azuar et al., (2019). Cholic Acid-based Delivery System for Vaccine Candidates against Group A Streptococcus. ACS Medicinal Chemistry Letters, 10: 1253-1529.

[0207] Beaudoin et al., (2016). ChAcNLS, a novel modification to antibody-conjugates permitting target cell-specific endosomal escape, localization to the nucleus and enhanced total intracellular accumulation. Molecular Pharmaceutics, 13(6): 1915-26.

[0208] Hanafi et al., (2018). Overview of Bile Acids Signaling and Perspective on the Signal of Ursodeoxycholic Acid, the Most Hydrophilic Bile Acid, in the Heart. Biomolecules, 8(4): 159.

[0209] Murakami et al., (2020). Bile acids and ceramide overcome the entry restriction for GII.3 human norovirus replication in human intestinal enteroids. Proceedings of the National Academy of Sciences USA. 117(3): 1700-1710.

[0210] Patel et al., (2017). Next generation approaches for tumor vaccination. Chinese Clinical Oncology. 6(2): 19.

[0211] Shivanna et al., (2014) The crucial role of bile acids in the entry of porcine enteric calicivirus. Virology 456-457, 268-278.

[0212] Shivanna et al., (2015). Ceramide formation mediated by acid sphingomyelinase facilitates endosomal escape of caliciviruses. Virology, 483, 218-228.

[0213] Smith et al., (2019). Alternative tumour-specific antigens. Nature Review Cancer. 19(8):465-478.

[0214] Sun et al., (2016). Factors influencing the nuclear targeting ability of nuclear localization signals. Journal of Drug Targeting, 24(10):927-933.

[0215] Tagliamonte et al., (2014). Antigen-specific vaccines for cancer treatment. Human Vaccines & Immunotherapeutics, 10(11):3332-3346. SEQUENCE LISTING <110> DEFENCE THERAPEUTICS INC. <120> Covalently modified antigens for improved immune response and / or stability <130> 20751-6 <150> US 63 / 127,731 <151> 2020-12-18 <150> US 63 / 202,047 <151> 2021-05-25 <160> 20 <170> PatentIn version 3.5 <210> 1 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> ChAcNLS <220> <221> modified_residue <222> (1)..(1) <223> ChAc <400> 1 Cys Gly Tyr Gly Pro Lys Lys Lys Arg Lys Val Gly Gly 1 5 10 <210> 2 <211> 385 <212> PRT <213> Artificial Sequence <220> <223> OVA <400> 2 Gly Ser Ile Gly Ala Ala Ser Met Glu Phe Cys Phe Asp Val Phe Lys 1 5 10 15 Glu Leu Lys Val His His Ala Asn Glu Asn Ile Phe Tyr Cys Pro Ile 20 25 30 Ala Ile Met Ser Ala Leu Ala Met Val Tyr Leu Gly Ala Lys Asp Ser 35 40 45 Thr Arg Thr Gln Ile Asn Lys Val Val Arg Phe Asp Lys Leu Pro Gly 50 55 60 Phe Gly Asp Ser Ile Glu Ala Gln Cys Gly Thr Ser Val Asn Val His 65 70 75 80 Ser Ser Leu Arg Asp Ile Leu Asn Gln Ile Thr Lys Pro Asn Asp Val 85 90 95 Tyr Ser Phe Ser Leu Ala Ser Arg Leu Tyr Ala Glu Glu Arg Tyr Pro 100 105 110 Ile Leu Pro Glu Tyr Leu Gln Cys Val Lys Glu Leu Tyr Arg Gly Gly 115 120 125 Leu Glu Pro Ile Asn Phe Gln Thr Ala Ala Asp Gln Ala Arg Glu Leu 130 135 140 Ile Asn Ser Trp Val Glu Ser Gln Thr Asn Gly Ile Ile Arg Asn Val 145 150 155 160 Leu Gln Pro Ser Ser Val Asp Ser Gln Thr Ala Met Val Leu Val Asn 165 170 175 Ala Ile Val Phe Lys Gly Leu Trp Glu Lys Ala Phe Lys Asp Glu Asp 180 185 190 Thr Gln Ala Met Pro Phe Arg Val Thr Glu Gln Glu Ser Lys Pro Val 195 200 205 Gln Met Met Tyr Gln Ile Gly Leu Phe Arg Val Ala Ser Met Ala Ser 210 215 220 Glu Lys Met Lys Ile Leu Glu Leu Pro Phe Ala Ser Gly Thr Met Ser 225 230 235 240 Met Leu Val Leu Leu Pro Asp Glu Val Ser Gly Leu Glu Gln Leu Glu 245 250 255 Ser lie lie Asn Phe Glu Lys Leu Thr Glu Trp Thr Ser Ser Asn Val 260 265 270 Met Glu Glu Arg Lys lie Lys Val Tyr Leu Pro Arg Met Lys Met Glu 275 280 285 Glu Lys Tyr Asn Leu Thr Ser Val Leu Met Ala Met Gly lie Thr Asp 290 295 300 Val Phe Ser Ser Ser Ala Asn Leu Ser Gly lie Ser Ser Ala Glu Ser 305 310 315 320 Leu Lys lie Ser Gin Ala Val His Ala Ala His Ala Glu lie Asn Glu 325 330 335 Ala Gly Arg Glu Val Val Gly Ser Ala Glu Ala Gly Val Asp Ala Ala 340 345 350 Ser Val Ser Glu Glu Phe Arg Ala Asp His Pro Phe Leu Phe Cys lie 355 360 365 Lys His lie Ala Thr Asn Ala Val Leu Phe Phe Gly Arg Cys Val Ser 370 375 380 Pro 385 <210> 3 <211> 1310 <212> PRT <213> Artificial Sequence <220> <223> SARS-CoV-2 Spike Protein <400> 3 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gin Cys Val 1 5 10 15 Asn Leu Thr Thr Arg Thr Gin Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 His Ser Thr Gin Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gin Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 Lys Val Cys Glu Phe Gin Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 130 135 140 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 145 150 155 160 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gin Pro Phe Leu 165 170 175 Met Asp Leu Glu Gly Lys Gin Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190 Val Phe Lys Asn He Asp Gly Tyr Phe Lys He Tyr Ser Lys His Thr 195 200 205 Pro He Asn Leu Val Arg Asp Leu Pro Gin Gly Phe Ser Ala Leu Glu 210 215 220 Pro Leu Val Asp Leu Pro He Gly He Asn He Thr Arg Phe Gin Thr 225 230 235 240 Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 245 250 255 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gin Pro 260 265 270 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr He Thr Asp Ala 275 280 285 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300 Ser Phe Thr Val Glu Lys Gly He Tyr Gin Thr Ser Asn Phe Arg Val 305 310 315 320 Gln Pro Thr Glu Ser lie Val Arg Phe Pro Asn lie Thr Asn Leu Cys 325 330 335 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 340 345 350 Trp Asn Arg Lys Arg lie Ser Asn Cys Val Ala Asp Tyr Ser Val Leu 355 360 365 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 370 375 380 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 385 390 395 400 Val lie Arg Gly Asp Glu Val Arg Gin lie Ala Pro Gly Gin Thr Gly 405 410 415 Lys lie Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430 Val lie Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440 445 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 450 455 460 Glu Arg Asp lie Ser Thr Glu lie Tyr Gin Ala Gly Ser Thr Pro Cys 465 470 475 480 Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 485 490 495 Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val 500 505 510 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525 Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540 Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu 545 550 555 560 Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val 565 570 575 Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 580 585 590 Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 595 600 605 Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile 610 615 620 His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser 625 630 635 640 Asn Val Phe Gin Thr Arg Ala Gly Cys Leu lie Gly Ala Glu His Val 645 650 655 Asn Asn Ser Tyr Glu Cys Asp lie Pro lie Gly Ala Gly lie Cys Ala 660 665 670 Ser Tyr Gin Thr Gin Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala 675 680 685 Ser Gin Ser lie lie Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser 690 695 700 Val Ala Tyr Ser Asn Asn Ser lie Ala lie Pro Thr Asn Phe Thr lie 705 710 715 720 Ser Val Thr Thr Glu lie Leu Pro Val Ser Met Thr Lys Thr Ser Val 725 730 735 Asp Cys Thr Met Tyr lie Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu 740 745 750 Leu Leu Gin Tyr Gly Ser Phe Cys Thr Gin Leu Asn Arg Ala Leu Thr 755 760 765 Gly lie Ala Val Glu Gin Asp Lys Asn Thr Gin Glu Val Phe Ala Gin 770 775 780 Val Lys Gln lie Tyr Lys Thr Pro Pro lie Lys Asp Phe Gly Gly Phe 785 790 795 800 Asn Phe Ser Gin lie Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser 805 810 815 Phe lie Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly 820 825 830 Phe lie Lys Gin Tyr Gly Asp Cys Leu Gly Asp lie Ala Ala Arg Asp 835 840 845 Leu lie Cys Ala Gin Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu 850 855 860 Leu Thr Asp Glu Met lie Ala Gin Tyr Thr Ser Ala Leu Leu Ala Gly 865 870 875 880 Thr lie Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gin lie 885 890 895 Pro Phe Ala Met Gin Met Ala Tyr Arg Phe Asn Gly lie Gly Val Thr 900 905 910 Gln Asn Val Leu Tyr Glu Asn Gin Lys Leu lie Ala Asn Gin Phe Asn 915 920 925 Ser Ala lie Gly Lys lie Gin Asp Ser Leu Ser Ser Thr Ala Ser Ala 930 935 940 Leu Gly Lys Leu Gin Asp Val Val Asn Gin Asn Ala Gin Ala Leu Asn 945 950 955 960 Thr Leu Val Lys Gin Leu Ser Ser Asn Phe Gly Ala He Ser Ser Val 965 970 975 Leu Asn Asp He Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gin 980 985 990 He Asp Arg Leu He Thr Gly Arg Leu Gin Ser Leu Gin Thr Tyr Val 995 1000 1005 Thr Gin Gin Leu He Arg Ala Ala Glu He Arg Ala Ser Ala Asn 1010 1015 1020 Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gin Ser Lys 1025 1030 1035 Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro 1040 1045 1050 Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val 1055 1060 1065 Pro Ala Gin Glu Lys Asn Phe Thr Thr Ala Pro Ala He Cys His 1070 1075 1080 Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn 1085 1090 1095 Gly Thr His Trp Phe Val Thr Gin Arg Asn Phe Tyr Glu Pro Gin 1100 1105 1110 Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val 1115 1120 1125 Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gin Pro 1130 1135 1140 Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn 1145 1150 1155 His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn 1160 1165 1170 Ala Ser Val Val Asn Ile Gin Lys Glu Ile Asp Arg Leu Asn Glu 1175 1180 1185 Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gin Glu Leu 1190 1195 1200 Gly Lys Tyr Glu Gin Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu 1205 1210 1215 Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met 1220 1225 1230 Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys 1235 1240 1245 Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro 1250 1255 1260 Val Leu Lys Gly Val Lys Leu His Tyr Thr Leu Glu Ser Gly Gly 1265 1270 1275 Gly Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser 1280 1285 1290 Gly Gly Gly Ser Gly Gly Ser Ser Ala Trp Ser His Pro Gln Phe 1295 1300 1305 Glu Lys 1310 <210> 4 <211> 1255 <212> PRT <213> Artificial Sequence <220> <223> SARS-CoV Spike protein <400> 4 Met Phe Ile Phe Leu Leu Phe Leu Thr Leu Thr Ser Gly Ser Asp Leu 1 5 10 15 Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln 20 25 30 His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg 35 40 45 Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser 50 55 60 Asn Val Thr Gly Phe His Thr lie Asn His Thr Phe Gly Asn Pro Val 65 70 75 80 Ile Pro Phe Lys Asp Gly lie Tyr Phe Ala Ala Thr Glu Lys Ser Asn 85 90 95 Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln 100 105 110 Ser Val lie lie lie Asn Asn Ser Thr Asn Val Val lie Arg Ala Cys 115 120 125 Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met 130 135 140 Gly Thr Gin Thr His Thr Met lie Phe Asp Asn Ala Phe Asn Cys Thr 145 150 155 160 Phe Glu Tyr lie Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser 165 170 175 Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly 180 185 190 Phe Leu Tyr Val Tyr Lys Gly Tyr Gin Pro lie Asp Val Val Arg Asp 195 200 205 Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro lie Phe Lys Leu Pro Leu 210 215 220 Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro 225 230 235 240 Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr 245 250 255 Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile 260 265 270 Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys 275 280 285 Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn 290 295 300 Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr 305 310 315 320 Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser 325 330 335 Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr 340 345 350 Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly 355 360 365 Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala 370 375 380 Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gin He Ala Pro Gly 385 390 395 400 Gln Thr Gly Val He Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe 405 410 415 Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn He Asp Ala Thr Ser 420 425 430 Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu 435 440 445 Arg Pro Phe Glu Arg Asp He Ser Asn Val Pro Phe Ser Pro Asp Gly 450 455 460 Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp 465 470 475 480 Tyr Gly Phe Tyr Thr Thr Thr Gly He Gly Tyr Gin Pro Tyr Arg Val 485 490 495 Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly 500 505 510 Pro Lys Leu Ser Thr Asp Leu He Lys Asn Gin Cys Val Asn Phe Asn 515 520 525 Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg 530 535 540 Phe Gin Pro Phe Gin Gin Phe Gly Arg Asp Val Ser Asp Phe Thr Asp 545 550 555 560 Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys 565 570 575 Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser 580 585 590 Glu Val Ala Val Leu Tyr Gin Asp Val Asn Cys Thr Asp Val Ser Thr 595 600 605 Ala Ile His Ala Asp Gin Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr 610 615 620 Gly Asn Asn Val Phe Gin Thr Gin Ala Gly Cys Leu Ile Gly Ala Glu 625 630 635 640 His Val Asp Thr Ser Tyr Gin Cys Asp Ile Pro Ile Gly Ala Gly Ile 645 650 655 Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser Gin Lys 660 665 670 Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala 675 680 685 Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile 690 695 700 Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser Val Asp Cys 705 710 715 720 Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu Leu 725 730 735 Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile 740 745 750 Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val Lys 755 760 765 Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe 770 775 780 Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr Lys Arg Ser Phe Ile 785 790 795 800 Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Met 805 810 815 Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu Ile 820 825 830 Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr 835 840 845 Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly Thr Ala 850 855 860 Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe 865 870 875 880 Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn 885 890 895 Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala 900 905 910 Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala Leu Gly 915 920 925 Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu 930 935 940 Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn 945 950 955 960 Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp 965 970 975 Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln 980 985 990 Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala 995 1000 1005 Thr Lys Met Ser Glu Cys Val Leu Gly Gin Ser Lys Arg Val Asp 1010 1015 1020 Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gin Ala Ala 1025 1030 1035 Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ser Gin 1040 1045 1050 Glu Arg Asn Phe Thr Thr Ala Pro Ala He Cys His Glu Gly Lys 1055 1060 1065 Ala Tyr Phe Pro Arg Glu Gly Val Phe Val Phe Asn Gly Thr Ser 1070 1075 1080 Trp Phe He Thr Gin Arg Asn Phe Phe Ser Pro Gin He He Thr 1085 1090 1095 Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val He Gly 1100 1105 1110 He He Asn Asn Thr Val Tyr Asp Pro Leu Gin Pro Glu Leu Asp 1115 1120 1125 Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser 1130 1135 1140 Pro Asp Val Asp Leu Gly Asp He Ser Gly He Asn Ala Ser Val 1145 1150 1155 Val Asn lie Gin Lys Glu lie Asp Arg Leu Asn Glu Val Ala Lys 1160 1165 1170 Asn Leu Asn Glu Ser Leu lie Asp Leu Gin Glu Leu Gly Lys Tyr 1175 1180 1185 Glu Gin Tyr lie Lys Trp Pro Trp Tyr Val Trp Leu Gly Phe lie 1190 1195 1200 Ala Gly Leu lie Ala lie Val Met Val Thr lie Leu Leu Cys Cys 1205 1210 1215 Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Ala Cys Ser Cys Gly 1220 1225 1230 Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys 1235 1240 1245 Gly Val Lys Leu His Tyr Thr 1250 1255 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> OT-I OVA Peptide <400> 5 Ser lie lie Asn Phe Glu Lys Leu 1 5 <210> 6 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> OT-II OVA Peptide <400> 6 Ile Ser Gin Ala Val His Ala Ala His Ala Glu lie Asn Glu Ala Gly 1 5 10 15 Arg <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> NLS from SV-40 large T antigen <400> 7 Pro Lys Lys Lys Arg Lys Val 1 5 <210> 8 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> GWG-SV40NLS <400> 8 Cys Gly Trp Trp Gly Tyr Gly Pro Lys Lys Lys Arg Lys Val Gly Gly 1 5 10 15 Trp Trp Gly <210> 9 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> hnRNPA1 M9 NLS <400> 9 Cys Ser Asn Phe Gly Pro Met Lys Gly Gly Asn Phe Gly Gly Arg Ser 1 5 10 15 Ser Gly Pro Tyr 20 <210> 10 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> hnRNP D NLS <400> 10 Cys Ser Gly Tyr Gly Lys Val Ser Arg Arg Gly Gly His Gin Asn Ser 1 5 10 15 Tyr Lys Pro Tyr 20 <210> 11 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> hnRNP M NLS <400> 11 Cys Asn Glu Lys Arg Lys Glu Lys Asn lie Lys Arg Gly Gly Asn Arg 1 5 10 15 Phe Glu Pro Tyr 20 <210> 12 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> PQBP-1 NLS <400> 12 Cys Ala Asp Arg Glu Glu Gly Lys Glu Arg Arg His His Arg Arg Glu 1 5 10 15 Glu Leu Ala Pro Tyr 20 <210> 13 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> NLS2-RG domain RPS17 <400> 13 Cys Asn Lys Arg Val Cys Glu Glu lie Ala lie lie Pro Ser Lys Lys 1 5 10 15 Leu Arg Asn Lys Gly Ser Gly Arg lie Gin Arg Gly Pro Val Arg Gly 20 25 30 lie Ser <210> 14 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> NLS1 RPS17 <400> 14 Cys Met Gly Arg Val Arg Thr Lys Thr Val Lys Lys Ala Ala Gly Gly 1 5 10 15 <210> 15 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> NLS2 RPS17 <400> 15 Cys Asn Lys Arg Val Cys Glu Glu lie Ala lie lie Pro Ser Lys Lys 1 5 10 15 Leu Arg Asn Lys 20 <210> 16 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> NLS3 RPS17 <400> 16 Cys Ser Lys Lys Leu Arg Asn Lys lie Ala Gly Tyr Val Thr His Leu 1 5 10 15 Met Lys Arg Ile 20 <210> 17 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> cMyc NLS <400> 17 Cys Gly Tyr Gly Pro Ala Ala Lys Arg Val Lys Leu Asp Gly Gly 1 5 10 15 <210> 18 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> HuR NLS <400> 18 Cys Gly Arg Phe Ser Pro Met Gly Val Asp His Met Ser Gly Leu Ser 1 5 10 15 Gly Val Asn Val Pro Gly 20 <210> 19 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Tus NLS <400> 19 Cys Gly Tyr Gly Lys Leu Lys Ile Lys Arg Pro Val Lys Gly Gly 1 5 10 15 <210> 20 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Nucleoplasmin NLS <400> 20 Cys Ala Val Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gin Ala Lys 1 5 10 15 Lys Lys Lys Leu Asp 20

Claims

1. A method for improving the immunogenicity of a polypeptide antigen, the method comprising providing a polypeptide antigen to be modified and covalently conjugating the polypeptide antigen to one or more bile acid-peptide moieties to produce a modified polypeptide antigen, the modified polypeptide antigen being conjugated to a sufficient number of bile acid-peptide moieties to trigger an improved adaptive immune response to the polypeptide antigen compared to a corresponding unmodified polypeptide antigen upon administration to a subject, wherein the peptides included in the bile acid-peptide moieties contain nuclear localization signals (NLS).

2. The method of claim 1, wherein the modified polypeptide antigen is conjugated with a sufficient number of bile acid-peptide portions to increase antigen presentation of the modified polypeptide antigen after intracellular delivery relative to the corresponding unmodified polypeptide antigen.

3. The method of claim 1, wherein the modified polypeptide antigen is conjugated with a sufficient number of bile acid-peptide moieties such that the modified polypeptide antigen exhibits higher thermal stability relative to the corresponding unmodified polypeptide antigen.

4. The method of claim 1, wherein the covalent conjugation of the polypeptide antigen to one or more bile acid-peptide moieties is performed by reacting the polypeptide antigen with a molar excess of the bile acid-peptide moieties.

5. The method of claim 4, wherein the polypeptide antigen reacts with a molar excess of the bile acid-peptide moiety of 2 to 100 times.

6. The method of claim 4, wherein the polypeptide antigen reacts with a 2 to 50 times molar excess of the bile acid-peptide moiety.

7. The method of claim 4, wherein the polypeptide antigen reacts with a 5 to 25 times molar excess of the bile acid-peptide moiety.

8. The method according to claim 1, wherein the average number of bile acid-peptide moieties conjugated to each modified polypeptide antigen is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

9. The method according to claim 1, wherein the bile acid is: cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), glycodeoxycholic acid (GDCA), glycocholic acid (GCA), taurocholic acid (TCA), glycochenodeoxycholic acid (GCDCA), taurodeoxycholic acid (TDCA), glycolithocholic acid (GLCA), taurolithocholic acid (TLCA), taurosudocholic acid (THDCA), taurochenodeoxycholic acid (TCDCA), ursolic acid (UCA), tauroursolic acid (TUDCA), ursolic acid (UDCA), or glycoursolic acid (GUDCA).

10. The method of claim 1, wherein the bile acid is an analogue of CA, CDCA, DCA, LCA, GDCA, GCA, TCA, GCDCA, TDCA, GLCA, TLCA, THDCA, TCDCA, UCA, TUDCA, UDCA, or GUDCA, wherein the analogue: induces endocytosis; triggers the accumulation of ceramide on the inner lobules of the endosome; or triggers increased acid sphingomyelinase (ASM)-mediated sphingomyelin cleavage to form ceramide.

11. The method according to claim 1, wherein the nuclear localization signal is: SV40 NLS as shown in SEQ ID NO: 1 or 7, GUG-SV40 NLS as shown in SEQ ID NO: 8, hnRNPA1 M9 NLS as shown in SEQ ID NO: 9, hnRNP D NLS as shown in SEQ ID NO: 10, hnRNP M NLS as shown in SEQ ID NO: 11, PQBP-1 NLS as shown in SEQ ID NO: 12, NLS2-RG domain RPS17 as shown in SEQ ID NO: 13, NLS1 RPS17 as shown in SEQ ID NO: 14, NLS2 RPS17 as shown in SEQ ID NO: 15, NLS3 RPS17 as shown in SEQ ID NO: 16, cMyc NLS as shown in SEQ ID NO: 17, HuR NLS as shown in SEQ ID NO: 18, Tus NLS as shown in SEQ ID NO: 19, or nucleoplasmic protein NLS as shown in SEQ ID NO:

20.

12. The method of claim 1, wherein the nuclear localization signal is a variant of an NLS having nuclear localization activity, the NLS comprising or consisting of an amino acid sequence of any one of SEQ ID NO: 7 to 20.

13. The method of claim 1, wherein the polypeptide antigen is conjugated to one or more bile acid-peptide portions via a linker.

14. The method of claim 13, wherein the connector is a bifunctional connector, a trifunctional connector, or a multifunctional connector.

15. The method of claim 1, wherein the modified polypeptide antigen molecule is conjugated to one or more bile acid-peptide portions via solvent-accessible functional groups of the polypeptide antigen.

16. The method of claim 1, wherein the polypeptide antigen is or includes tumor-associated antigens (TAAs), tumor-specific antigens (TSAs), tumor-derived cell lysates, tumor-derived exosomes, neoantigens, viral antigens, bacterial antigens, fungal antigens, or other antigens associated with a disease or condition that can be treated by vaccination and / or immunotherapy.

17. The method of claim 1, wherein the polypeptide antigen is or includes the SARS-CoV spike protein or an antigenic fragment thereof.

18. An immunogenic composition comprising: Modified peptide antigens and pharmaceutically acceptable excipients and / or adjuvants, wherein the modified peptide antigen comprises a peptide antigen covalently conjugated to a sufficient number of bile acid-peptide moieties to trigger an improved adaptive immune response to the peptide antigen compared to a corresponding unmodified peptide antigen upon administration to a subject, wherein the peptides contained in the bile acid-peptide moieties contain nuclear localization signals (NLS); or A cell population comprising a modified polypeptide antigen and pharmaceutically acceptable excipients and / or adjuvants, wherein the modified polypeptide antigen comprises a polypeptide antigen covalently conjugated to a sufficient number of bile acid-peptide moieties to trigger an improved adaptive immune response to the polypeptide antigen compared to a corresponding unmodified polypeptide antigen upon administration to a subject, wherein the peptides contained in the bile acid-peptide moieties contain nuclear localization signals (NLS).

19. The immunogenic composition of claim 18, wherein the cell population comprises dendritic cells, B cells, T cells, macrophages, engineered antigen-presenting cells, cells expressing MHC class I, cells expressing MHC class II, or any combination thereof.

20. Use of the immunogenic composition of claim 18 or 19 for the preparation of a medicament that triggers an enhanced adaptive immune response against an unmodified polypeptide antigen of interest in a subject.

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