The present description relates to covalently modified antigens to enhance or modify their immunogenicity and/or stability. More specifically, the present description relates to polypeptide antigens covalently conjugated to one or more steroid acid moieties for improved cellular immunity and/or improved thermal stability.

BACKGROUND

While subunit vaccines based on polypeptide antigens are generally considered amongst the safest vaccines, such antigens may not elicit sufficiently strong immune responses 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 strict refrigeration requirements is a hurdle to their deployment on a global scale. Thus, methods of improving the immunogenicity, efficacy, and stability of polypeptide antigen-based vaccines would be highly desirable.

SUMMARY

In a first aspect, described herein is a method of improving polypeptide antigen immunogenicity and/or stability, 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 upon intracellular delivery relative to a polypeptide antigen lacking said modification, wherein the modified polypeptide antigen triggers an improved adaptive immune response to said polypeptide antigen upon administration to a subject as compared to a 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 that of the polypeptide antigen prior to conjugation.

In further aspects, 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. In a further 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 an immunogenic composition as described herein to the subject

In a further aspect, described herein is a method for treating or preventing a disease or disorder amenable to treatment by vaccination and/or immunotherapy, the method comprising administering an immunogenic composition as described herein to the subject.

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

In a further aspect, described herein is a method for treating or preventing a disease or disorder amenable to treatment by vaccination and/or immunotherapy, the method comprising administering an immunogenic composition as described herein to the subject.

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

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

In a further 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 greater stability (e.g., thermal stability) than that of the polypeptide antigen prior to conjugation.

General Definitions

Headings, and other identifiers, e.g., (a), (b), (i), (ii), etc., are presented merely for ease of reading the specification and claims. The use of headings or other identifiers in the specification or claims does not necessarily require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.

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

As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “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.

As used herein, the expression “consisting essentially of’ or “consists essentially of’ refers to those elements required for a given embodiment. The expression permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention. In the context of modified polypeptide antigens described herein, the expressions “consisting essentially of’ or “consists essentially of’ refer to the elements required to improve polypeptide antigen immunogenicity as compared to an unmodified antigen (e.g., by improving antigen presentation by professional antigen-presenting cells). For greater clarity, the expressions do not exclude the possibility that other additional non-essential ingredients (e.g., excipients, fillers, stabilizers, or inert components) that do not materially change the function or ability of the steroid acid-peptide moieties to improve polypeptide antigen immunogenicity.

The term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed in order to determine the value. In general, the terminology “about” is meant to designate a possible variation of up to 10%. Therefore, a variation of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10% of a value is included in the term “about”. Unless indicated otherwise, use of the term “about” before a range applies to both ends of the range.

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

BRIEF DESCRIPTION OF THE DRAWINGS

In the appended drawings:

Fig. 1 shows the biochemical characterization of the ChAcNLS-antigen formulation. Fig. 1A is a schematic diagram representing covalent binding of a given antigen to a ChAcNLS moiety. Fig. IB shows a representative Coomassie blue staining displaying unmodified OVA (line 1), or ChAcNLS conjugated to OVA at a molar ratio of 25x (line 2) or 50x (line 3). Fig. 1C shows the amino acid sequence of chicken OVA. Lysine residues that are predicted to be accessible for ChAcNLS conjugation (>50%) are highlighted in black. The three lysine residues predicted to be weakly accessible are underlined. Fig. ID shows a ribbon structure of the OVA protein with lysine residues that are predicted to be highly (blue), moderate (green) or poorly (yellow) accessible lysine residues. Fig. IE shows a representative Western blot displaying unmodified OVA (line 1), ChAcNLS-OVA at a ratio of 25x (line 2), and ChAcNLS-OVA at a ratio of 50x (line 3). Fig. IF shows an intrinsic tryptophan fluorescence (ITF) analysis of nOVA or ChAcNLS-OVA (cOVA) at various ChAcNLS to OVA ratios in response to thermal stress. Figs. 1G and 1H shows the effect of various cOVA variants on the efficacy of antigen presentation by DCs. Fig. 1G shows a representative schematic diagram of the different variants tested in Fig. 1H. Fig. 1H shows the response quantification using the SIINFEKL-specific B3Z cell line cocultured with DCs treated with the different variants. For this panel, n=5/group with *** p<0.001 when compared to the nOVA group.

Fig. 2 shows the results of an antigen cross-presentation assay. Fig. 2A shows schematically the antigen classical (MHC-II) and cross-presentation (MHC-I) assays used to assess OVA-responding OT-I (CD8) and OT-II (CD4) T cells. Fig. 2B shows the IFN-gamma produced by OT-I-derived CD8 T cells incubated with DCs and either naked OVA (nOVA) or ChAcNLS-OVA (cOVA). Fig. 2C shows the IL-2 levels produced by OT-II-derived CD4 T cells incubated with DCs and either naked OVA (nOVA) or ChAcNLS-OVA (cOVA). Fig. 2D shows the IFN-gamma production for the experiment of Fig. 2C. Fig. 2E shows the results of a representative flow-cytometry experiment investigating OVA-DQ™ (grey peak) versus ChAcNLS-OVA-DQ (red peak) processing by DCs at different time points. Fig. 2F shows the quantification of the mean fluorescent intensity (MFI) of the OVA-DQ/ChAcNLS-OVA-DQ signals shown in Fig. 2E. For this experiment, n=5/group with ***p<0.001. Fig. 2G shows a representative experiment of Gal3-GFP-expressing DC2.4 cells treated with nOVA (upper pictogram) versus cOVA (lower pictogram). White arrows point to some of the damaged endosomes.

Fig. 3 shows the syngeneic prophylactic vaccination against T-cell lymphoma. Fig. 3A is a schematic representation of the timeline used for prophylactic vaccination using the OVA protein. Fig. 3B-3C shows assessment of tumor growth volume (Fig. 3B) and survival (Fig. 3C) of animals challenged with the EG.7 tumor following prophylactic vaccination using naked OVA (nOVA; green)- or ChAcNLS- OVA (cOVA; red)-pulsed DCs. Non-immunized mice injected with EG.7 are shown as “Ctl” (black). Fig. 3D shows the antibody titers of vaccinated animals quantified by ELISA. Fig. 3E shows the quantification of central memory (T _cm ) and effector memtory (T _e ff) CD4 T cells derived from mice immunized with nOVA-/cOVA-pulsed DCs from vaccinated animals of this study. Fig. 3F shows the quantification of central memory (T _cm ) and effector memtory (Ten) CD 8 T cells derived from mice immunized with nOVA-/cOVA-pulsed DCs from vaccinated animals of this study. Fig. 3G shows the Luminex™ analysis of cytokine/chemokine production in response to in vitro re-stimulation of T cells isolated from vaccinated animals of this study. Cytokines/chemokines with the highest fold change were boxed. For panels Fig. 3B, 3C, 3D and 3E, n=10/group with ***P<0.001.

Fig. 4 shows the immunity assessment following direct injection of the OVA protein. Fig. 4A is a schematic representation of the timeline used for prophylactic vaccination using the OVA protein with or without vaccine adjuvants. Fig. 4B shows average tumor measurements in animals immunized using naked OVA (green; nOVA) (Ipg), ChAcNLS-OVA (red; cOVA) ( 1 ng). cOVA ( 1 gg) with AddaS03™ adjuvant (blue), and cOVA ( 1 pg) with AddaVax™ adjuvant (purple). Non-immunized mice injected with EG.7 are shown as “Ctl” (black). Fig. 4C shows the survival results from the experiment shown in Fig. 4A. Fig. 4D shows quantification of antibody titers from the experiment shown in Fig. 4A. For this experiment, n=10/group with *P<0.05.

Fig. 5 shows the therapeutic vaccination against T-cell lymphoma. Fig. 5A is a schematic representation of the timeline used for therapeutic vaccination. Fig. 5B-5C shows tumor growth volume (Fig. 5B) and survival (Fig. 5C) of animals challenged with the EG.7 tumor following syngeneic therapeutic vaccination using anti-PD-1 alone (“aPD-1”), naked OVA (“nOVA”)- or ChAcNLS-OVA (“cOVA”)-pulsed DCs, with (“+ aPD-1”) or without anti-PD-1. Non-immunized mice injected with EG.7 are shown as “Ctl”. Fig. 5D-5E show assessments of tumor growth volume (Fig. 5D) and survival (Fig. 5E) of animals challenged with the EG.7 tumor following allogeneic therapeutic vaccination anti-PD-1 (dashed black), ChAcNLS-OVA (“cOVA”)-pulsed DCs with anti-PD-1 at varying cell numbers (3K, purple; 30K, blue; 100K, green; 300K, red) or without anti-PD-1 (300K, orange). For all panels, n=10/group.

Fig. 6 shows the tumor lysate-based therapeutic vaccination against T-cell lymphoma. Fig. 6A is schematic representation of the timeline used for allogeneic therapeutic vaccination. Fig. 6B-6C shows assessments of tumor growth volume (Fig. 6B) and survival (Fig. 6C) of animals challenged with the EL4 tumor following immunization with anti-PD-1 (dashed black; “aPD-1”), BALB/c-derived allogeneic DCs pulsed with 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). Fig. 6D shows a schematic representation of the experimental design of the tumor-mfiltrating lymphocytes (TILs) study. Fig. 6E shows an analysis of various immune cells in tumors derived from all groups shown in Figs. 6B and 6C. Fig. 6F shows the assessment of the CD8/Treg ratio in the tumors depicted in Figs. 6B and 6C. For Figs. 6B and 6C, n=10/group. For Figs. 6E-6G, n=5/group with 772 *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 7 shows the SARS-CoV-2 Spike protein used for the formulation of ChAcNLS-Spike-CoV- 2. Fig. 7A shows a schematic diagram of the ribbon structure of SARS-CoV-2 Spike protein (Wuhan strain with D614G mutation) with lysine residues that are predicted to be highly (blue), moderate (green) or poorly (yellow) accessible lysine residues. Fig. 7B shows the amino acid sequence of SARS-CoV-2 Spike protein. Lysine residues that are predicted to be accessible for ChAcNLS conjugation (>50%) are highlighted in black. The lysine residues predicted to be weakly accessible are underlined. Fig. 8 shows the evaluation of immunogenicity of the ChAcNLS-Spike-CoV-2 vaccine using different CoV-2 Spike protein domains. Fig. 8A shows the antibody titers from mice vaccinated with the 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. Mice were given an additional boost injection at 17 weeks. IgG antibody titers were measured by ELISA. Fig. 8B shows the different isotype titers from the study of Fig. 8A. Fig. 8C shows the antibody titers from mice vaccinated with the Sl-RBD portion of the CoV-2 Spike protein, “naked” Sl-RBD-CoV-2 (unconjugated; nSl-RBD- CoV-2; black bars) or ChAcNLS-Sl-RBD-CoV-2 (“cSl-RBD-CoV-2”; grey bars) were injected in the presence of AddaS03 or AddaVax adjuvants, or alone. IgG antibody titers were measured by ELISA. Fig. 8D shows the antibody titers from mice vaccinated at week 18with 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) were injected in the presence of AddaS03 or AddaVax adjuvants, or alone. IgG antibody titers were measured by ELISA. Fig. 8E shows the results of an in vitro infectivity neutralization assay used to assess the neutralizing capacity to the generated antibodies. Antibodies isolated from cSpike- CoV-2 -immunized mice were more efficient at inhibiting viral infection of HEK cells, as compared to nSpike-CoV-2, as shown with the NT ₅ o titers. Forthis panel, n=5/group with *P<0.05, **P<0.01 and ***P<0.001.

Fig. 9 shows the cytokine profiling by Luminex™ following T-cell re-stimulation in vitro. Fig. 9A shows the cytokine profiling using T cells derived from mice vaccinated with nSpike-CoV-2 with two different adjuvants. Fig. 9B shows the cytokine profiling using T cells derived from mice vaccinated with cSpike-CoV-2 with two different adjuvants.

Fig. 10 shows the evaluation of the vaccine immunogenicity in rabbits. Fig. 10A shows a schematic diagram of the experimental design. Three doses of cSpike-CoV-2 were tested in this experiment. Fig. 10B shows antibody titers assessed on sera collected every 2 weeks, n=3/group with ***p<0.001.

Fig. 11 shows the evaluation of the therapeutic efficacy of cSpike-CoV-2 in a challenge model. Fig. 11A shows a schematic diagram of the experimental design for the vaccine efficacy study in hamsters. Fig. 11B shows antibody titers in response to the cSpike-CoV-2 vaccine mixed with the FDA- approved (GMP grade) MONTANIDE™ISA 720 VG adjuvant or AddaS03. The vaccines were tested using excess ratios of ChAcNLS to Spike-CoV-2 protein, 10X and 50X.

Fig. 12 shows the evaluation of the cross-reactivity of generated antibodies against various SARS-CoV-2 variants. Fig. 12A shows a schematic diagram of the SARS-CoV-2 Spike variants used in the study, as well as the different mutations in the RBD domains. Fig. 12B shows antibody titers against the different RBD domains using sera isolated from cSpike-CoV-2 -vaccinated mice with or without adjuvants. Fig. 12C shows the percentage of cross-reactivity with all tested variants based on data shown in Fig. 12B. Fig. 12D shows the neutralization levels obtained against various viral variants. Data presented in this figure are conducted with an n=5/group and with *P<0.05, **P<0.01 and ***P<0.001.

Fig. 13 shows the evaluation of the immunogenicity of the cSpike-CoV-2 vaccine using the Indian (IN) CoV-2 Spike protein variant (cSpike-CoV-2 -IN) in the presence of the Eurocine™ adjuvant. Fig. 13A shows a schematic representation of the schedule used for cSpike-CoV-2 -IN vaccination. Figs. 13B and C show IgG and IgA titers, respectively, in sera of cSpike-CoV-2 -IN vaccinated mice as compared to a saline control. For Fig. 13C, analysis was conducted on samples collected at week 5. Fig. 13D and E show IgG and IgA titer analysis in the bronchoalveolar lavage fluid (BALF) of cSpike-CoV- 2-IN-vaccinated mice at week 6. For this study, *P<0.05 and * *P<0.01. The two-way ANOVA test was applied for Panel B. The one-way ANOVA (Bonferroni test) was conducted for studies in panel C, D and E.

Fig. 14 shows the cytokine/chemokine analysis of the cSpike-CoV-2-IN vaccine using the Indian (IN) CoV-2 Spike protein variant. Luminex™ analysis of cytokine (Fig. 14A) and chemokine (Fig. 14B) response following in vitro splenocyte re -stimulation using recombinant Spike protein for three days is shown. Cytokines or chemokines depicted by gray highlighting have significant fluctuations compared to control (ctl; saline) animals. Units shown are in pg/mL.

Fig. 15 shows the cross-reactivity of sera-derived IgGs from mice vaccinated with cSpike-CoV- 2 -IN vaccine using the Indian (IN) CoV-2 Spike protein variant with various CoV-2 Spike protein variants. The original Wuhan strain Spike protein was used as a comparative to the remaining variants. For this assay, n=5/group with *P<0.05 against the original SARS-COV2 strain.

Figs. 16A-16D show representative SDS-PAGE gels of the different bile acid-NLS-OVA conjugate preparations using 10X or 50X excess molar ratios of bile acid-NLS reactants to OVA antigen.

Fig. 17 shows the effect on antigen presentation by dendritic cells of OVA antigen conjugated to different types of bile acid-NLS moieties (0.1 mg/mL). 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). OD570 levels represent the level of OVA presentation, 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); glycoursodeoxycholic acid (GUDCA); deoxycholic acid (DCA); glycocholic acid (GCA); and lithocholic acid (LCA).

Fig. 18 shows the effect on antigen presentation by B cells of OVA antigen conjugated to different types of bile acid-NLS moieties (0.1 mg/mL). 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). OD570 levels represent the level of OVA presentation, 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); glycoursodeoxycholic acid (GUDCA); deoxycholic acid (DCA); glycocholic acid (GCA); and lithocholic acid (LCA).

SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form created November 1, 2021. The computer readable form is incorporated herein by reference .

DETAILED DESCRIPTION

Described herein are compositions, cells, and methods relating to improving or modifying the adaptive immune response to polypeptide antigens and/or to improving the stability of polypeptide antigens. In some aspects, the present invention stems from the demonstration herein that conjugating a polypeptide antigen to steroid acid moieties triggers improved cellular immunity, or improved cellular and humoral immunity, against the antigen. In some aspects, the present invention stems from the demonstration herein that conjugating a polypeptide antigen to steroid acid moieties improves the stability (e.g., against thermal stress) of the polypeptide antigen. In some embodiments, the polypeptide antigens described herein may be covalently conjugated via functionalized linkers to the steroid acid moieties or to steroid acid-peptide moieties. Advantageously, when the polypeptide antigens are conjugated to steroid acid-peptide moieties, the peptide may be designed to comprise one or more domains imparting a desired functionality to the modified polypeptide antigen (e.g., protein transduction and/or subcellular targeting), which may further enhance immunogenicity.

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 steroid acid moieties 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 upon administration to a subject (e.g., as compared to a corresponding unmodified polypeptide antigen). In some embodiments, the modified polypeptide antigen is conjugated to a number of steroid acid moieties that is sufficient to increase endocytosis and/or endosomal escape of the modified polypeptide antigen (e.g., as compared to a corresponding unmodified polypeptide antigen) upon intracellular delivery. In some embodiments, the modified polypeptide antigen triggers an improved adaptive immune response (e.g., improved cellular and/or humoral immune response) against the polypeptide antigen upon administration to a subject as compared to a corresponding unmodified polypeptide antigen.

Polypeptide antigens are normally captured by antigen-presenting cells (e.g., dendritic cells) but are initially entrapped in endosomes. Endosomal maturation towards lysosomes results in a decrease in pH and an activation of proteolytic enzymes that mediate non-specific antigen degradation. As a result, some of the antigen fragments generated may then pass through endosomal pores to reach the cytosol where further antigen degradation takes place by the proteasomal machinery prior to MHC class I presentation. Although this process occurs naturally, the generated antigen fragments that ultimately leave the endosomes may be small and/or damaged, rendering them unsuitable for proteasomal degradation, thereby precluding their MHC class I presentation and thus cellular immunity based thereon. Without being bound by theory, the increased endosomal escape of the modified polypeptide antigens described herein may enable antigens (or larger antigen fragments) to reach the cytosol in a more native conformation. As a result, proteasomal degradation of these more native antigens may result in a higher number of immunogenic and/or stable peptides presented via MHC class I at the surface of antigen- presenting cells, thereby eliciting potent T-cell activation.

As used herein, “polypeptide antigen” refers to an immunogenic peptide-linked chain of amino acids of any length, but generally at least 8, 9, 10, 11, or 12 amino acids long. For greater clarity, polypeptide antigens referred to herein exclude antigen-binding antibodies or fragments thereof. As used herein, a “protein antigen” refers to a polypeptide antigen having a length of at least 50 amino acid residues, while a “peptide antigen” refers to a polypeptide antigen having a length of less than 50 amino acid residues. For greater clarity, polypeptides, proteins, and peptides described herein may or may not comprise any type of modification (e.g., chemical or post-translational modifications such as acetylation, phosphorylation, glycosylation, sulfatation, sumoylation, prenylation, ubiquitination, etc.) or incorporate one or more synthetic or non-natural amino acids, to the extent that the modification or synthetic or nonnatural amino acids does not destroy the antigenicity of the polypeptide antigen or the desired functionality of the peptide (or domain comprised therein).

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

In some embodiments, polypeptide antigens described herein may be a protein antigen. In some embodiments, protein antigens may advantageously comprise a plurality of available functional groups to which the steroid acid or steroid acid-peptide moieties may be conjugated. In contrast, peptide antigens may not comprise a sufficient number of functional groups for steroid acid conjugation. Furthermore, steroid acid-peptide antigen conjugates may undesirably self-assemble into rod-like nanoparticles, as reported in Azuar et al., 2019, in which the hydrophobic steroid acid groups from different modified peptide antigens aggregate and are sequestered internally, thereby preventing their ability to interact with the membrane and mediate endosomal escape. Insufficient endosomal escape may not negatively affect MHC class II presentation and thus may benefit humoral immunity, but is unlikely to benefit cellular immunity (Azuar et al., 2019).

In some embodiments, the protein antigens described herein may comprise (or may be engineered to comprise) between 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) available for conjugation to the steroid acid or steroid-peptide moieties described herein. In some embodiments, the polypeptide antigen may be a protein antigen 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 may be a protein antigen having 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 may comprise one or more MHC class I epitopes and/or MHC class II epitopes.

In some embodiments, polypeptide antigens described herein may be or may comprise a tumor- associated antigen (TAA), tumor-specific antigen (TSA), a neoantigen, a viral antigen, a bacterial antigen, a fungal antigen, an antigen associated with a disease or disorder amenable to treatment by vaccination and/or immunotherapy; or any antigenic fragment thereof. In some embodiments, polypeptide antigens described herein may be or may comprise the 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 may be a single-nucleotide variant antigen, a mutational frameshift antigen, 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 may be a viral-derived cancer antigen, such as from human papillomavirus (HPV), cytomegalovirus, or Epstein-Barr virus (EBV). In some embodiments, the TAA may be or may comprise 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, polypeptide antigens described herein may be or may comprise cell lysates or other material derived from a tumor such as tumor-derived exosomes.

In some embodiments, the polypeptide antigens may be conjugated to a steroid acid moiety that enhances endocytosis and/or endosomal escape of internalized cargoes. Without being bound by theory, steroid acids (e.g., bile acids and bile acid analogs) have been shown to be utilized/exploited by viruses to facilitate their infection of host cells, such as by increasing their endocytic uptake and/or endosomal escape to gain access to the cytosol (Shivanna et al., 2014; Shivanna et al., 2015; Murakami et al., 2020). For example, bile acids have been shown to trigger the enzyme acid sphingomyelinase (ASM) to cleave sphingomyelin to ceramide on the inner leaflet of endosomes. Increased amounts of ceramide destabilize membranes and facilitate endosomal escape. In some embodiments, steroid acids suitable for conjugation to the polypeptide antigens described herein comprise those that trigger ceramide accumulation on the inner leaflet of endosomes, thereby destabilizing endosomal membranes and facilitating endosomal escape of the modified polypeptide antigen upon intracellular delivery. In some embodiments, steroid acids suitable for conjugation to the polypeptide antigens described herein comprise those that trigger increased acid sphingomyelinase (ASM)-mediated cleavage of sphingomyelin to form ceramide.

In some embodiments, a steroid acid suitable for conjugation to a polypeptide antigen 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 may be or comprise: cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), glycodeoxycholic acid (GDCA), glycocholic acid (GCA), taurocholic acid (TCA), glycodeoxycholic acid (CDCA), glycochenodeoxycholic acid (GCDCA), taurodeoxycholic acid (TDCA), glycolithocholic acid (GLCA), taurolithocholic acid (TLCA), taurohyodeoxycholic acid (THDCA), taurochenodeoxycholic acid (TCDCA), ursocholic acid (UCA), tauroursodeoxycholic acid (TUDCA), ursodeoxycholic acid (UDCA), glycoursodeoxycholic acid (GUDCA), or any analog thereof that: induces endocytosis; triggers ceramide accumulation on the inner leaflet of endosomes; triggers increased acid sphingomyelinase (ASM)-mediated cleavage of sphingomyelin to form ceramide; and/or has a hydrophobicity greater than that of cholic acid.

Hydrophobic bile acids such as GCDCA, TCA, GCA, and CA (but not hydrophilic bile acids such as UDCA) were shown to increase GII.3 human norovirus infection and replication in host intestinal cells by enhancing endosomal uptake and endosomal escape via ASM-mediated ceramide accumulation on the apical membrane (Murakami et aL, 2020). In some embodiments, a steroid acid suitable for conjugation to a polypeptide antigen described herein comprises or consists of a bile acid or bile acid analog that is more hydrophobic than cholic acid. In some embodiments, a steroid acid suitable for conjugation to a polypeptide antigen 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).

In some embodiments, the average number of steroid acid moieties per modified polypeptide antigen may be modified, for example, based on the type of steroid acid and/or type of polypeptide antigen selected (e.g., amino acid length, structure, number of available functional groups). In some embodiments, the polypeptide antigen may be reacted with a molar excess of steroid acid or steroid acid- peptide moieties to maximize the number of steroid acid moieties conjugated. In some embodiments, the polypeptide antigen may be reacted with a limiting amount of steroid acid or steroid acid-peptide moieties to control or limit the number of steroid acid moieties conjugated. In some embodiments, each modified polypeptide antigen molecule may 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 may be conjugated to the steroid acid (or steroid acid-peptide) moiety at solvent- accessible amine (e.g., primary amine) and/or sulfhydryl group of the polypeptide antigen. In some embodiments, the modified polypeptide antigen molecule may be conjugated to the steroid acid (or steroid acid-peptide) moiety at any other chemical or functional group present on or engineered into the polypeptide antigen. It is understood that the maximum number of steroid acid moieties comprised 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 the steroid acid or steroid acid-peptide moiety) may be pre-functionalized for example with a bifunctional, trifunctional, or multi-functional linker group, prior to the reaction conjugating the polypeptide antigen to the steroid acid or steroid acid-peptide moiety.

In some embodiments, the steroid acids described herein may be comprised in a steroid acid- peptide moiety. In some embodiments, the steroid acid may be pre-conjugated to the peptide, for example 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 may then be conjugated to the steroid acid-peptide moiety via the peptide, such as at an N- or C-terminal residue of the peptide.

In some embodiments, the peptide may be a non -immunogenic peptide. In some embodiments, the peptide may be a water-soluble peptide, wherein conjugation of the peptide to the steroid acid increases the water solubility of the steroid acid-peptide moiety as compared to the steroid acid moiety alone. In some embodiments, the peptide may be a cationic peptide (e.g., that promotes interaction the plasma and/or endosomal membrane).

In some embodiments, the peptide may comprise one or more domains that impart an additional functionality to the modified polypeptide antigen. As used herein, a “domain” generally refers to a part of a protein having a particular functionality. Some domains conserve their function when separated from the rest of the protein, and thus can be used in a modular fashion. The modular characteristic of many protein domains can provide flexibility in terms of their placement within the peptides described herein. However, some domains may perform better when engineered at certain positions of the peptide (e.g., at the N- or C-terminal region, or therebetween). The position of the domain within its endogenous protein may be an indicator of where the domain should be engineered within the peptide.

In some embodiments, the peptide may comprise a protein transduction domain (PTD) that stimulates endocytosis, endosomal formation, or intracellular delivery in a non-cell-specific manner. In some embodiments, the peptide may comprise a subcellular targeting signal promoting targeting of the modified polypeptide antigen to a specific subcellular compartment. In some embodiments, the peptide may comprise a nuclear localization signal (NLS) that targets the modified polypeptide antigen to the nucleus. Interestingly, while targeting to the cytosolic compartment may be expected to be advantageous given that proteosome-mediated MHC class I peptide epitope processing occurs in the cytosol, results shown herein surprisingly demonstrate that modified polypeptide antigens comprising a nuclear localization signal triggered a striking increase in antigen immunogenicity. In some embodiments, the nuclear localization signals described herein may comprise or be derived from the NLS from SV-40 large T-antigen (e.g., PKKKRKV; SEQ ID NO: 7) or from other classical NLSs. In some embodiments, the nuclear localization signals described herein may comprise or be derived from non-classical NLS (e.g., acidic M9 domain in the hnRNP Al protein; the sequence KIPIK in yeast transcription repressor Mata2; PY-NLS; ribosomal NLS; or the complex signals of U snRNPs). In some embodiments, the nuclear localization signal described herein comprises or consists essentially of the amino acid sequence of any one 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 localisation signal which is SV40 NLS (e.g., comprised in SEQ ID NO: 1 or 7), GWG-SV40NLS (e.g., comprised in SEQ ID NO: 8), hnRNPAl M9 NLS (e.g, comprised in SEQ ID NO: 9), hnRNP D NLS (e.g., comprised in SEQ ID NO: 10), hnRNP M NLS (e.g., comprised in SEQ ID NO: 11), PQBP-1 NLS (e.g., comprised in SEQ ID NO: 12), NLS2-RG Domain RPS17 (e.g., comprised in SEQ ID NO: 13), NLS1 RPS17 (e.g., comprised in SEQ ID NO: 14), NLS2 RPS17 (e.g., comprised in SEQ ID NO: 15), NLS3 RPS17 (e.g., composed in SEQ ID NO: 16), cMyc NLS (e.g., comprised in SEQ ID NO: 17), HuRNLS (e.g., comprised in SEQ ID NO: 18), Tus NLS (e.g., comprised in SEQ ID NO: 19), or Nucleoplasmin NLS (e.g., comprised in SEQ ID NO: 20) In some instances, the SEQ ID NOs referred to above comprise an N-terminal cysteine residue that was used to facilitate conjugation to the polypeptide antigen (e.g., the thiol group of the N- terminal cysteine residue). Thus, in some embodiments, the NLS sequences referred to herein may exclude the N-terminal cysteine residue comprised in any one of SEQ ID NOs: 1 and 7-20. In some embodiments, other functional groups added or inserted (e.g., towards the N to C terminal portions of the peptides described herein) to facilitate steroid acid-peptide conjugation to a given polypeptide antigen are also envisaged (e.g., carboxyl groups, synthetic amino acids, etc.).

In some embodiments, the nuclear localization signals described herein may comprise the general consensus sequence: (i) K(K7R)X(K/R); (ii) ( K/R)( K/R)X , , 12(K/R) . wherein (K/R) ₃ /5 represents three lysine or arginine residues out of five consecutive amino acids; (iii) KRXio 12KRRK; (iv) KRXio i2K(K/R)(K/R); or (v) KRXio i2K(K/R)X(K/R), wherein X is any ammo acid (Sun et al., 2016).

In some embodiments, modified polypeptide antigens described herein may exhibit increased cytosolic delivery, as compared to a corresponding unmodified polypeptide antigen. In some embodiments, modified polypeptide antigens described herein may exhibit increased total cellular delivery of the modified polypeptide antigen, as compared to a corresponding unmodified polypeptide antigen. In some embodiments, modified polypeptide antigens described herein may exhibit enhanced cellular immunity against the polypeptide antigen, as compared to a corresponding unmodified polypeptide antigen. In some embodiments, modified polypeptide antigens described herein exhibit increased IFN-gamma production by CD8+ T cells upon exposure to said polypeptide antigen, as compared to a corresponding unmodified polypeptide antigen. In some embodiments, modified polypeptide antigens described herein exhibit enhanced humoral immunity against said polypeptide antigen, as compared to a corresponding unmodified polypeptide antigen. In some embodiments, modified polypeptide antigens described herein trigger an increased variety (or biodiversity) of antibody species against the polypeptide antigen, as compared to a corresponding unmodified polypeptide antigen (e.g., including antibodies against epitopes that are poorly immunogenic).

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

In some aspects, described herein is an immunogenic composition comprising: a modified polypeptide antigen described herein or produced by a method as described herein, or a population of cells as described herein, or any combination thereof, and a pharmaceutically acceptable excipient and/or adjuvant (e.g., vaccine adjuvant suitable for human or animal use). In some embodiments, the adjuvant may 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 may be a therapeutic or prophylactic vaccine (e.g., anti -cancer vaccine, anti-viral vaccine, or anti-bacterial vaccine). In some embodiments, modified polypeptide antigens described herein may enable a decrease in the quantity of antigen and/or antigen-presenting cells formulated in an immunogenic composition (e.g., vaccine) required to generate an immune response, as compared to the quantity when a corresponding unmodified polypeptide antigen lacking steroid-acid conjugation is used.

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

In a further aspect, described herein is a method for treating or preventing a disease or disorder amenable to treatment by vaccination and/or immunotherapy, the method comprising administering an immunogenic composition as described herein to the subject.

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

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

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 that of 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 upon intracellular delivery relative to a polypeptide antigen lacking said modification. In embodiments, the modified polypeptide antigen is a modified polypeptide antigen as defined herein.

ITEMS

In various aspects, described herein are one or more of the following items:

1. A method of improving polypeptide antigen immunogenicity, 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, the modified polypeptide antigen being conjugated to a sufficient number of steroid acid moieties to increase endosomal escape of the modified polypeptide antigen upon intracellular delivery relative to a polypeptide antigen lacking said modification, wherein the modified polypeptide antigen triggers an improved adaptive immune response to said polypeptide antigen upon administration to a subject as compared to a corresponding unmodified polypeptide antigen.

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

3. The method of item 1 or 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.

4. The method of any one of items 1 to 3, wherein the polypeptide antigen comprises one or more MHC class I epitopes and/or MHC class II epitopes.

5. The method of any one of items 1 to 4, wherein the polypeptide antigen is or comprises a tumor-associated antigen (TAA), tumor-specific antigen (TSA), a neoantigen, a viral antigen, a bacterial antigen, a fungal antigen, an antigen associated with a disease or disorder amenable to treatment by vaccination and/or immunotherapy; or any antigenic fragment thereof. The method of any one of items 1 to 5, wherein the polypeptide antigen is or comprises a corona viral antigen (e.g., SARS-CoV-2 Spike protein (SEQ ID NO: 3) or SARS-CoV Spike protein (SEQ ID NO: 4) or an antigenic fragment thereof; or a cancer antigen, such as a singlenucleotide variant antigen, a mutational frameshift antigen, 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, cell lysates or other material derived from a tumor (e.g., tumor-derived exosomes). The method of any one of items 1 to 6, wherein the steroid acid triggers ceramide accumulation on the inner leaflet of endosomes, thereby destabilizing endosomal membranes and facilitating endosomal escape of the polypeptide antigen upon intracellular delivery. The method of any one of items 1 to 7, wherein the steroid acid triggers increased acid sphingomyelinase (ASM)-mediated cleavage of sphingomyelin to form ceramide. The method of any one of items 1 to 8, wherein the steroid acid is a bile acid. The method of any one of items 1 to 9, wherein the steroid acid is a primary bile acid or a secondary bile acid. The method of any one of items 1 to 10, wherein the steroid acid is or comprises : (a) a bile acid which is: cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), glycodeoxycholic acid (GDCA), glycocholic acid (GCA), taurocholic acid (TCA), glycodeoxycholic acid (CDCA), glycochenodeoxycholic acid (GCDCA), taurodeoxycholic acid (TDCA), glycolithocholic acid (GLCA), taurolithocholic acid (TLCA), taurohyodeoxycholic acid (THDCA), taurochenodeoxycholic acid (TCDCA), ursocholic acid (UCA), tauroursodeoxycholic acid (TUDCA), ursodeoxycholic acid (UDCA), or glycoursodeoxycholic acid (GUDCA); (b) an analog of the bile acid of (a) that: induces endocytosis; triggers ceramide accumulation on the inner leaflet of endosomes; triggers increased acid sphingomyelinase (ASM)-mediated cleavage of sphingomyelin to form ceramide; and/or has a hydrophobicity greater than that of 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) to (c). 12. The method of any one of items 1 to 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.

13. The method of any one of items 1 to 12, wherein the modified polypeptide antigen molecule is conjugated to the steroid acid at solvent-accessible amine (e.g., primary amine) and/or sulfhydryl groups of the polypeptide antigen.

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

15. The method of any one of items 1 to 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 said peptide, such as at an N- or C-terminal residue).

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

17. The method of item 16, wherein the subcellular targeting signal is a nuclear localization signal, such as a classical NLS (e.g., NLS from SV-40 large T-antigen (e.g., PKKKRKV; SEQ ID NO: 7) or from other classical NLSs) or a non-classical NLS (e.g., acidic M9 domain in the hnRNP Al protein; the sequence KIPIK in yeast transcription repressor Mata2; PY-NLS; ribosomal NLS; and the complex signals of U snRNPs).

18. The method of item 16 or 17, wherein the nuclear localization signal is a/an: SV40 NLS (e.g., comprised in SEQ ID NO: 1 or 7), GWG-SV40NLS (e.g., comprised in SEQ ID NO: 8), hnRNPAl M9 NLS (e.g., comprised in SEQ ID NO: 9), hnRNP D NLS (e g., comprised in SEQ ID NO: 10), hnRNP M NLS (e.g., comprised in SEQ ID NO: 11), PQBP-1 NLS (e.g, comprised in SEQ ID NO: 12), NLS2-RG Domain RPS17 (e.g., comprised in SEQ ID NO: 13), NLS1 RPS17 (e.g., comprised in SEQ ID NO: 14), NLS2 RPS17 (e.g., comprised in SEQ ID NO: 15), NLS3 RPS17 (e.g., comprised in SEQ ID NO: 16), cMyc NLS (e.g., comprised in SEQ ID NO: 17), HuRNLS (e.g., comprised in SEQ ID NO: 18), Tus NLS (e.g., comprised in SEQ ID NO: 19), or Nucleoplasmin NLS (e.g., comprised in SEQ ID NO: 20) ; or 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 to 20. The method of any one of items 1 to 18, wherein the modified polypeptide antigen triggers: (i) increased cytosolic delivery of the modified polypeptide antigen, as compared to a corresponding unmodified polypeptide antigen; (ii) increased total cellular delivery of the modified polypeptide antigen, as compared to a corresponding unmodified polypeptide antigen; (iii) enhanced cellular immunity against said polypeptide antigen, as compared to a corresponding unmodified polypeptide antigen; (iv) increased IFN-gamma production by CD8+ T cells upon exposure to said polypeptide antigen, as compared to a corresponding unmodified polypeptide antigen; (v) enhanced humoral immunity against said polypeptide antigen, as compared to a corresponding unmodified polypeptide antigen; (vi) an increased variety of antibody species against the polypeptide antigen, as compared to a corresponding unmodified polypeptide antigen; or (vii) any combination of (i) to (vi). A population of cells (e.g., in vitro or ex vivo) comprising the modified polypeptide antigen produced by the method of any one of items 1 to 19, or the modified polypeptide antigen as defined in any one of items 1 to 19. The population of cells of item 20, which comprises immune cells (e.g., T cells), antigen- presenting cells (e.g., dendritic cells, macrophages, engineered antigen-presenting cells), MHC class I-expressing cells, MHC class Il-expressing cells, or any combination thereof. An immunogenic composition comprising: the modified polypeptide antigen produced by the method of any one of items 1 to 19, the modified polypeptide antigen as defined in any one of items 1 to 19, the population of cells of item 20or 21, or any combination thereof; and a pharmaceutically acceptable excipient and/or adjuvant (e.g., an emulsion adjuvant, oil-in-water emulsion adjuvant, or a squalene -based oil-in-water emulsion adjuvant). The immunogenic composition of item 22, which is a therapeutic or prophylactic vaccine (e.g., anti-cancer vaccine, anti-viral vaccine, or anti-bacterial vaccine). A method for triggering an enhanced adaptive immune response in a subject against an unmodified polypeptide antigen of interest, the method comprising administering the immunogenic composition of item 22 or 23 to the subject. A method for vaccinating a subject against an infectious disease, the method comprising administering the immunogenic composition of item 22 or 23 to the subject, wherein the polypeptide antigen comprises an antigenic fragment of a pathogen (e g., virus, bacteria, fungus) causing the infectious disease. A method for treating cancer in a subject, the method comprising administering the immunogenic composition of item 22 or 23 to the subject. The method of item 26, wherein the method is combined with immune-checkpoint inhibitor therapy. A modified polypeptide antigen as defined in any one of items 1 to 19, or produced by the method of any one of items 1 to 19, for use in generating an immune response in a subject or for the manufacture of an immunogenic composition for generating an immune response in a subject. Use of the modified polypeptide antigen as defined in any one of items 1 to 19, the modified polypeptide antigen produced by the method of any one of items 1 to 19, the population of cells as defined in item 20 or 21, or the immunogenic composition as defined in item 22 or 23, for generating an immune response in a subject or for the manufacture of a medicament (e.g., vaccine) for generating an immune response in a subject. The modified polypeptide antigen for the use of item 28, or the use of item 29, wherein the immune response comprises enhanced cellular immunity against said polypeptide antigen, increased IFN-gamma production by CD8+ T cells upon exposure to said polypeptide antigen, enhanced humoral immunity against said polypeptide antigen, or any combination thereof, as compared to that generated from a corresponding unmodified polypeptide antigen. 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 that of the polypeptide antigen prior to conjugation. The method of item 31, wherein the number of steroid acid moieties conjugated to the polypeptide antigen is sufficient to increase endosomal escape of the modified polypeptide antigen upon intracellular delivery relative to a polypeptide antigen lacking said modification. The method of item 31 or 32, wherein the modified polypeptide antigen is as defined in any one of items 3 to 19. A method of improving polypeptide antigen immunogenicity, 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 said polypeptide antigen upon administration to a subject as compared to a corresponding unmodified polypeptide antigen, wherein the peptide comprised in the bile acid-peptide moiety comprises a nuclear localization signal (NLS). 35. The method of item 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 upon intracellular delivery relative to a corresponding unmodified polypeptide antigen.

36. The method of item 34 or 35, wherein the modified polypeptide antigen is conjugated to a sufficient number of bile acid-peptide moieties such that the modified polypeptide antigen exhibits greater thermal stability relative to a corresponding unmodified polypeptide antigen.

37. The method of any one of items 34 to 36, wherein covalently conjugating 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 moiety.

38. The method of item 37, wherein the polypeptide antigen is reacted with between a 2-fold and 100-fold molar excess of the bile acid-peptide moiety.

39. The method of item 37, wherein the polypeptide antigen is reacted with between a 2-fold and 50-fold molar excess of the bile acid-peptide moiety.

40. The method of item 37, wherein the polypeptide antigen is reacted with between a 5-fold and 25-fold molar excess of the bile acid-peptide moiety.

41. The method of any one of items 34 to 40, wherein the mean number of bile acid-peptide moieties conjugated per modified polypeptide antigen is at least labout , 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 is between about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 and n, wherein n is the total number of accessible sites on the polypeptide antigen available for conjugation.

42. The method of any one of items 34 to 41, 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), glycodeoxycholic acid (CDCA), glycochenodeoxycholic acid (GCDCA), taurodeoxycholic acid (TDCA), glycolithocholic acid (GLCA), taurolithocholic acid (TLCA), taurohyodeoxycholic acid (THDCA), taurochenodeoxycholic acid (TCDCA), ursocholic acid (UCA), tauroursodeoxycholic acid (TUDCA), ursodeoxycholic acid (UDCA), or glycoursodeoxycholic acid (GUDCA).

43. The method of any one of items 34 to 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 ceramide accumulation on the inner leaflet of endosomes; or triggers increased acid sphingomyelinase (ASM)-mediated cleavage of sphingomyelin to form ceramide.

44. The method of any one of items 34 to 43, wherein the nuclear localization signal is a/an: SV40 NLS (SEQ ID NO: 1 or 7), GWG-SV40NLS (SEQ ID NO: 8), hnRNPAl 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), HuRNLS (SEQ ID NO: 18), Tus NLS (SEQ ID NO: 19), or Nucleoplasmin NLS (SEQ ID NO: 20)

45. The method of any one of items 34 to 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 to 20.

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

47. The method of item 46, wherein the linker is a bifunctional linker, trifunctional linker, or multifunctional linker.

48. The method of any one of items 34 to 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.

49. The method of any one of item 34 to 48, wherein the polypeptide antigen is or comprises a tumor-associated antigen (TAA), tumor-specific antigen (TSA), cell lysate derived from a tumor, tumor-derived exosomes, a neoantigen, a viral antigen, a bacterial antigen, a fungal antigen, or other antigen associated with a disease or disorder amenable to treatment by vaccination and/or immunotherapy.

50. The method of any one of items 34 to 49, wherein the polypeptide antigen is or comprises a SARS-CoV Spike protein or an antigenic fragment thereof.

51. An immunogenic composition comprising: the modified polypeptide antigen produced by the method of any one of items 34 to 50 or a population of cells comprising the modified polypeptide antigen produced by the method of any one of items 34 to 50, and a pharmaceutically acceptable excipient and/or adjuvant.

52. The immunogenic composition of item 51, wherein the population of cells comprises dendritic cells, B cells, T cells, macrophages, engineered antigen-presenting cells, MHC class I- expressing cells, MHC class Il-expressing cells, or any combination thereof. 53. A method for triggering an enhanced adaptive immune response in a subject against an unmodified polypeptide antigen of interest, the method comprising administering the immunogenic composition of item 52 to the subject.

EXAMPLES

Example 1: General Materials and Methods Animals and ethics

Six to eight week-old BALB/c mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA) whereas C57BL/6 mice of similar age were purchased from Charles River (Montreal, QC, Canada). Littermate mice were interbred 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 Care Committee of Uni versite de Montreal.

Cell lines and reagents

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

Generation of bone marrow derived DCs

Mouse bone marrow derived DCs (BMDCs) were generated by flushing the whole marrow from mouse femurs using 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 p-mercaptoethanol. Following red blood cell lysis, cells were then cultured in media supplemented with 50 ng/mL murine recombinant GM-CSF. The media was changed on days 2, 4, 6 and 8. On day 9, the media was replaced to include recombinant murine GM-CSF and LPS from Escherichia coli Ol l i (1 ng/mL) to stimulate DC maturation. Mature DCs were assessed by flow cytometry for their surface expression of CD3, CD19, NK1.1, CD11c, CD80, CD86, and I-A ^b . Modeling accessible Lysine in Protein Antigen

Antigen 3D structure was modeled using the RCSB PDB and Swiss-Model Expasy™ free access software. Accessible amino acids representing the lysine residues were identified and highlighted according to their rate of accessibility (blue: high; green: medium and yellow: poor).

Cancer cell lysate preparation

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. The cells were then subjected to 5 rounds of freeze and thaw in liquid nitrogen/boiling water, respectively. To remove large particles, the lysate was shredded using a G26 needle, passed through a 70 pm cell strainer, 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.

Generation of the ChAcNLS-antigen formulations

ChAcNLS was synthesized as previously described in Beaudoin et al., 2016 unless otherwise indicated. OVA, OVA-DQ, or cancer cell lysate were solubilized at 1-10 mg/mL in sterile PBS with or without other formulation components, but free of amine (NEE) or sulfhydryl (SEI) groups. The SM(PEG)4 cross-linker was added to the reaction for Ih using different molar excess ratios (5x, lOx, 25x, Ox). The free SM(PEG)4 cross-linker was discarded by Centricon™ filtration and Sephadex™ column. ChAcNLS was added in the same molar excess ratios and incubated for Ih to obtain different amounts of ChAcNLS moieties linked per antigen. Unless otherwise specified, the cOVA conjugates tested in the Examples were produced using 5 OX molar excess ratios. Free unlinked ChAcNLS was removed by centricon filtration and Sephadex column. ChAcNLS-modified antigens were concentrated in sterile PBS to obtain final concentration 5-10 mg/mL as determined by UV absorbance.

To evaluate ChAcNLS loading, 10 pg of OVA or ChAcNLS-OVA conjugates were separated under reducing conditions on a 12% polyacrylamide gel and stained with Coomassie brilliant blue R- 250™ (Bio-Rad, Mississauga, ON, Canada). The migration distance in the gel relative to the blue dye front (Rf) was measured and the numbers of ChAcNLS moieties introduced into OVA were categorized into low, medium, and high ChAcNLS loads, estimated by reference to a logarithm plot of molecular weight versus 1/Rf for Kaleidoscope pre-stained standards (Bio-Rad) electrophoresed under identical conditions. In addition, western blot analysis against OVA was performed to confirm the Coomassie results.

Biochemical characterization of ChAcNLS-OVA

A series of tests including: 1) Differential Scanning Calorimetry or Dynamic Light Scattering, 2) Circular Dichroism (CD) Far and Near UV Spectra Scans and Fourier Transform Infrared Spectroscopy (FTIR), 3) Size Exclusion Chromatography with Multi Angle Laser Light Scattering, 4) Intrinsic Tryptophan Fluorescence (ITF), 5) Peptide Mapping (Reference Standard Characterization by LC- MS/MS), and 6) Intact and Subunit Molecular Weight via LC-MS were conducted by Charles River (Wilmington, MA, USA) to characterize the ChAcNLS-OVA modified antigens.

Generation of the bile acid-NLS moieties

Bile acid-NLS moieties were synthesized similar to the synthesis of cholic acid-NLS (ChAcNLS) as previously described in Beaudoin et al., 2016 unless otherwise specified. 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) that comprises a nuclear localization signal from SV40 large T-antigen (SEQ ID NO: 7) flanked by linker amino acids.

Assessment of Intrinsic Tryptophan Fluorescence (ITF)

An Applied Photophysics (Leatherhead, Surrey, UK) Chirascan™ QI 00 circular dichroism (CD) spectrometer was used for intrinsic tryptophan fluorescence (ITF) analysis and a VWR digital heatblock (Radnor, PA) was used for dry block temperature incubations. The Chirscan QI 00 autosampler rack cooling system was used for all 4°C incubations. Data was 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 to 0.8 mg/mL in PBS from stock concentrations in the range of 4 to 5 mg/mL. Diluted samples were then analyzed for ITF without exposure to thermal stress (Native) or after ten minutes of thermal stress by dry block incubation. An aliquot of each diluted sample was incubated at 4°C, a second aliquot was incubated at 37°C, while a third aliquot was incubated at 80°C. BSA, diluted to 0.8 mg/mL, was included with the samples under each of the thermal conditions described above. All samples were re-equilibrated to room temperature after incubation. ITF Analysis was performed in 8 triplicates by excitation at 280 nm with an emission scan range of 200 - 600 nm with a bandwidth of 1.0 nm, a Time-per point of 1 s, and a Step of 0.5. The triplicate spectra were blank subtracted, averaged, and converted from units of mdeg to relative fluorescence intensity using MATLAB software. Diluted BSA solutions were assayed as controls preceding and following the sample sequence.

DC2.4 transfection and assessment of damaged endosomes by microscopy

For this assay, 15 x 10 ³ DC2.4 cells were seeded on a sterile cover slide in a 24-well plate. Two days following transfection of DC2.4 cells with the eGFP-hGal3 mammalian expression vector, 0.1 mg/mL of nOVA or cOVA was added to cells then incubated for 3h at 37°C. The cells were then washed twice to remove excess protein prior to being mounted on a slide. The slides were viewed by fluorescent microscopy (Nikon, Eclipse™ Ti2-U) and the results analyzed using the Image J™ software. Phenotypic assessment of generated BMDCs by flow cytometry

To assess the expression of cell surface markers, BMDCs were incubated with various antibodies diluted according to manufacturer’s instructions using the staining buffer (PBS containing 2% FBS) for 30 min at 4°C in the dark. After extensive washing using the staining buffer, the cells were re-suspended in 400 pL of staining buffer. The samples were acquired by BD FACSDiva™ on CANTOII™, then analyzed using FlowJo™ vlO.

Monitoring antigen processing

To evaluate OVA processing, cells were incubated with 10 pg/mL OVA-DQ (with or without ChAcNLS modification) at 37°C. 30 minutes later, cells were washed, and regular media was added. At the end of the indicated incubation time, cells were collected and washed with cold PBS containing 2% FBS. Fluorescence was monitored by analyzing the cells by flow cytometry.

Antigen presentation assay

To evaluate antigen cross-presentation, cells were seeded at 25 x 10 ³ cells per well in 24-well plate (Coming; Massachusetts, United States), then pulsed with the antigens at different concentrations for 3 h. At the end of the pulsing period, the 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 mouse, respectively, using T- cell isolation kits according to the manufacturer’s protocol. After 72 hours, supernatants were collected and used to quantify cytokine production by commercial enzyme-linked immunosorbent assays (ELISAs).

For the B3Z assay, 5 x 10 ⁴ DCs were first pulsed with the selected proteins or cOVA variants for 3 h followed by washing prior to adding 5 x 10 ⁴ B3Z cells. The cells were incubated for 17-19 h prior to their lysis and incubation for another 4-6 h at 37°C with a Chlorophenol red-P-D-galactopyranoside (CPRG) solution. The optical density signal was detected using a SynergyHl™ microplate reader (Biotek, Winooski, VT, United States).

Quantification of antibody titer by ELISA

Nunc MaxiSorp™ plates were coated overnight with 1 pg OVA diluted in coating buffer at 4°C. The following day, the plates were washed then blocked with 3% skim milk for 1 h at room temperature. Following that step, the plates were washed prior to adding the diluted sera (two-fold dilutions were prepared). Following a 2-h incubation period, the plates were washed prior to adding the secondary HRP- linked anti-mouse IgG antibody at a dilution of 1: 1000. Two hours later, the plates were washed then incubated at room temperature with HRP for 10-20 min. Following HRP quenching, the signal was detected using a Synergy ™H1 microplate reader (Biotek; Winooski, VT, United States). Immunizations and tumor challenge

For prophylactic vaccinations, female C57BL/6 mice (n=10/group) were subcutaneously (SC)- injected at Day 0 and 14 with OVA/OVA-ChAcNLS (1 pg/dose), 10 ⁴ BMDCs pulsed with the OVA formulations (0.1 mg/mL), or tumor lysate (0.1 mg/mL). Two weeks following the second vaccination, mice were subcutaneously (SC) challenged with 5 x 10 ⁵ EG.7 or EL4 cells and tumor growth was assessed overtime. To evaluate antigen-specific CD8 T-cell activation, splenocytes isolated from immunized mice were first stimulated in vitro with 1 pg/mL OVA then the supernatant collected three days later to assess cytokine/chemokine production by Luminex™.

For therapeutic vaccinations, female C57BL/6 mice (n=10/group) received a SC injection of 5 x 10 ⁵ EL4 or EG.7 cells at Day 0. Five days later (appearance of palpable tumors ~ 40-60 mm ³ ), mice were SC-injected with 3 x 10 ⁴ OVA-/OVA-ChAcNLS or tumor lysate-/ChAcNLS-lysate-pulsed BMDCs (two injections; 1 week apart). Control animals received 5 x 10 ⁵ tumor cells alone. Treated animals were followed thereafter for tumor growth. For therapeutic vaccination in combination with immune- checkpoint inhibitors (e.g., aPD-1), mice received SC-injections of the antibody or its isotype at 200 pg/per dose every 2 days for a total of 6 doses over two weeks. A similar approach was conducted for allogeneic dosing vaccination in BALB/c mice.

Analysis of turn or- infiltrating immune cells

Following their resection, tumor masses were first weighed then cut into smaller pieces_with surgical scissors in 4-5 ml of Master Mix containing 2mg/ml of Collagenase D,_2 mg/mL of collagenase IV, and 100 pg/mL of DNase type IV mixed in DMEM_supplemented with 5% FBS. The mix was then stirred in a cell culture incubator at 37°C._After 30 min of incubation, 10 mL of DMEM was added to neutralize the enzymaticjeaction. The digested solution was filtered using a 70 pm cell strainer and all retained_fragments at the top of the strainer were smashed with a plunger followed by addition of 1-2 DMEM to wash the strainer. Collected cells were then centrifuges for 5 min at 1200 rpm (4°C), treated with a red blood cell lysis buffer for 1 min then resuspended in 3-4 mL of DMEM supplemented with 5% FBS. Following cell washing, the pellet was resuspended in DMEM supplemented with 5% FBS prior to initiate cell staining for flow cytometry analysis.

Antigen-presentation assay using the B3Z reporter system

Various bile acid-NLS conjugates were screened using the B3Z reporter system. The B3Z cell line is a T-cell hybridoma specific for the H2-Kb-SIINFEKL complex. Once activated via its TCR, the LacZ reporter gene (under the NF AT promoter control) is expressed. Briefly, 1.5 x 10 ⁵ BMDCs or isolated B cells were co-cultured with 5 x 10 ⁴ B3Z cells treated with ovalbumin (OVA)-bile acid-NLS conjugates for overnight at 37 °C with 5% CO2. The following day, all cells were washed twice with PBS (pH 7.4), and the cell pellets were lysed by adding 100 pL of a lysis buffer containing 0. 15 mM chlorophenol red-beta-D-galactopyranoside (CPRG) substrate (Calbiochem, La Jolla, CA), 0.125% NP40 (EMD Sciences, La Jolla, CA), 9 mM MgCL (Aldrich, USA) and 100 mM 2-mercaptoethanol in PBS After a 5- or 24-h incubation at 37 °C, absorbance was taken at 570 nm with 636 nm as the reference wavelength. For these experiments, bile acid-NLS-OVA conjugates were re-suspended in PBS (pH 7.3) at 0.1 mg/mL (prepared with 10X molar ratio of bile acid-NLS moiety to OVA) and OVA alone was resuspended at 5 mg/mL.

Statistical analysis - values were calculated using the one-way analysis of variance (ANOVA). Results are represented as average mean with S.D. error bars, and statistical significance is represented with asterisks: *P<0.05, **P<0.01, ***P<0.001.

Example 2: Biochemical characterization of the ChAcNLS-antigen formulation

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. The peptide ( CGY GPKKKRKV GG ; SEQ ID NO: 1) comprised a nuclear localization signal (underlined) from SV40 large T-antigen flanked by linker amino acids. Multiple ChAcNLS moieties were then conjugated to the epsilon-amino groups of accessible lysme residues of the prototypical polypeptide antigen OVA (SEQ ID NO: 2; Fig. 1C). A schematic diagram representing covalent binding of a given antigen to an ChAcNLS moiety is shown in Fig. 1A. ChAcNLS-OVA was then biochemically characterized, as described in Example 1, and binding was confirmed by Coomassie blue staining (Fig. IB) and Western blot (Fig. IE). Biochemical characterizations revealed that ChAcNLS-OVA conjugated at a ratio of 25x (Fig. IB, line 2) had an average of about four ChAcNLS moieties conjugated per OVA corresponding to a MW increase of about 8,6 kDa. ChAcNLS-OVA conjugated at a ratio of 50x (Fig. IB, line 3) had an average of about eight ChAcNLS moieties conjugated per OVA corresponding to a MW of about 19.2 kDa. A ribbon structure of the OVA protein with lysine residues that are predicted to be highly (in blue), moderate (green) or poorly (yellow) accessible lysine residues is shown in Fig. ID.

Furthermore, to assess the overall stability of ChAcNLS-OVA (cOVA), ITF analysis was conducted to measure its unfolding following thermal stress. In this assay, changes in peak shifts or intensities are indicative of unfolding as polypeptide residues may become solvent-exposed and undergo change in orientation (Fig. IF). When different cOVA ratios were assayed under native or thermally variable conditions, nOVA underwent complete denaturation at 80°C along with partial reduction in peak intensity observed for the 50X cOVA (Fig. IF). No changes in ITF spectral measures were observed for the other cOVA samples suggesting that conjugation with the ChAcNLS moieties greatly increased antigen stability.

An antigen presentation assay using the SIINFEKL-specific B3Z cell line was then conducted to compare different OVA conjugates. As shown schematically in Fig. 1G, the different conjugates tested included: cholic acid-NLS moiety (“ChAcNLS”); OVA conjugated to cholic acid moieties without an NLS peptide (“ChAc-OVA”); OVA conjugated to cholic acid-NLS moieties via a PEG4 bifunctional linker (“Q1ACNLS-PEG4-OVA” or “cOVA”); and OVA conjugated to cholic acid-NLS moieties via a PEG. bifunctional linker (“ChAcNLS-PEGs-OVA”). As shown in Fig. 1H, conjugating OVA to cholic acid moieties alone without an NLS peptide (“ChAc-OVA”) did not lead to improved antigen presentation as compared to naked OVA (“nOVA”) alone. Strikingly, OVA conjugated with 50X molar excess of ChAcNLS moieties via a PEG ₄ bifunctional linker (“cOVA (50X)”) exhibited the same level of antigen presentation as the SIINFEKL positive control peptide (“SIINFEKL”). Interestingly, comparable levels of antigen presentation were obtained by reducing the molar excess of ChAcNLS moieties to 5X, 10X and 25X [“cOVA (ChAcNLS-PEG4-OVA)”], although this heightened antigen presentation was lost at a 2X molar excess of ChAcNLS moieties. Antigen presentation levels comparable to the SIINFEKL positive control were also observed for OVA conjugated with 2X to 25X molar excess of ChAcNLS moieties via a PEGs bifunctional linker (“ChAcNLS-PEGs-OVA”). Lastly, antigen presentation levels higher than nOVA but below that of the SIINFEKL positive control peptide were observed using OVA conjugated with 5X and 10X molar excess of ChAcNLS moieties via a much longer PEG24 bifanctional linker (“ChAcNLS-PEG24-OVA”), but the increase in antigen presentation over the nOVA was lost at molar excesses of 2X and 25X (data not shown).

Example 3: In vitro cross presentation of ChAcNLS-OVA

To generate BMDCs, femur and tibias of female C57BL/6 or BALB/c mice were flushed to collect total nucleated cells. Cells were then plated for 8 days with recombinant GM-CSF (10 ng/mL) and replaced every 2 days. LPS was added on day 9 to trigger DC maturation prior to antigen pulsing. Maturation of the BMDCs was confirmed by flow cytometry. No T cells, B cells or NK cells were detected at Day 9 and more than 80% of BMDCs expressed CD1 lc+, CD80+, CD86+, and I-Ab+. BMDCs were then incubated with either naked OVA (nOVA) or ChAcNLS-OVA (cOVA) at varying concentrations, and either CD4+ T cells from OT-II transgenic mice or CD8+ T cells from OT-I transgenic mice were added.

Fig. 2A is a schematic diagram showing the set-up of the antigen cross-presentation of OVA peptides (i.e., SIINFEKL [OT-I peptide for CD8+ T cell; SEQ ID NO: 5] or ISQAVHAAHAEINEAGR [OT-II peptide for CD4+ T cells; SEQ ID NO: 6]) used to assess OVA-re spending OT-I (CD8) and OT- II (CD4) T cell activation.

Fig. 2B shows the amount of IFN-gamma 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-gamma than when incubated with the naked antigen (nOVA). Figs. 2C and 2D shows the amount of IL-2 and IFN-gamma, respectively, produced using OT-II-derived CD4 T cells, which is a measure of classical MHC class II presentation activity. Strikingly, CD4+ T cells incubated with BMDCs and cOVA produced significantly more IFN-gamma than when incubated with the naked antigen (nOVA). In light of these observations, intracellular processing of captured OVA was then monitored. For this purpose, the ChAcNLS was cross-linked onto OVA-DQ prior to pulsing ex vivo generated primary bone marrow-derived DCs. Although an increase in differences could be depicted for both antigen conditions 3h post-DC pulsing, the signal intensity in DCs treated with ChAcNLS-linked OVA-DQ was significantly higher 6 h post-pulsing compared to nOVA (Fig. 2E and F). Interestingly, no differences in signal intensity could be detected between nOVA pulsing at 3 or 6 h suggesting a signal saturation (Fig. 2E). Nevertheless, these observations correlate with the antigen presentation assays using primary DCs co-cultured with OT-I (CD8) (Fig. 2B) or OT-II (CD4) T cells (Fig. 2C and D).

To determine if cOVA enhances endosome-to-cytosol escape, a Galectin-3 (Gal3) expression system was used as a marker of damaged endo-membranes. More specifically, Gal3 exhibits high affinity towards -galactoside conjugates, which are normally present on the cell surface, Golgi apparatus and in the lumens of endocytic compartments. Therefore, when expressed under normal conditions, Gal3 is evenly distributed across the cytoplasm. Conversely, induction of endosomal membrane rupture allows Gal3 to access and bind luminal glycoproteins. We thus transiently transfected the DC2.4 cell line with a construct expressing the Gal3 as a fusion with the enhanced green fluorescent protein (eGFP-Gal3) to evaluate its distribution pattern. As anticipated, the GFP signal was diffusely distributed throughout the cytosol following treatment of eGFP-Gal3-expressing DC2.4 cells with nOVA (Fig. 2G - upper panel). In contrast, pulsing of DC2.4 with cOVA induces the appearance of several puncta clearly indicating signal re-localization to damages endosomes (Fig. 2G - lower panel).

Example 4: In vivo anti-cancer activity

To determine the effectiveness of ChAcNLS-modified OVA as a prophylactic vaccine, mice were vaccinated with cOVA as either a cell-based or a stand-alone vaccine. For the cell-based vaccine, BMDCs pulsed with either nOVA or cOVA were subcutaneously injected into mice before implantation of EG.7 lymphoma cells, followed by a re-challenge. The immunization scheme is depicted in Fig. 3A. Strikingly, mice vaccinated with BMDCs pulsed with cOVA did not show any tumor growth and had a 100% survival rate, whereas control (unvaccinated) and mice vaccinated with BMDCs pulsed with nOVA developed large tumors and were more susceptible to death (Fig. 3B and 3C). Furthermore, mice vaccinated with BMDCs pulsed with cOVA developed higher antibody titers (Fig. 3D). In addition, the level of CD4 effector (CD44hiCD62Llo) and CD8 central (CD44hiCD62Lhi) and effector memory T cells was substantially higher in the cOVA-DC group (Fig. 3E and F). Finally, Luminex™ analysis of cytokines/chemokines derived from in vitro re-stimulated T cells show elevated levels of IFN-gamma in the cOVA group compared to nOVA-injected mice (Fig. 3G). Similar data were also observed for macrophage -inflammatory protein (MIP)-ip and MIP-2, two strong chemoattractants for monocytes/macrophages, NK cells and neutrophils, as well as interleukin (IL)-6 and IL- 10, two cytokines known to support B-cell differentiation and antibody production (Fig. 3G). Altogether, the improved immune responses observed in animals vaccinated with cOVA-pulsed DCs is consistent with their acquired resistance to multiple EG.7 re-challenges and durable survival benefits.

In a similar immunization scheme, mice were vaccinated with either cOVA or nOVA alone (not BMDC-pulsed) before implantation of EG.7 lymphoma cells (Fig. 4A). As shown in Fig. 4B, mice vaccinated with cOVA developed smaller tumors, had significantly increased survival rates (Fig. 4C) and antibody response (Fig. 4D) in comparison to mice vaccinated with nOVA or unvaccinated control mice. Vaccination using the two squalene-based oil-in-water emulsion adjuvants, AddaS03™ or AddaVax™, both improved the immune response potency with AddaVax triggering superior effect in cOVA- vaccinated mice.

Example 5: In vivo therapeutic vaccination against T-cell lymphoma

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

Mice immunized with BMDCs pulsed with cOVA had significantly smaller tumors (Fig. 5B) and increased survival rates (Fig. 5C) compared to mice immunized with anti-PD-1 antibody alone or BMDCs pulsed with nOVA. Strikingly, mice treated with a combination therapy of anti-PD-1 Ab and BMDCs pulsed with cOVA showed synergistic efficacy in treating T cell lymphoma in mice, as shown by the decrease in tumor volumes and increase in survival rates. This synergistic effect was directly correlated to the number of BMDCs pulsed with cOVA immunized in mice (Fig. 5D and 5E).

Finally, ChAcNLS was covalently linked to EL4 T cell lymphoma lysates to determine the effect of an antigen specific therapeutic vaccine. Mice implanted with EL4 T cell lymphoma cells were immunized with BMDCs pulsed with either EL4 lysates alone or ChAcNLS-EL4 lysates, in the presence or absence of anti-PD-1 antibody (Fig. 6A). Similar to with cOVA, mice immunized with BMDCs pulsed with ChAcNLS-EL4 lysates had significantly smaller tumors and increased survival rates compared to mice immunized with anti-PD-1 Ab alone or BMDCs pulsed with EL4 lysates independent of the presence of anti-PD-1 Ab (Fig. 6B and 6C). Of note, a synergistic effect combining BMDCs pulsed with ChAcNLS-EL4 lysates and anti-PDl Ab treatment was seen, as tumor growth in the mice plateaued at around 36 days and the mice had a 70% survival rate at the conclusion of the study (54 days). These observations were further supported by tumor-infiltrating lymphocyte (TIL) analysis (Fig. 6E), which revealed enhanced recruitment of CD8, NK and CD11c immune effector cells in the ChAcNLS- EL4 lysate (“cLysate”)-pulsed DCs/PD-1 (Fig. 6E). In sharp contrast, the level of regulatory CD4 T cells (Tregs) was greatly diminished in the same group (Fig. 6E), bolstering the idea that combining cLysate- pulsed DCs to PD-1 favors inflammation by tipping the balance in favour of CD8 T cells versus suppressive Tregs infiltration (Fig. 6F). Overall, these findings indicate that "off-the-shelf allogeneic DCs treated with the ChAcNLS- EL4 lysate formulation can be effectively exploited as universal vaccines to trigger potent anti-tumoral responses.

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

To determine the effectiveness of bile acid-conjugated polypeptide antigens as therapeutic vaccines for microbial infections, particularly viral infections such as with SARS-CoV-2, a vaccine composed of ChAcNLS covalently linked to SARS-CoV-2 Spike protein was constructed, similar to the construction of the cOVA vaccine as described in Examples 1, 2, and 4.

Fig. 7A and 7B shows the SARS-CoV-2 Spike protein used for the formulation of ChAcNLS- Spike-CoV-2, and a schematic diagram of the ribbon structure of SARS-CoV-2 Spike protein (Wuhan strain D614G) with lysine residues that are predicted to be highly (blue), moderate (green) or poorly (yellow) accessible lysine residues, similarly to OVA. As better depicted by the amino acid sequence of SARS-CoV-2 Spike protein (SEQ ID NO: 3; Fig. 7B), over 50% of the lysine residues are predicted to be accessible (highlighted in black) for ChAcNLS conjugation.

Mice were vaccinated with the 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 adjuvants (Fig. 8A). Elevated IgG titers against the Spike protein were observed in mice vaccinated with cSpike-CoV-2, in comparison to unconjugated nSpike-CoV-2, in the presence of either adjuvant. These IgG antibodies were mostly of IgGl isotype, however, significant levels of IgG2a and IgG2b were observed (Fig. 8B). To evaluate the immunogenicities of the different domains of SARS-CoV-2 Spike protein, mice were also vaccinated with the unconjugated or conjugated vaccines containing Sl-RBD or S2 portions. Antibody titers from mice vaccinated with the S 1-RBD and S2 portions of the CoV-2 Spike protein, in the presence of AddaS03 or AddaVax adjuvants were significantly elevated, as shown in Fig. 8C and 8D.

To evaluate whether the anti-Spike IgG antibodies from vaccinated mice possessed neutralizing activity, an in vitro infectivity neutralization assay was developed using Spike 1 -pseudotyped viral -like particles and HEK cells. As show in Fig. 8E, sera from mice vaccinated with cSpike-CoV-2 were more efficient at inhibiting viral infection of HEK cells, and therefore had stronger neutralizing activity, as compared to nSpike-CoV-2, as shown with by NT50 titers.

Vaccines composed of Spike protein conjugated to ChAcNLS were shown to be efficient in generating a strong humoral response. To determine whether the same vaccines were efficient at generating a cellular response, cytokine profiling following T-cell re-stimulation in vitro was assessed in mice vaccinated with nSpike-CoV-2 or cSpike-CoV-2 in the presence of two different of adjuvants. Results are shown in Fig. 9A and 9B. In mice vaccinated with cSpike-CoV-2, a strong IFN-y response was observed with either adjuvant, as compared to vaccination with nSpike-CoV-2, which is indicative of a strong and consistent Thl response required for control of viral infections.

To further evaluate the immunogenicity of the SARS-CoV-2 vaccines, rabbits and hamsters were vaccinated with different doses of cSpike-CoV-2 in the presence of different adjuvants. Fig. 10 shows a schematic diagram (Fig. 10A) of the vaccination design in rabbits with cSpike-CoV-2 and IgG titers (Fig. 10B) at different timepoints and doses. Fig. 11 shows the evaluation of the therapeutic efficacy of cSpike- CoV-2 in a challenge model. Fig. 11 shows a schematic diagram (Fig. HA) of the experimental design for the vaccine efficacy study in hamsters and IgG antibody titers (Fig. 11B) in response to the cSpike- CoV-2 vaccine mixed with the FDA-approved (GMP grade) MONTANIDE™ ISA 720 VG adjuvant or AddaS03. Here, the vaccines were tested using excess molar ratios of ChAcNLS to Spike-CoV-2 protein, 10X and 50X. Prior to the third dose, hamsters were challenged intranasally with the SARS-CoV-2 Delta variant. In general, the vaccines at all doses were well tolerated by rabbits and hamsters and generated strong humoral responses.

Finally, to evaluate whether vaccination with cSpike-CoV-2 is protective against different SARS- CoV-2 variant infections, sera from vaccinated mice were tested for cross-reactivity against Spike protein from the California, Brazil, South Africa, UK, Indian, and Delta strains, which possess specific mutations in the RBD (Fig. 12A) with respect to the “wild-type” Wuhan strain. As shown in Figs. 12B and 12C, sera from mice immunized with cSpike-CoV-2 were significantly cross-reactive with the Spike protein from every SARS-CoV-2 variant tested. Furthermore, antibodies from sera of vaccinated mice were protective and had strong neutralization activity against UK, Brazil, South Africa, Delta, and California SARS-CoV-2 strains, as shown by the in vitro neutralization assay (Fig. 12D)

Overall, these findings indicate that the ChAcNLS-CoV-2-Spike protein formulations can be effectively exploited as universal vaccines to trigger potent antiviral responses.

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

To determine whether SARS-CoV-2 vaccines using Spike protein derived from different variants would be effective using the same formulation, cSpike-CoV-2-IN vaccine was produced using the Spike protein from the Indian variant.

Mice were vaccinated with different doses of the cSpike-CoV-2-IN vaccine or with saline (control), and elevated IgG titers in the sera and BALF were observed at different time points (Fig. 13). Furthermore, a strong cellular response was also observed by the detection of different cytokine and chemokine levels in T cells from mice vaccinated with cSpike-CoV-2-IN (Fig. 14).

Finally, sera from mice vaccinated with c Spike-Co V-2-IN vaccine were cross-reactive with Spike proteins from all of the different SARS-CoV-2 variants tested (Fig. 15).

Overall, these findings indicate that the ChAcNLS can be adapted to Spike proteins from different SARS-CoV-2 -variants to formulate an effective vaccine that triggers a broad, protective, and potent antiviral response.

Example 8: Enhanced antigen presentation of antigen conjugated to different bile acid-NLS conjugates

Different bile acid-NLS conjugates conjugated to OVA were produced and evaluated for their ability to enhance DC or B cell antigen presentation of OVA in a B3Z reporter assay. Bile acid-NLS- OVA conjugates were produced at 10X or 50X molar excess ratios of bile acid-NLS to OVA, as shown by SDS-PAGE in Fig. 16, but only the results with conjugates produced at a 10X molar excess ratio are shown in the antigen presentation assays of Fig. 17 (dendritic cells) and Fig. 18 (B cells). From the relative migration distances (Rf) calculated from the SDS-PAGE results (e.g., Fig. 16): 10X molar excess of bile acid-NLS reactant resulted in about 1-4 bile acid-NLS moieties conjugated per OVA molecule; 25X molar excess of bile acid-NLS reactant resulted in about 5-9 bile acid-NLS moieties conjugated per OVA molecule; and 50X molar excess of bile acid-NLS reactant resulted in about 12-20 bile acid-NLS moieties conjugated per OVA molecule.

Dendritic cells as antigen presenting cells As shown in Fig. 17, all of the different bile acid-NLS conjugates tested enhanced the presentation by BMDCs of OVA at a level similar to or greater than that of CA-SV40 (i.e., ChAcNLS; indicated as a broken line in Fig. 17). Strikingly, the increase in antigen presentation observed for all the bile acid-NLS conjugates tested in Fig 17 was superior to that of the “OVA alone” control despite the fact that a fifty fold higher concentration of OVA antigen was used (5 mg/mL) as compared to for the OVA conjugates (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 markedly enhanced OVA presentation compared to CA-SV40 (Fig. 17). Furthermore, a negative control in which OVA was conjugated to SV40 peptide alone (without a bile acid) and used at a more comparable concentration of 0.1 mg/mL (“SV40” in Fig. 17) produced a similar result to the “PBS” negative control. The bile acid- NLS conjugate “SV40-CA” differs from the conjugate “CA-SV40” mainly in the placement of the cholic acid group. That is, in the “SV40-CA” conjugate, the cholic acid group is conjugated to the C terminus of the SV40 NLS peptide via a C-terminal lysine residue added to t4he SV40 NLS peptide. As shown in Fig. 17, both the conjugates “SV40-CA” and “CA-SV40” yielded similar B3Z responses, which suggests that the placement of the cholic acid group with respect to the peptide moiety does not affect the immunostimulatory effect of the bile acid-NLS moieties.

B cells as antigen presenting cells

As shown in Fig. 18, the majority of the different bile acid-NLS conjugates tested yielded comparable or enhanced antigen presentation by B cells as compared to a fifty fold higher dose of the OVA alone (5 mg/mL) control or as 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-hnRNPAl M9, CA-cMyc, GDCA-SV40, GUDCA- PQBP1, and CDCA-NLS3-RPS17 markedly enhanced OVA presentation as compared to CA-SV40.

Overall, these findings support the versality of a variety of 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 the polypeptide antigens, which can be the mostly costly ingredient of a subunit vaccine to manufacture.

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