Methods for the sole use of polymersome-associated adjuvants to stimulate immune responses

Abstract

The present invention relates to eliciting an immune response in a subject by the sole administration of an adjuvant, where the adjuvant is associated with a population of polymersomes.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to European Patent Application No. 21154745, filed with the European Patent Office on February 2, 2021, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing in computer readable format, which is incorporated herein by reference.

[0003] Technical Field The present invention relates to a method of eliciting an immune response in a subject by the use of an adjuvant alone, wherein the adjuvant is associated with one or more polymersome populations. [Background technology]

[0004] Background of the Invention Immunization is a well-established process, but different immunogens or antigens elicit different responses. For example, membrane proteins form one class of antigens that elicit low-level responses, which means that a large amount of membrane protein is required to generate or induce a desired level of immune response. Membrane proteins are notoriously difficult to synthesize and are insoluble in water in the absence of detergents. Therefore, membrane proteins are expensive and difficult to obtain in sufficient quantities for immunization. Furthermore, membrane proteins require proper folding to function properly. The immunogenicity of properly folded native membrane proteins is typically much better than their solubilized forms, which may not be folded in a physiologically meaningful manner. Therefore, even though adjuvants can be used to boost the immunogenicity of such solubilized antigens, it is an inefficient method without significant advantages (e.g., WO2014 / 077781A1).

[0005] To increase the chances of isolating antibodies that may be effective in vivo, transfected cells and lipid-based systems have been used to present membrane protein antigens. However, these systems are often unstable (e.g., sensitive to oxidation), laborious, and costly. Moreover, for such membrane protein antigens, the current state of the art is to use inactive virus-like particles for immunization.

[0006] Meanwhile, vaccines are the most effective method for preventing infectious diseases (e.g., Liu et al., 2016). Currently, the majority of licensed vaccines are made from either live or killed viruses. Although such vaccines are effective in generating humoral immunity (antibody-mediated responses) to prevent viral spread and cellular entry, concerns remain regarding the safety of such vaccines. Over the past few decades, scientific advances have helped overcome these issues by engineering non-replicating recombinant virus vaccine vectors. In parallel, protein-based or subunit antigens have been explored as safer alternatives. However, the immunity (both humoral and cellular responses) elicited by such protein-based vaccines is typically insufficient. Several approaches have been used to improve the immunogenic properties of antigens. For example, microencapsulation of antigens in polymers has been extensively studied, which has enhanced immunogenicity, but antigen aggregation and denaturation remain unresolved (e.g., Hilbert et al., 1999). Furthermore, to elicit more pronounced humoral and cellular responses, adjuvants (e.g., oil-in-water emulsions or polymer emulsions) [e.g., US9636397B2, US2015 / 0044242A1] are used together with antigens. Despite these advances, they have low uptake and cross-presentation efficiency. To facilitate cross-presentation, virus-like particles have been developed that mimic the immune system's characteristics during viral infection. Synthetic architectures, such as liposomes, in which antigens are encapsulated, are particularly attractive. Liposomes are self-assembled monolayer structures made of lipids, and cationic liposomes are more attractive and promising as delivery vehicles due to their efficient uptake by antigen-presenting cells (APCs) [e.g., Maji et al., 2016].Furthermore, liposomes can also be loaded with immunomodulators, such as monophosphoryl lipid A (MPL) and CpG oligodeoxynucleotides, which are toll-like receptor (TLR) agonists that stimulate immune cells via receptors. Despite these advantageous properties of such delivery vehicles, one of the limiting factors is the stability of liposomes in the presence of serum components. This issue can be somewhat alleviated by PEGylation and loading with high-melting-point lipids. One well-characterized example is the interbilayered-crosslinked multilamellar vesicle (ICMV), which is formed by stabilizing multilamellar vesicles with short covalent lipid-linking bridges (e.g., Moon et al., 2011). Other nanoparticle architectures have also led to successful immunizations using nanodiscs (e.g., Kuai et al., 2017) or pH-sensitive particles (e.g., Luo et al., 2017). However, such strategies still require adjuvants or are not very efficient outside the prototypical ovalbumin (OVA) model.

[0007] Additionally, polymersomes have been proposed as a stable alternative to liposomes and have been used to incorporate membrane proteins to induce immune responses (e.g., Quer et al., 2011, WO2014 / 077781A1). Protein antigens have also been encapsulated in the chemically modified (but oxidation-sensitive) membrane of polymersomes to deliver antigens and adjuvants to dendritic cells (e.g., Stano et al., 2013).

[0008] Even with this advancement made through the use of polymers, there remains a need to provide alternative methods of eliciting an immune response (e.g., in the absence of antigen), especially for the treatment and / or prevention of infectious diseases, cancer, and autoimmune diseases. Summary of the Invention

[0009] The present invention relates to methods of inducing an immune response in a subject by the sole administration of one or more adjuvants associated with one or more polymersome populations. The present invention further relates to methods of sole administration of one or more adjuvants of the present invention, wherein said sole administration is characterized in that an antigen is not administered to said subject in combination (e.g., co-administered) with said one or more adjuvants (e.g., simultaneously or at different times). The present invention further relates to an adjuvant for use in inducing an immune response in a subject, said use comprising the sole administration of said adjuvant, wherein said adjuvant is associated with one or more polymersome populations.

[0010] The present application therefore fulfills the above-mentioned need by providing two distinct polymersome populations that improve the immunogenic properties of an antigen when administered, methods for producing such two polymersome populations, and compositions comprising such two polymersome populations, as described herein below, characterized in the claims, and illustrated by the accompanying examples and figures.

[0011] Sequence Listing Overview As used herein, references are made to UniProtKB accession numbers (unless otherwise indicated or otherwise specific, as available at http: / / www.uniprot.org / , e.g., UniProtKB Release 2017_12).

[0012] SEQ ID NO:1 is the amino acid sequence of the tumor neoantigen polypeptide Reps1 P45A from the MC-38 mouse model of colon cancer.

[0013] SEQ ID NO:2 is the amino acid sequence of the tumor neo-antigenic peptide Adpgk R304M derived from the MC-38 mouse model of colon cancer.

[0014] SEQ ID NO:3 is the amino acid sequence of the tumor neo-antigen peptide Dpagt1 V213L derived from the MC-38 mouse model of colon cancer.

[0015] SEQ ID NO:4 is the amino acid sequence of chicken ovalbumin (OVA), UniProtKB accession number P01012.

[0016] SEQ ID NO:5 is the amino acid sequence of influenza A virus (A / New York / 38 / 2016 (H1N1)) hemagglutinin, UniProtKB accession number A0A192ZYK0.

[0017] SEQ ID NO:6 is the amino acid sequence of influenza A virus (A / Swine / 4 / Mexico / 2009 (H1N1)) hemagglutinin, UniProtKB accession number D2CE65.

[0018] SEQ ID NO:7 is the amino acid sequence of influenza A virus (A / Puerto Rico / 8 / 1934 (H1N1)) hemagglutinin.

[0019] SEQ ID NO:8 is the amino acid sequence of influenza A virus (A / California / 07 / 2009 (H1N1)) hemagglutinin.

[0020] SEQ ID NO:9 is the amino acid sequence of the tumor neoantigen polypeptide CD8 Trp2 173-196 from the B16-F10 mouse model of melanoma.

[0021] SEQ ID NO:10 is the amino acid sequence of the tumor neoantigen polypeptide CD4 M30 Kif18b K739N from the melanoma B16-F10 mouse model.

[0022] SEQ ID NO:11 is the amino acid sequence of the tumor neoantigen polypeptide CD4 M44 Cpsf3l D314N from the melanoma B16-F10 mouse model.

[0023] SEQ ID NO:12 is the amino acid sequence of the soluble portion (amino acid residues 19 to 1327) of the porcine epidemic diarrhea virus (PEDv) spike protein (S protein) (UniProtKB accession number V5TA78).

[0024] SEQ ID NO:13 is the amino acid sequence of the S1 region (amino acid residues 19 to 739) of the PEDv spike protein (S protein).

[0025] SEQ ID NO:14 is the amino acid sequence of the S2 region (amino acid residues 739 to 1327) of the PEDv spike protein (S protein).

[0026] SEQ ID NO:15 is the amino acid sequence of enhanced green fluorescent protein (eGFP).

[0027] SEQ ID NO:16 is the sequence of the CD8 T cell peptide epitope (SIINFEKL).

[0028] SEQ ID NO:17 is the sequence of a CD8 T cell peptide epitope (SVYDFFVWL).

[0029] SEQ ID NO:18 is a class B CpG oligodeoxynucleotide CpG ODN1826 (5'-tccatgacgttcctgacgtt-3') available from InvivoGen.

[0030] SEQ ID NO:19 is the amino acid sequence of the SARS-CoV-2 spike protein according to UniProtKB accession number P0DTC2.

[0031] SEQ ID NO:20 is the amino acid sequence of the SARS-CoV-2 spike protein according to GenBank accession number QII57278.1.

[0032] SEQ ID NO:21 is the amino acid sequence of the SARS-CoV-2 spike protein according to GenBank accession number YP_009724390.1.

[0033] SEQ ID NO:22 is the amino acid sequence of the SARS-CoV-2 spike protein according to GenBank accession number QIO04367.1.

[0034] SEQ ID NO:23 is the amino acid sequence of the SARS-CoV-2 spike protein according to GenBank accession number QHU79173.2.

[0035] SEQ ID NO:24 is the amino acid sequence of the SARS-CoV-2 spike protein according to GenBank accession number QII87830.1.

[0036] SEQ ID NO:25 is the amino acid sequence of the SARS-CoV-2 spike protein according to GenBank accession number QIA98583.1.

[0037] SEQ ID NO:26 is the amino acid sequence of the SARS-CoV-2 spike protein according to GenBank accession number QIA20044.1.

[0038] SEQ ID NO:27 is the amino acid sequence of the SARS-CoV-2 spike protein according to GenBank accession number QIK50427.1.

[0039] SEQ ID NO:28 is the amino acid sequence of the SARS-CoV-2 spike protein according to GenBank accession number QHR84449.1.

[0040] SEQ ID NO:29 is the amino acid sequence of the SARS-CoV-2 spike protein according to GenBank accession number QIQ08810.1.

[0041] SEQ ID NO:30 is the amino acid sequence of the SARS-CoV-2 spike protein according to GenBank accession number QIJ96493.1.

[0042] SEQ ID NO:31 is the amino acid sequence of the SARS-CoV-2 spike protein according to GenBank accession number QIC53204.1.

[0043] SEQ ID NO:32 is the amino acid sequence of the SARS-CoV-2 spike protein according to GenBank accession number QHZ00379.1.

[0044] SEQ ID NO:33 is the amino acid sequence of the SARS-CoV-2 spike protein according to GenBank accession number QHS34546.1.

[0045] SEQ ID NO:34 is the amino acid sequence of a soluble fragment of the SARS-CoV-2 spike protein corresponding to positions 16 to 1213 of UniProtKB accession number P0DTC2.

[0046] SEQ ID NO:35 is the amino acid sequence of a soluble fragment of the SARS-CoV-2 spike protein corresponding to positions 14 to 1204 of UniProtKB accession number P0DTC2.

[0047] SEQ ID NO:36 is the amino acid sequence of a soluble fragment of the SARS-CoV-2 spike protein.

[0048] SEQ ID NO:37 is the amino acid sequence of a soluble fragment of the SARS-CoV-2 spike protein corresponding to positions 16 to 685 of UniProtKB accession number P0DTC2.

[0049] SEQ ID NO:38 is the amino acid sequence of a soluble fragment of the SARS-CoV-2 spike protein corresponding to positions 686 to 1213 of UniProtKB accession number P0DTC2.

[0050] SEQ ID NO:39 is the amino acid sequence of a soluble fragment of the SARS-CoV-2 spike protein corresponding to positions 646 to 1204 of UniProtKB accession number P0DTC2.

[0051] SEQ ID NO:40 is the amino acid sequence of a soluble fragment of the SARS-CoV-2 spike protein.

[0052] SEQ ID NO:41 is the amino acid sequence of a soluble fragment of the SARS-CoV-2 spike protein corresponding to positions 318 to 524 of UniProtKB accession number P0DTC2.

[0053] SEQ ID NO:42 is the amino acid sequence of the MERS-CoV spike protein according to UniProtKB accession number K0BRG7.

[0054] SEQ ID NO:43 is the amino acid sequence of a soluble fragment of the MERS-CoV spike protein corresponding to positions 1 to 1297 of UniProtKB accession number K0BRG7.

[0055] SEQ ID NO:44 is the amino acid sequence of a soluble fragment of the MERS-CoV spike protein corresponding to positions 18 to 725 of UniProtKB accession number K0BRG7.

[0056] SEQ ID NO:45 is the amino acid sequence of a soluble fragment of the MERS-CoV spike protein corresponding to positions 726 to 1296 of UniProtKB accession number K0BRG7.

[0057] SEQ ID NO:46 is the amino acid sequence of a soluble fragment of the MERS-CoV spike protein corresponding to positions 377 to 588 of UniProtKB accession number K0BRG7.

[0058] SEQ ID NO:47 is the amino acid sequence of the SARS-CoV-1 spike protein according to UniProtKB accession number P59594.

[0059] SEQ ID NO:48 is the amino acid sequence of a soluble fragment of the SARS-CoV-1 spike protein corresponding to positions 14 to 1195 of UniProtKB accession number P59594.

[0060] SEQ ID NO:49 is the amino acid sequence of a soluble fragment of the SARS-CoV-1 spike protein corresponding to positions 14 to 667 of UniProtKB accession number P59594.

[0061] SEQ ID NO:50 is the amino acid sequence of a soluble fragment of the SARS-CoV-1 spike protein corresponding to positions 668 to 1195 of UniProtKB accession number P59594.

[0062] SEQ ID NO:51 is the amino acid sequence of a soluble fragment of the SARS-CoV-1 spike protein corresponding to positions 306 to 527 of UniProtKB accession number P59594.

[0063] SEQ ID NO:52 is the amino acid sequence of the furin cleavage site of the SARS-CoV-2 spike protein.

[0064] SEQ ID NO:53 is the amino acid sequence of the mutant furin cleavage site of the SARS-CoV-2 spike protein.

[0065] SEQ ID NO:54 is the amino acid sequence of the foldon domain.

[0066] SEQ ID NO:55 is the amino acid sequence of the GCN4 domain.

[0067] SEQ ID NO:56 is the amino acid sequence of the immunosilenced GCN4 domain.

[0068] SEQ ID NO:57 is the amino acid sequence of the honeybee melittin leader sequence.

[0069] SEQ ID NO:58 is the amino acid sequence of the furin cleavage site of the MERS-CoV spike protein.

[0070] SEQ ID NO:59 is the amino acid sequence of the mutant furin cleavage site of the MERS-CoV spike protein.

[0071] SEQ ID NO:60 is the amino acid sequence of the furin cleavage site of the SARS-CoV-1 spike protein.

[0072] SEQ ID NO:61 is the amino acid sequence of the mutant furin cleavage site of the SARS-CoV-1 spike protein.

[0073] SEQ ID NO:62 is the nucleotide sequence of the CpG oligonucleotide ODN2006.

[0074] SEQ ID NO:63 is the nucleotide sequence of the CpG oligonucleotide ODN2007.

[0075] SEQ ID NO:64 is the nucleotide sequence of the CpG oligonucleotide ODN2216.

[0076] SEQ ID NO:65 is the amino acid sequence of a soluble fragment of the SARS-CoV-2 spike protein corresponding to positions 19 to 1204 of UniProtKB accession number P0DTC2, but containing a mutant furin cleavage site.

[0077] SEQ ID NO:66 is the amino acid sequence of the SARS-CoV-2 spike protein, corresponding to positions 19 to 1273 of UniProtKB accession number P0DTC2, but containing a mutant furin cleavage site.

[0078] SEQ ID NO:67 is the nucleotide sequence of the CpG oligonucleotide ODN 2135.

[0079] SEQ ID NO:68 is the nucleotide sequence of an exemplary CpG oligonucleotide ODN.

[0080] SEQ ID NO:69 is the nucleotide sequence of an exemplary CpG oligonucleotide ODN.

[0081] SEQ ID NO:70 is the nucleotide sequence of an exemplary CpG oligonucleotide ODN.

[0082] SEQ ID NO:71 is an exemplary nucleotide sequence of a CpG oligonucleotide ODN class A (taken from https: / / www.invivogen.com / cpg-odns-classes and illustrated in Figure 31).

[0083] SEQ ID NO:72 is an exemplary nucleotide sequence of a CpG oligonucleotide ODN class B (taken from https: / / www.invivogen.com / cpg-odns-classes and illustrated in Figure 31).

[0084] SEQ ID NO:73 is an exemplary nucleotide sequence of a CpG oligonucleotide ODN class C (taken from https: / / www.invivogen.com / cpg-odns-classes and illustrated in Figure 31).

[0085] SEQ ID NO:74 is an exemplary nucleotide sequence of a CpG oligonucleotide ODN class B (ODN2007).

[0086] SEQ ID NO:75 is an exemplary nucleotide sequence of a CpG oligonucleotide ODN class B (ODN2006).

[0087] SEQ ID NO:76 is an exemplary nucleotide sequence of a CpG oligonucleotide ODN (ODN2014).

[0088] SEQ ID NO:77 is an exemplary nucleotide sequence of a CpG oligonucleotide ODN (ODN2135). [Brief explanation of the drawings]

[0089] [Figure 1] FIG. 1 is a schematic diagram of immunization with polymersomes of the present invention encapsulating antigens and measuring humoral and cellular responses. [Figure 2A] Figure 2 shows dynamic light scattering results for polymersomes of the present invention. Figure 2A shows a dynamic light scattering plot of OVA-encapsulated polymersomes, a monodisperse population of 173.1 nm (diameter). Figure 2B shows a table of the average diameter (Z-average) measured by DLS for various polymersomes encapsulating various antigens. The names of the formulations shown in parentheses, such as "ACM-OVA," "ACM-CpG," "ACM-OVA-CpG," and "ACM-Trp2," are used in other sections of this application. [Figure 2B] See legend to Figure 2A. [Figure 3] 1 shows the elution profile of OVA-encapsulated polymersomes in size exclusion chromatography. [Figure 4] 1 shows sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of OVA-encapsulated polymersomes. [Figure 5A] Figure 5 shows the results of encapsulating nucleic acids (here, a gene encoding enhanced green fluorescent protein (eGFP)) into polymersomes of the present invention and cellular uptake of the nucleic acid-encapsulated polymers. Figure 5A shows the fluorescence intensity uptake of various polymersomes into cells and eGFP expression based on DNA encapsulated in the polymersomes. Figure 5B shows a fluorescent image of a cell transfected with DNA-encapsulated polymersomes. Figure 5C shows a fluorescent image of a cell transfected with DNA-encapsulated polymersomes. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 6] Figure 1 shows antibody titers from the serum of mice immunized with PBS, OVA alone, OVA with SAS adjuvant, and OVA-encapsulated polymersomes without adjuvant. Only ACM encapsulating OVA (hereinafter, "ACM" refers to the polymersomes of the present invention) was able to induce IgG titers. [Figure 7] Figure 1 shows antibody titers from mouse sera immunized with PBS, HA alone, and HA-encapsulated polymersomes without adjuvant. HA-encapsulated ACM (polymersomes of the present invention) was able to induce IgG titers. [Figure 8] Results are shown for the MC-38 mouse tumor model. Tumor volume was monitored in mice immunized with free peptide (open circles), peptide encapsulated in ACM (filled squares, polymersomes of the present invention), or peptide encapsulated in ACM with anti-PD1 antibody treatment (filled triangles). Tumor growth was modified by peptide encapsulated in ACM (polymersomes of the present invention) compared to free peptide, and was further enhanced by the addition of anti-PD1 antibody. No adjuvant was added to any group. [Figure 9]Figure 9 shows IgG antibody titers and virus neutralization responses (against the PEDv USA / Colorado / 2013 (CO / 13) strain) from sera of mice immunized with PBS and a soluble fragment of PEDv S protein encapsulated in polymersomes used as described herein ("spike protein-encapsulated polymersomes"), as well as, for comparison, sera of mice immunized with killed PED virus ("killed PEDv") and ACM polymersomes alone (i.e., no antigen, "polymersomes alone"). From the IgG titers in Figure 9, it is clear that both the ACM-encapsulated PEDv S protein fragment and the killed virus induced IgG titers. The virus neutralization data demonstrate that only the ACM-encapsulated PEDv S protein produced significant neutralization titers, while the negative control (ACM polymersomes without any antigen) and the killed PED virus showed negligible neutralization. [Figure 10] Virus neutralization data (against the PEDv USA / Colorado / 2013 (CO / 13) strain) from sera generated from mice after immunization with PBS and various polymersomes (e.g., BD21 (described below), PDMS46-PEO37 (simply referred to as "PDMS" in the figures), PDMS46-PEO37 with DSPE-PEG (distearoylphosphatidylethanolamine [DSPE] polyethylene glycol) as an added lipid, and polyethylene glycol-polylactic acid (PLA-PEG) with asolectin lipid (a commercially available phospholipid derived from soybean) encapsulating either the full-length soluble PED spike protein ("BD21 with soluble S protein") or its S1 or S2 fragment (all other cases)) are shown. It is clear from Figure 10 that the group of mice immunized with the PBS sample does not show any virus neutralization, whereas the polymersome formulations, irrespective of whether they encapsulate the full-length protein or its fragment, all show varying degrees of virus neutralization. [Figure 11]Figure 11 shows IgA antibody titers from pigs orally immunized with PEDv S protein encapsulated in ACM without adjuvant. Titers are from fecal swabs. As can be seen in Figure 11, the titers increase over time, demonstrating that polymersomes of the present invention encapsulating PEDv S protein and administered orally can induce an immune response in pigs. [Figure 12] Schematic representation of the porcine epidemic diarrhea virus (PEDv) spike protein (S protein) (UniProtKB accession number V5TA78) and soluble fragments of SEQ ID NO:12 (amino acid residues 19-1327), SEQ ID NO:13 (amino acid residues 19-739), and SEQ ID NO:14 (amino acid residues 739-1327), which were used for encapsulation of soluble S protein in polymersomes and subsequent immunization / vaccination of mice and pigs as described herein. [Figure 13] Figure 13 shows tumor growth curves after prophylactic vaccination with ACM OVA formulations. Tumor growth curves for mice inoculated with 10 B16-OVA cells after administration of different OVA formulations. Figure 13A shows the PBS group, the free OVA and CpG administration group, and the ACM-encapsulated OVA and free CpG co-administration group. Figure 13B shows the PBS group, the co-administered ACM-encapsulated OVA and ACM-encapsulated CpG, and the ACM encapsulated with OVA and CpG. [Figure 14A] Figure 14 shows tumor growth curves after therapeutic vaccination with ACM OVA formulations. Tumor growth curves for mice vaccinated with different ACM OVA formulations followed by inoculation with 10 B16-OVA cells. Figure 14A) PBS group, free OVA and CpG administration group, and ACM-encapsulated OVA and free CpG co-administration group. B) PBS group, free OVA and CpG-encapsulated ACM, and ACM encapsulated with OVA and CpG co-administered ACM. C) OVA-specific CD8 T cells quantified using dextramer specific for the CD8 T cell SIINFEKL (SEQ ID NO: 16) peptide epitope. [Figure 14B] See legend to Figure 14A. [Figure 14C] See legend to Figure 14A. [Figure 15A] Figure 15A shows tumor growth curves of therapeutic vaccination of mice with the ACM melanoma B16F10 formulation inoculated with 10 B16F10 cells. Figure 15A shows the PBS group, co-administered free Trp2 (SEQ ID NO:9) and CpG, co-administered Trp2-encapsulated ACM and CpG, co-administered free Trp2 and ACM-encapsulated CpG, and co-administered ACM-encapsulated Trp2 and ACM-encapsulated CpG. Figure 15B shows Trp2-specific CD8-specific T cells quantified in the blood using pentamer specific for the CD8 T cell SVYDFFVWL (SEQ ID NO:17) peptide epitope. Figure 15C shows CD8 T cell infiltration in the tumor. [Figure 15B] See legend to Figure 15A. [Figure 15C] See legend to Figure 15A. [Figure 16] 1 shows the dynamic light scattering (DLS) spectrum of OVA-conjugated ACM. [Figure 17] Characterization of OVA-conjugated ACM is shown. Figure 17A shows the size-exclusion chromatography (SEC) profile of OVA-conjugated ACM, and Figure 17B shows SDS-PAGE of a sample from the SEC peak loaded and stained using silver staining. [Figure 18] 1 shows the DLS spectrum of HA-conjugated ACM. [Figure 19] Immunoblot of ACM-conjugated HA samples showing different mobilities of coupled and free HA. [Figure 20] SEC profile of HA-conjugated ACM (mAU, light grey trace) overlaid with ELISA signal (OD450, black trace) performed on all collected fractions. [Figure 21]Antibody titers from the sera of C57B1 / 6 mice immunized with PBS, free OVA, free OVA and SAS, BD21-encapsulated OVA, and BD21-conjugated OVA are shown (p<0.01). [Figure 22] Antibody titers from the sera of Balb / c mice immunized with PBS, free HA, BD21-encapsulated HA, and BD21-conjugated HA are shown. [Figure 23A] Figure 23A is a schematic representation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S protein) (UniProtKB accession number: P0DTC2) and soluble fragments of SEQ ID NO:34 (amino acid residues 16-1213), SEQ ID NO:37 (amino acid residues 16-685), and SEQ ID NO:38 (amino acid residues 685-1213). According to UniProtKB, amino acids 1214-1234 form the transmembrane region, and 1235-1273 form the intraviral region. The endpoints of the S1 and S2 segments, the transmembrane region, and / or the intraviral region may vary depending on the prediction software. Figure 23B depicts a protocol for immunizing mice with ACM encapsulating the SARS-CoV-2 spike protein. Figure 23C shows IgG titers measured in Balb / c mice on day 35 immunized with the following formulations: BD21-encapsulated soluble S1 and S2 segments co-administered with adjuvant (group 1), BD21-encapsulated soluble S1 and S2 segments (group 2), BD21-encapsulated soluble S2 segment co-administered with encapsulated adjuvant (group 3), and PBS as a negative control (group 4). [Figure 23B] See legend to Figure 23A. [Figure 23C] See legend to Figure 23A. [Figure 24]Figure 24A shows the protocol and results of immunization of mice with full-length soluble encapsulated SARS-CoV-2 spike protein encapsulated in ACM. Figure 24B shows the immunization protocol. Figure 24B shows the IgG antibody titers against SARS-CoV-2 spike protein 28 days after the first immunization for the four groups. The following formulations were prepared: (i) free recombinant spike protein ("fSpike"), (ii) BD21 polymersome-encapsulated spike protein ("ACM-Spike"), (iii) a mixture of free spike protein and free CpG adjuvant ("fSpike fCpG"), and (iv) a mixture of BD21 polymersome-encapsulated spike protein and BD21 polymersome-encapsulated CpG ("ACM-Spike ACM-CpG"). [Figure 25] 1 shows the results of a virus neutralization assay (PEDv) after immunization of guinea pigs with ACM encapsulating PEDv S2 spike protein mixed with ACM encapsulating CpG administered by different routes. [Figure 26] Figure 26 shows the protocol and results of immunization of mice with ACM encapsulating the MERS spike protein. Figure 26A shows the immunization protocol, Figure 26B shows the results of ELISA for the MERS-CoV spike protein S1 domain, and Figure 26C shows the results of a virus neutralization assay (MERS-CoV). [Figure 27] 1 shows the results of a virus neutralization assay for mice immunized with ACM encapsulating either the S1 or S2 domain of the PEDv spike protein or a mixture of the S1 and S2 domains (PEDv). [Figure 28] Figure 1 shows the results of CpG treatment, demonstrating the successful in vitro induction of cytokines by ACM-CpG adjuvant based on the innate immune response. [Figure 29]Activation of cDCs with free CpG or ACM-CpG. a, b. Representative histograms showing the expression of CD86 and CD80 activation markers in cDC1 and cDC2. Mice were SC injected with PBS, empty ACM, free CpG, or ACM-CpG, and cDCs from inguinal lymph nodes were examined 2 days later. c, d. Comparison of CD86+ and CD80+ cDC1 and cDC2 between treatment groups. [Figure 30] Cytokine profiles of free CpG-B or ACM-encapsulated CpG-B. a-c. IL-6 production by human PBMCs after incubation with free CpG-A, free CpG-B, or ACM-CpG-B. d-f. IFN-α production. Individual dose-response curves are shown. Healthy donor identification numbers are indicated in the lower right. [Figure 31] Exemplary CpG ODN classes are adapted from https: / / www.invivogen.com / cpg-odns-classes. Exemplary CpG-A ODNs are characterized by their inclusion of a PO central CpG-containing palindromic motif and a PS-modified 3' poly-G string. Exemplary CpG-B ODNs are characterized by their inclusion of a complete PS backbone with one or more CpG dinucleotides. Exemplary CpG-C ODNs are characterized by their combination of features from both class A and class B. [Figure 32] Individual species reactivity patterns to each CpG ODN (adapted from Rankin et al., 2001). a Plots were shaded if the mean proliferative response of a particular ODN and species was at least 50% of the value for the ODN that was maximally stimulatory for that species. DETAILED DESCRIPTION OF THE INVENTION

[0090] Detailed Description of the Invention In the following detailed description, references are made to the accompanying examples and drawings, which set forth, by way of illustration, specific details and embodiments that may be employed in practicing the invention. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments may be utilized, such that structural, logical, and optional changes may be made without departing from the scope of the invention. The various aspects of the invention described herein are not necessarily mutually exclusive, as aspects of the invention may be combined with one or more other aspects to form new embodiments of the invention.

[0091] The present invention relates to a method for inducing an immune response (e.g., an innate immune response) in a subject by the sole administration of one or more adjuvants associated with one or more polymersome populations. This essentially means that one or more adjuvants of the present invention can be individually encapsulated, for example, within one or more polymersome populations of the present invention, and the polymersomes can be administered to a subject, thereby inducing an immune response (e.g., an innate immune response). In the context of the present invention, the sole administration of one or more adjuvants can be characterized in that an antigen (e.g., an antigen capable of inducing an immune response, e.g., an antibody-mediated immune response) is not administered (e.g., co-administered) in combination with the one or more adjuvants associated with one or more polymersome populations. Thus, the sole administration can be characterized in that the antigens are not administered to the subject simultaneously (at the same time) or at different times. In a preferred embodiment, one or more adjuvants of the present invention are encapsulated in polymersomes of the present invention. In another preferred embodiment, the adjuvant of the present invention is selected from the group consisting of CpG oligodeoxynucleotides (i.e., CpG ODN, such as any suitable CpG ODN described herein), or bacterial and mycobacterial cell walls and proteins.

[0092] The present invention is based on the surprising discovery that two distinct populations of polymersomes, where one population of polymersomes is associated with only an antigen and a second population of polymersomes is associated with only an adjuvant, when administered together, improve the immune response to an antigen, thereby providing an immunization or therapeutic effect against, for example, an infectious disease or cancer. (See, e.g., Examples 7-9 or Example 19 herein, where Example 8 shows that administration of a first population of polymersomes encapsulating an antigen together with a second, separate population of polymersomes encapsulating CpG (an adjuvant) produces an immune response in mice that correlates with both tumor burden and T cell infiltration; Example 9 shows that administration of the immunogenic tumor neo-antigen Trp2 peptide encapsulated in a first population of polymersomes together with CpG oligonucleotides (an adjuvant) encapsulated in a second (separate) population produces a much stronger anti-tumor response compared to, for example, free Trp2 peptide; and Example 19 shows that the immune response to the spike protein of Sars-CoV-2 virus is strongest when the spike protein of Sars-CoV-2 is encapsulated in a first population of polymersomes and the CpG oligonucleotides (an adjuvant) are encapsulated in a second (separate) population.) The discovery that two distinct polymersome populations result in an improved immune response has the additional advantage of allowing the two polymersome populations to be produced separately / independently from one another. This, in turn, simplifies GMP production of, for example, each vaccine or therapeutic composition, because a first population of polymersomes, e.g., those comprising an antigen encapsulated in the polymersomes or conjugated to the surface of the polymersomes, can be produced under standardized GMP conditions, while a second population of polymersomes, e.g., those comprising an adjuvant encapsulated in the polymersomes or conjugated to the surface of the polymersomes, can also be produced under standardized conditions. These two populations can then be combined in the manufacturing process (to obtain a composition combining both polymersome populations for co-administration) or administered separately to a subject.Such a drug / vaccine manufacturing process is much easier to manage than, for example, encapsulating both an antigen and an adjuvant in the same polymersome population.

[0093] The antigen can be associated with the first population of polymersomes through any possible interaction between the antigen and the first population of polymersomes. For example, the antigen can be encapsulated within the first population of polymersomes, as described in co-pending PCT application PCT / EP2019 / 051853, filed January 25, 2019, the entire contents of which are incorporated herein by reference. Alternatively, the antigen can be incorporated into the surrounding membrane of a polymersome of the first population of polymersomes, as described in international application WO2014 / 077781. The antigen can also be covalently conjugated to the outer surface of the polymersomes of the first population of polymersomes, as described in co-pending European Patent Application No. 18193946.3, filed September 12, 2018, the entire contents of which are incorporated herein by reference.

[0094] Furthermore, antigens can be conjugated to the outer surface of the polymersomes of the first polymersome population via non-covalent bonds. Examples of such non-covalent bonds include electrostatic interactions, such as salt bridges, between positively and negatively charged residues present on the surface of the polymersome or antigen. For example, a salt bridge can be formed between a positively charged amino group (NH) and a negatively charged carboxylic acid group (COOH). Another example of such a non-covalent interaction between the first polymersome population and an antigen is a binding pair between streptavidin and biotin, avidin and biotin, streptavidin and a streptavidin-binding peptide, or avidin and an avidin-binding peptide. For example, polymersomes with biotin groups located on the polymersome surface can be prepared as described in Broz et al., "Cell targeting by a generic receptor-targeted polymer nanocontainer platform," Journal of Controlled Release. 2005;102(2):475-488, and can be reacted with antigens conjugated to streptavidin or avidin. Non-covalent biotin-streptavidin conjugates of polymersomes and antigens can also be prepared as described in Egli et al., "Functionalization of Block Copolymer Vesicle Surfaces Polymers," 2011, 3(1), 252-280. In this context, the term "antigen associated with a first population of polymersomes" as used herein does not mean that only one specific antigen is associated with a first population of polymersomes, but also encompasses the possibility that two or more antigens, e.g., two or more antigens, can be associated with a first population of polymersomes. As a specific example, for example, two or more immunogenic peptides can be associated with a first population of polymersomes of the present invention. It is also possible to associate one or more immunogenic peptides and the respective nucleic acid molecules encoding those peptides with a first population of polymersomes as used herein.As used herein, the term "antigen associated with a first population of polymersomes" also means that two or more first polymersome populations can be used, each carrying a different antigen. For example, two different antigenic peptides can be used, each of which can be associated with a separate first polymersome population of the invention.

[0095] The adjuvant can be associated with the second population of polymersomes through any possible interaction in the same way that an antigen can be associated with the first population of polymersomes. That is, the adjuvant can be encapsulated within the first population of polymersomes, as described in co-pending PCT application PCT / EP2019 / 051853, filed January 25, 2019, the entire contents of which are incorporated herein by reference. Alternatively, the adjuvant can be incorporated into the surrounding membrane of the polymersomes of the first population of polymersomes, as described in International Application WO2014 / 077781. Specific examples of adjuvants that are / can be incorporated into the surrounding membrane of polymersomes (of the second polymersome population) include synthetic monophosphoryl lipid A (see Cluff, "Monophosphoryl Lipid A (MPL) as an Adjuvant for Anti-Cancer Vaccines: Clinical Results," in Lipid A in Cancer Therapy, edited by Jean-Francois Jeannin (2009, Landes Bioscience and Springer)), polysorbate 80, alpha-DL-tocopherol, dioleoyl-3-trimethylammonium propane (DOTAP), and the cationic lipid 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM) (see Bernstein et al., "The Adjuvant CLDC Increases Protection of a Herpes Simplex Type 2 Glycoprotein D Vaccine in Guinea Pigs," Vaccine. May 2010). 7;28(21):3748-3753, or the synthetic amphiphile dimethyldioctadecylammonium (DDA) (see Smith Korsholm et al., "The adjuvant mechanism of cationic dimethyldioctadecylammonium liposomes," Immunology, 121, 216-226), are just a few examples.In this regard, it is clear that one or more adjuvants can be present in the polysomes of the second polymersome population referred to herein. For example, the second polymersome population can include an encapsulated adjuvant, such as a CpG oligonucleotide, and an adjuvant incorporated into the surrounding membrane of the polymersome, such as monophosphoryl lipid A or DOTAP (although in accordance with the disclosure above, the second polymersome population does not include an antigen, i.e., the second polymersome population does not contain any antigen).

[0096] In line with the above, it is of course possible to conjugate the adjuvant to the outer surface of the polymersomes of the first polymersome population via a covalent bond, as described in co-pending European Patent Application No. 18193946.3, filed September 12, 2018, the entire contents of which are incorporated herein by reference. Alternatively, the conjugation of the adjuvant to the outer surface of the polymersomes may be achieved via a non-covalent bond, such as a biotin-streptavidin interaction. It should be noted here that CpG oligonucleotides, such as the class B CpG oligodeoxynucleotide CpG ODN1826 (5'-tccatgacgttcctgacgtt-3', SEQ ID NO:18), are available in biotinylated form and can therefore be readily reacted with biotinylated polymersomes "modified" with streptavidin, as described in Broz et al., "Journal of Controlled Release. 2005," supra. It is also clear from this example that the second polymersome population can carry more than one adjuvant, for example, a CpG oligonucleotide covalently or non-covalently conjugated to the outer surface of the polymersome and an additional adjuvant, such as monophosphoryl lipid A or DOTAP, incorporated into the surrounding membrane of the polymersome. It is further clear that the same adjuvant can be associated with the second polymersome population in a different way, for example, a CpG oligonucleotide can be covalently or non-covalently conjugated to the outer surface of the polymersome at the same time as being encapsulated in the polymersome. This allows for greater amounts of adjuvant to be administered, if desired.

[0097] In line with the above disclosure, any type of first polymersome population can be used for administration with any type of second polymersome population, regardless of how the antigen and adjuvant are associated with the first and second polymersome populations. For example, a first population of polymersomes can have an antigen encapsulated within the polymersomes, and a second population of polymersomes can have an adjuvant encapsulated within the polymersomes. Alternatively, a first population of polymersomes can have an antigen covalently or noncovalently conjugated to the outer surface of the polymersomes, while a second population of polymersomes can have an adjuvant covalently or noncovalently conjugated to the outer surface of the polymersomes. As yet another purely illustrative example, a first population of polymersomes can have an antigen incorporated into the surrounding membrane of the polymersomes, and a second population of polymersomes can have an adjuvant incorporated into the surrounding membrane of the polymer. As a further specific example, a first population of polymersomes can have an antigen encapsulated within the polymersomes, while a second population of polymersomes can have (a) an adjuvant covalently or non-covalently conjugated to the outer surface of the polymersomes, or (b) an adjuvant incorporated into the surrounding membrane of the polymersomes. As yet another specific example, a first population of polymersomes can have an antigen covalently conjugated to the outer surface of the polymersomes, and a second population of polymersomes can have an adjuvant encapsulated within the polymersomes.

[0098]

[0013] Now, to elaborate on the administration of the two polymersome populations of the present invention, the first population of polymersomes and the second population of polymersomes can be administered simultaneously (i.e., at the same time) or at different times. When the two populations are administered simultaneously, the two polymersome populations can be administered together (i.e., by co-administration). In this case, the two polymersome populations are mixed or blended together prior to administration, so that they are present in the same composition, e.g., a pharmaceutically acceptable carrier (e.g., a physiological buffer or a solid formulation suitable for oral administration). However, when administered at the same time, it is also possible to administer each of the two polymersome populations separately. In this case, the two polymersome populations can, of course, not be mixed with each other prior to administration, e.g., administered by two or more separate injections.

[0099] The two polymersome populations are known to elicit an immune response in a subject and can be administered to a selected subject by any method suitable for administering the polymersome populations to a given subject. When immunizing fish or agricultural animals such as chickens, pigs, or sheep, oral administration can be used, for example, and it can be advantageous to formulate a composition containing the two polymersome populations of the present invention as a food additive. Alternatively, intradermal administration utilizing a syringe gun or jet injector can be used for agricultural animals. Invasive and non-invasive administration can be used for humans. Suitable routes of administration for both humans and non-human animals include, but are not limited to, oral administration, intranasal administration, administration to a mucosal surface, inhalation, intraperitoneal administration, subcutaneous administration, intravenous administration, or intramuscular administration.

[0100] To elaborate on the conjugation of antigens and / or adjuvants to the outer surface of polymersomes of either the first or second polymersome population, the covalent bond can be any suitable covalent bond capable of conjugating an antigen (e.g., an antigen of the present invention) or an adjuvant to the outer surface of a polymersome of the present invention. Conjugation reactions that result in the covalent bonds of the present invention are well known in the art (e.g., NHS-EDC conjugation, reductive amination conjugation, sulfhydryl conjugation, "click" and "photoclick" conjugation, pyrazoline conjugation, etc.). Non-limiting examples of such covalent bonds and methods for their generation are listed herein below. Thus, in some aspects, the covalent bond for conjugating the antigen or adjuvant of the invention to the outer surface of the polymersome of the invention comprises (i) an amide moiety (e.g., as described in the Examples section herein), and / or (ii) a secondary amine moiety (e.g., as described in the Examples section herein), and / or (iii) a 1,2,3-triazole moiety (e.g., as described in van Dongen et al., 2008, Macromol. Rapid Communications, 2008, 29, 321-325), preferably the 1,2,3-triazole moiety is a 1,4-disubstituted[1,2,3]triazole moiety or a 1,5-disubstituted[1,2,3]triazole moiety (e.g., as described in Boren et al., 2008), and / or (iv) a pyrazoline moiety (e.g., as described in de Hoog et al., Polym. Chem., 2012, 3, 302-306), and / or an ether moiety. In this regard, it should be noted that the formation of conjugation / covalent bonds between the outer surface of a polymersome and an antigen may require modification of both the polymersome and the antigen, e.g., a protein. In addition to the classical chemical conjugation chemistries (reactions) described above, it is also possible to form covalent bonds between the outer surface of a polymersome and an antigen by enzymatic reactions.

[0101] In some aspects, the present invention relates to NHS-EDC conjugation (i.e., conjugation based on N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)), which is one of the exemplary alternative methods for conjugating antigens to polymersomes of the present invention. In this method, a carboxylic acid group reacts with EDC to generate an intermediate O-acylisourea, which then reacts with a primary amine to form an amide moiety with the carboxyl group.

[0102] In some aspects, the present invention relates to reductive amination conjugation, another exemplary alternative method for conjugating antigens or adjuvants to the polymersomes of the present invention, in which an aldehyde-containing compound is conjugated to an amine-containing compound to form a Schiff base intermediate, which then undergoes reduction to form a stable secondary amine moiety.

[0103] In some aspects, the present invention relates to sulfhydryl conjugation, another exemplary alternative method for conjugating antigens or adjuvants to the polymersomes of the present invention, in which sulfhydryl (-SH)-containing compounds (e.g., those present in the side chain of cysteine) are conjugated to sulfhydryl-reactive chemical groups (e.g., maleimides) by alkylation or disulfide exchange to form thioether or disulfide bonds, respectively.

[0104] In some aspects, the present invention relates to another exemplary alternative method for conjugating antigens to the polymersomes of the present invention, the so-called "click" reaction (also known as "azide-alkyne cycloaddition") on the polymersome surface (e.g., as described in van Dongen et al., 2008, supra). In this method, an aqueous solution of an azide-functionalized antigen (e.g., a polypeptide) is added to a dispersion of polymersomes. A premixed aqueous solution of Cu(II)SO4·5H2O and sodium ascorbate and a bathophenanthroline ligand are then added to the resulting dispersion of polymersomes and allowed to stand at 4°C for 60 hours. The dispersion is then filtered through a 100 nm cutoff and centrifuged to dryness to generate the 1,2,3-triazole moiety. In this context, copper-catalyzed azide-alkyne cycloaddition (also known as CuAAC) specifically allows the synthesis of 1,4-disubstituted regioisomers, while ruthenium-catalyzed azide-alkyne cycloaddition (also known as RuAAC) (e.g., using Cp as catalyst) has been shown to be effective. * It should also be noted that the use of RuCl(PPh3)2 allows the production of 1,5-disubstituted triazoles (see R. Johansson, Johan & Beke-Somfai, Tamas & Said Stalsmeden, Anna & Kann, Nina. (2016). Ruthenium-Catalyzed Azide Alkyne Cycloaddition Reaction: Scope, Mechanism, and Applications. Chemical Reviews. 116.10.1021 / acs.chemrev.6b00466).

[0105] In some aspects, the present invention relates to another exemplary alternative method for conjugating antigens to the polymersomes of the present invention: the photoinduced generation of a nitrile imine intermediate (e.g., generated from a bisaryl-tetrazole) and its cycloaddition to an alkene (the so-called photoinduced cycloaddition or "photoclick" reaction, e.g., as described in de Hoog et al., 2011, supra). In this method, an ABA block copolymer is methacrylate (MA)- or hydroxyl-terminated with tetrazole via the photoinduced generation of a nitrile imine intermediate to produce ABA polymersomes containing MA-ABA copolymers and hydroxyl-terminated ABA copolymers, which are then reacted with a tetrazole-containing antigen (HRP) under UV irradiation to generate a pyrazoline moiety.

[0106] The covalent bond conjugating the antigen or adjuvant to the outer surface of the polymersome can be formed between an atom / group of a molecule, such as an amphiphilic polymer, that is part of (present in) the surrounding membrane of the polymersome. Alternatively, the covalent bond between the antigen or antigen and the outer surface of the polymer is formed via a linker moiety attached to a molecule that is part of (present in) the surrounding membrane of the polymersome. The linker may have any suitable length and can have a length of one main chain atom (e.g., when the linker is a simple carbonyl group (C=O) that results in an amide or ester moiety that forms the covalent bond). Specific examples of such "one atom / linker moiety" having a length of one main atom are BD 21In the Examples, modification of the amphiphilic polymer BD21 with Dess-Martin periodinane to obtain -CHO (i.e., a terminal aldehyde group) is performed, which is then used to form an amine bond with a selected antigen (hemagglutinin is used as an example of an antigen in the Experimental Section for purely illustrative purposes). Alternatively, the linker moiety can have a length of several hundred or more main chain atoms, such as a polyethylene glycol (PEG) moiety, which is commonly used for conjugation (covalent coupling) of polypeptides to molecules of interest. As a purely illustrative example, see the distearoylphosphatidylethanolamine [DSPE] polyethylene glycol (DSPE-PEG) conjugate described below and used in the Examples. The DSPE-PEG(3000) linker moiety used in the Examples has approximately 65 ethylene oxide (CH2-CH2-O) subunits, thus the PEG moiety alone has approximately 325 main chain atoms, for a total length of approximately 408 main chain atoms. In line with the above exemplary embodiments, the linker moiety may comprise 1 to about 550 main chain atoms, 1 to about 500 main chain atoms, 1 to about 450 main chain atoms, 1 to about 350 main chain atoms, 1 to about 300 main chain atoms, 1 to about 250 main chain atoms, 1 to about 200 main chain atoms, 1 to about 150 main chain atoms, 1 to about 100 main chain atoms, 1 to about 50 main chain atoms, 1 to about 30 main chain atoms, 1 to about 20 main chain atoms, 1 to about 15 main chain atoms, or 1 to about 12 main chain atoms, or 1 to about 10 main chain atoms, where a main chain atom is a carbon atom optionally replaced with one or more heteroatoms selected from the group consisting of N, O, P, and S.

[0107] According to the above disclosure, the linker moiety can be a peptide linker or a linear or branched hydrocarbon-based linker. The linker moiety may also be a copolymer with different block lengths. The linker moiety used in the present invention can include a membrane anchoring domain that integrates the linker moiety into the membrane of the polymersome. Such a membrane anchoring domain can include a lipid such as a phospholipid or glycolipid. The glycolipid used in the membrane anchoring domain can include glycophosphatidylinositol (GPI), a widely used membrane anchoring domain (see, for example, International Patent Applications WO2009 / 127537 and WO2014 / 057128). The phospholipid used in the linker of the present invention can be a sphingophospholipid or a glycerophospholipid. In a specific example of such a linker, the sphingophospholipid can include distearoylphosphatidylethanolamine [DSPE] conjugated to polyethylene glycol (PEG) (DSPE-PEG) as the membrane anchoring domain. In such conjugates, DSPE-PEG can contain any suitable number of ethylene oxide units, for example, from 2 to about 500. Specific examples include DSPE-PEG(1000), DSPE-PEG(2000), or DSPE-PEG(3000), but these are just a few examples. Alternatively, the phospholipid (sphingolipid or glycerophospholipid) can contain cholesterol as a membrane anchor domain. Cholesterol-based membrane anchor domains are described, for example, in Achalkumar et al., "Cholesterol-based anchors and tethers for phospholipid bilayers and for model biological membranes," Soft Matter, 2010, 6, 6036-6051.In exemplary embodiments, the linker portion of such a membrane anchor domain may comprise from 1 to about 550 main chain atoms, from 1 to about 500 main chain atoms, from 1 to about 450 main chain atoms, from 1 to about 350 main chain atoms, from 1 to about 300 main chain atoms, from 1 to about 250 main chain atoms, from 1 to about 200 main chain atoms, from 1 to about 150 main chain atoms, from 1 to about 100 main chain atoms, from 1 to about 50 main chain atoms, from 1 to about 30 main chain atoms, from 1 to about 20 main chain atoms, from 1 to about 15 main chain atoms, or from 1 to about 12 main chain atoms, or from 1 to about 10 main chain atoms, wherein a main chain atom is a carbon atom optionally replaced with one or more heteroatoms selected from the group consisting of N, O, P, and S.

[0108] Any type of polymersome can be used in the present invention, as long as it can function as a carrier for an associated antigen or adjuvant. The polymersome can be, for example, an oxidation-sensitive polymersome, as described in Stano et al., "Tunable T cell immunity toward a protein antigen using polymersomes vs. solid-core nanoparticles," Biomaterials 34 (2013): 4339-4346, or U.S. Patent No. 8,323,696 to Hubbel. Alternatively, the polymersome may be oxidation-insensitive. Regardless of chemical stability (including the possibility of oxidation sensitivity or insensitivity), in the present invention, a polymersome is a vesicle with a polymer membrane, typically formed by self-assembly of a dilute solution of one or more amphiphilic block copolymers, which can be of various types, such as diblock and triblock (ABA or ABC), but not necessarily. The polymersome of the present invention can also be formed from tetrablock or pentablock copolymers. While triblock copolymers often shield the central block from the environment by its neighboring blocks, diblock copolymers achieve a similar effect by self-assembling into bilayers and arranging the two hydrophobic blocks in a tail-to-tail configuration. In most cases, vesicle membranes have an insoluble middle layer and a soluble outer layer. The driving force behind self-assembly of polymersome formation is believed to be microphase separation of the insoluble blocks, which tend to associate to shield themselves from contact with water. The polymersomes of the present invention have remarkable properties due to the large molecular weight of the constituent copolymers. Vesicle formation is favored as the total molecular weight of the block copolymers increases. As a result, the diffusion of (polymeric) amphiphiles is significantly reduced compared to vesicles formed by lipids and surfactants. The reduced mobility of polymer chains aggregated into vesicle structures allows for the formation of stable polymersome morphologies.Unless otherwise specified, the terms "polymersome" and "vesicle" used herein are construed as being similar and may be used interchangeably. Importantly, the polymersomes of the present invention can be formed from one type of block copolymer or from two or more types of block copolymers. That is, polymersomes can also be formed from a mixture of polymersomes and thus can contain two or more types of block copolymers. In some aspects, the polymersomes of the present invention are oxidatively stable.

[0109] In some aspects, the present invention relates to a method for eliciting an immune response in a subject against a soluble (e.g., solubilized) encapsulated antigen. The method is suitable for injecting a composition comprising a polymersome (e.g., a carrier or vehicle) having a membrane (e.g., a surrounding membrane) of an amphiphilic polymer into the subject. The composition comprises a soluble (e.g., solubilized) antigen encapsulated by the membrane (e.g., the surrounding membrane) of the amphiphilic polymer of the polymersome of the present invention. The antigen can be one or more of the following: (i) a polypeptide, (ii) a carbohydrate, (iii) a polynucleotide (e.g., the polynucleotide is not an antisense oligonucleotide, and preferably the polynucleotide is a DNA molecule or a messenger RNA (mRNA) molecule), or a combination of (i) and / or (ii) and / or (iii).

[0110] In some further aspects, the present invention relates to polymersomes capable of eliciting a CD8(+) T cell-mediated immune response.

[0111] In some aspects, the present invention relates to polymersomes capable of targeting lymph node-resident macrophages and / or B cells. Exemplary, non-limiting targeting mechanisms envisioned by the present invention include (i) delivery of encapsulated antigens (e.g., polypeptides) to dendritic cells (DCs) for T cell activation (CD4 and / or CD8). Another mechanism is (ii) delivery of whole, folded antigens (e.g., proteins) that will find their way to DCs and also trigger titers (B cells).

[0112] In some aspects, the present invention relates to polymersomes encapsulating an antigen selected from the group consisting of (i) an autoantigen, (ii) a non-autoantigen, (iii) a non-autoimmunogen, and (iv) an autoimmunogen. Thus, the products and methods of the present invention are suitable for use in the context of induced tolerance (e.g., in clinical settings), for example, when targeting autoimmune diseases.

[0113] In some aspects, the present invention relates to polymersomes of the present invention comprising lipid polymers.

[0114] The polymersomes of the present invention may also co-encapsulate (i.e., encapsulate in addition to the antigen) one or more adjuvants, including synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs that can trigger cells expressing Toll-like receptor 9 (including human plasmacytoid dendritic cells and B cells) to mount an innate immune response characterized by the production of Th1 and proinflammatory cytokines, cytokines such as interleukin-1, interleukin-2, or interleukin-12, keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, to name just a few.

[0115] The polymersomes of the present invention can be of any size as long as they are capable of eliciting an immune response. For example, the polymersomes may have a diameter greater than 70 nm. The polymersome diameter may range from about 100 nm to about 1 μm, or from about 100 nm to about 750 nm, or from about 100 nm to about 500 nm. The polymersome diameter may further range from about 125 nm to about 175 nm, or from about 125 nm to about 250 nm, or from about 140 nm to about 240 nm, or from about 150 nm to about 235 nm, or from about 170 nm to about 230 nm, or from about 220 nm to about 180 nm, or from about 190 nm to about 210 nm. The polymersome diameter may be, for example, about 200 nm, about 205 nm, or about 210 nm. When used as (first and second) populations to elicit an immune response, the polymersome populations are typically monodisperse. The average diameter of the polymersome population used is typically greater than 70 nm, or greater than 120 nm, or greater than 125 nm, or greater than 130 nm, or greater than 140 nm, or greater than 150 nm, or greater than 160 nm, or greater than 170 nm, or greater than 180 nm, or greater than 190 nm (see also FIG. 2 in this regard). The average diameter of the polymersome population may be, for example, within the ranges of the individual polymersomes described above. That is, the average diameter of the polymersome population may be in the range of 100 nm to about 1 μm, or in the range of about 100 nm to about 750 nm, or in the range of about 100 nm to about 500 nm, or in the range of about 125 nm to about 250 nm, about 140 nm to about 240 nm, about 150 nm to about 235 nm, about 170 nm to about 230 nm, or about 220 nm to about 180 nm, or about 190 nm to about 210 nm. The average diameter of a population of polymersomes can be, for example, about 200 nm, about 205 nm, or about 210 nm. The diameter can be determined, for example, by a dynamic light scattering (DLS) instrument using the preferred DLS parameter Z-average (d, nm). Z-average size is the intensity weighted harmonic mean particle diameter (see Examples 1 and 2).In this regard, it should be noted that U.S. Patent No. 8,323,696 to Hubbel et al. requires that polymersome aggregates / clusters have an average diameter of less than 70 nm to be able to induce an immune response. Similarly, Stano et al., 2013, supra, while hoping to use smaller polymersomes, technical limitations led them to use polymersomes with a diameter of 125 nm ± 15 nm to induce an immune response. Thus, it is surprising that polymersome aggregates / clusters of the present invention, for example, with an average diameter greater than 150 nm, are able to induce both cellular and humoral immune responses (see "Examples"). Such polymersome aggregates may be in a form suitable for inducing an immune response, for example, by injection or oral administration.

[0116] In some aspects, the present invention relates to compositions of the present invention suitable for intradermal, intraperitoneal, subcutaneous, intravenous, or intramuscular injection, or non-invasive administration of antigens of the present invention, such as oral administration, inhalation administration, or nasal administration. The compositions may comprise polymersomes (e.g., carriers) of the present invention having a membrane (e.g., a surrounding membrane) of an amphiphilic polymer. The compositions may further comprise a soluble (e.g., solubilized) antigen encapsulated by the amphiphilic polymer membrane of the polymersome. The compositions of the present invention may be used for therapeutic purposes (e.g., treating a diseased subject or preventing disease, such as by vaccination), or for antibody discovery, vaccine discovery, or targeted delivery.

[0117] In some aspects, the polymersomes of the invention have hydroxyl groups on their surface. In some further aspects, the polymersomes of the invention do not have hydroxyl groups on their surface.

[0118] In the context of this application, the term "encapsulated" means surrounded by a membrane (e.g., the membrane of a polymersome of the invention) (e.g., contained within the lumen of the polymersome). The term "encapsulated" with respect to an antigen further means that the antigen is not incorporated into, covalently attached to, or conjugated to a membrane (e.g., the membrane of a polymersome of the invention). With respect to the compartmentalization of the polymersome vesicle structures described herein, the term "encapsulated" means that the inner vesicle is completely contained within the outer vesicle and surrounded by the vesicle membrane of the outer vesicle. The closed space surrounded by the vesicle membrane of the outer vesicle forms one compartment. The closed space surrounded by the vesicle membrane of the inner vesicle forms another compartment.

[0119] In the context of this application, the term "single administration" may refer to a type of administration characterized in that the antigen is not administered in combination (e.g., simultaneously or at different times (e.g., before and / or after) (e.g., co-administered)) with one or more adjuvants of the invention. In a preferred embodiment, the single administration of one or more adjuvants of the invention elicits a natural immune response including, for example, the production and / or secretion of interleukin-6 (IL-6).

[0120] In the context of this application, the term "adjuvant" as used herein can refer to a compound or substance that can induce, enhance, or improve an immune response with or without an antigen. Adjuvants of the present invention further include immunostimulants that can induce an immune response (e.g., an innate immune response) without an antigen. Exemplary adjuvants of the present invention include, but are not limited to, compounds or substances that can induce, enhance, or improve an immune response in the presence of an antigen.

[0121] For purposes of this application, the term "innate immune response" as used herein may refer to an immune response characterized by, by way of example only, the production of cytokines such as Th1 cytokines and pro-inflammatory cytokines, interleukin-1, interleukin-2, interleukin-6 or interleukin-12, keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor.

[0122] For purposes of this application, the term "antigen" refers to any substance that can be specifically bound by components of the immune system. Only antigens that are capable of eliciting (or eliciting or inducing) an immune response are considered immunogenic and are called "immunogens." Exemplary, non-limiting antigens are polypeptides derived from the soluble portion of a protein, hydrophobic polypeptides that have been made soluble for inclusion, and aggregated polypeptides that are soluble as aggregates. Antigens can originate within the body ("self-antigens") or from the external environment ("non-self").

[0123] Membrane proteins form a class of antigens that typically generate low-level immune responses. Of particular interest, soluble (e.g., solubilized) membrane proteins (MPs) and membrane-associated peptides (MAPs) and fragments (i.e., portions) thereof (e.g., antigens referred to herein) can be encapsulated by polymersomes, which allows them to fold in a physiologically meaningful manner. This strongly boosts the immunogenicity of such antigens, so that a smaller amount of the corresponding antigen can be used to generate the same level of immune response compared to the free antigen. Furthermore, the larger size of polymersomes (compared to free membrane proteins) allows them to be more easily detected by the immune system.

[0124] For purposes of this application, the term "B16 peptide" refers to any neoantigen polypeptide derived from a spontaneous C57BL / 6-derived B16 melanoma model (e.g., the B16-F10 melanoma mouse model), including, but not limited to, peptides of SEQ ID NOs:9, 10, and 11.

[0125] For purposes of this application, the term "MC38 peptide" refers to any neoantigenic polypeptide derived from the MC38 mouse model of colon cancer, including, but not limited to, peptides of SEQ ID NOs: 1, 2, and 3.

[0126] For purposes of this application, the term "influenza hemagglutinin (HA)" refers to a glycoprotein found on the surface of influenza viruses. HA has at least 18 different antigens, all of which are within the scope of the present invention. These subtypes are designated H1 through H18. Non-limiting examples of "influenza hemagglutinin (HA)" subtype H1 include polypeptides of SEQ ID NOs: 5, 6, 7, and 8.

[0127] As used herein, the term "swine influenza hemagglutinin (HA)" refers to a glycoprotein found on the surface of swine influenza viruses, a family of influenza viruses unique to pigs. A non-limiting example of a "swine influenza hemagglutinin (HA)" is subtype H1 of SEQ ID NO:6.

[0128] For purposes of this application, the term "coronavirus" refers to viruses in the subfamily of the Coronaviridae family, a family of enveloped, positive-sense, single-stranded RNA viruses. Coronaviruses can cause disease in mammals and birds. This subfamily includes four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. In humans, coronaviruses can cause respiratory tract infections, which can be mild or fatal, such as SARS, MERS, and COVID-19. Human pathogenic coronaviruses generally belong to the Alphacoronavirus or Betacoronavirus genera. Examples of viruses in the Alphacoronavirus genus include PEDv, transmissible gastroenteritis virus (TGEV), feline coronavirus (FCoV), including feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV), canine coronavirus (CCoV), or the human pathogenic coronaviruses human coronavirus 229E (HCoV-229E) and human coronavirus NL63 (HCoV-NL63). Within the genus Betacoronavirus, the subgenera Sarbecovirus and Merbecovirus are most relevant to the present disclosure and include the species SARS-CoV-1, SARS-CoV-2, and MERS-CoV. Other human pathogenic Betacoronaviruses are human coronavirus OC43 (HCoV-OC43) and human coronavirus HKU1 (HCoV-HKU1). Human pathogenic coronaviruses are reviewed in Corman VM, Muth D, Niemeyer D, Drosten C., Hosts and Sources of Endemic Human Coronaviruses. Adv Virus Res. 2018;100:163-188.

[0129] For the purposes of this application, the term "spike protein" refers to a glycoprotein present on the surface of the viral capsid or viral envelope. The spike protein binds to a specific receptor on the host cell and is therefore important for both host specificity and viral infectivity.

[0130] For purposes of this application, the term "PEDv S protein" refers to the spike glycoprotein present on the surface of the porcine epidemic diarrhea virus (PEDV), a member of the swine coronavirus family. Non-limiting examples of soluble "PEDv S protein" that may be used herein include the entire soluble fragment of the porcine epidemic diarrhea virus (PEDv) spike protein (S protein) (UniProtKB Accession No. V5TA78) consisting of the S1 and S2 regions having the amino acid sequence of SEQ ID NO:12, a soluble fragment of the S1 region of SEQ ID NO:13, or a soluble fragment of the S2 region of SEQ ID NO:14. It is, of course, also possible to use a soluble fragment of the entire S1 and S2 regions, or shorter fragments of only either the S1 or S2 regions (see FIG. 12 in this regard). It is, of course, also possible to use a fragment containing a portion of S1 and a portion of S2, e.g., amino acids 500-939 of the deposited sequence of the spike protein, in the polymersomes of the present invention. It should also be noted that the polymersomes of the present invention may encapsulate one or more different soluble fragments of the spike protein, such as the S1 region, the S2 region, and / or the entire S1 and S2 regions. In exemplary embodiments of the polymersomes of the present invention, the polymersomes encapsulate one type of soluble fragment (e.g., the S1 region only), two different types of soluble fragments (e.g., the S1 and S2 regions), three different types of soluble fragments (the S1 region, the S2 region, and the entire soluble fragment of S1 and S2 of SEQ ID NO:12 (amino acid residues 19-1327)), or even four different types of fragments (e.g., the S1 region, the S2 region, and the entire soluble fragment of S1 and S2 of SEQ ID NO:12 (amino acid residues 19-1327), and a fourth type of fragment as described above containing a portion of S1 and a portion of S2, e.g., amino acids 500-939 of the spike protein sequence).It should also be noted here that the polymersomes of the present invention encapsulating one or more different soluble fragments of the spike protein are used in a preferred embodiment as an oral vaccine against porcine epidemic diarrhea virus.

[0131] For purposes of this application, the term "MERS-CoV S protein" or "MERS-CoV spike protein" refers to the spike glycoprotein present on the surface of the human pathogenic coronavirus, Middle East Respiratory Syndrome-associated coronavirus (MERS-CoV). The MERS-CoV spike protein of the present disclosure has the sequence set forth in UniProtKB Accession No.: K0BRG7 version 40 as of February 26, 2020 (GenBank Accession No. AFS88936, version AFS88936.1) or SEQ ID NO: 42. Non-limiting examples of soluble "MERS-CoV S proteins" that can be used in the present invention include the entire soluble fragment of the S1 and S2 regions of the MERS-CoV spike protein (S protein), which can correspond to amino acids 1-1297 of the MERS-CoV spike protein or have the amino acid sequence set forth in SEQ ID NO: 43. Non-limiting examples of soluble "MERS-CoV S proteins" that can be used in the present invention include the S1 region, which corresponds to amino acids 18-725 of the MERS-CoV spike protein (S protein) or has the amino acid sequence of SEQ ID NO:44. Non-limiting examples of soluble "MERS-CoV S proteins" that can be used in the present invention also include a soluble fragment of the S2 region, which can correspond to amino acids 726-1296 of the MERS-CoV spike protein (S protein) or has the amino acid sequence of SEQ ID NO:45. It is, of course, also possible to use shorter fragments of the entire S1 and S2 regions, or of either the S1 or S2 region alone; for example, a fragment can include the receptor binding domain (RBD), which corresponds to amino acids 377-588 of the MERS-CoV spike protein or has the amino acid sequence of SEQ ID NO:46. It is also noted herein that the polymersomes of the present invention may encapsulate one or more different soluble fragments of the spike protein, such as the S1 region, the S2 region, or soluble fragments thereof, the entire soluble fragments of the S1 and S2 regions, and / or the RBD.In exemplary embodiments of the polymersomes of the invention, the polymersomes encapsulate therein one type of soluble fragment (e.g., only the entire soluble fragment of the S1 and S2 regions), two different types of soluble fragments (e.g., the entire soluble fragment of the S1 and S2 regions and a soluble fragment of the entire S1 region or the S2 region), three different types of soluble fragments (a soluble fragment of the S1 region, a soluble fragment of the S2 region, and the entire soluble fragment of S1 and S2 of SEQ ID NO:42 (amino acid residues 1-1297)), or even four different types of fragments (e.g., a soluble fragment of the S1 region, a soluble fragment of the S2 region, the entire soluble fragment of S1 and S2 of SEQ ID NO:42 (amino acid residues 1-1297), and a fourth type, the RBD). In a preferred embodiment, the polymersomes of the invention encapsulate therein a soluble fragment comprising, consisting essentially of, or consisting of the S1 region corresponding to amino acid residues 18-725 of the full-length MERS-CoV spike protein. In a preferred embodiment, the polymersomes of the invention have encapsulated therein a soluble fragment comprising, consisting essentially of, or consisting of a soluble fragment of the S2 region corresponding to amino acid residues 726-1296 of the full-length MERS-CoV spike protein. In a preferred embodiment, the polymersomes of the invention have encapsulated therein a soluble fragment comprising, consisting essentially of, or consisting of the S1 and S2 regions corresponding to amino acid residues 1-1297 of the full-length MERS-CoV spike protein. In a preferred embodiment, the polymersomes of the invention have encapsulated therein a fragment comprising, consisting essentially of, or consisting of the S1 and S2 regions corresponding to amino acid residues 1-1327 of the full-length MERS-CoV spike protein.In this context, "consisting essentially of" means that the N- and / or C-terminal ends of the fragment may vary within a limited range, e.g., by up to 25 amino acid positions, e.g., by up to 20 amino acid positions, e.g., by up to 15 amino acid positions, by up to 10 amino acid positions, by up to 5 amino acid positions, by up to 4 amino acid positions, by up to 3 amino acid positions, by up to 2 amino acid positions, or by up to 1 amino acid position. In one specific example, a fragment consisting essentially of amino acids 726-1296 of the full-length MERS-CoV spike protein may consist of amino acids 716-1296, 736-1296, 726-1286, or 726-1306, 716-1286, 736-1286, 736-1306, or 716-1306 of the full-length MERS-CoV spike protein.

[0132] The MERS-CoV spike protein of the present disclosure may also include variants of the above-described sequences. These variants include natural variants from other isolates of MERS-CoV as well as artificial modifications that can be introduced into the sequence of the MERS-CoV S protein. In one specific example, mutations can be introduced to alter the formation of the expressed protein. To this end, the furin cleavage site located at positions 754-757 of SEQ ID NO:42 can be mutated. Reduced post-expression cleavage can be achieved by reducing the basicity of this amino acid sequence. For example, residues arginine 754 and / or 757 can be mutated to a less basic amino acid, such as glycine (position numbers correspond to the amino acid sequence shown in SEQ ID NO:42) or another less basic amino acid. Thus, a furin cleavage site having a native sequence of RSVR (SEQ ID NO:58) can be mutated to GSVG (SEQ ID NO:59). Further modifications can include the addition of a trimerization domain, preferably to the C-terminus of the protein, which can help increase the native fold of the S1 and / or S2 domains. Such trimerization domains can include a Foldon domain (e.g., SEQ ID NO:54), a GCN4-based trimerization domain (e.g., SEQ ID NO:55 or SEQ ID NO:56), or other motifs known to those of skill in the art. Additionally, secretory leader sequences can be added to the N-terminus of the protein, which can improve production and / or subsequent processing, such as isolation and purification. One example of such a leader sequence is the honeybee melittin leader sequence (SEQ ID NO:57). Other useful leader sequences are known to those of skill in the art. Thus, the soluble fragments of spike proteins disclosed herein also include highly identical variants of the specific sequences of soluble fragments of spike proteins explicitly or implicitly disclosed herein.For example, variants having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a soluble fragment of the spike protein of the present disclosure, particularly a soluble fragment of the MERS-CoV S protein of the present disclosure. In one specific example, a soluble fragment of the S fragment of the present disclosure can comprise, consist essentially of, or consist of a sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a sequence selected from the group consisting of SEQ ID NOs:43-46.

[0133] Alternatively or additionally, the polymersomes of the present disclosure may encapsulate one or more nucleic acids, such as mRNA, self-amplifying mRNA, or DNA encoding one or more MERS-CoV spike proteins or soluble fragments thereof of the present disclosure.

[0134] It is also noted herein that in a preferred embodiment, the polymersomes of the present invention encapsulating one or more different soluble fragments of the MERS-CoV spike protein and / or nucleic acids encoding same or the full-length MERS-CoV spike protein are used as vaccines against human diseases, in particular infections caused by human pathogenic coronaviruses, in particular Middle East Respiratory Syndrome (MERS). Accordingly, the polymersomes of the present invention encapsulating one or more different soluble fragments of the MERS-CoV spike protein and / or nucleic acids encoding same or the full-length MERS-CoV spike protein may be used to treat (including prevent) fever, cough, sputum, shortness of breath, pneumonia, and / or acute respiratory distress syndrome (ARDS).

[0135] In a preferred embodiment, the polymersomes of the present invention encapsulating one or more different soluble fragments of the MERS-CoV spike protein and / or nucleic acids encoding same or the full-length MERS-CoV spike protein are administered intramuscularly. In a preferred embodiment, the polymersomes of the present invention encapsulating one or more different soluble fragments of the MERS-CoV spike protein and / or nucleic acids encoding same or the full-length MERS-CoV spike protein are administered intranasally. In a preferred embodiment, the polymersomes of the present invention encapsulating one or more different soluble fragments of the MERS-CoV spike protein and / or nucleic acids encoding same or the full-length MERS-CoV spike protein are administered by inhalation.

[0136] For purposes of this application, the term "SARS-CoV-2 S protein" or "SARS-CoV-2 spike protein" refers to the spike glycoprotein present on the surface of the human pathogenic coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 spike protein of the present disclosure has the sequence set forth in UniProtKB Accession No. P0DTC2 Version 1 on April 22, 2020 (GenBank Accession No. MN908947, Version MN908947.3) or SEQ ID NO: 19. Non-limiting examples of soluble "SARS-CoV-2 S proteins" that can be used in the present invention include the entire soluble fragment consisting of the S1 and S2 regions of the SARS-CoV-2 spike protein (S protein), corresponding to amino acids 16-1213, 14-1204, or 19-1204 of the SARS-CoV-2 spike protein, or having the amino acid sequence set forth in SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:65. Non-limiting examples of soluble "SARS-CoV-2 S proteins" that can be used in the present invention also include the S1 region, corresponding to amino acids 16-685 of the SARS-CoV-2 spike protein (S protein), or having the amino acid sequence of SEQ ID NO:37. Non-limiting examples of soluble "SARS-CoV-2 S proteins" that can be used in the present invention also include the S2 region corresponding to amino acids 686-1213 or 646-1204 of the SARS-CoV-2 spike protein (S protein), or having the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. It is of course also possible to use shorter fragments of the entire S1 and S2 regions, or of either the S1 or S2 region alone, such as the amino acid sequence from 318 to 524 of the SARS-CoV-2 protein (SEQ ID NO: 41; see Figure 23A in this regard) as the receptor-binding domain.In one embodiment, a shorter fragment of the S2 region comprises, consists essentially of, or consists of amino acids corresponding to positions 686-1204 of SEQ ID NO:19. In one embodiment, a soluble fragment of the spike protein comprises, consists essentially of, or consists of amino acids corresponding to positions 646-1204 of SEQ ID NO:19. In one embodiment, a soluble fragment of the spike protein comprises, consists essentially of, or consists of the sequence set forth in any of SEQ ID NOs:34-36 and SEQ ID NO:65. It should also be noted herein that the polymersomes of the present invention may encapsulate one or more different soluble fragments of the spike protein, such as the S1 region or fragments thereof, the S2 region or fragments thereof, and / or fragments thereof comprising the entire S1 and S2 regions or portions of the S1 and S2 regions. In exemplary embodiments of the polymersomes of the present invention, the polymersomes of the present invention encapsulate within them one type of soluble fragment (e.g., the S1 region alone or a fragment thereof), two different types of soluble fragments (e.g., the S1 and S2 regions or fragments of the S1 and / or S2 regions), three different types of soluble fragments (the S1 region or a fragment thereof, the S2 region or a fragment thereof, and the entire soluble fragment of S1 and S2 of SEQ ID NO:19), or four different types of fragments (e.g., the S1 region or a fragment thereof, the S2 region or a fragment thereof, the entire soluble fragment of S1 and S2 of SEQ ID NO:19 or a fragment thereof comprising a portion of the S1 region and a portion of the S2 region, and a fourth type of fragment as described above containing a portion of S1 and a portion of S2, e.g., amino acids 14-1204 of the spike protein sequence).

[0137] Variants of the SARS-CoV-2 S protein may be identified, for example, by GeneBank Accession No. QII57278.1 (SEQ ID NO:20), GeneBank Accession No. YP_009724390.1 (SEQ ID NO:21), GeneBank Accession No. QIO04367.1 (SEQ ID NO:22), GeneBank Accession No. QHU79173.2 (SEQ ID NO:23), GeneBank Accession No. QII87830.1 (SEQ ID NO:24), GeneBank Accession No. QIA98583.1 (SEQ ID NO:25), GeneBank Accession No. QIA20044.1 (SEQ ID NO:26), GeneBank Accession No. QIK50427.1 (SEQ ID NO:27), GeneBank Accession No. QHR84449.1 (SEQ ID NO:28), Several variants are known in the art, including those under GeneBank Accession No. QIQ08810.1 (SEQ ID NO: 28), GeneBank Accession No. QIJ96493.1 (SEQ ID NO: 30), GeneBank Accession No. QIC53204.1 (SEQ ID NO: 31), GeneBank Accession No. QHZ00379.1 (SEQ ID NO: 32), and GeneBank Accession No. QHS34546.1 (SEQ ID NO: 33). Compared to SEQ ID NO: 19, these variants contain mutations at sequence positions 28, 49, 74, 145, 157, 181, 221, 307, 408, 528, 614, 655, 797, and 930. Further modifications can be introduced into the sequence of the SARS-CoV-2 S protein. In one embodiment, mutations can be introduced to alter the formation of the expressed protein. To this end, the furin cleavage site located at positions 679-685 of SEQ ID NO:19 can be mutated. Reduced post-expression cleavage can be achieved by reducing the basicity of this amino acid sequence.For example, residues Pro681, Arg682, and / or Arg683 can be mutated to less basic amino acids, e.g., Pro681 → Asn, Arg682 → Gln, and / or Arg683 → Ser (position numbers correspond to the amino acid sequence shown in SEQ ID NO:19), or other less basic amino acids. Thus, a furin cleavage site with the native sequence NSPRRAR (SEQ ID NO:52) can be mutated to the sequence NSNQSAR (SEQ ID NO:53). A specific example of a soluble fragment of the SARS-CoV-2 spike protein with a mutant furin cleavage site is shown in SEQ ID NO:65. A specific example of a SARS-CoV-2 spike protein with a mutant furin cleavage site is shown in SEQ ID NO:66. Further modifications can include adding a trimerization domain, preferably to the C-terminus of the protein, which can help increase the native fold of the S1 and / or S2 domains. Such trimerization domains include the Foldon domain (e.g., as described in Guethe et al., J. Mol. Biol. (2004) 337, 905-915). TIFF2024505964000001.tif4128), including GCN4-based trimerization domains and immunosilenced variants thereof (e.g., as described in Sliepen et al. J. Biol. Chem. (2015) 290(12):7436-7442). TIFF2024505964000002.tif11162), or other motifs known to those skilled in the art. Additionally, secretory leader sequences may be added to the N-terminus of the protein, which may improve production and / or subsequent processing, such as isolation and purification. One specific example of such a leader sequence is the honeybee melittin leader sequence. TIFF2024505964000003.tif4128. Other useful leader sequences are known to those skilled in the art. Thus, soluble fragments of spike proteins of the present disclosure also include highly identical variants of the specific sequences of soluble fragments of spike proteins explicitly or implicitly disclosed herein. For example, variants having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to soluble fragments of spike proteins of the present disclosure, particularly to soluble fragments of the SARS-CoV-2 S protein of the present disclosure. In one embodiment, a soluble fragment of the S fragment of the present disclosure can comprise, consist essentially of, or consist of a sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a sequence selected from the group consisting of positions 16-1213, 16-685, 686-1213, 686-1204, 646-1204, or 14-1204 of SEQ ID NO: 19 (SARS-CoV-2 spike protein). In another embodiment, a soluble fragment of the S fragment of the present disclosure can have a sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a sequence selected from the group consisting of positions 16-1213, 16-685, 686-1213, 686-1204, 646-1204, or 14-1204 of SEQ ID NO: 19 (SARS-CoV-2 spike protein).

[0138] In a preferred embodiment, a polymersome of the invention has encapsulated therein a soluble fragment comprising, consisting essentially of, or consisting of an S1 region corresponding to amino acid residues 16-685 of the full-length SARS-CoV-2 spike protein as set forth in SEQ ID NO: 19, or having the amino acid sequence of SEQ ID NO: 37. In a preferred embodiment, a polymersome of the invention has encapsulated therein a soluble fragment comprising, consisting essentially of, or consisting of an S2 region corresponding to amino acid residues 686-1213 of the full-length SARS-CoV-2 spike protein as set forth in SEQ ID NO: 19, or having the amino acid sequence of SEQ ID NO: 38. In a preferred embodiment, the polymersomes of the invention have encapsulated therein a soluble fragment comprising, consisting essentially of, or consisting of the S1 and S2 regions corresponding to amino acid residues 16-1213 of the full-length SARS-CoV-2 spike protein shown in SEQ ID NO:19, or having the amino acid sequence of SEQ ID NO:34. In a preferred embodiment, the polymersomes of the invention have encapsulated therein a soluble fragment comprising, consisting essentially of, or consisting of the amino acid sequence corresponding to amino acid residues 686-1204 of the full-length SARS-CoV-2 spike protein shown in SEQ ID NO:19. In a preferred embodiment, the polymersomes of the invention have encapsulated therein a soluble fragment comprising, consisting essentially of, or consisting of the amino acid sequence corresponding to amino acid residues 646-1204 of the full-length SARS-CoV-2 spike protein shown in SEQ ID NO:19, or having the amino acid sequence of SEQ ID NO:39. In a preferred embodiment, the polymersomes of the invention have encapsulated therein a soluble fragment comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to amino acid residues 14 to 1204 of the full-length SARS-CoV-2 spike protein as set forth in SEQ ID NO:19, or having the amino acid sequence of SEQ ID NO:35.In a preferred embodiment, the polymersomes of the invention have encapsulated therein a soluble fragment comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to amino acid residues 19 to 1204 of the full-length SARS-CoV-2 spike protein as set forth in SEQ ID NO:19, or having the amino acid sequence of SEQ ID NO:65. In a preferred embodiment, the polymersomes of the invention comprise, consist essentially of, or encapsulate a soluble fragment thereof having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a sequence selected from the group consisting of sequences corresponding to positions 16-1213, 16-685, 686-1213, 686-1204, 646-1204, 14-1204, or 19-1204 of SEQ ID NO: 19 (SARS-CoV-2 spike protein). In a preferred embodiment, the polymersomes of the present invention encapsulate therein a soluble fragment that comprises, consists essentially of, or consists of a sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 36, 40, and / or 65. In this context, "consisting essentially of" means that the N- and / or C-terminal points of the fragment may vary within a limited range, for example, by up to 25 amino acid positions, for example, by up to 20 amino acid positions, for example, by up to 15 amino acid positions, by up to 10 amino acid positions, by up to 5 amino acid positions, by up to 4 amino acid positions, by up to 3 amino acid positions, by up to 2 amino acid positions, or by up to 1 amino acid position. In one specific example, a fragment consisting essentially of amino acids 646 to 1204 of the full-length SARS-CoV-2 spike protein can consist of amino acids 641 to 1204, 651 to 1204, 646 to 1209, or 646 to 1199, 641 to 1209, or 651 to 1199 of the full-length SARS-CoV-2 spike protein.

[0139] Alternatively or additionally, the polymersomes of the present disclosure may encapsulate one or more nucleic acids, such as mRNA, self-amplifying mRNA, or DNA encoding one or more SARS-CoV-2 spike proteins or soluble fragments thereof of the present disclosure.

[0140] It is also noted herein that in a preferred embodiment, polymersomes of the invention encapsulating one or more different soluble fragments of the SARS-CoV-2 spike protein and / or nucleic acids encoding same are used as vaccines against human diseases, particularly infections caused by human pathogenic coronaviruses, coronavirus disease 2019 (COVID-19). Accordingly, polymersomes of the invention encapsulating one or more different soluble fragments of the SARS-CoV-2 spike protein and / or nucleic acids encoding same may be used to treat (including prevent) fever, cough, shortness of breath, pneumonia, organ failure, acute respiratory distress syndrome (ARDS), fatigue, muscle pain, diarrhea, sore throat, loss of smell, and / or abdominal pain.

[0141] In a preferred embodiment, polymersomes of the present invention encapsulating one or more different soluble fragments of the SARS-CoV-2 spike protein and / or nucleic acids encoding same or the full-length SARS-CoV-2 spike protein are administered intramuscularly. In a preferred embodiment, polymersomes of the present invention encapsulating one or more different soluble fragments of the SARS-CoV-2 spike protein and / or nucleic acids encoding same or the full-length SARS-CoV-2 spike protein are administered intranasally. In a preferred embodiment, polymersomes of the present invention encapsulating one or more different soluble fragments of the SARS-CoV-2 spike protein and / or nucleic acids encoding same or the full-length SARS-CoV-2 spike protein are administered by inhalation.

[0142] For purposes of this application, the term "SARS-CoV-1 S protein" or "SARS-CoV-1 spike protein" refers to the spike glycoprotein present on the surface of the human pathogenic coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV or SARS-CoV-1). The SARS-CoV-1 spike protein of the present disclosure has the sequence set forth in UniProtKB Accession No. P59594 version 134 dated December 11, 2019, or SEQ ID NO: 48. Non-limiting examples of soluble "SARS-CoV-1 S proteins" that can be used in the present invention include the entire soluble fragment of the S1 and S2 regions of the SARS-CoV-1 spike protein (S protein), which may correspond to amino acids 14-1195 of the SARS-CoV-1 spike protein, or having the amino acid sequence set forth in SEQ ID NO: 48. Non-limiting examples of soluble "SARS-CoV-1 S proteins" that can be used in the present invention include the S1 region, which corresponds to amino acids 14-667 of the SARS-CoV-1 spike protein (S protein) or has the amino acid sequence of SEQ ID NO:49. Non-limiting examples of soluble "SARS-CoV-1 S proteins" that can be used in the present invention also include a soluble fragment of the S2 region, which can correspond to amino acids 668-1198 of the SARS-CoV-1 spike protein (S protein) or has the amino acid sequence of SEQ ID NO:50. It is, of course, also possible to use shorter fragments of the entire S1 and S2 regions, or of either the S1 or S2 regions alone; for example, a fragment can include the receptor binding domain (RBD), which corresponds to amino acids 306-527 of the SARS-CoV-1 spike protein or has the amino acid sequence of SEQ ID NO:51. It is also noted herein that the polymersomes of the present invention may encapsulate one or more different soluble fragments of the spike protein, such as the S1 region, the S2 region, or soluble fragments thereof, the entire soluble fragments of the S1 and S2 regions, and / or the RBD.In exemplary embodiments of the polymersomes of the invention, the polymersomes of the invention encapsulate within them one type of soluble fragment (e.g., only the entire soluble fragment of the S1 and S2 regions), two different types of soluble fragments (e.g., the entire soluble fragment of the S1 and S2 regions and a soluble fragment of the entire S1 region or the S2 region), three different types of soluble fragments (a soluble fragment of the S1 region, a soluble fragment of the S2 region, and the entire soluble fragment of S1 and S2 of SEQ ID NO:47 (amino acid residues 14-1195)), or even four different types of fragments (e.g., a soluble fragment of the S1 region, a soluble fragment of the S2 region, the entire soluble fragment of S1 and S2 of SEQ ID NO:47 (amino acid residues 14-1195), and a fourth type, the RBD). In a preferred embodiment, the polymersomes of the invention encapsulate therein a soluble fragment comprising, consisting essentially of, or consisting of the S1 region corresponding to amino acid residues 14-667 of the full-length SARS-CoV-1 spike protein. In a preferred embodiment, the polymersomes of the invention encapsulate therein a soluble fragment comprising, consisting essentially of, or consisting of the S2 region corresponding to amino acid residues 668-1195 of the full-length SARS-CoV-1 spike protein. In a preferred embodiment, the polymersomes of the invention encapsulate therein a soluble fragment comprising, consisting essentially of, or consisting of the S1 and S2 regions corresponding to amino acid residues 14-1195 of the full-length SARS-CoV-1 spike protein. In a preferred embodiment, the polymersomes of the invention encapsulate within them fragments of, consisting essentially of, or consisting of the S1 and S2 regions corresponding to amino acid residues 14 to 1255 of the full-length SARS-CoV-1 spike protein.In this context, "consisting essentially of" means that the N-terminal and / or C-terminal points of the fragment may vary within a limited range, for example by up to 25 amino acid positions, for example by up to 20 amino acid positions, for example by up to 15 amino acid positions, up to 10 amino acid positions, up to 5 amino acid positions, up to 4 amino acid positions, up to 3 amino acid positions, up to 2 amino acid positions, or up to 1 amino acid position.

[0143] The SARS-CoV-1 spike protein of the present disclosure may also include variants of the aforementioned sequences. These variants include naturally occurring variants of other SARS-CoV-1 isolates as well as artificial modifications that can be introduced into the sequence of the SARS-CoV-1 S protein. In one specific example, mutations can be introduced to alter the formation of the expressed protein. To this end, the furin cleavage site located at positions 761-767 of SEQ ID NO:47 can be mutated. Reduced post-expression cleavage can be achieved by reducing the basicity of this amino acid sequence. For example, residues Arg764 and / or Arg767 can be mutated to a less basic amino acid, such as Gly (position numbers correspond to the amino acid sequence shown in SEQ ID NO:47) or another less basic amino acid. Thus, a furin cleavage site with a native sequence of EQDRNTR (SEQ ID NO:60) can be mutated to the sequence EQDGNTG (SEQ ID NO:61). Further modifications can include the addition of a trimerization domain, preferably to the C-terminus of the protein, which can help increase the native fold of the S1 and / or S2 domains. Such trimerization domains can include a Foldon domain (e.g., SEQ ID NO:54), a GCN4-based trimerization domain (e.g., SEQ ID NO:55 or SEQ ID NO:56), or other motifs known to those of skill in the art. Additionally, secretory leader sequences can be added to the N-terminus of the protein, which can improve production and / or subsequent processing, such as isolation and purification. One example of such a leader sequence is the honeybee melittin leader sequence (SEQ ID NO:57). Other useful leader sequences are known to those of skill in the art. Thus, the soluble fragments of spike proteins disclosed herein also include highly identical variants of the specific sequences of soluble fragments of spike proteins explicitly or implicitly disclosed herein.For example, variants having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a soluble fragment of the spike protein of the present disclosure, particularly a soluble fragment of the SARS-CoV-1 S protein of the present disclosure. In one specific example, a soluble fragment of the S fragment of the present disclosure can comprise, consist essentially of, or consist of a sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a sequence selected from the group consisting of SEQ ID NOs:48-51.

[0144] Alternatively or additionally, the polymersomes of the present disclosure may encapsulate one or more nucleic acids, such as mRNA, self-amplifying mRNA, or DNA encoding one or more SARS-CoV-1 spike proteins or soluble fragments thereof of the present disclosure.

[0145] It is also noted herein that in a preferred embodiment, the polymersomes of the invention encapsulating one or more different soluble fragments of the SARS-CoV-1 spike protein and / or nucleic acids encoding same or the full-length SARS-CoV-1 spike protein are used as vaccines against human diseases, particularly infections caused by human pathogenic coronaviruses, and in particular severe acute respiratory syndrome (SARS). Accordingly, the polymersomes of the invention encapsulating one or more different soluble fragments of the SARS-CoV-1 spike protein and / or nucleic acids encoding same or the full-length SARS-CoV-1 spike protein may be used to treat (including prevent) fever, muscle aches, lethargy, cough, sore throat, shortness of breath, pneumonia, and / or acute respiratory distress syndrome (ARDS).

[0146] In a preferred embodiment, polymersomes encapsulating one or more different soluble fragments of the SARS-CoV-1 spike protein and / or nucleic acids encoding same or the full-length SARS-CoV-1 spike protein are administered intramuscularly. In a preferred embodiment, polymersomes of the invention encapsulating one or more different soluble fragments of the SARS-CoV-1 spike protein and / or nucleic acids encoding same or the full-length SARS-CoV-1 spike protein are administered intranasally. In a preferred embodiment, polymersomes of the invention encapsulating one or more different soluble fragments of the SARS-CoV-1 spike protein and / or nucleic acids encoding same or the full-length SARS-CoV-1 spike protein are administered by inhalation.

[0147] In the context of this application, the term "oxidative stability" refers to a measure of the resistance of polymersomes (or corresponding polymers or membranes) to oxidation, using, for example, the method described by Scott et al., 2012. In this method, polymersomes with encapsulated antigens are incubated in a 0.5% solution of hydrogen peroxide, and the amount of free (released) antigen can be quantified by UV / fluorescence HPLC. Polymersomes that release a substantial portion or all of the encapsulated antigen under these oxidizing conditions are considered to be oxidation-sensitive. Another method for determining whether a block copolymer, and therefore the resulting polymersome, is oxidatively stable or oxidation-sensitive is described in column 16 of U.S. Pat. No. 8,323,696. According to this method, polymers with oxidation-sensitive functional groups are chemically modified with a mild oxidizing agent, and the test is enhanced solubility in 10% hydrogen peroxide in vitro for 20 hours. For example, poly(propylene sulfide) (PPS) is an oxidation-sensitive polymer (see, for example, Scott et al., 2012, supra, and U.S. Pat. No. 8,323,696). PPS can serve as a standard for determining whether a polymer of interest and each polymersome of interest are oxidation-susceptible or oxidation-stable. For example, if the polymersome of interest releases the same or more antigens, or about 90% or more, or about 80% or more, or about 70% or more, or about 60% or more, compared to the amount released from a PPS polymersome containing the same antigen, the polymersome is considered oxidation-susceptible. If the polymersome of interest releases less than about 0.5%, or about 1.0%, or about 2%, or about 5%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50% of the antigens, compared to the amount released from a PPS polymersome containing the same antigen, the polymersome is considered oxidation-stable.Accordingly, in line with this, PPS polymersomes as described in U.S. Pat. No. 8,323,696 or PPS-bl-PEG polymersomes, such as those made of poly(propylene sulfide) (PPS) and poly(ethylene glycol) (PEG) as components as described in Stano et al., are not oxidatively stable polymersomes within the meaning of the present invention. Similarly, PPS30-PEG17 polymersomes are not oxidatively stable polymersomes within the meaning of the present invention. Other non-limiting examples of oxidative stability measurements include measuring stability in the presence of serum components (e.g., components of mammalian serum, e.g., human serum), or measuring stability, for example, inside endosomes.

[0148] For purposes of this application, the term "reductive stability" refers to a measure of a polymersome's resistance to reduction in a reducing environment.

[0149] As used herein, the term "serum" refers to plasma from which clotting proteins have been removed.

[0150] For purposes of this application, the term "oxidation-independent release" refers to release of polymersome contents without, or essentially without, oxidation of the polymer forming the polymersome.

[0151] The term "polypeptide" is used herein equivalently to the term "protein." Proteins (including fragments thereof, preferably biologically active fragments, and peptides, usually those having fewer than 30 amino acids) comprise one or more amino acids coupled to one another via covalent peptide bonds (resulting in a chain of amino acids). As used herein, the term "polypeptide" refers to a group of molecules, e.g., those consisting of more than 30 amino acids. Polypeptides can further form multimers, i.e., those consisting of two or more polypeptide molecules, such as dimers, trimers, and higher oligomers. The polypeptide molecules forming such dimers, trimers, etc. may or may not be identical. Thus, the corresponding higher-order structures of such multimers are referred to as homodimers or heterodimers, homotrimers or heterotrimers, etc. One example of a heteromultimer is an antibody molecule, which in its native form consists of two identical light polypeptide chains and two identical heavy polypeptide chains. The terms "polypeptide" and "protein" also refer to naturally modified polypeptides / proteins, where the modification is achieved by post-translational modifications such as glycosylation, acetylation, phosphorylation, etc. Such modifications are well known in the art.

[0152] As used herein, the term "sugar" refers to compounds such as aldoses and ketoses having the stoichiometric formula C(HO) (e.g., and therefore "carbohydrates"). The collective term "sugar" includes, but is not limited to, monosaccharides, oligosaccharides, and polysaccharides, as well as substances derived from monosaccharides by reduction of the carbonyl group (alditols), oxidation of one or more terminal groups to carboxylic acids, or replacement of one or more hydroxy groups with groups such as hydrogen atoms, amino groups, or thiol groups. Derivatives of these compounds are also included.

[0153] For purposes of this application, the term "polynucleotide" (also referred to as "nucleic acid," which can be used interchangeably with the term "polynucleotide") refers to a polymer composed of nucleotide units that may be hydrolyzable, for example, to certain pyrimidine or purine bases (usually adenine, cytosine, guanine, thymine, uracil), d-ribose or 2-deoxy-d-ribose, and phosphate. Non-limiting examples of "polynucleotides" include DNA molecules (e.g., cDNA or genomic DNA), RNA (mRNA), combinations thereof, or hybrid molecules composed of DNA and RNA. Nucleic acids can be double-stranded or single-stranded and can contain both double- and single-stranded fragments. Most preferred are double-stranded DNA molecules and mRNA molecules.

[0154] As used herein, the term "antisense oligonucleotide" refers to a nucleic acid polymer, at least a portion of which is complementary to a nucleic acid present in a normal cell or a diseased cell. Exemplary "antisense oligonucleotides" include antisense RNA, siRNA, and RNAi.

[0155] As used herein, the term "CD8(+) T cell-mediated immune response" refers to an immune response mediated by cytotoxic T cells (TCs, also known as cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, CD8(+) T cells, or killer T cells). Examples of cytotoxic T cells include, but are not limited to, antigen-specific effector CD8(+) T cells. For the T cell receptor (TCR) to bind to class I MHC molecules, it must be associated with a glycoprotein called CD8, which binds to the constant portion of class I MHC molecules. These T cells are therefore called CD8(+) T cells. Once activated, TC cells undergo "clonal expansion" with the help of the cytokine interleukin-2 (IL-2), a growth and differentiation factor for T cells. This increases the number of cells specific to the target antigen, which can then migrate throughout the body in search of antigen-positive somatic cells.

[0156] As used herein, the term "clonal expansion of antigen-specific CD8(+) T cells" refers to an increase in the number of CD8(+) T cells specific for a target antigen.

[0157] As used herein, the term "cellular immune response" refers to an immune response that does not involve antibodies, but rather involves the activation of phagocytes, antigen-specific cytotoxic T lymphocytes, and the release of various cytokines in response to an antigen.

[0158] As used herein, the term "cytotoxic phenotype of antigen-specific CD8(+) T cells" refers to a set of observable characteristics of antigen-specific CD8(+) T cells that are related to their cytotoxic function.

[0159] As used herein, the term "lymph node-resident macrophages" refers to macrophages, large white blood cells that are an integral part of our immune system and reside in lymph nodes, small, bean-like glands found throughout the body, where they engulf particles and then digest them using a process called phagocytosis.

[0160] As used herein, the term "humoral immune response" refers to an immune response mediated by macromolecules found in extracellular fluids, such as secreted antibodies, complement proteins, and certain antimicrobial peptides. Aspects involving antibodies are often referred to as antibody-mediated immunity.

[0161] As used herein, the term "B cells," also known as B lymphocytes, are a type of white blood cell of the lymphocyte subtype. They function in the humoral immune component of the adaptive immune system by secreting antibodies.

[0162] As used herein, an "antibody" is a protein comprising one or more polypeptides (including one or more binding domains, preferably antigen-binding domains) substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. "Immunoglobulin" (Ig) is used interchangeably with "antibody" herein. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. In particular, as used herein, an "antibody" is typically a tetrameric, glycosylated protein composed of two light (L) chains, each approximately 25 kDa, and two heavy (H) chains, each approximately 50 kDa. Two types of light chains, called lambda and kappa, can be found in antibodies. Depending on the amino acid sequence of the heavy chain constant domain, immunoglobulins can be assigned to five major classes: A, D, E, G, and M. Some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. IgG is preferred in the context of the present invention. Antibodies relevant to the present invention also include those having an IgE constant domain or a portion thereof that is bound by Fc epsilon receptor I. IgM antibodies consist of five basic heterotetrameric units and an additional polypeptide called the J chain, containing 10 antigen-binding sites. On the other hand, IgA antibodies contain two to five basic four-chain units, which can multimerize to form multivalent aggregates in combination with the J chain. In the case of IgG, the four-chain unit is generally approximately 150,000 daltons. Each light chain contains an N-terminal variable (V) domain (VL) and a constant (C) domain (CL). Each heavy chain contains an N-terminal V domain (VH), three or four C domains (CH), and a hinge region. The constant domains are not directly involved in binding the antibody to an antigen, but can exhibit various effector functions, such as participating in antibody-dependent cellular cytotoxicity (ADCC).If the antibody is to exert ADCC, it is preferable that the antibody be of the IgG1 subtype; IgG4 subtypes will not have the ability to exert ADCC.

[0163] The term "antibody" also includes, but is not limited to, monoclonal antibodies, monospecific antibodies, multispecific or polyspecific antibodies, such as bispecific antibodies, humanized antibodies, camelized antibodies, human antibodies, single-chain antibodies, chimeric antibodies, synthetic antibodies, recombinant antibodies, hybrid antibodies, mutated antibodies, grafted antibodies, and in vitro-generated antibodies; chimeric or humanized antibodies are preferred. The term "humanized antibody" is generally defined as an antibody in which the CDRs encoding the specificities of the HC and LC have been transferred onto a suitable human variable framework ("CDR-grafting"). The term "antibody" also encompasses scFvs, single-chain antibodies, diabodies or tetrabodies, domain antibodies (dAbs), and nanobodies. In the context of the present invention, the term "antibody" also encompasses dimeric, trimeric, or multimeric forms having several antigen-binding sites, or bifunctional, trifunctional, or multifunctional antibodies.

[0164] Furthermore, the term "antibody" as used in the present invention also relates to derivatives (including fragments) of the antibodies described herein. "Derivatives" of antibodies include amino acid sequences that have been altered by introducing substitutions, deletions, or additions of amino acid residues. In addition, derivatives also encompass antibodies that have been modified by the covalent attachment of any type of molecule to the antibody or protein. Examples of such molecules include, but are not limited to, sugars, PEG, hydroxyl groups, ethoxy groups, carboxyl groups, or amine groups. In summary, covalent modification of antibodies can lead to, but is not limited to, glycosylation, PEGylation, acetylation, phosphorylation, amidation, etc.

[0165] Antibodies related to the present invention are preferably "isolated" antibodies. "Isolated," when used to describe antibodies disclosed herein, refers to an antibody that has been identified, separated, and / or recovered from components of its production environment. Preferably, an isolated antibody is free from association with any other components from its production environment. Contaminating components of the production environment, such as those resulting from recombinant, transfected cells, are materials that would typically interfere with diagnostic or therapeutic uses of the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody will be (1) purified sufficiently to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator, or (2) purified to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue staining or, preferably, silver staining. Ordinarily, however, an isolated antibody will be prepared by at least one purification step.

[0166] The term "essentially non-immunogenic" means that the block copolymers or amphiphilic polymers of the present invention do not elicit an adaptive immune response, i.e., the block copolymers or amphiphilic polymers exhibit less than 30%, preferably less than 20%, more preferably less than 10%, and particularly preferably less than 9, 8, 7, 6, or 5% of the immune response compared to the encapsulated immunogen.

[0167] The term "essentially non-antigenic" means that the block copolymers or amphiphilic polymers of the present invention do not specifically bind to certain products of adaptive immunity (e.g., T cell receptors or antibodies), i.e., the block copolymers or amphiphilic polymers exhibit less than 30%, preferably less than 20%, more preferably less than 10%, and particularly preferably less than 9, 8, 7, 6, or 5% binding compared to the encapsulated antigen.

[0168] Typically, binding affinities of 10 -6Binding is considered specific when the binding affinity is greater than M. Preferably, the binding affinity is about 10 -11 ~10 -8 M(KD), preferably about 10 -11 ~10 -9 Binding is considered specific when M. If necessary, binding conditions can be varied to reduce non-specific binding without substantially affecting specific binding.

[0169] The term "amino acid" or "amino acid residue" typically refers to an amino acid having its art-recognized definition, e.g., an amino acid selected from the group consisting of alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (He or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V), although modified, synthetic, or rare amino acids may be used if desired. In general, amino acids can be classified as having nonpolar side chains (e.g., Ala, Cys, He, Leu, Met, Phe, Pro, Val), negatively charged side chains (e.g., Asp, Glu), positively charged side chains (e.g., Arg, His, Lys), or uncharged polar side chains (e.g., Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr).

[0170] "Effector cells," preferably human effector cells, are leukocytes that express one or more FcRs and perform effector function. Preferably, these cells express at least FcγRm and perform ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from native sources, such as blood.

[0171] The term "immunizing" refers to one or more steps of administering to a human or non-human animal one or more antigens so that antibodies can be raised in the animal.

[0172] Specifically, non-human animals are immunized, preferably at least twice, and more preferably at least three times, with a polypeptide (antigen), optionally mixed with an adjuvant. An "adjuvant" is a non-specific stimulator of the immune response. Adjuvants can take the form of compositions containing either or both of the following components: (a) a substance (e.g., mineral oil, alum, aluminum hydroxide, liposomes, or surfactants (e.g., pluronic polyols)) intended to form a deposit that protects the antigen from rapid catabolism; and (b) a substance that non-specifically stimulates the immune response of the immunized host animal (e.g., by increasing lymphokine levels therein).

[0173] As used herein, "cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can lead to the formation of malignant tumors or cells that invade neighboring tissues and can metastasize to distant parts of the body via the lymphatic system or bloodstream.

[0174] Non-limiting examples of cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-NSCLC, glioma, gastrointestinal cancer, renal cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon cancer, and head and neck cancer (or cancers), gastric cancer, germ cell tumors. tumors, pediatric sarcomas, sinonasal natural killer tumors, melanoma (e.g., metastatic melanoma, e.g., subcutaneous or intraocular melanoma), bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumors, ureteral cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma , squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers such as those induced by asbestos, virus-associated cancers (e.g., human papillomavirus (HPV)-associated tumors), hematological malignancies derived from either of the two major blood cell lineages, i.e., myeloid lineage (producing granulocytes, erythrocytes, platelets, macrophages, and mast cells) or lymphoid lineage (producing B cells, T cells, NK cells, and plasma cells), such as all types of leukemias, lymphomas, and myelomas, e.g., acute, chronic, Lymphocytic and / or myeloid leukemias, such as acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), undifferentiated AML (M0), myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cellular maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilicity [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), solitary granulocytic sarcoma, and chloroma;Lymphomas, e.g., Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell lymphoma, T-cell lymphoma, lymphoplasmacytic lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki1+) large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma Lymphoma, T-lymphoblastic lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL), hematopoietic neoplasms of the lymphatic system, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sézary syndrome), and lymphoplasmacytic lymphoma (LPL) with Waldenström's macroglobulinemia; myelomas, such as IgG myeloma, light-chain myeloma, non-secretory myeloma, smoldering myeloma (also called asymptomatic myeloma), solitary plasmacytoma, and multiple myeloma; chronic lymphocytic leukemia (CLL); hairy cell lymphoma; hematopoietic tumors of the myeloid lineage; fibrosarcoma; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, such as melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular carcinoma, and teratocarcinoma, hematopoietic tumors of the lymphatic system, including T-cell tumors and B-cell tumors, T-cell disorders such as, but not limited to, T-prolymphocytic leukemia (T-PLL), including the small cell and cerebriform cell types; large granular lymphocyte leukemia (LGL), preferably of the T-cell type; a / d T-NHL hepatosplenic lymphoma; peripheral / retrothymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; head and neck cancer, renal cancer, rectal cancer, thyroid cancer;and acute myeloid lymphoma, as well as any combination of the foregoing cancers. The methods described herein can also be used to treat metastatic cancers, refractory cancers (e.g., cancers that are refractory to previous immunotherapy, such as with CTLA-4 or PD-1 or PD-L1 blocking antibodies), and recurrent cancers.

[0175] The term "subject" is intended to encompass living organisms. Examples of subjects include mammals, such as humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. However, subjects (animals) can also be non-mammals, such as birds or fish. In some preferred embodiments of the present invention, the subject is a human, while in other preferred embodiments, the subject can be an agricultural animal, which can be either a mammal or a non-mammal. Examples of such non-mammals include birds (e.g., poultry such as chickens, ducks, geese, or turkeys), fish (e.g., farmed fish such as salmon, trout, or tilapia), or crustaceans (e.g., shrimp or prawns). Examples of mammalian (livestock) animals include goats, sheep, cows, horses, pigs, or donkeys. Other mammals include, for example, cats, dogs, mice, and rabbits. In exemplary embodiments, the polymersomes of the present invention are used to vaccinate or immunize the above-mentioned agricultural animals, both mammalian and non-mammalian agricultural animals (birds, fish, crustaceans), against viral infections (see the Examples). Thus, in such cases, the polymersomes of the present invention may encapsulate soluble full-length viral proteins or soluble fragments of full-length viral proteins therein.

[0176] For use in vaccinating both humans and non-human animals, the polymersomes of the present invention or compositions comprising the polymersomes of the present invention can be dissolved in an appropriate (pharmaceutically acceptable) buffer, such as phosphate-buffered saline (PBS) or 0.9% saline (an isotonic solution of 0.90% w / v NaCl with an osmolality of 308 mOsm / L), and orally administered to each subject (see again the Examples). The polymersomes can be further mixed with an adjuvant. When administered orally, the adjuvant can help protect the polymersomes from the acidic environment of the stomach. Such adjuvants can be water-miscible or capable of forming water-oil emulsions, such as oil-in-water emulsions or water-in-oil emulsions. Specific examples of such adjuvants are oil-in-water emulsions, water-in-oil emulsions, monophosphoryl lipid A, and / or trehalose dicorynomycolate, where the oil preferably comprises, consists essentially of, or consists of mineral oil, simethicone, Span 80, squalene, and combinations thereof. Further specific examples are monophosphoryl lipid A (e.g., from Salmonella Minnesota), trehalose dicorynomycolate, or mixtures thereof, which may be in the form of an oil-in-water (e.g., squalene) emulsion. The emulsion may include an emulsifier (e.g., a polysorbate, e.g., Polysorbate 80). Alternatively, polymersomes can be modified, such as by coating with natural polymers, or formulated into particles of natural polymers such as alginate or chitosan, or into particles of synthetic polymers such as poly(d,l-lactide-co-glycolide) (PLG), poly(d,l-lactic-co-glycolic acid) (PLGA), poly(g-glutamic acid) (g-PGA) [31,32] or poly(ethylene glycol) (PEG). These particles can be either micrometer-range particles ("macrobeads") or nanoparticles, or nanoparticles incorporated into macrobeads, all of which are well known in the art.See, for example, Hari et al., "Chitosan / calcium-alginate beads for oral delivery of insulin," Applied Polymer Science, Volume 59, Issue 11, 14 March 1996, pp. 1795-1801; Sosnik's review, "Alginate Particles as Platform for Drug Delivery by the Oral Route: State-of-the-Art," ISRN Pharmaceutics Volume 2014, Article ID 926157; Machado et al., "Encapsulation of DNA in Macroscopic and Nanosized Calcium Alginate Gel Particles," Langmuir 2013, 29, pp. 15926-15935; International Patent Application WO2015 / 110656; Liang Zhao et al., "Nanoparticle vaccines," Vaccine 32 (2014) 327-337; or Li et al., "Chitosan-Alginate Nanoparticles as a Novel Drug Delivery System," Applied Polymer Science, Volume 59, Issue 11, 14 March 1996, pp. 1795-1801. See "Delivery System for Nifedipine" Int J Biomed Sci vol. 4 no. 3 September 2008, 221-228.In exemplary embodiments of these polymersomes and oral formulations, the polymersomes used for vaccination encapsulate viral antigens, including influenza hemagglutinin, swine influenza hemagglutinin, foot-and-mouth disease (FMD) viral proteins such as VP1, VP2, or VP3 coat proteins (the VP1 coat protein contains the major antigenic determinant of the FMD virion, and therefore variations in its sequence likely account for the high antigenic diversity of this virus), ovalbumin (OVA), spike proteins, such as the soluble portion of a spike protein of a human pathogenic coronavirus, such as the porcine epidemic diarrhea (PED) virus spike protein, the MERS-CoV spike protein, the SARS-CoV-2 spike protein, or the SARS-CoV-1 spike protein. As is evident from the use of polymersomes containing soluble portions of FMD viral proteins such as influenza hemagglutinin or the VP1, VP2, or VP3 coat proteins, viral diseases can affect any animal, including birds and mammals, and the mammal can be a human.

[0177] The term "effective amount" or "effective dosage" is defined as an amount sufficient to achieve a desired effect or an amount sufficient to at least partially achieve a desired effect. The term "therapeutically effective amount" is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Amounts effective for this use will depend on the severity of the infection and the general state of the subject's own immune system. The term "patient" includes human and other mammalian subjects receiving either prophylactic or therapeutic treatment.

[0178] The appropriate dosage or therapeutically effective amount of the antibody or antigen-binding portion thereof will depend on the condition being treated, the severity of the condition, previous treatments, and the patient's clinical history and response to the therapeutic agent. The appropriate dose can be adjusted according to the judgment of the attending physician so that it can be administered to the patient once or as a series of doses. The pharmaceutical composition can be administered as the sole therapeutic agent or in combination with additional therapies as needed.

[0179] If the pharmaceutical composition is lyophilized, the lyophilized material is first reconstituted in a suitable liquid prior to administration, such as bacteriostatic water for injection (BWFI), saline, phosphate buffered saline (PBS), or the same formulation in which the protein was contained prior to lyophilization.

[0180] Pharmaceutical compositions for injection can be presented in unit dosage form, for example, in ampoules or in multi-dose containers with added preservatives.In addition, several new drug delivery approaches have been developed, and the pharmaceutical compositions of the present invention are suitable for administration using these new methods, for example, pen-type injectors such as Inject-ease, Genject, Genen, and needleless devices such as MediJector and BioJector.The pharmaceutical compositions can also be adapted to administration methods that have not yet been discovered.See also Langer, 1990, Science, 249:1527-1533.

[0181] Pharmaceutical compositions can be prepared for intranasal or inhalation administration, for example, for local administration to the respiratory tract and / or lungs. Means and devices for administering substances by inhalation are known to those skilled in the art and are disclosed, for example, in WO94 / 017784A and Elphick et al. (2015) Expert Opin Drug Deliv, 12, 1375-87. Such means and devices include nebulizers, metered-dose inhalers, powder inhalers, and nasal sprays. Other suitable means and devices for inhalation administration of drugs or vaccines are known in the art. The preferred route for local administration to the respiratory tract and / or lungs is by aerosol inhalation. Pulmonary drug delivery, i.e., by inhalation of aerosols (which can also be used for intranasal administration) or by intratracheal instillation, is reviewed, for example, in Patton, JS, et al. (2004) Proc. Amer. Thoracic Soc., 1, 338-344. Nebulizers are useful for generating aerosols from solutions, while metered dose inhalers, dry powder inhalers, etc. are effective for generating small particle aerosols. Thus, the pharmaceutical composition can be formulated in the form of an aerosol (mixture), spray, mist, or powder.

[0182] Pharmaceutical compositions against mucosal pathogens, such as respiratory coronaviruses like SARS-CoV-2, MERS, or SARS-CoV1, should confer long-lasting protective immunity at both the systemic and mucosal levels. Therefore, pharmaceutical compositions of the present disclosure may preferably be prepared for mucosal administration, such as by inhalation or intranasal administration. As shown in Example 14, intranasal administration of a coronavirus vaccine can induce not only a mucosal immune response but also a systemic immune response. Pharmaceutical compositions of the present disclosure may also preferably be prepared for systemic administration, such as intramuscular administration.

[0183] A nebulizer is a drug delivery device used to administer medication in the form of a mist that is inhaled into the lungs. Various types of nebulizers are known to those skilled in the art, including jet nebulizers, ultrasonic nebulizers, vibrating mesh technology, and soft mist inhalers. Some nebulizers provide a continuous flow of atomized solution; that is, they provide continuous spray over a long period of time, regardless of whether the subject inhales it. Other nebulizers are breath-activated; that is, the subject only receives a dose if they inhale it themselves. The vaccines of the present invention, particularly vaccines for human pathogenic coronavirus infections such as MERS, COVID-19, or SARS, can be formulated for use in a nebulizer and can be contained in or administered using a nebulizer.

[0184] A metered dose inhaler (MDI) is a device that delivers a specific amount of medication to the lungs in the form of a short burst of liquid aerosolized medication. Such metered dose inhalers generally consist of three main components: a canister containing the formulation to be administered, a metering valve that allows a metered amount of the formulation to be dispensed per actuation, and an actuator (or mouthpiece) that allows the patient to operate the device and direct the liquid aerosol to the lungs. The vaccines of the present invention, particularly vaccines for human pathogenic coronavirus infections such as MERS, COVID-19, or SARS, can be formulated for use in MDIs and can be contained in or administered using MDIs, particularly canisters for MDIs.

[0185] A dry powder inhaler (DPI) is a device that delivers medication to the lungs in the form of a dry powder. Dry powder inhalers are an alternative to aerosol-based inhalers, such as metered-dose inhalers. The medication is typically held in a capsule for manual loading or in a dedicated blister pack placed inside the inhaler. The vaccines of the present invention, particularly vaccines for human pathogenic coronavirus infections such as MERS, COVID-19, or SARS, can be formulated for use in a DPI and can be contained in or administered using a DPI, particularly a capsule or blister pack for an MDI.

[0186] Nasal sprays can be used for nasal administration, in which a drug is insufflated into the nose. The vaccines of the present invention, particularly vaccines for human pathogenic coronavirus infections such as MERS, COVID-19, or SARS, can be formulated as nasal sprays and contained in nasal spray bottles or administered as nasal sprays.

[0187] Pharmaceutical compositions can also be formulated as depot preparations. Such long-acting formulations can be administered by implantation (e.g., subcutaneous, intraligamentous or intratendinous, subsynovial or intramuscular), subsynovial injection or intramuscular injection. Thus, for example, the formulation can be modified with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resin, or modified as sparingly soluble derivatives, for example, as sparingly soluble salts.

[0188] The pharmaceutical compositions may be in a variety of conventional depot forms used for administration to provide the reactive composition, including, for example, solid, semi-solid and liquid dosage forms such as solutions or suspensions, slurries, gels, creams, balms, emulsions, lotions, powders, sprays, foams, pastes, ointments, salves, balms and drops.

[0189] If desired, pharmaceutical compositions can be presented in a vial, a pack or a dispenser device, which can contain one or more unit dosage forms containing active ingredients.In one embodiment, the dispenser device can include a syringe with a single dose of liquid formulation that can be immediately injected.The syringe can be provided with instructions for administration.

[0190] Pharmaceutical compositions may further comprise additional pharmaceutically acceptable components. The proteins described herein may also contain other pharmaceutically acceptable carriers, excipients, or stabilizers, such as those described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), as long as they do not adversely affect the desired characteristics of the formulation. As used herein, "pharmaceutically acceptable carrier" refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and may include additional buffering agents; preservatives; cosolvents; antioxidants, such as ascorbic acid and methionine; chelating agents, such as EDTA; metal complexes (e.g., Zn-protein complexes); biodegradable polymers, such as polyesters; salt-forming counterions, such as sodium, polyhydric sugar alcohols; amino acids, such as alanine, glycine, asparagine, 2-phenylalanine, and threonine; sugars or sugar alcohols, such as lactitol, stachyose, mannose, sorbose, xylose, These include ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol; sulfur-containing reducing agents, such as glutathione, thioctic acid, sodium thioglycolate, thioglycerol, [alpha]-monothioglycerol, and sodium thiosulfate; low molecular weight proteins, such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; and hydrophilic polymers, such as polyvinylpyrrolidone.

[0191] The formulations described herein are useful as pharmaceutical compositions in the treatment and / or prevention of the pathological medical conditions described herein in patients in need thereof. The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Treatment includes the application or administration of a formulation to the body, isolated tissue, or cells of a patient with a disease / disorder, a symptom of a disease / disorder, or a predisposition to a disease / disorder, with the intent to cure, heal, alleviate, relieve, alter, correct, improve, ameliorate, or affect the disease, symptom of a disease, or predisposition to a disease.

[0192] As used herein, the terms "treat" and "treatment" refer to administering a therapeutically effective amount of a pharmaceutical composition of the present invention to a subject. A "therapeutically effective amount" refers to an amount of a pharmaceutical composition or antibody sufficient to treat or ameliorate a disease or disorder, delay the onset of a disease, or provide some therapeutic benefit in the treatment or management of a disease.

[0193] As used herein, the term "prevention" refers to the use of an agent to prevent the onset of a disease or disorder. A "prophylactically effective amount" defines the amount of an active component or pharmaceutical agent sufficient to prevent the onset or recurrence of a disease.

[0194] As used herein, the terms "disorder" and "disease" are used interchangeably to refer to a condition in a subject. In particular, the term "cancer" is used interchangeably with the term "tumor."

[0195] As used herein, the term "CpG oligonucleotide" may refer to any synthetic or natural oligodeoxynucleotide (ODN) containing an unmethylated CpG motif (e.g., as described in Bode et al., CpG DNA as a vaccine adjuvant. Expert Rev Vaccines. 2011 April;10(4):499-511). Thus, in the present invention, any suitable CpG oligonucleotide (e.g., as described in Rankin et al., 2001 (Antisense and nucleic acid drug development 11:333-340 (2001), especially Tables 1 and 2 therein, and as described in Figure 32 herein) may be used, for example, in combination with a suitable animal. The CpG oligonucleotide may belong to any of three major classes of (stimulatory) CpG ODNs that have been identified, for example, based on structural features and activity on human peripheral blood mononuclear cells (PBMCs), particularly B cells and plasmacytoid dendritic cells (pDCs). The three classes are class A (e.g., PS-PO (phosphorothioated phosphodiester) backbone; also known as type D), class B (e.g., PS (phosphorothioated) backbone; also known as type K), and class C (e.g., PS (phosphorothioated) backbone). CpG-A ODNs are typically characterized by a palindromic motif containing a PO (phosphodiester) central CpG and a PS-modified (i.e., phosphorothioate-modified) 3' poly-G string, while CpG-B ODNs contain a complete PS backbone with one or more CpG dinucleotides. CpG-C ODNs combine features of both class A and class B CpG oligonucleotides. An exemplary CpG ODN of the present invention is further illustrated in Figure 31 herein (adapted from https: / / www.invivogen.com / cpg-odns-classes).A preferred CpG-A ODN of the present invention can primarily induce IFN-α production from plasmacytoid dendritic cells (pDCs) rather than stimulating TLR9-dependent NF-κB signaling and inflammatory cytokine (e.g., IL-6) production. A preferred CpG-B ODN of the present invention can primarily activate B cells and TLR9-dependent NF-κB signaling rather than stimulating IFN-α secretion. A preferred CpG-C ODN of the present invention can (i) primarily induce IFN-α production from plasmacytoid dendritic cells (pDCs) rather than stimulating TLR9-dependent NF-κB signaling and inflammatory cytokine (e.g., IL-6) production, and (ii) primarily activate B cells and TLR9-dependent NF-κB signaling rather than stimulating IFN-α secretion.

[0196] Kits of the present invention will typically include the above-described container and one or more other containers containing materials desirable from a commercial and user standpoint, such as buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0197] For purposes of this application, the term "liposome" refers to a spherical vesicle having at least one lipid bilayer.

[0198] For purposes of this application, the term "endosome" refers to a membrane-bound compartment (i.e., vacuole) within a eukaryotic cell into which endocytosed material is delivered.

[0199] For the purposes of this application, the term "late endosome" refers to a pre-lysosomal endocytic organelle that is distinguished from early endosomes by a lower luminal pH and different protein composition. Late endosomes are more spherical than early endosomes, are mostly juxtanuclear, and are concentrated near microtubule-organizing centers.

[0200] For purposes of this application, the term "T helper cells" (also called TH cells or "effector CD4(+) T cells") refers to T lymphocytes that assist other white blood cells in immunological processes, including B cell maturation into plasma cells and memory B cells and activation of cytotoxic T cells and macrophages. These cells are also known as "CD4(+) T cells" because they express the CD4 glycoprotein on their surface. Helper T cells are activated when presented with, for example, peptide antigens, by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs).

[0201] As used herein, the term "autoantigen" refers to any molecule or chemical group in one organism that acts as an antigen in inducing antibody formation in another organism, but to which the healthy immune system of the parent organism is tolerant.

[0202] The term "% identity" used herein refers to the percentage of identical amino acid residues at corresponding positions in sequences when comparing two amino acid sequences through optimal sequence alignment, as exemplified by the ClustalW method or ClustalX method available at www.clustal.org or equivalent techniques.Therefore, align both sequences (reference sequence and target sequence), identify the identical amino acid residues between both sequences, and divide the total number of identical amino acids by the total number of amino acids (amino acid length).The result of this division is a percentage value, that is, percent identity value / percent identity.

[0203] Because the immunization method of the present invention can be carried out by using a full-length soluble encapsulated antigen (e.g., a protein) or a fragment of said protein in a synthetic environment that allows its proper folding, the probability of isolating an antibody capable of detecting the corresponding antigen (e.g., a membrane protein) in vivo will be increased. Moreover, the immunization and antibody generation can be carried out without any prior knowledge of the membrane protein structure, which would otherwise be required when using a peptide-based immunization approach.

[0204] Furthermore, compared to other techniques, the method of the present invention allows for the rapid and cost-effective production of membrane proteins encapsulated in an oxidation-stable membrane environment.

[0205] In some aspects, the present invention relates to a method for eliciting an immune response to an antigen (e.g., an immunogen) in a subject. The method can include administering to the subject a composition comprising a polymersome of the present invention having a membrane (e.g., a surrounding membrane) of an amphiphilic polymer. The composition further comprises a soluble antigen encapsulated by the amphiphilic polymer membrane of the polymersome of the present invention. The immunogen can be a membrane-bound protein. In some further aspects, the polymersome of the present invention comprises a lipid polymer. Administration can be carried out by any suitable method, such as oral administration, topical administration, topical administration to the respiratory tract, topical administration to the lungs, inhalation administration, intranasal administration, or injection.

[0206] Those skilled in the art can determine and adjust the frequency of administration (e.g., oral administration or injection) depending on the level of response desired. For example, a subject (which may include a mammal) can be given a weekly or biweekly administration (e.g., oral administration or injection) of a polymersome of the invention. The immune response can be measured by quantifying the blood concentration level (titer) of antibodies in a mammal to an initial amount of antigen encapsulated in a polymersome of the invention (see "Examples").

[0207] Polymersome structures can include amphiphilic block copolymers that self-assemble into vesicles to encapsulate various antigens (e.g., soluble proteins), which are encapsulated by solvent rehydration methods, direct dispersion, or spontaneous self-assembly (e.g., Example 1 described herein).

[0208] In the context of this application, the term "soluble antigen" as used herein refers to an antigen that can be dissolved or liquefied. As a specific example, a soluble antigen can consist of amino acids from the extracellular and / or intracellular regions of a membrane protein. However, a soluble antigen can also contain amino acids from the extracellular and / or intracellular regions of a membrane protein and one or more additional amino acids belonging to the transmembrane region of the membrane protein, as long as the antigen can still be dissolved or liquefied. As a specific example, a soluble fragment of the MERS-CoV spike protein of SEQ ID NO:43, which contains one amino acid (position 1297) belonging to the transmembrane region, is a soluble antigen within the meaning of the present disclosure. However, it is envisioned that a soluble antigen preferably lacks at least a portion of the transmembrane region or the entire transmembrane region. The term "soluble antigen" also encompasses "solubilized" antigens, i.e., antigens that have been solubilized or increased in solubility, especially in water, by the action of detergents or other agents. Exemplary, non-limiting soluble antigens of the present invention include polypeptides derived from insoluble portions of proteins, hydrophobic polypeptides that have been made soluble for inclusion, and aggregated polypeptides that are soluble as aggregates.

[0209] In some aspects, antigens (e.g., membrane proteins) of the present invention are solubilized using detergents, surfactants, temperature changes, or pH changes. The vesicle structure provided by the amphiphilic block copolymer allows the antigen (e.g., membrane protein) to fold in a physiologically correct, functional form, thereby enabling the immune system of the target mammal to detect the antigen and thereby generate a strong immune response.

[0210] In some aspects, injection of the compositions of the present invention can include intraperitoneal injection, subcutaneous or intravenous injection, intramuscular injection, or non-invasive administration. In other aspects, injection of the compositions of the present invention can include intradermal injection.

[0211] In some other aspects, the level of immune response can be further enhanced or boosted by including an adjuvant in a composition comprising the polymersomes of the present invention. The adjuvant can be an encapsulated adjuvant or a non-encapsulated adjuvant. The adjuvant can be mixed with the polymersomes of the present invention or the combinations of the present invention. The adjuvant can be soluble in water or in the form of a water-oil emulsion. In such aspects, the polymersomes and adjuvant can be administered to a subject simultaneously.

[0212] In some aspects, the block copolymers or amphiphilic polymers of the polymersomes of the invention are not immunostimulants or adjuvants.

[0213] In some other aspects, the block copolymers or amphiphilic polymers of the present invention are immunostimulants and / or adjuvants.

[0214] In some further aspects, the polymersomes of the present invention are immunogenic.

[0215] In some further aspects, the polymersomes of the invention are non-immunogenic.

[0216] In some aspects, the adjuvant may be administered separately from the administration of the composition of the present invention comprising the polymersome of the present invention. The adjuvant may be administered before, simultaneously with, or after the administration of the composition comprising the polymersome encapsulating the antigen of the present invention. For example, the adjuvant may be injected into a subject after the injection of the composition comprising the polymersome encapsulating the antigen of the present invention. In some aspects, the adjuvant may be encapsulated in a polymersome together with the antigen. In other preferred aspects, the adjuvant is encapsulated in a separate polymersome. That is, the adjuvant is encapsulated separately from the antigen, so that the antigen is encapsulated in one type of polymersome and the adjuvant is encapsulated in a second type of polymersome. It should be noted here that the adjuvant and polymersome may be encapsulated in a polymersome formed from the same amphiphilic polymer. Each antigen and CpG oligodeoxynucleotide as an exemplary adjuvant See Examples 7-9 or 14 or 18 of the present application, where TIFF2024505964000004.tif11157 are both encapsulated in BD21 polymersomes. Alternatively, the amphiphilic polymer used to encapsulate the antigen may be different from the amphiphilic polymer used to encapsulate the adjuvant. As a purely illustrative example, the antigen may be encapsulated in BD21 polymersomes while the adjuvant is encapsulated in PDMS. 12 -PEO 46 Polymersomes or PDMS 47 PEO 36 It may be encapsulated in a polymersome.

[0217] Any known adjuvant can be used in the present invention, and those skilled in the art will readily recognize and understand that the type of adjuvant injected may depend on the type of antigen used to elicit an immune response. The adjuvant may be an antigen derived from bacteria, viruses, or fungi. The adjuvant may be a nucleic acid, such as CpG oligodeoxynucleotides (also known as "CpG ODNs" or referred to herein as "CpG"). CpG molecules are naturally occurring oligonucleotides derived from bacteria that contain unmethylated CpG dinucleotides in a specific sequence context (CpG motif). These CpG motifs occur 20 times more frequently in bacterial DNA than in mammalian DNA. CpG ODNs are recognized by Toll-like receptor 9 (TLR9) to provide strong immunostimulatory effects and are widely available commercially. Specific examples of commercially available CpG ODNs include those with the sequence ODN2006 (SEQ ID NO:62, commercially available from Miltenyi Biotech as catalog number 130-100-106), a 24-mer having the sequence TIFF2024505964000005.tif4128; ODN2007, a 22-mer with sequence TIFF2024505964000006.tif4128 TIFF2024505964000007.tif4128, a 20-mer having the sequence ODN1826 listed above, or and ODN2216, a 20-mer with TIFF2024505964000008.tif4128. All three are commercially available from InvivoGen. As with natural DNA molecules, the bases are linked together by phosphodiester (PO) bonds. However, these bonds are susceptible to degradation by nucleases. When used as adjuvants without any protective elements, natural CpG molecules have a very short half-life in the body. To avoid this short half-life, the phosphodiester bond can be replaced with a phosphorothioate bond by changing one of the oxygen atoms to a sulfur atom. This substitution prevents degradation by nucleases and extends the half-life of the modified CpG. For example, the CpG molecules ODN2006, ODN2007, and ODN1826 are provided with a fully phosphorothioate backbone to make them nuclease-resistant. Alternatively, CpG is encapsulated in cationic liposomes to avoid degradation by nucleases. In addition to CpG, many other widely used Toll-like receptor agonists, such as polyinosinic:polycytidylic acid (poly(I:C)) (TLR3), lipopolysaccharide (LPS) (TLR4), and monophosphoryl lipid (MPL) (TLR5), can be used as one or more adjuvants in the present invention. Furthermore, components derived from bacterial or mycobacterial cell walls, such as those present in the Sigma Adjuvant System or Freund's adjuvant, or proteins such as keyhole limpet hemocyanin (KLH) are further examples of adjuvants that can also be used in the present invention. Further examples of suitable adjuvants that can be used in the present invention include the Sigma Adjuvant System (SAS), simethicone, or alpha-tocopherol. Other antigen-adjuvant pairs are also suitable for use in the methods of the present invention.

[0218] In this context, the term "adjuvant" as used herein is not limited to pharmacological or immunological agents that modify the effects of other agents (such as the adjuvants described above), but refers to "any substance that stimulates the action of the immune system." Thus, checkpoint inhibitors that stimulate the action of the immune system are also encompassed by the term adjuvant as used herein. For example, PD-L1 present on the cell surface binds to PD1 on the surface of immune cells, inhibiting immune cell activity. Thus, for example, an antibody that binds to either PD-1 or PD-L1 and blocks the interaction between PD1 and PD-L1 is a "positive checkpoint inhibitor" because it can enable T cells to attack tumors.

[0219] In some aspects, membrane proteins used as antigens in the present invention can include fragments or extracellular domains of transmembrane proteins. Antigens can also be (full-length) transmembrane proteins, G protein-coupled receptors, neurotransmitter receptors, kinases, porins, ABC transporters, ion transporters, acetylcholine receptors, and cell adhesion receptors. Membrane proteins can be fused or coupled to a tag, or can be tagless. When membrane proteins are tagged, the tag can be selected from well-known affinity tags, such as VSV, His-tag, Strep-tag®, Flag-tag, intein-tag, or GST-tag, or anti-affinity binding pair partners, such as biotin or avidin, or labels such as fluorescent labels, enzyme labels, NMR labels, or isotopic labels.

[0220] In some aspects, the membrane protein or a fragment (or portion) thereof may be prepared prior to encapsulation or may be encapsulated simultaneously with production of the protein in a cell-free expression system, which may be an in vitro transcription-translation system.

[0221] The cell-free expression system can also be a eukaryotic cell-free expression system, such as the TNT system based on rabbit reticulocytes, wheat germ extract or insect extract, a prokaryotic cell-free expression system or an archaeal cell-free expression system.

[0222] The antigens of the present disclosure, or fragments (or portions) thereof, can be produced in vivo. For example, the antigens or fragments (or portions) thereof can be produced in a bacterial or eukaryotic host organism and isolated from the host organism or a culture thereof. Antigens or fragments (or portions) thereof can also be produced in vitro, for example, by using an in vitro translation system. A preferred expression system is the baculovirus expression system. The use of baculovirus protein expression systems is often overlooked because it is perceived as slow and expensive. However, one major advantage of the baculovirus system is the ability to generate and maintain cell lines independent of the virus. This allows for the rapid production of new subunit antigens without the need for regulatory approval for new cell lines, a useful tool given the rapid sequence changes of viruses such as MERS-CoV and SARS-CoV-1. Furthermore, the baculovirus system produces antigens with novel glycosylation profiles compared to mammalian systems, which have been shown to enhance immune responses. For example, in the case of SARS-CoV-1 and MERS-CoV, the entire soluble (S1-S2) domain of the spike protein can be expressed in Sf9 cells. When immunized into Balb / c mice, these proteins, whether administered alone or with alum or Matrix M1 adjuvants, exhibit high virus-neutralizing titers, which can persist for at least 45 days. Thus, antigens of the present disclosure are preferably produced using eukaryotic host cells, preferably insect cells, such as Sf9 cells, or preferably using a baculovirus expression system.

[0223] As described above, polymersomes can be formed from amphiphilic diblock or triblock copolymers. In various aspects, the amphiphilic polymer can include at least one carboxylic acid, amide, amine, alkylene, dialkylsiloxane, ether, or alkylene sulfide monomer unit.

[0224] In some aspects, the amphiphilic polymer can be a polyether block selected from the group consisting of an oligo(oxyethylene) block, a poly(oxyethylene) block, an oligo(oxypropylene) block, a poly(oxypropylene) block, an oligo(oxybutylene) block, and a poly(oxybutylene) block. Further examples of blocks that can be included in the polymer include, but are not limited to, poly(acrylic acid), poly(methyl acrylate), polystyrene, poly(butadiene), poly(2-methyloxazoline), poly(dimethylsiloxane), poly(e-caprolactone), poly(propylene sulfide), poly(N-isopropylacrylamide), poly(2-vinylpyridine), poly(2-(diethylamino)ethyl methacrylate), poly(2-diisopropylamino)ethyl methacrylate), poly(2-methacryloyloxy)ethyl phosphorylcholine, poly(isoprene), poly(isobutylene), poly(ethylene-co-butylene), and poly(lactic acid). Examples of suitable amphiphilic polymers include poly(ethylethylene)-b-poly(ethylene oxide) (PEE-b-PEO), poly(butadiene)-b-poly(ethylene oxide) (PBD-b-PEO), poly(styrene)-b-poly(acrylic acid) (PS-PAA), poly(dimethylsiloxane)-poly(ethylene oxide) (herein referred to as PDMS-PEO), also known as poly(dimethylsiloxane-b-ethylene oxide), poly(dimethylsiloxane)-poly(acrylic acid) (PDMS-PAA), e.g., PMOXA used by May et al., 2013. 20 -PDMS 54 -PMOXA 20Examples of block copolymers include, but are not limited to, poly(2-methyloxazoline)-b-poly(dimethylsiloxane)-b-poly(2-methyloxazoline) (PMOXA-bPDMS-bPMOXA), poly(2-methyloxazoline)-b-poly(dimethylsiloxane)-b-poly(ethylene oxide) (PMOXA-b-PDMS-b-PEO), poly(ethylene oxide)-b-poly(propylene sulfide)-b-poly(ethylene oxide) (PEO-b-PPS-b-PEO), and poly(ethylene oxide)-poly(butylene oxide) block copolymers, including triblock copolymers such as (ABA). Block copolymers can be further specified by the average block length of each block contained in the copolymer. Thus, PB M PEO N indicates the presence of a polybutadiene block (PB) of length M and a polyethylene oxide (PEO) block of length N. M and N are independently selected integers, which may be selected, for example, within the range of about 6 to about 60. Thus, PB 35 PEO 18 indicates the presence of polybutadiene blocks of average length 35 and polyethylene oxide blocks of average length 18. In certain aspects, the PB-PEO diblock copolymer comprises 5-50 blocks PB and 5-50 blocks PEO. Similarly, PB 10 PEO 24 indicates the presence of polybutadiene blocks of average length 10 and polyethylene oxide blocks of average length 24. Specific examples of suitable PB-PEO diblock copolymers that can be used in the present invention include the diblock copolymer PBD 21 -PEO 14 (also commercially available) and [PBD] 21 -[PEO] 12 (See WO2014 / 077781A1 and Nallani et al., 2011). As a further example, E O B Pindicates the presence of an ethylene oxide block (E) of length O and a butadiene block (B) of length P. Thus, O and P are independently selected integers, for example, ranging from about 10 to about 120. Thus, E 16 E 22 indicates the presence of ethylene oxide blocks of average length 16 and butadiene blocks of average length 22.

[0225] Considering now another preferred block copolymer used to form the polymersomes of the present invention, poly(dimethylsiloxane-b-ethylene oxide) (PDMS-PEO), it is noted that both linear and comb-shaped PDMS-PEO can be used here (see Gaspard et al., "Mechanical Characterization of Hybrid Vesicles Based on Linear Poly(Dimethylsiloxane-b-Ethylene Oxide) and Poly(Butadiene-b-Ethylene Oxide) Block Copolymers," Sensors 2016, 16(3), 390, which describes polymersomes formed from PDMS-PEO).

[0226] The structure of linear PDMS-PEO is shown below as formula (I): The structure of the comb-shaped PDMS-PEO is shown as formula (II): TIFF2024505964000010.tif42128. PDMS according to structural formula (I) n -PEO m The term "polydimethylsiloxane" refers to the presence of a polydimethylsiloxane (PDMS) block of length n and a polyethylene oxide (PEO) block of length m, where m and n are independently selected integers and can each be selected, for example, in the range of about 5 or about 6 to about 100, about 5 to about 60, or about 6 to about 60, or about 5 to 50. For example, PDMS 12 -PEO 46 or PDMS 47 PEO36 Linear PDMS-PEO, such as PDMS-PEO, is commercially available from Polymer Source Inc., Dorval, Quebec (Montreal), Canada. Thus, PDMS-PEO block copolymers can include 5-100 block PDMS and 5-100 block PEO, 6-100 block PDMS and 6-100 block PEO, 5-100 block PDMS and 5-60 block PEO, or 5-60 block PDMS and 5-60 block PEO.

[0227] In accordance with the above, in one aspect, the present invention relates to a method for eliciting an immune response in a subject, the method comprising administering to the subject a polymersome formed from PDMS-PEO carrying an antigen. The antigen can be associated / physically linked to the PDMS-PEO polymersome in any suitable manner. For example, the PDMS-PEO polymersome can have a soluble antigen encapsulated therein, as described in the present invention. Alternatively or additionally, the polymersome can have an antigen incorporated / incorporated into the surrounding membrane of the polymersome, as described in WO 2014 / 077781 A1. In this case, the antigen is a membrane protein whose transmembrane domain(s) is incorporated into the surrounding membrane of the PDMS-PEO-polymersome. Incorporation can be achieved as described in WO 2014 / 077781 A1 or Nallani et al., "Proteopolymersomes: in vitro production of a membrane protein in polymersome membranes," Biointerphases, 1 December 2011, page 153. When an antigen is encapsulated in a PDMS-PEO polymersome, it can be a soluble antigen selected from the group consisting of polypeptides, carbohydrates, polynucleotides, and combinations thereof. The present invention further relates to a method for producing such encapsulated antigens in PDMS-PEO polymersomes and the polymersomes produced by said method.

[0228] The present invention further relates to compositions comprising PDMS-PEO polymersomes loaded with an antigen. Again, in these compositions, the antigen can be associated / physically linked to the PDMS-PEO polymersome in any suitable manner. For example, the PDMS-PEO polymersomes can have a soluble antigen encapsulated therein, as described herein. Alternatively or additionally, the polymersomes can have an antigen incorporated / incorporated into the surrounding membrane of the polymersome, as described in WO 2014 / 077781 A1. The present invention also relates to vaccines comprising such PDMS-PEO polymersomes loaded with an antigen, methods for eliciting an immune response, or methods for treating, ameliorating, preventing, or diagnosing cancer, autoimmune diseases, or infectious diseases, comprising administering the antigen-loaded PDMS-PEO polymersomes to a subject in need thereof.

[0229] In accordance with the above, the present invention also relates to the in vitro and in vivo use of PDMS-PEO polymersomes carrying (or transporting) antigens in a form suitable for eliciting an immune response. The antigen can be encapsulated in the PDMS-PEO polymersome or incorporated into the surrounding membrane of the polymersome, as described, for example, in WO 2014 / 077781 A1.

[0230] Another preferred block copolymer is poly(dimethylsiloxane)-poly(acrylic acid) (PDMS-PAA), which denotes the presence of poly(dimethylsiloxane) (PDMS) blocks of length M and poly(acrylic acid) (PAA) blocks of length N. M -PAA NM and N are independently selected integers, for example, selected from the range of about 5 to about 100, and represent the average length of the blocks. The PDMS-PAA preferably contains 5 to 100 blocks of PDMS and 5 to 100 blocks of PAA. The PDMS-PAA preferably contains 5 to 50, preferably 10 to 40 blocks of PDMS and / or 5 to 30, preferably 5 to 25, preferably 5 to 20 blocks of PAA. The PDMS-PAA preferably contains PDMS 30 -PAA 14 , PDMS 15 -PAA7 or PDMS 34 -PAA 16 is selected from the group consisting of:

[0231] In certain aspects, the polymersomes of the present invention may contain one or more compartments (also referred to as "multicompartments"). The compartmentalization of the polymersome vesicle structure allows for the coexistence of complex reaction pathways in living cells and serves to separate many activities spatially and temporally within the cell. Thus, two or more types of antigens may be encapsulated in the polymersomes of the present invention. Different antigens may have the same or different isoforms. Each compartment may be formed from the same or different amphiphilic polymers. In various aspects, two or more different antigens are incorporated into the surrounding membrane of the amphiphilic polymer. Each compartment may encapsulate at least one of a peptide, a protein, and a nucleic acid. The peptide, protein, polynucleotide, or carbohydrate may be immunogenic.

[0232] Further details of suitable multi-compartmental polymersomes can be found in WO20121018306, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0233] Additionally, polymersomes may be free-standing or immobilized on a surface, such as those described in WO2010 / 1123462, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0234] When a polymersome carrier contains two or more compartments, the compartments can include an outer block copolymer vesicle and at least one inner block copolymer vesicle encapsulated inside the outer block copolymer vesicle. In some aspects, the block copolymers of the outer and inner vesicles each include a polyether block, such as a poly(oxyethylene) block, a poly(oxypropylene) block, and a poly(oxybutylene) block. Further examples of blocks that can be included in the copolymer include, but are not limited to, poly(acrylic acid), poly(methyl acrylate), polystyrene, poly(butadiene), poly(2-methyloxazoline), poly(dimethylsiloxane), poly(L-isocyanoalanine(2-thiophen-3-yl-ethyl)amide), poly(e-caprolactone), poly(propylene sulfide), poly(N-isopropylacrylamide), poly(2-vinylpyridine), poly(2-(diethylamino)ethyl methacrylate), poly(2-(diisopropylamino)ethyl methacrylate), poly(2-(methacryloyloxy)ethyl phosphorylcholine), and poly(lactic acid).Examples of suitable outer and inner vesicles include poly(ethylethylene)-b-poly(ethylene oxide) (PEE-b-PEO), poly(butadiene)-b-poly(ethylene oxide) (PBD-b-PEO), poly(styrene)-b-poly(acrylic acid) (PS-b-PAA), poly(ethylene oxide)-poly(caprolactone) (PEO-b-PCL), poly(ethylene oxide)-poly(lactic acid) (PEO-b-PLA ), poly(isoprene)-poly(ethylene oxide) (PI-b-PEO), poly(2-vinylpyridine)-poly(ethylene oxide) (P2VP-b-PEO), poly(ethylene oxide)-poly(N-isopropylacrylamide) (PEO-b-PNIPAm), poly(ethylene glycol)-poly(propylene sulfide) (PEG-b-PPS), poly(dimethylsiloxane)-poly(acrylic acid) (PDMS-PAA), Poly(methylphenylsilane)-poly(ethylene oxide) (PMPS-b-PEO-b-PMPS-b-PEO-b-PMPS), poly(2-methyloxazoline)-b-poly-(dimethylsiloxane)-b-poly(2-methyloxazoline) (PMOXA-b-PDMS-b-PMOXA), poly(2-methyloxazoline)-b-poly(dimethylsiloxane)-b-poly(ethylene oxide) (PMOXA-b-PDMS-b- Examples of block copolymers include, but are not limited to, poly(ethylene oxide)-b-poly(propylene sulfide)-b-poly(ethylene oxide) (PEO-b-PPS-b-PEO), poly(ethylene oxide)-poly(butylene oxide) (PEO-b-PBO) block copolymers. Block copolymers can be further specified by the average number of each block contained in the copolymer. Thus, PS. M -PIAT Nindicates the presence of a polystyrene block (PS) having M repeating units and a poly(L-isocyanoalanine(2-thiophen-3-yl-ethyl)amide) (PIAT) block having N repeating units. M and N are independently selected integers, and may be selected, for example, within the range of about 5 to about 95. Therefore, PS 40 -PIAT 50 indicates the presence of PS blocks with an average of 40 repeat units and PIAT blocks with an average of 50 repeat units.

[0235] In some aspects, the polymersomes of the present disclosure contain lipids, preferably mixed with block copolymers or amphiphilic polymers. The lipid content is typically low compared to the amount of block copolymers or amphiphilic polymers. Typically, the lipid will comprise up to about 50%, up to about 45%, up to about 40%, up to about 35%, up to about 30%, up to about 20%, up to about 15%, up to about 10%, up to about 5%, up to about 2%, up to about 1%, up to about 0.5%, up to about 0.2%, or up to about 0.1% of the components forming the polymersome membrane (percentages are given by weight). The addition of lipids can improve encapsulation efficiency. The lipids can be synthetic lipids, natural lipids, lipid mixtures, or combinations of synthetic and natural lipids. Non-limiting examples of lipids include phospholipids, such as phosphatidylcholines, e.g., POPC, lecithin, cephalin, or phosphatidylinositol, or lipid mixtures containing phospholipids, such as soybean phospholipids, e.g., asolectin. Further non-limiting examples of lipids include cholesterol, cholesterol sulfate, and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). The lipid is preferably non-antigenic. In some aspects, the polymersomes of the present disclosure contain less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.2%, less than about 0.1%, or are essentially free of saponin (percentages given by weight).

[0236] In some aspects, the present invention relates to a method for producing an encapsulated antigen in a polymersome, the method comprising the steps of: (i) dissolving an amphiphilic polymer of the present invention in chloroform, wherein the amphiphilic polymer is preferably polybutadiene-polyethylene oxide (BD); (ii) drying the dissolved amphiphilic polymer to form a polymer film; (iii) adding a solubilized antigen to the dried amphiphilic polymer film of step (ii), wherein the antigen is selected from the group consisting of: (a) a polypeptide, preferably a polypeptide that is an antigen of the present invention; (b) a carbohydrate; (c) a combination of (a) and / or (b) and / or (c); (iv) rehydrating the polymer film of step (iii) to form polymer vesicles; (v) optionally filtering the polymer vesicles of step (iv) to purify the polymer vesicles into monodisperse vesicles; and / or (vi) optionally isolating the polymer vesicles of step (iv) or step (v) from non-encapsulated antigen.

[0237] In some other aspects, the present invention relates to other methods for producing encapsulated antigens in polymersomes, including methods based on mixing a non-aqueous solution of a polymer with an aqueous solution of an antigen, sonicating the corresponding polymer-antigen mixed solution, or extruding the corresponding polymer-antigen mixed solution. Exemplary methods include those described in Rameez et al., Langmuir 2009 and Neil et al., Langmuir 2009, 25(16), 9025-9029.

[0238] In some aspects, the present invention relates to methods of eliciting an immune response in a subject by the sole administration of one or more adjuvants associated with one or more polymersome populations.

[0239] In some aspects, a single administration is an administration characterized in that the antigen is not administered to the subject in combination with the one or more adjuvants.

[0240] In some aspects, the single administration of the present invention is one or more of the following: (i) a prophylactic single administration (e.g., for antiviral and / or immunomodulatory prophylaxis); (ii) a therapeutic single administration (e.g., for antiviral and / or immunomodulatory treatment); (iii) a single administration to reduce stress levels in a subject (e.g., to reduce stress levels during transportation, transport, and / or mixing of cattle with other animals); or (iv) any combination of (i)-(iii).

[0241] In some aspects, one or more adjuvants of the invention are independently associated with the same inventive polymersome population or different inventive polymersome populations.

[0242] In some aspects, one or more adjuvants of the present invention are independently associated with the one or more polymersome populations by one or more of the following means: (i) encapsulating the one or more adjuvants within the one or more polymersome populations; (ii) incorporating and / or embedding the one or more adjuvants in the surrounding membrane of the one or more polymersome populations; (iii) covalently conjugating the one or more adjuvants to the outer surface of the one or more polymersome populations; (iv) non-covalently conjugating the one or more adjuvants to the outer surface of the one or more polymersome populations; and / or (v) any combination of (i)-(iv).

[0243] In some aspects, the one or more adjuvants are independently selected from the group consisting of: (i) CpG oligodeoxynucleotides (i.e., CpG ODNs) described herein; and (ii) non-antigenic components derived from bacterial and mycobacterial cell walls and proteins.

[0244] In some aspects, the polymersome is an oxidatively stable polymersome.

[0245] In some aspects, the immune response comprises stimulating the production and / or secretion of one or more cytokines, preferably the immune response is an innate immune response.

[0246] In some aspects, the immune response comprises stimulating the production and / or secretion of one or more cytokines, including stimulating the production and / or secretion of interleukin-6 (IL-6), preferably wherein the production and / or secretion of interleukin-6 (IL-6) predominates over the production and / or secretion of interleukin-12 (IL-12), and more preferably wherein the production and / or secretion of interleukin-6 (IL-6) is not accompanied by the production and / or secretion of interleukin-12 (IL-12).

[0247] In some aspects, the single route of administration is selected from the group consisting of oral administration, intranasal administration, administration to a mucosal surface, inhalation, intradermal administration, intraperitoneal administration, subcutaneous administration, intravenous administration, and intramuscular administration.

[0248] In some aspects, the subject is selected from the group consisting of (i) a mammal, preferably wherein the mammal is a human, cat, dog, cow, goat, sheep, cow, or pig, and (ii) a non-mammal.

[0249] In some aspects, the methods of the invention can be used to treat a variety of diseases, including cancer (e.g., sarcoma, fibrosarcoma), atopic dermatitis, African swine fever, avian influenza, bovine spongiform encephalopathy, brucellosis, cattle tick, chronic wasting disease, swine fever, contagious equine metritis, equine herpesvirus, equine infectious anemia, equine piroplasmosis, equine viral arteritis, foot and mouth disease, Johne's disease, Mycoplasma ovipneumoniae, porcine epidemic diarrhea virus, pseudorabies, rabbit hemorrhagic disease virus, Schmallenberg virus, scrapie, spring viremia of carp, influenza A virus in swine, tuberculosis, vesicular stomatitis, West Nile virus, stress-related diseases (e.g., pasteurellosis, Mannheimia haemolyticus), and / or other diseases. and / or methods of prevention against a disease selected from the group consisting of: feline calicivirus, coronavirus, herpesvirus, canine parvovirus, porcine postweaning diarrhea, cattle, horse and / or kitten upper respiratory disease complex.

[0250] Compared to existing uptake and cross-presentation vehicles and methods based on them, the polymersomes of the present invention have, among others, the following advantages, which are also aspects of the present invention: The polymersomes are highly efficient in uptake and cross-presentation to the immune system. · CD8 immune response (+) including T cell-mediated immune responses, Polymersomes are oxidatively stable, A stronger humoral response compared to that generated by free antigen-based techniques with or without adjuvant; The immune response induced by the polymersomes of the present invention can be further boosted using adjuvants. The polymers of the polymersomes of the present invention are inherently robust and can be adapted or functionalized to increase circulation time in the body. The polymersomes of the present invention are stable in the presence of serum components. Polymersome polymers are inexpensive and rapidly synthesized. The amount of antigen required to induce an immune response by the method of the present invention is less compared to free antigen-based techniques with or without adjuvant. The method of eliciting an immune response in a subject by the sole administration of one or more adjuvants of the present invention allows for improved induction of an immune response in the absence of antigen (e.g., for antiviral and / or immunomodulatory prophylaxis and / or treatment). ·ACM encapsulation enhanced the biological function of CpG. ACM-CpG exhibited superior adjuvant activity compared to free CpG. ACM-CpG induced a broader cytokine profile than free CpG. ACM-CpG encapsulated in polymersomes of the present invention composed of BD21 (85 mol%) and DOTAP (15 mol%), when administered at a single dose of about 7.5 μg to about 12.5 μg of CpG per kg, generated significant immune responses, including therapeutic and / or prophylactic modalities, against highly infectious pathogens (e.g., bacteria and / or viruses, such as Calcici virus and / or FHV (feline herpesvirus)) in cats and dogs (see, e.g., Examples 23-24 herein).

[0251] The present invention is also characterized by the following items. 1. A polymersome (e.g., an oxidatively stable polymersome comprising a soluble encapsulated antigen), The soluble encapsulated antigen is (i) a polypeptide, (ii) carbohydrates; (iii) a polynucleotide that is preferably not an antisense oligonucleotide, and more preferably is a DNA molecule or an mRNA molecule; (iv) a combination of (i) and / or (ii) and / or (iii) selected from the group consisting of Polymersomes. 2. Polymersomes Conjugate with CD8 (+) Item 10. The polymersome of any one of the preceding items, which is capable of inducing a T cell-mediated immune response, preferably in vivo, ex vivo or in vitro. 3. The antigen comprises a soluble portion of a membrane protein (MP) or membrane-associated peptide (MAP), preferably the antigen comprises a soluble portion of influenza hemagglutinin, swine influenza hemagglutinin, ovalbumin (OVA), a spike protein such as a porcine epidemic diarrhea virus spike protein, a spike protein of a human pathogenic coronavirus such as a MERS-CoV spike protein, a SARS-CoV-2 spike protein, or a SARS-CoV-1 spike protein, a B16 peptide, or an MC38 peptide, more preferably the antigen is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NOs:12-14, SEQ ID NOs:43-46, and SEQ ID NOs:34-41, SEQ ID NOs: The polymersome of any one of the preceding items, comprising a polypeptide that is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to a polypeptide sequence selected from the group consisting of SEQ ID NO:48-51, SEQ ID NO:65. 4. The polymersome of any one of the preceding paragraphs, wherein the polymersome is stable in the presence of serum components, preferably said stability being in vivo, ex vivo or in vitro. 5. The polymersome of any one of the preceding paragraphs, wherein the polymersome is stable within an endosome, preferably said stability being in vivo, ex vivo or in vitro. 6. The polymersome of any one of the preceding paragraphs, wherein the polymersome has improved oxidative stability compared to the corresponding oxidative stability of a liposome, preferably wherein the improved stability is improved stability in vivo, ex vivo or in vitro. 7. Polymersomes release their contents, including soluble encapsulated antigens, in an oxidation-independent manner to target CD8 (+) Item 10. The polymersome of any one of the preceding items, which is capable of triggering a T cell-mediated immune response, preferably wherein said release is in vivo, ex vivo or in vitro release. 8. Polymersomes Conjugate with CD8 (+) 2. The polymersome of any one of the preceding items, which is capable of eliciting a cellular immune response, including a T cell-mediated immune response, preferably wherein the immune response is an in vivo, ex vivo or in vitro immune response. 9. The polymersome has the ability to induce a cellular and / or humoral immune response, and the cellular immune response is CD8 (+) The polymersome of any one of the preceding items, comprising a T cell mediated immune response, preferably wherein the immune response is an in vivo, ex vivo or in vitro immune response. 10. The polymersome according to any one of the preceding items, wherein the humoral immune response comprises the production of specific antibodies, more preferably said immune response is an in vivo, ex vivo or in vitro immune response. 11. Polymersomes bind to effector CD4 (+) The polymersome according to any one of the preceding items, which is capable of enhancing the frequency of T cells, preferably said enhancement being in vivo, ex vivo or in vitro. 12. The polymersome of any one of the preceding items, wherein the cellular immune response comprises a T cell-mediated immune response, preferably wherein the immune response is an in vivo, ex vivo or in vitro immune response. 13. Polymersomes increase antigen-specific CD8 activity compared to free antigen (+) The polymersome of any one of the preceding items, which is capable of enhancing clonal expansion of T cells, preferably wherein said expansion is in vivo, ex vivo or in vitro expansion. 14. Polymersomes bind antigen-specific effector CD8 (+) The polymersome according to any one of the preceding items, which is capable of inducing T cells, preferably said induction being in vivo, ex vivo or in vitro. 15. Polymersomes bind antigen-specific CD8 (+) Item 10. The polymersome of any one of the preceding items, which is capable of enhancing the cytotoxic phenotype of T cells, preferably said enhancement being in vivo, ex vivo or in vitro. 16. The polymersome of any one of the preceding items, wherein the polymersome is capable of targeting lymph node resident macrophages and / or B cells, preferably said targeting being in vivo, ex vivo or in vitro targeting. 17. A polymersome according to any one of the preceding items, wherein the polymersome is reduction-stable, preferably the polymersome is reduction-stable in the presence of serum components, and more preferably the reduction-stability is in vivo, ex vivo or in vitro. 18. A polymersome according to any one of the preceding items, wherein the polymersome has reduced permeability, preferably the reduced permeability is compared to the corresponding permeability of a liposome, and more preferably the permeability is in vivo, ex vivo or in vitro. 19. The polymersome according to any one of the preceding items, wherein the polymersome is capable of releasing its contents within an endosome, preferably the endosome is a late endosome, and more preferably the release is in vivo, ex vivo or in vitro. 20. The polymersome of any one of the preceding items, having one or more of the following capabilities: (i) the ability to induce a cellular immune response, preferably the cellular immune response being CD8 (+) and more preferably, the cellular immune response is a CD8 (+) and most preferably, the cellular immune response is a response to a soluble portion of influenza hemagglutinin, swine influenza hemagglutinin, ovalbumin (OVA), a spike protein such as a porcine epidemic diarrhea virus spike protein, a spike protein of a human pathogenic coronavirus such as a MERS-CoV spike protein, a SARS-CoV-2 spike protein, or a SARS-CoV-1 spike protein, a B16 peptide, or an MC38 peptide, and even most preferably, the cellular immune response is a response to a soluble portion of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NOs:12-14, SEQ ID NOs:43-46, SEQ ID NOs:34-41, SEQ ID NOs: the ability to elicit a cellular immune response that is in response to a polypeptide that is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to a polypeptide sequence selected from the group consisting of SEQ ID NOs:48-51, and SEQ ID NO:65; (ii) the ability to release polymersome contents within an endosome, preferably the endosome is a late endosome, more preferably the contents comprise influenza hemagglutinin, swine influenza hemagglutinin, ovalbumin (OVA), a spike protein, such as a porcine epidemic diarrhea virus spike protein, a spike protein of a human pathogenic coronavirus, such as a MERS-CoV spike protein, a SARS-CoV-2 spike protein, or a SARS-CoV-1 spike protein, a soluble portion of a B16 peptide or an MC38 peptide, and most preferably the contents are SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NOs:12-14, SEQ ID NOs:43-46, SEQ ID NOs: the ability to release polymersome contents within an endosome comprising a polypeptide that is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to a polypeptide sequence selected from the group consisting of SEQ ID NOs:34-41, SEQ ID NOs:48-51, and SEQ ID NO:65; (iii) Oxidation-independent release of polymersome contents to CD8 (+)and the ability to trigger a T cell-mediated immune response, preferably the content comprises a soluble portion of influenza hemagglutinin, swine influenza hemagglutinin, ovalbumin (OVA), a spike protein, such as a porcine epidemic diarrhea virus spike protein, a spike protein of a human pathogenic coronavirus, such as a MERS-CoV spike protein, a SARS-CoV-2 spike protein, or a SARS-CoV-1 spike protein, a B16 peptide, or an MC38 peptide, more preferably the content comprises a soluble portion of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12-14, SEQ ID NO:43-46, SEQ ID NO:34-41, SEQ ID NO: and SEQ ID NO: 48-51, and SEQ ID NO: 65. (+) the ability to trigger a T-cell-mediated immune response; (iv) the ability to stimulate an immune response to an antigen, preferably the antigen comprises a soluble portion of influenza hemagglutinin, swine influenza hemagglutinin, ovalbumin (OVA), a spike protein, such as a porcine epidemic diarrhea virus spike protein, a spike protein of a human pathogenic coronavirus, such as a MERS-CoV spike protein, a SARS-CoV-2 spike protein, or a SARS-CoV-1 spike protein, a B16 peptide, or an MC38 peptide, more preferably the antigen is SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NOs:12-14, SEQ ID NOs:43-46, SEQ ID NOs:34-41, SEQ ID NOs: the ability to stimulate an immune response to an antigen, including a polypeptide that is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to a polypeptide sequence selected from the group consisting of SEQ ID NOs:48-51, and SEQ ID NO:65; (v) CD8 (+)the ability to trigger cross-protection induced by a T cell-mediated immune response, preferably the response is a response to a soluble portion of influenza hemagglutinin, swine influenza hemagglutinin, a spike protein such as a porcine epidemic diarrhea virus spike protein, a spike protein of a human pathogenic coronavirus such as a MERS-CoV spike protein, a SARS-CoV-2 spike protein, or a SARS-CoV-1 spike protein, ovalbumin (OVA), a B16 peptide, or an MC38 peptide, more preferably the response is a response to a soluble portion of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NOs:12-14, SEQ ID NOs:43-46, SEQ ID NOs:34-41, SEQ ID NOs: and CD8 responses to a polypeptide that is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to a polypeptide sequence selected from the group consisting of SEQ ID NOs: 48-51, and SEQ ID NO: 65. (+) the ability to trigger cross-protection induced by T cell-mediated immune responses; (vi) the ability to deliver a peptide or protein to antigen-presenting cells (APCs), preferably the peptide or protein comprises or is derived from a soluble portion of influenza hemagglutinin, swine influenza hemagglutinin, ovalbumin (OVA), a spike protein, such as a porcine epidemic diarrhea virus spike protein, a spike protein of a human pathogenic coronavirus, such as a MERS-CoV spike protein, a SARS-CoV-2 spike protein, or a SARS-CoV-1 spike protein, a B16 peptide, or an MC38 peptide, more preferably the peptide or protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NOs:12-14, SEQ ID NOs:43-46, SEQ ID NOs: the ability to deliver a peptide or protein to antigen-presenting cells, comprising or derived from a polypeptide that is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to a polypeptide sequence selected from the group consisting of SEQ ID NOs: 34-41, 48-51, and 65; (vii) CD8 (+) T cell-mediated immune response and / or CD4 (+)and the ability to trigger an immune response, including a T cell-mediated immune response, preferably the response is a response to a soluble portion of influenza hemagglutinin, swine influenza hemagglutinin, ovalbumin (OVA), a spike protein, such as a porcine epidemic diarrhea virus spike protein, a spike protein of a human pathogenic coronavirus, such as a MERS-CoV spike protein, a SARS-CoV-2 spike protein, or a SARS-CoV-1 spike protein, a B16 peptide, or an MC38 peptide, and more preferably the response is a response to a soluble portion of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NOs:12-14, SEQ ID NOs:43-46, SEQ ID NOs:34-41, SEQ ID NOs: and CD8 responses to a polypeptide that is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to a polypeptide sequence selected from the group consisting of SEQ ID NOs: 48-51, and SEQ ID NO: 65. (+) T cell-mediated immune response and / or CD4 (+) the ability to trigger immune responses, including T-cell-mediated immune responses; (viii) the ability to stimulate an immune response in a subject, preferably the response is a response to a soluble portion of influenza hemagglutinin, swine influenza hemagglutinin, ovalbumin (OVA), a spike protein, such as a porcine epidemic diarrhea virus spike protein, a spike protein of a human pathogenic coronavirus, such as a MERS-CoV spike protein, a SARS-CoV-2 spike protein, or a SARS-CoV-1 spike protein, a B16 peptide, or an MC38 peptide, and more preferably the response is a response to a soluble portion of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NOs:12-14, SEQ ID NOs:43-46, SEQ ID NOs:34-41, SEQ ID NOs: the ability to stimulate an immune response in a subject to a polypeptide that is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to a polypeptide sequence selected from the group consisting of SEQ ID NOs:48-51, and SEQ ID NO:65; (ix) the ability to immunize a non-human animal, preferably the immunization is against influenza hemagglutinin, swine influenza hemagglutinin, ovalbumin (OVA), a spike protein, such as a porcine epidemic diarrhea virus spike protein, a spike protein of a human pathogenic coronavirus, such as a MERS-CoV spike protein, a SARS-CoV-2 spike protein, or a SARS-CoV-1 spike protein, a soluble portion of a B16 peptide or an MC38 peptide, and more preferably the immunization is against SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NOs:12-14, SEQ ID NOs:43-46, SEQ ID NOs:34-41, SEQ ID NOs: the ability to immunize a non-human animal against a polypeptide that is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to a polypeptide sequence selected from the group consisting of SEQ ID NOs:48-51, and SEQ ID NO:65; (x) the polymersome has an altered antigenicity compared to the corresponding antigenicity of the antigen without the polymersome, preferably the antigen is influenza hemagglutinin, swine influenza hemagglutinin, ovalbumin (OVA), a spike protein such as a porcine epidemic diarrhea virus spike protein, a spike protein of a human pathogenic coronavirus such as a MERS-CoV spike protein, a SARS-CoV-2 spike protein, or a SARS-CoV-1 spike protein, a soluble portion of a B16 peptide or an MC38 peptide, more preferably the antigen is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NOs:12-14, SEQ ID NOs:43-46, SEQ ID NOs: having altered antigenicity, which is a polypeptide that is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to a polypeptide sequence selected from the group consisting of SEQ ID NOs:34-41, SEQ ID NOs:48-51, and SEQ ID NO:65; (xi) the polymersome has altered immunogenicity compared to the corresponding immunogenicity of the antigen without the polymersome, preferably wherein the immunogen is influenza hemagglutinin, swine influenza hemagglutinin, ovalbumin (OVA), a spike protein such as a porcine epidemic diarrhea virus spike protein, a spike protein of a human pathogenic coronavirus such as a MERS-CoV spike protein, a SARS-CoV-2 spike protein, or a SARS-CoV-1 spike protein, a soluble portion of a B16 peptide or an MC38 peptide, more preferably wherein the immunogen is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NOs:12-14, SEQ ID NOs:43-46, SEQ ID NOs: and having altered immunogenicity, wherein the polypeptide is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to a polypeptide sequence selected from the group consisting of SEQ ID NOs:34-41, SEQ ID NOs:48-51, and SEQ ID NO:65. 21. The polymersome of any one of the preceding items, having one or more of the following properties: (i) the polymersome comprises an oxidatively stable membrane, and / or (ii) the polymersome is synthetic, and / or (iii) the polymersomes are free of unencapsulated antigen or are mixed with free unencapsulated antigen, and / or (iv) the polymersome comprises a membrane of an amphiphilic polymer, and / or (v) the polymersome comprises an amphiphilic synthetic block copolymer that forms a vesicle membrane; and / or (vi) the polymersome has a diameter of greater than 70 nm, preferably in the range of about 100 nm to about 1 μm, or about 120 nm to about 250 nm, or about 125 nm to about 250 nm, about 140 nm to about 240 nm, about 150 nm to about 235 nm, about 170 nm to about 230 nm, or about 220 nm to about 180 nm, or about 190 nm to about 210 nm; and / or (vii) the polymersome has a vesicular morphology; (viii) The polymersomes are self-assembling. 22. The polymersome according to item 21, wherein the polymersome is in the form of a polymersome aggregate, and the average diameter of the polymersome aggregate is in the range of about 100 nm to about 1 μm, or about 100 nm to about 750 nm, or about 100 nm to about 500 nm, or about 120 nm to about 250 nm, or about 125 nm to about 250 nm, about 140 nm to about 240 nm, about 150 nm to about 235 nm, about 170 nm to about 230 nm, or about 220 nm to about 180 nm, or about 190 nm to about 210 nm. 23. The polymersome of any one of the preceding items, wherein the antigen is an immunogen. 24. The polymersome of any one of the preceding items, wherein the antigen is selected from the group consisting of (i) an autoantigen, (ii) a non-autoantigen, (iii) a non-autoimmunogen, and (iv) an autoimmunogen. 25. The antigen is: (i) a polypeptide that is at least 80% or more (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to a viral polypeptide sequence, preferably wherein the viral polypeptide sequence is influenza hemagglutinin or swine influenza hemagglutinin, and more preferably wherein the viral polypeptide sequence is selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8; (ii) a polypeptide that is at least 80% or more (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to a bacterial polypeptide sequence; (iii) a polypeptide that is at least 80% or more (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to a mammalian or avian polypeptide sequence, preferably the mammalian or avian polypeptide sequence is at least 80% or more (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to ovalbumin (OVA), spike protein, B16 peptide, or MC38 peptide, more preferably the mammalian or avian polypeptide sequence is SEQ ID NO:4, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NOs:12-14, SEQ ID NOs:43-46, SEQ ID NOs: A polypeptide selected from the group consisting of SEQ ID NOs: 34-41, SEQ ID NOs: 48-51, and SEQ ID NO: 65. The polymersome of any one of the preceding items, selected from the group consisting of: 26. The polymersome of any one of the preceding items, wherein the mammalian polypeptide sequence is selected from the group consisting of human, rodent, rabbit and equine polypeptide sequences. 27. The polymersome of any one of the preceding items, wherein the antigen is an antibody or a fragment thereof. 28. The antigen is: (i) influenza hemagglutinin (HA), preferably selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8; (ii) swine influenza hemagglutinin (HA), preferably SEQ ID NO:6; (iii) ovalbumin (OVA), preferably SEQ ID NO:4; (iv) spike proteins, such as porcine epidemic diarrhea virus (PED) spike proteins, spike proteins of human pathogenic coronaviruses, such as MERS-CoV spike proteins, SARS-CoV-2 spike proteins, or SARS-CoV-1 spike proteins, preferably SEQ ID NOs: 12-14, 43-46, 34-41, 48-51, and 65; (v) B16 peptide, preferably selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; (vi) MC38 peptides, preferably selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3; (vii) B16 peptide and MC38 peptide, preferably the peptides are independently selected from the group: (i) SEQ ID NO: 1-3 and (ii) SEQ ID NO: 9-11; The polymersome of any one of the preceding items, selected from the group consisting of: 29. The polymersome of any one of the preceding items, selected from the group consisting of cationic, anionic and nonionic polymersomes, and mixtures thereof. 30. The polymersome according to any one of the preceding items, wherein the block copolymer or amphiphilic polymer is essentially non-immunogenic or essentially non-antigenic, preferably the block copolymer or amphiphilic polymer is non-immunogenic or non-antigenic. 31. The polymersome of any one of the preceding items, wherein the block copolymer or amphiphilic polymer is oxidatively stable. 32. The polymersome of any one of the preceding items, wherein the block copolymer or amphiphilic polymer is not an immunostimulant or adjuvant. 33. The polymersome of any one of the preceding items, wherein the amphiphilic polymer comprises a diblock or triblock (ABA or ABC) copolymer. 34. The polymersome of any one of the preceding items, wherein the amphiphilic polymer comprises the copolymer poly(N-vinylpyrrolidone)-b-PLA. 35. The polymersome of any one of the preceding items, wherein the amphiphilic polymer comprises at least one carboxylic acid, amide, amine, alkylene, dialkylsiloxane, ether, or alkylene sulfide monomer unit. 36. The polymersome of any one of the preceding items, wherein the amphiphilic polymer is a polyether block selected from the group consisting of an oligo(oxyethylene) block, a poly(oxyethylene) block, an oligo(oxypropylene) block, a poly(oxypropylene) block, an oligo(oxybutylene) block, and a poly(oxybutylene) block. 37. The polymersome of any one of the preceding items, wherein the amphiphilic polymer is a poly(butadiene)-poly(ethylene oxide) (PB-PEO) diblock copolymer. 38. The polymersome of any one of the preceding items, wherein the PB-PEO diblock copolymer comprises 5-50 block PB and 5-50 block PEO. 39. The polymersome of any one of the preceding items, wherein the amphiphilic polymer is a poly(dimethylsiloxane)-poly(ethylene oxide) (PDMS-PEO) diblock copolymer, preferably the PDMS-PEO diblock copolymer preferably comprising 5-100 blocks of PDMS and 5-100 blocks of PEO. 40. The polymersome of any one of the preceding items, wherein the polymersome can be composed of a block copolymer or amphiphilic polymer alone, or a block copolymer or amphiphilic polymer mixed with a lipid. 41. The polymersome of any one of the preceding items, wherein the lipid is composed of a synthetic lipid or a natural lipid, or a mixture of synthetic lipids or a mixture of natural lipids, or a combination of synthetic and natural lipids. 42. The polymersome of any one of the preceding items, wherein the amphiphilic polymer is a poly(lactide)-poly(ethylene oxide) / 1-palmitoyl-2-oleyl-sn-glycero-3-phospho-L-serine (PLA-PEO / POPC) copolymer, preferably wherein the PLA-PEO / POPC has a ratio of PLA-PEO to POPC (e.g., PLA-PEO / POPC) of 50:50 or more (e.g., 50 / 50 or 75 / 25 or 90 / 10). 43. The polymersome of any one of the preceding items, wherein the amphiphilic polymer is a poly(caprolactone)-poly(ethylene oxide) / 1-palmitoyl-2-oleyl-sn-glycero-3-phospho-L-serine (PCL-PEO / POPC) copolymer, preferably having a ratio of PCL-PEO to POPC (e.g., PCL-PEO / POPC) of 50:50 or more (e.g., 50 / 50 or 75 / 25 or 90 / 10). 44. The polymersome of any one of the preceding items, wherein the amphiphilic polymer is polybutadiene-polyethylene oxide (BD) or poly(dimethylsiloxane)-poly(ethylene oxide) (PDMS-PEO) diblock copolymer or poly(dimethylsiloxane)-poly(acrylic acid) (PDMS-PAA) diblock copolymer. 45. Polymersomes are made of diblock copolymers (PBD) 21 -PEO 14 (referred to herein as "BD21"), PDMS 47 -PEO 36 (PDMS-PEO) or triblock copolymer PMOXA 12 -PDMS 55 -PMOXA 12 The polymersome of any one of the preceding items, comprising: 46. ​​The polymersome of any one of the preceding items, comprising one or more compartments. 47. The polymersome of any one of the preceding items, wherein the polymersome comprises one or more compartments, each of which encapsulates at least one peptide, protein, and nucleic acid, preferably wherein at least one of the peptide, protein, and nucleic acid is immunogenic or antigenic, and more preferably wherein each of the one or more compartments is composed of the same or different amphiphilic polymers. 48. The polymersome comprises two or more compartments, the compartments comprising an outer block copolymer vesicle and at least one inner block copolymer vesicle, the at least one inner block copolymer vesicle being encapsulated within the outer block copolymer vesicle, and preferably the outer block copolymer vesicle is (i) poly[styrene-b-poly(L-isocyanoalanine(2-thiophen-3-yl-ethyl)amide)] (PS-PIAT), (ii) poly(butadiene)-poly(ethylene oxide) (PBD-PEO); (iii) poly(ethylene oxide)-poly(caprolactone) (PEO-PCL); (iv) poly(ethylethylene)-poly(ethylene oxide) (PEE-PEO); (v) poly(ethylene oxide)-poly(lactic acid) (PEO-PLA); (vi) poly(isoprene)-poly(ethylene oxide) (PI-PEO); (vii) poly(2-vinylpyridine)-poly(ethylene oxide) (P2VP-PEO); (viii) poly(ethylene oxide)-poly(N-isopropylacrylamide) (PEO-PNIPAm); (ix) poly(styrene)-poly(acrylic acid) (PS-PAA); (x) poly(ethylene glycol)-polypropylene sulfide (PEG-PPS), (xi) poly(2-methyloxazoline)-poly(dimethylsiloxane)-poly(2-methyloxazoline) (PMOXA-PDMS-PMOXA); (xii) poly(ethylene oxide)-poly(dimethylsiloxane)-poly(2-methyloxazoline) (PEO-PDMS-PMOXA); (xiii) poly(methylphenylsilane)-poly(ethylene oxide) (PMPS-PEO-PMPS-PEO-PMPS), and (xiv) Poly(dimethylsiloxane)-poly(acrylic acid) (PDMS-PAA) and more preferably, the at least one inner block copolymer vesicle is a polymersome formed from a copolymer independently selected from the group consisting of (xv) poly[styrene-b-poly(L-isocyanoalanine(2-thiophen-3-yl-ethyl)amide)] (PS-PIAT), (xvi) poly(butadiene)-poly(ethylene oxide) (PBD-PEO), (xvii) poly(ethylene oxide)-poly(caprolactone) (PEO-PCL); (xviii) poly(ethylethylene)-poly(ethylene oxide) (PEE-PEO); (xix) poly(ethylene oxide)-poly(lactic acid) (PEO-PLA), (xx) poly(isoprene)-poly(ethylene oxide) (PI-PEO), (xxi) poly(2-vinylpyridine)-poly(ethylene oxide) (P2VP-PEO), (xxii) poly(ethylene oxide)-poly(N-isopropylacrylamide) (PEO-PNIPAm), (xxiii) poly(styrene)-poly(acrylic acid) (PS-PAA); (xxiv) poly(ethylene glycol)-polypropylene sulfide) (PEG-PPS), (xxv) poly(2-methyloxazoline)-poly(dimethylsiloxane)-poly(2-methyloxazoline) (PMOXA-PDMS-PMOXA), (xxvi) poly(ethylene oxide)-poly(dimethylsiloxane)-poly(2-methyloxazoline) (PEO-PDMS-PMOXA), (xxvii) poly(methylphenylsilane)-poly(ethylene oxide) (PMPS-PEO-PMPS-PEO-PMPS), and (xxviii) Poly(dimethylsiloxane)-poly(acrylic acid) (PDMS-PAA) The polymersome according to any one of the preceding items, which is a polymersome formed from a copolymer independently selected from the group consisting of: 49. The polymersome of any one of the preceding items, comprising a lipid polymer. 50. The polymersome of any one of the preceding items, further comprising an encapsulated adjuvant. 51. A method for producing an encapsulated antigen in a polymersome, comprising the steps of: (i) dissolving an amphiphilic polymer in chloroform, preferably the amphiphilic polymer is polybutadiene-polyethylene oxide (BD); (ii) drying the dissolved amphiphilic polymer to form a polymer film; (iii) adding the solubilized antigen to the dry amphiphilic polymer film of step (ii), wherein the antigen is (a) a polypeptide, preferably the polypeptide antigen is as described in any one of the preceding items, more preferably the polypeptide antigen comprises influenza hemagglutinin, swine influenza hemagglutinin, ovalbumin (OVA), a spike protein such as a porcine epidemic diarrhea virus spike protein, a spike protein of a human pathogenic coronavirus such as a MERS-CoV spike protein, a SARS-CoV-2 spike protein, or a SARS-CoV-1 spike protein, a soluble portion of a B16 peptide or an MC38 peptide, most preferably the polypeptide antigen is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NOs:12-14, SEQ ID NOs:43-46, SEQ ID NOs:34-41, SEQ ID NOs: a polypeptide that is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to a polypeptide sequence selected from the group consisting of SEQ ID NOs:48-51, and SEQ ID NO:65; (b) carbohydrates, (c) a polynucleotide that is not an antisense oligonucleotide and is preferably a DNA molecule or an mRNA molecule; (d) a combination of (a) and / or (b) and / or (c) a step selected from the group consisting of: (iv) rehydrating the polymer film of step (iii) to form polymer vesicles; (v) optionally filtering the polymer vesicles of step (iv) to purify the polymer vesicles into monodisperse vesicles; and / or (vi) optionally isolating the polymer vesicles of step (iv) or step (v) from unencapsulated antigen. 52. A method for producing an encapsulated antigen in a polymersome according to any one of the preceding items, wherein the polymersome is a polymersome according to any one of the preceding items. 53. A polymersome produced by the method for producing an encapsulated antigen in a polymersome described in any one of the preceding items. 54. A composition comprising a polymersome according to any one of the preceding items. 55. The composition of any one of the preceding items, which is a pharmaceutical composition or a diagnostic composition. 56. The composition of any one of the preceding items, which is an immunogenic composition, an antigenic composition or an immunotherapeutic composition. 57. The composition of any one of the preceding items, further comprising one or more immunostimulants and / or one or more adjuvants. 58. The composition of any one of the preceding items, which is a vaccine. 59. The composition of any one of the preceding items, formulated for intradermal, intraperitoneal, intramuscular, subcutaneous, intravenous injection, or non-invasive administration to a mucosal surface. 60. An isolated antigen-presenting cell or hybridoma cell exposed to a polymersome or composition according to any one of the preceding items. 61. The antigen-presenting cell of any one of the preceding items, comprising a dendritic cell. 62. The antigen-presenting cell of any one of the preceding items, comprising a macrophage. 63. The antigen-presenting cell of any one of the preceding items, which comprises a B cell. 64. A vaccine comprising the polymersome, composition, antigen-presenting cell or hybridoma according to any one of the preceding items, and further comprising a pharmaceutically acceptable excipient or carrier. 65. (i) The antigen comprises influenza hemagglutinin (HA) and the vaccine is an influenza vaccine, preferably wherein the influenza hemagglutinin (HA) is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to a polypeptide selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8; (ii) the antigen comprises swine influenza hemagglutinin (HA) and the vaccine is a swine influenza vaccine, preferably the swine influenza hemagglutinin (HA) is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO:6; (iii) the antigen comprises ovalbumin (OVA) and the vaccine is a cancer vaccine, preferably wherein the ovalbumin (OVA) is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO:4; (iv) the antigen comprises a spike protein (PEDv S), and the vaccine is a PED vaccine, preferably wherein the porcine epidemic diarrhea virus spike protein (S protein) is at least 80% or more (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to the polypeptide of SEQ ID NO: 12-14; (v) the antigen comprises a B16 peptide, and the vaccine is a cancer vaccine, preferably the peptide is selected from the group consisting of SEQ ID NOs: 9 to 11; (vi) The antigen comprises an MC38 peptide, and the vaccine is a cancer vaccine, preferably the peptide is selected from the group consisting of SEQ ID NOs: 1 to 3; (vii) the antigen comprises a B16 peptide and an MC38 peptide, and the vaccine is a cancer vaccine, preferably the peptides are independently selected from the group: (i) SEQ ID NOs: 1-3 and (ii) SEQ ID NOs: 9-11; (viii) the antigen is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to a polypeptide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and the vaccine is a cancer vaccine; (ix) the antigen comprises a MERS-CoV spike protein, a SARS-CoV-2 spike protein, or a SARS-CoV-1 spike protein that is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to a polypeptide sequence selected from the group consisting of a spike protein of a human pathogenic coronavirus, such as SEQ ID NOs: 43-46, SEQ ID NOs: 34-41, SEQ ID NOs: 48-51, and SEQ ID NO: 65, and the vaccine is against a human pathogenic coronavirus, such as MERS-CoV, SARS-CoV-2, or SARS-CoV-1; The vaccine according to any one of the preceding items. 66. A kit comprising the polymersome, composition, antigen-presenting cell, hybridoma or vaccine of any one of the preceding items. 67. A method for inducing an immune response in a subject (e.g., a human), comprising the steps of: (i) providing to said subject the polymersome, composition, antigen-presenting cell, hybridoma or vaccine according to any one of the preceding items; (ii) administering the polymersome, composition, antigen-presenting cell, hybridoma or vaccine to the subject, preferably by intradermal, intraperitoneal, intramuscular, subcutaneous, intravenous injection, or non-invasive administration to a mucosal surface. 68. The method of eliciting an immune response according to any one of the preceding items, wherein the immune response is a broad immune response. 69. Immune response is CD8 (+) T cell-mediated immune response and / or CD4 (+) A method of eliciting an immune response according to any preceding item, comprising a T cell-mediated immune response. 70. A method for treating or preventing an infectious disease, cancer or autoimmune disease in a subject (e.g., a human) in need thereof, comprising administering to the subject a therapeutically effective amount of a polymersome, composition, antigen-presenting cell, hybridoma or vaccine described in any one of the preceding items, preferably wherein the infectious disease is a viral or bacterial infectious disease. 71. A method for immunizing a non-human animal, comprising the steps of: (i) providing a polymersome, composition, antigen-presenting cell, hybridoma, or vaccine according to any one of the preceding paragraphs; (ii) immunizing a non-human animal with the polymersome, composition, antigen-presenting cell, hybridoma, or vaccine. 72. A method for preparing an antibody, comprising the steps of: (i) immunizing a non-human mammal with the polymersome, composition, antigen-presenting cell, hybridoma or vaccine of any one of the preceding items; (ii) isolating the antibody obtained in step (i). 73. The method of any one of the preceding items, wherein the antibody is a monoclonal antibody (mAb). 74. A polymersome, composition, antigen-presenting cell, hybridoma or vaccine according to any one of the preceding items for use as a medicament. 75. The polymersome, composition, antigen-presenting cell, hybridoma, or vaccine of any preceding item for use in one or more of the following methods: (i) methods for discovering and / or screening and / or preparing antibodies; (ii) methods for discovering and / or screening and / or preparing vaccines; (iii) a method for producing or preparing an immunogenic or immunostimulatory composition; (iv) A method for targeted delivery of proteins and / or peptides, preferably the targeted delivery is targeted delivery of an antigenic protein and / or peptide according to any one of the preceding items, more preferably the antigenic protein and / or peptide comprises a soluble portion of a membrane protein (MP) or a membrane-associated peptide (MAP), most preferably the antigen comprises a soluble portion of influenza hemagglutinin, swine influenza hemagglutinin, ovalbumin (OVA), a spike protein such as porcine epidemic diarrhea virus spike protein, a spike protein of a human pathogenic coronavirus such as MERS-CoV spike protein, SARS-CoV-2 spike protein or SARS-CoV-1 spike protein, B16 peptide or MC38 peptide, even most preferably the antigen is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ a polypeptide sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NOs:12-14, SEQ ID NOs:43-46, SEQ ID NOs:34-41, SEQ ID NOs:48-51 and SEQ ID NO:65, wherein the polypeptide sequence is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to a polypeptide sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NOs:12-14, SEQ ID NOs:43-46, SEQ ID NOs:34-41, SEQ ID NOs:48-51 and SEQ ID NO:65, and most preferably wherein targeted delivery is performed in a subject; (v) A method for stimulating an immune response to an antigen, preferably the antigen is an antigen according to any one of the preceding paragraphs, more preferably the antigen comprises influenza hemagglutinin, swine influenza hemagglutinin, ovalbumin (OVA), a spike protein such as a porcine epidemic diarrhea virus spike protein, a spike protein of a human pathogenic coronavirus such as a MERS-CoV spike protein, a SARS-CoV-2 spike protein, or a SARS-CoV-1 spike protein, a soluble portion of a B16 peptide or an MC38 peptide, and most preferably the antigen is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NOs:12-14, SEQ ID NOs:43-46, SEQ ID NOs:34-41, SEQ ID NOs:35-42, SEQ ID NOs:36-37, SEQ ID NOs:38-43, SEQ ID NOs:39-44, SEQ ID NOs:45-46, SEQ ID NOs:47-48, SEQ ID NOs:49-50, SEQ ID NOs:51-52, SEQ ID NOs:53-54, SEQ ID NOs:55-56, SEQ ID NOs:57-58, SEQ ID NOs:59-60, SEQ ID NOs:61-62, SEQ ID NOs:63-64, SEQ ID NOs:64-65, SEQ ID NOs:65-66, SEQ ID NOs:66-67, SEQ ID NOs:67-70, SEQ ID NOs:68-71, SEQ ID NOs:69-72, a polypeptide sequence that is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to a polypeptide sequence selected from the group consisting of SEQ ID NOs: 48-51, and SEQ ID NO: 65, and most preferably for use in stimulating an immune response to said antigen in a subject; (vi) CD8 (+) A method for triggering cross-protection induced by a T cell-mediated immune response, preferably a CD8 against an antigen according to any one of the preceding items. (+)A method for triggering cross-protection induced by a T cell-mediated immune response, further preferably wherein the antigen comprises a soluble portion of influenza hemagglutinin, swine influenza hemagglutinin, ovalbumin (OVA), a spike protein, such as a porcine epidemic diarrhea virus spike protein, a spike protein of a human pathogenic coronavirus, such as a MERS-CoV spike protein, a SARS-CoV-2 spike protein, or a SARS-CoV-1 spike protein, a B16 peptide, or an MC38 peptide, and most preferably wherein the antigen is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NOs:12-14, SEQ ID NOs:43-46, SEQ ID NOs:34-41, SEQ ID NOs: a polypeptide sequence selected from the group consisting of SEQ ID NOs:48-51, and SEQ ID NO:65, which is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to a polypeptide sequence selected from the group consisting of SEQ ID NOs:48-51, and SEQ ID NO:65; (vii) A method for delivering a peptide and / or protein to an antigen-presenting cell (APC) according to any one of the preceding paragraphs, wherein preferably the peptide and / or protein is an antigen according to any one of the preceding paragraphs, more preferably the antigen comprises influenza hemagglutinin, swine influenza hemagglutinin, ovalbumin (OVA), a spike protein, such as a porcine epidemic diarrhea virus spike protein, a spike protein of a human pathogenic coronavirus, such as a MERS-CoV spike protein, a SARS-CoV-2 spike protein, or a SARS-CoV-1 spike protein, a soluble portion of a B16 peptide or an MC38 peptide, and most preferably the antigen is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12-14, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, a polypeptide sequence selected from the group consisting of SEQ ID NOs:43-46, SEQ ID NOs:34-41, SEQ ID NOs:48-51, and SEQ ID NO:65, which is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to a polypeptide sequence selected from the group consisting of SEQ ID NOs:43-46, SEQ ID NOs:34-41, SEQ ID NOs:48-51, and SEQ ID NO:65; (viii) CD8 (+) T cell-mediated immune response and / or CD4 (+)10. A method for triggering an immune response, including a T cell-mediated immune response, preferably wherein the response is a response to an antigen according to any one of the preceding items, more preferably wherein the antigen comprises influenza hemagglutinin, swine influenza hemagglutinin, ovalbumin (OVA), a spike protein, such as a porcine epidemic diarrhea virus spike protein, a spike protein of a human pathogenic coronavirus, such as a MERS-CoV spike protein, a SARS-CoV-2 spike protein, or a SARS-CoV-1 spike protein, a soluble portion of a B16 peptide or an MC38 peptide, more preferably wherein the response is a response to an antigen selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NOs:12-14, SEQ ID NOs:43-46, SEQ ID NOs: a method in which the response is to an antigen that is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to a polypeptide sequence selected from the group consisting of SEQ ID NOs:34-41, SEQ ID NOs:48-51, and SEQ ID NO:65; (ix) A method for treating, ameliorating, preventing, or diagnosing an infectious disease, preferably wherein the infectious disease is a viral infectious disease or a bacterial infectious disease, more preferably wherein the viral infectious disease is selected from the group consisting of influenza infection, respiratory syncytial virus infection, and herpes virus infection; (x) a method for the treatment, amelioration, prevention or diagnosis of cancer or autoimmune disease; (xi) a method for sensitizing cancer cells to chemotherapy; (xii) a method for inducing apoptosis in cancer cells; (xiii) a method for stimulating an immune response in a subject; (xiv) methods for immunizing non-human animals; (xv) methods for preparing hybridomas; (xvi) the method according to any one of the preceding items, (xvii) The method according to any one of (i) to (xvi), which is an in vivo and / or ex vivo and / or in vitro method; (xviii) The method according to any one of (i) to (xvii) above, wherein the antigen is heterologous to the environment in which the antigen is used. 76. Use of the polymersome, composition, antigen-presenting cell, hybridoma or vaccine according to any one of the preceding items for one or more of the following: (i) To discover and / or screen and / or prepare antibodies; (ii) to discover and / or screen and / or prepare vaccines; (iii) to produce or prepare an immunogenic or immunostimulatory composition; (iv) for targeted delivery of proteins and / or peptides, preferably targeted delivery of antigenic proteins and / or peptides, more preferably targeted delivery is carried out in a subject; (v) for stimulating an immune response to an antigen, preferably for use in stimulating an immune response to an antigen in a subject; (vi) CD8 (+) To trigger cross-protection induced by T cell-mediated immune responses, (vii) for delivery of peptides or proteins to antigen-presenting cells (APCs), preferably wherein the peptide or protein is an antigen, and more preferably wherein the peptide or protein is immunogenic or immunotherapeutic; (viii) CD8 (+) T cell-mediated immune response and / or CD4 (+) To trigger immune responses, including T cell-mediated immune responses, (ix) A method for treating, ameliorating, preventing, or diagnosing an infectious disease, wherein the infectious disease is preferably a viral infectious disease or a bacterial infectious disease, and more preferably the viral infectious disease is selected from the group consisting of influenza infection, respiratory syncytial virus infection, and herpes virus infection, (x) for the treatment, amelioration, prevention, or diagnosis of cancer or autoimmune disease; (xi) to sensitize cancer cells to chemotherapy; (xii) for the induction of apoptosis in cancer cells; (xiii) to stimulate an immune response in a subject; (xiv) for immunizing non-human animals; (xv) for the preparation of hybridomas, (xvi) In the method according to any one of the preceding items, (xvii) For the use according to any one of (i) to (xvi), which is in vivo and / or ex vivo and / or in vitro use, (xviii) For the use according to any one of (i) to (xvii) above, wherein the antigen is heterologous to the environment in which the antigen is used. 77. A method for inducing an immune response in a subject, comprising administering to the subject polymersomes formed from PDMS-PEO carrying an antigen. 78. The method of item 77, wherein the antigen is encapsulated within a PDMS-PEO polymersome. 79. The method according to item 78, wherein the antigen encapsulated in the PDMS-PEO polymersome is a soluble antigen. 80. The method of item 79, wherein the antigen is selected from the group consisting of a polypeptide, a carbohydrate, a polynucleotide, and a combination thereof. 81. The method of item 77, wherein the antigen is incorporated into the surrounding membrane of the PDMS-PEO polymersome. 82. Antigen-loaded PDMS-PEO polymersomes. 83. The polymersome according to item 82, wherein the antigen is encapsulated within the PDMS-PEO polymersome. 84. The polymersome according to item 83, wherein the antigen encapsulated in the PDMS-PEO polymersome is a soluble antigen. 85. The polymersome according to item 84, wherein the antigen is selected from the group consisting of polypeptides, carbohydrates, polynucleotides and combinations thereof. 86. The polymersome according to item 85, wherein the antigen is incorporated into the surrounding membrane of the PDMS-PEO polymersome. 87. The polymersome according to item 86, wherein the antigen is a membrane-bound protein or lipid antigen. 88. The polymersome according to item 87, wherein the membrane-bound protein is selected from the group consisting of transmembrane proteins, G protein-coupled receptors, neurotransmitter receptors, kinases, porins, ABC transporters, ion transporters, acetylcholine receptors and cell adhesion receptors. 89. A pharmaceutical composition comprising a polymersome according to any one of items 82 to 88. 90. Use in vitro and in vivo of PDMS-PEO according to any one of items 82 to 88 or the pharmaceutical composition according to item 89 for inducing an immune response. 91. To induce an immune response, (i) a polypeptide, (ii) carbohydrates; (iii) a polynucleotide that is preferably not an antisense oligonucleotide, and more preferably is a DNA molecule or an mRNA molecule; or (iv) a combination of (i) and / or (ii) and / or (iii) 51. Use of a polymersome having a diameter of about 120 nm or more, preferably a polymersome according to any one of items 1 to 50, comprising a soluble encapsulated antigen selected from the group consisting of: 92. The use according to item 91, wherein the diameter of the polymersome is in the range of about 120 nm to about 1 μm, or about 140 nm to about 750 nm, or about 120 nm to about 500 nm, or about 140 nm to about 250 nm, about 120 nm to about 240 nm, about 150 nm to about 235 nm, about 170 nm to about 230 nm, or about 220 nm to about 180 nm, or about 190 nm to about 210 nm. 93. To induce an immune response (i) a polypeptide, (ii) carbohydrates; (iii) a polynucleotide that is preferably not an antisense oligonucleotide, and more preferably is a DNA molecule or an mRNA molecule; or (iv) a combination of (i) and / or (ii) and / or (iii) 51. Use of an ensemble of polymersomes having an average diameter of about 120 nm or more, preferably an ensemble of polymersomes as defined in any one of items 1 to 50, comprising a soluble encapsulated antigen selected from the group consisting of: 94. The use according to item 93, wherein the average diameter of the polymersome aggregates is in the range of about 120 nm to about 1 μm, or about 120 nm to about 750 nm, or about 120 nm to about 500 nm, or about 120 nm to about 250 nm, about 120 nm to about 240 nm, about 150 nm to about 235 nm, about 170 nm to about 230 nm, or about 220 nm to about 180 nm, or about 190 nm to about 210 nm. 95. The use of any one of items 91 to 94, wherein the subject is vaccinated against a viral infection. 96. The use according to any one of items 91 to 95, wherein the polymersome or polymersome aggregate is administered by a route of administration selected from the group consisting of oral administration, intranasal administration, administration to a mucosal surface, inhalation, intradermal administration, intraperitoneal administration, subcutaneous administration, intravenous administration and intramuscular administration. 97. The use according to any one of items 91 to 96, wherein the subject is a mammal, including a human, or a non-mammal. 98. The use according to item 97, wherein the subject is a mammal and is to be vaccinated against a disease selected from the group consisting of cancer, viral infections, and bacterial infections. 99. The use according to item 98, wherein the subject is a human and is to be vaccinated against a coronavirus infection. 100. The use according to item 99, wherein the coronavirus is pathogenic to humans. 101. The use according to item 99 or 100, wherein the coronavirus is a beta-coronavirus. 102. The use according to any one of items 99 to 101, wherein the coronavirus is a sarbecovirus or a merbecovirus. 103. 20. The use according to item 99, wherein the coronavirus is MERS-CoV, SARS-CoV-2 or SARS-CoV-1. 104. The use according to item 97, wherein the subject is a non-mammalian animal and is to be vaccinated against a disease selected from the group consisting of viral infections and bacterial infections. 105. The use according to item 104, wherein the non-mammalian animal is a bird (e.g., a poultry such as a chicken, duck, goose, or turkey), a fish, or a crustacean. 106. The use according to item 105, wherein the bird is a chicken, duck, goose or turkey. 107. The use according to item 105, wherein the fish is salmon, trout or tilapia. 108. The use according to item 105, wherein the crustacean is a shrimp, a prawn, or a crab. 109. The use according to item 97, wherein the mammal is a goat, sheep, cow, or pig. 110. The use according to item 109, wherein the animal is a pig and is vaccinated against porcine epidemic diarrhea virus. 111. The use according to item 109, wherein the animal is an ungulate and is vaccinated against foot-and-mouth disease virus. 112. Polymersomes are diblock copolymers (PBD) 21 -PEO 14The polymersome of any one of the preceding items, comprising (BD21) and DOTAP (e.g., BD21 at an 85 molar concentration) and DOTAP (at a 15 molar concentration), wherein the polymersome encapsulates at least one adjuvant, such as a CpG oligonucleotide. 113. (i) the at least one adjuvant is independently associated with the same or a different polymersome population, preferably the at least one adjuvant is a mixture of adjuvants, more preferably the at least one adjuvant is a CpG oligonucleotide, such as one selected from the same or a different CpG class, e.g., a CpG oligonucleotide having a sequence as set forth in SEQ ID NO: 18, 62-64, and 67-77, or any other suitable CpG ODN described herein, or a CpG selected from CpG classes A, B, and C, and / or (ii) the CpG oligodeoxynucleotide is selected from the group consisting of CpG-A ODN, CpG-B ODN, and CpG-C ODN (e.g., those depicted in Figure 31 herein), more preferably the CpG-A The polymersome of any one of the preceding items, wherein the ODN is capable of primarily inducing IFN-α production from plasmacytoid dendritic cells (pDCs) rather than stimulating TLR9-dependent NF-κB signaling and inflammatory cytokine (e.g., IL-6) production, and / or the CpG-B ODN is capable of primarily activating B cells and TLR9-dependent NF-κB signaling rather than stimulating IFN-α secretion, and / or the CpG-C ODN is capable of (a) primarily inducing IFN-α production from plasmacytoid dendritic cells (pDCs) rather than stimulating TLR9-dependent NF-κB signaling and inflammatory cytokine (e.g., IL-6) production, and (b) primarily activating B cells and TLR9-dependent NF-κB signaling rather than stimulating IFN-α secretion. 114. A polymersome according to any one of the preceding items for use as a medicament and / or in therapy. 115. Cancer (e.g., sarcoma, fibrosarcoma), atopic dermatitis, African swine fever, avian influenza, bovine spongiform encephalopathy, brucellosis, cow tick, chronic wasting disease, swine fever, contagious equine metritis, equine herpesvirus, equine infectious anemia, equine piroplasmosis, equine viral arteritis, foot-and-mouth disease, Johne's disease, Mycoplasma obineumoniae, porcine epidemic diarrhea virus, pseudorabies, rabbit hemorrhagic disease virus, Schmallenberg virus, scrapie, spring viremia of carp, influenza A virus in pigs, tuberculosis, vesicular stomatitis, West Nile virus, stress-related diseases (e.g., pasteurellosis, Mannheimia haemolytica and coccidiosis) 10. The polymersome of any one of the preceding items for use in treatment and / or prophylaxis against a disease selected from the group consisting of feline upper respiratory infection (URI), feline upper respiratory tract disease (FeURTD), feline herpesvirus (FHV), canine parvovirus (CPV), feline urinary incontinence virus (HUV), feline urinary incontinence virus (HUV), feline urinary incontinence virus (FH ... 116. A polymersome according to any one of the preceding items for use in inducing an immune response in a subject, the use comprising administering the polymersome alone to a subject in need thereof. 117. The polymersome of any one of the preceding items, wherein the single administration comprises one or more administrations of polymersomes encapsulating CpG oligonucleotides, wherein the CpG oligonucleotides are administered at a single dose of about 7.5 to about 12.5 μg of CpG per kg of subject body weight (e.g., per administration). 118. A polymersome according to any one of the preceding items, wherein the single administration comprises one or more administrations of a polymersome encapsulating at least one CpG oligonucleotide, and wherein a single dose of the CpG oligonucleotide (e.g., per administration) is from about 7.5 μg to about 12.5 μg of CpG per kg of body weight of the subject. 119. A method for inducing an immune response (e.g., an innate immune response) in a subject by the sole administration of one or more adjuvants (e.g., at least one adjuvant), wherein the one or more adjuvants (e.g., at least one adjuvant) are associated with one or more polymersome populations. 120. The method of any one of the preceding items, wherein the single administration is an administration in which the antigen is not administered (e.g., co-administered) to the subject in combination (e.g., simultaneously or at different times) with the one or more adjuvants (e.g., at least one adjuvant). 121. Single administration of: (i) prophylactic administration, preferably for antiviral and / or immunomodulatory prophylaxis; (ii) therapeutic administration, preferably for antiviral and / or immunomodulatory treatment; (iii) administration to reduce stress levels in a subject (e.g., to reduce stress levels during transportation and / or transport of cattle and / or when mixing cattle with other animals); (iv) Any combination of (i) to (iii) The method of any one of the preceding items, wherein the method is one or more of: 122. (i) the one or more adjuvants (e.g., at least one adjuvant) are independently associated with the same or different polymersome populations, preferably the one or more adjuvants are different adjuvants, and more preferably the different adjuvants are different CpGs selected from the same or different CpG classes, e.g., CpGs having SEQ ID NO: 18, 62-64, and 67-77, or any other suitable CpG ODN described herein, or CpGs selected from CpG classes A, B, and C; and / or (ii) the CpG oligodeoxynucleotide is selected from the group consisting of CpG-A ODN, CpG-B ODN, and CpG-C ODN (e.g., those depicted in Figure 31 herein), and more preferably, CpG-A ODN can primarily induce IFN-α production from plasmacytoid dendritic cells (pDCs) rather than stimulating TLR9-dependent NF-κB signaling and inflammatory cytokine (e.g., IL-6) production, and / or CpG-B ODN can primarily activate B cells and TLR9-dependent NF-κB signaling rather than stimulating IFN-α secretion, and / or CpG-C ODN can primarily activate B cells and TLR9-dependent NF-κB signaling rather than stimulating IFN-α secretion. ODNs can (a) primarily induce IFN-α production from plasmacytoid dendritic cells (pDCs) rather than stimulating TLR9-dependent NF-κB signaling and inflammatory cytokine (e.g., IL-6) production, and (b) primarily activate B cells and TLR9-dependent NF-κB signaling rather than stimulating IFN-α secretion. The method described in any one of the preceding items. 123. The method of any preceding paragraph, wherein the one or more adjuvants (e.g., at least one adjuvant) are independently associated with the one or more polymersome populations by one or more of the following means: (i) encapsulating the one or more adjuvants (e.g., at least one adjuvant) within the one or more polymersome populations; (ii) incorporating and / or embedding the one or more adjuvants (e.g., at least one adjuvant) in the surrounding membrane of the one or more polymersome populations; (iii) covalently conjugating the one or more adjuvants (e.g., at least one adjuvant) to the outer surface of the one or more polymersome populations; (iv) non-covalently conjugating the one or more adjuvants (e.g., at least one adjuvant) to the outer surface of the one or more polymersome populations; and / or (v) Any combination of (i) to (iv). 124. The one or more adjuvants (e.g., at least one adjuvant): (i) CpG oligodeoxynucleotides (i.e., CpG ODNs, such as any suitable CpG ODNs described herein), preferably CpG classes A, B, and C, more preferably CpGs having SEQ ID NOs: 18, 62-64, and 67-77; (ii) non-antigenic components derived from bacterial and mycobacterial cell walls and proteins The method of any one of the preceding items, wherein the medicament is independently selected from the group consisting of: 125. The method of any one of the preceding items, wherein the polymersome is an oxidatively stable polymersome. 126. The method of any one of the preceding paragraphs, wherein the immune response comprises stimulating the production and / or secretion of one or more cytokines. 127. The method of any one of the preceding items, wherein the immune response comprises stimulating the production and / or secretion of one or more cytokines, including stimulating the production and / or secretion of interleukin-6 (IL-6, e.g., UniProtKB-P08505), preferably wherein the production and / or secretion of interleukin-6 (IL-6) predominates over the production and / or secretion of interleukin-12 (IL-12, e.g., UniProtKB-P43431 and / or Q3ZAX5), and more preferably wherein the production and / or secretion of interleukin-6 (IL-6) is not accompanied by the production and / or secretion of interleukin-12 (IL-12). 128. The method of any one of the preceding items, wherein the single route of administration is selected from the group consisting of oral administration, intranasal administration, administration to a mucosal surface, inhalation, intradermal administration, intraperitoneal administration, subcutaneous administration, intravenous administration, and intramuscular administration. 129. The method of any one of the preceding items, wherein the subject is a mammal, preferably a human, cat (e.g., in combination with CpGs of SEQ ID NO:74, 75, 76 or 77), dog (e.g., in combination with CpGs of SEQ ID NO:74, 75, 76 or 77), cow, goat, sheep (e.g., in combination with CpGs of SEQ ID NO:74, 75, 76 or 77), dairy cow, pig, horse (e.g., in combination with CpGs of SEQ ID NO:75, 76 or 77), or non-mammalian animal (other suitable CpGs are known in the art, for example, from Rankin et al., 2001 or Figure 32 herein). 130. Cancer (e.g., sarcoma, fibrosarcoma), atopic dermatitis, African swine fever, avian influenza, bovine spongiform encephalopathy, brucellosis, cow tick, chronic wasting disease, swine fever, contagious equine metritis, equine herpesvirus, equine infectious anemia, equine piroplasmosis, equine viral arteritis, foot-and-mouth disease, Johne's disease, Mycoplasma obineumoniae, porcine epidemic diarrhea virus, pseudorabies, rabbit hemorrhagic disease virus, Schmallenberg virus, scrapie, spring viremia of carp, influenza A virus in swine, tuberculosis, vesicular stomatitis, West Nile virus, stress-related diseases (e.g., The method of any one of the preceding paragraphs is a method for treating and / or preventing a disease selected from the group consisting of: viral diseases (e.g., antiviral treatment or prevention), immune diseases (e.g., immunomodulatory treatment or prevention), feline calicivirus, coronavirus, herpesvirus, canine parvovirus, porcine postweaning diarrhea, bovine, equine and / or kitten upper respiratory disease syndrome, feline upper respiratory infection (URI), feline upper respiratory tract disease (FeURTD), feline herpesvirus (FHV), canine parvovirus (CPV). 131. The method of any one of the preceding paragraphs, wherein the polymersome has one or more of the following properties: i) comprising an oxidation-stable film; ii) is synthetic; iii) free of encapsulated and associated antigens; iv) comprising a film of an amphiphilic polymer; and / or v) comprising an amphiphilic synthetic block copolymer that forms a vesicle membrane; and / or vi) have a diameter of more than 50 nm (e.g., more than 70 nm), preferably the diameter is in the range of about 100 nm to about 1 μm, or about 100 nm to about 750 nm, or about 100 nm to about 500 nm, or about 125 nm to about 250 nm, about 140 nm to about 240 nm, about 150 nm to about 235 nm, about 170 nm to about 230 nm, or about 220 nm to about 180 nm, or about 190 nm to about 210 nm, and most preferably the diameter is about 200 nm; and / or vii) having a vesicular form; viii) is self-assembling; ix) essentially non-immunogenic or essentially non-antigenic, preferably the block copolymer or amphiphilic polymer is non-immunogenic or non-antigenic. 132. (i) the one or more polymersome populations comprise or are formed from amphiphilic polymers, including or consisting of diblock or triblock (ABA or ABC) copolymers; and / or (ii) the one or more polymersome populations comprise a lipid polymer; The method described in any one of the preceding items. 133. (a) The amphiphilic polymer comprises the copolymer poly(N-vinylpyrrolidone)-b-PLA; (b) the amphiphilic polymer is a poly(butadiene)-poly(ethylene oxide) (PB-PEO) diblock copolymer, or the amphiphilic polymer is a poly(dimethylsiloxane)-poly(ethylene oxide) (PDMS-PEO) diblock copolymer or a poly(dimethylsiloxane)-poly(acrylic acid) (PDMS-PAA), and the PB-PEO diblock copolymer preferably comprises 5-50 blocks PB and 5-50 blocks PEO, or the PB-PDMS diblock copolymer preferably comprises 5-100 blocks PDMS and 5-100 blocks PEO; (c) the amphiphilic polymer is a poly(lactide)-poly(ethylene oxide) / 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (PLA-PEO / POPC) copolymer, preferably having a ratio of PLA-PEO to POPC (e.g., PLA-PEO / POPC) of 75 to 25 (e.g., 75 / 25); (d) the amphiphilic polymer is a poly(caprolactone)-poly(ethylene oxide) / 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (PCL-PEO / POPC) copolymer, preferably having a ratio of PCL-PEO to POPC (e.g., PCL-PEO / POPC) of 75 to 25 (e.g., 75 / 25); (e) the amphiphilic polymer is polybutadiene-polyethylene oxide (BD), and / or (f) the first and / or second population of polymersomes are polymerized with a diblock copolymer PBD 21 -PEO 14 (BD21) and / or triblock copolymer PMOXA 12 -PDMS 55 -PMOXA 12 Including, The method described in any one of the preceding items. 134. (g) The first and / or second population of polymersomes comprises a diblock copolymer PBD. 21 -PEO 14 (BD21) and DOTAP (e.g., BD21 (85 mol%) and DOTAP (15 mol%). The method of any preceding item, wherein the single administration comprises one or more administrations of polymersomes encapsulating a CpG oligonucleotide, wherein the CpG oligonucleotide is administered in a single dose of about 7.5 to about 12.5 μg of CpG per kg of body weight of the subject (e.g., per administration). 135. The method of any one of the preceding items, wherein the single administration comprises one or more administrations of the first and / or second population of polymersomes encapsulating at least one CpG oligonucleotide, wherein the single dose of CpG oligonucleotide is about 7.5 μg to about 12.5 μg of CpG per kg of body weight of the subject (e.g., per administration). 136. An adjuvant for use in eliciting an immune response in a subject, said use comprising administration of said adjuvant alone, wherein said adjuvant is associated with one or more polymersome populations. 137. An adjuvant for use in eliciting an immune response in a subject according to any one of the preceding items, wherein said use is in accordance with a method according to any one of the preceding claims. 138. An adjuvant for use in eliciting an immune response in a subject according to any one of the preceding items, wherein the sole administration is characterized in that the antigen is not administered to the subject in combination with the one or more adjuvants. 139. Single administration of: (i) prophylactic administration, preferably for antiviral and / or immunomodulatory prophylaxis; (ii) therapeutic administration, preferably for antiviral and / or immunomodulatory treatment; (iii) administration to reduce stress levels in a subject (e.g., to reduce stress levels during transportation, transport, and / or mixing of cattle with other animals); (iv) Any combination of (i) to (iii) 10. The adjuvant for use in eliciting an immune response in a subject according to any one of the preceding items, wherein the adjuvant is one or more of: 140. An adjuvant for use in eliciting an immune response in a subject according to any one of the preceding items, wherein the adjuvant is independently associated with the same or a different polymersome population, and preferably the adjuvant is a class A, B, or C CpG oligonucleotide. 141. An adjuvant for use in eliciting an immune response in a subject according to any one of the preceding items, which is (i) a class A, B or C CpG oligonucleotide, or (ii) a mixture comprising at least one class A, B or C CpG oligonucleotide. 142. An adjuvant for use in eliciting an immune response in a subject according to any one of the preceding items, wherein the CpG oligonucleotide is selected from the group of oligonucleotides having the sequences set forth in SEQ ID NOs: 18, 62-64, and 67-77. 143. An adjuvant for use in eliciting an immune response in a subject according to any one of the preceding items, independently associated with the one or more polymersome populations by one or more of the following means: (i) encapsulating the adjuvant within the one or more polymersome populations; (ii) incorporating and / or embedding the adjuvant in the surrounding membrane of the one or more polymersome populations; (iii) covalently conjugating the adjuvant to the outer surface of the one or more polymersome populations; (iv) non-covalently conjugating the adjuvant to the outer surface of the one or more polymersome populations; and / or (v) Any combination of (i) to (iv). 144. (i) CpG oligodeoxynucleotides (i.e., CpG ODNs), preferably CpG oligodeoxynucleotides selected from the group consisting of CpG classes A, B, and C, more preferably CpG oligodeoxynucleotides selected from the group consisting of CpG oligodeoxynucleotides having SEQ ID NOs: 18, 62-64, and 67-77; (ii) non-antigenic components derived from bacterial and mycobacterial cell walls and proteins An adjuvant for use in eliciting an immune response in a subject according to any one of the preceding items, independently selected from the group consisting of: 145. An adjuvant for use in eliciting an immune response in a subject according to any one of the preceding items, wherein the polymersome is an oxidatively stable polymersome. 146. An adjuvant for use in eliciting an immune response in a subject according to any one of the preceding items, wherein the immune response comprises stimulating the production and / or secretion of one or more cytokines, preferably wherein the immune response is an innate immune response. 147. An adjuvant for use in eliciting an immune response in a subject according to any one of the preceding items, wherein the immune response comprises stimulating the production and / or secretion of one or more cytokines, including stimulating the production and / or secretion of interleukin-6 (IL-6), preferably wherein the production and / or secretion of interleukin-6 (IL-6) predominates over the production and / or secretion of interleukin-12 (IL-12), and more preferably wherein the production and / or secretion of interleukin-6 (IL-6) is not accompanied by the production and / or secretion of interleukin-12 (IL-12). 148. An adjuvant for use in eliciting an immune response in a subject according to any one of the preceding items, wherein the single route of administration is selected from the group consisting of oral administration, intranasal administration, administration to a mucosal surface, inhalation, intradermal administration, intraperitoneal administration, subcutaneous administration, intravenous administration, and intramuscular administration. 149. The subject (i) a mammal (e.g., suitable CpGs are known in the art, e.g., from Rankin et al., 2001 or Figure 32 herein), preferably the mammal is a human, cat (e.g., in combination with SEQ ID NO: 74, 75, 76 or 77), dog (e.g., in combination with SEQ ID NO: 74, 75, 76 or 77), cow, goat, sheep (e.g., in combination with SEQ ID NO: 74, 75, 76 or 77), dairy cow, horse (e.g., in combination with SEQ ID NO: 75, 76 or 77) or pig, and (ii) non-mammalian animals; 10. An adjuvant for use in eliciting an immune response in a subject according to any one of the preceding items, selected from the group consisting of: 150. An adjuvant for use in eliciting an immune response in a subject according to any one of the preceding items, wherein the use is pharmaceutical and / or therapeutic use. 151. The use is in the treatment of cancer (e.g., sarcoma, fibrosarcoma), atopic dermatitis, African swine fever, avian influenza, bovine spongiform encephalopathy, brucellosis, cow tick, chronic wasting disease, swine fever, contagious equine metritis, equine herpesvirus, equine infectious anemia, equine piroplasmosis, equine viral arteritis, foot-and-mouth disease, Johne's disease, Mycoplasma obinexus, porcine epidemic diarrhea virus, pseudorabies, rabbit hemorrhagic disease virus, Schmallenberg virus, scrapie, spring viremia of carp, influenza A virus in swine, tuberculosis, vesicular stomatitis, West Nile virus, stress-related diseases (e.g., pasteurellosis, Mannheimia hemorrhagica), 10. The adjuvant for use in eliciting an immune response in a subject according to any one of the preceding items, wherein the adjuvant is for use in a method for treating and / or preventing a disease selected from the group consisting of: feline upper respiratory infection (URI), feline upper respiratory tract disease (FeURTD), feline herpesvirus (FHV), canine parvovirus (CPV), feline rabies, ... 152. An adjuvant for use in eliciting an immune response in a subject according to any one of the preceding items, wherein the polymersome has one or more of the following properties: i) comprising an oxidation-stable film; ii) is synthetic; iii) free of encapsulated and associated antigens; iv) comprising a membrane of an amphiphilic polymer; v) containing an amphiphilic synthetic block copolymer that forms a vesicle membrane; vi) having a diameter of more than 70 nm, preferably the diameter is in the range of about 100 nm to about 1 μm, or about 100 nm to about 750 nm, or about 100 nm to about 500 nm, or about 125 nm to about 250 nm, about 140 nm to about 240 nm, about 150 nm to about 235 nm, about 170 nm to about 230 nm, or about 220 nm to about 180 nm, or about 190 nm to about 210 nm, and most preferably the diameter is about 200 nm; vii) having a vesicular form; viii) is self-assembling, and / or ix) essentially non-immunogenic or essentially non-antigenic, preferably the block copolymer or amphiphilic polymer is non-immunogenic or non-antigenic. 153. (i) the one or more polymersome populations comprise or are formed from amphiphilic polymers, including or consisting of diblock or triblock (ABA or ABC) copolymers; and / or (ii) the one or more polymersome populations comprise a lipid polymer; An adjuvant for use in eliciting an immune response in a subject according to any one of the preceding items. 154. (a) The amphiphilic polymer comprises the copolymer poly(N-vinylpyrrolidone)-b-PLA; (b) the amphiphilic polymer is a poly(butadiene)-poly(ethylene oxide) (PB-PEO) diblock copolymer, or the amphiphilic polymer is a poly(dimethylsiloxane)-poly(ethylene oxide) (PDMS-PEO) diblock copolymer or a poly(dimethylsiloxane)-poly(acrylic acid) (PDMS-PAA), and the PB-PEO diblock copolymer preferably comprises 5-50 blocks PB and 5-50 blocks PEO, or the PB-PDMS diblock copolymer preferably comprises 5-100 blocks PDMS and 5-100 blocks PEO; (c) the amphiphilic polymer is a poly(lactide)-poly(ethylene oxide) / 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (PLA-PEO / POPC) copolymer, preferably having a ratio of PLA-PEO to POPC (e.g., PLA-PEO / POPC) of 75 to 25 (e.g., 75 / 25); (d) the amphiphilic polymer is a poly(caprolactone)-poly(ethylene oxide) / 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (PCL-PEO / POPC) copolymer, preferably having a ratio of PCL-PEO to POPC (e.g., PCL-PEO / POPC) of 75 to 25 (e.g., 75 / 25); (e) the amphiphilic polymer is polybutadiene-polyethylene oxide (BD), and / or (f) the first and / or second population of polymersomes are polymerized with a diblock copolymer PBD 21 -PEO 14 (BD21) and / or triblock copolymer PMOXA 12 -PDMS 55 -PMOXA 12 Including, (g) the first and / or second population of polymersomes are diblock copolymers PBD 21 -PEO 14 (BD21) and DOTAP (e.g., 85 mol % BD21 and 15 mol % DOTAP), An adjuvant for use in eliciting an immune response in a subject according to any one of the preceding items. 155. An adjuvant for use in eliciting an immune response in a subject according to any one of the preceding items, wherein the single administration comprises one or more administrations of the first and / or second population of polymersomes (e.g., the first and / or second population of polymersomes described in any one of the preceding items) encapsulating at least one CpG oligonucleotide, wherein a single dose (e.g., per administration) of the CpG oligonucleotide is from about 7.5 μg to about 12.5 μg per kg of body weight of the subject. 156. (i) CpG oligonucleotides of class A, B or C, preferably selected from the group of oligonucleotides having the sequences set forth in SEQ ID NOs: 18, 62-64 and 67-77; or (ii) a mixture comprising at least one class A, B, or C CpG oligonucleotide; 4. The adjuvant for use in eliciting an immune response in a subject according to any one of the preceding items, 157. The method or adjuvant of any preceding item, wherein the polymersome is a polymersome of any preceding item. 158. The method or adjuvant of any one of the preceding items, wherein the adjuvant is an adjuvant of any one of the preceding items. 159. Cancer (e.g., sarcoma, fibrosarcoma), atopic dermatitis, African swine fever, avian influenza, bovine spongiform encephalopathy, brucellosis, cow tick, chronic wasting disease, swine fever, contagious equine metritis, equine herpesvirus, equine infectious anemia, equine piroplasmosis, equine viral arteritis, foot-and-mouth disease, Johne's disease, Mycoplasma obineumoniae, porcine epidemic diarrhea virus, pseudorabies, rabbit hemorrhagic disease virus, Schmallenberg virus, scrapie, spring viremia of carp, influenza A virus in swine, tuberculosis, vesicular stomatitis, West Nile virus, stress-related diseases (e.g., pasteurellosis) , Mannheimia haemolytica and coccidiosis), viral diseases (e.g., antiviral treatment or prevention), immune diseases (e.g., immunomodulatory treatment or prevention), feline calicivirus, coronavirus, herpesvirus, canine parvovirus, porcine postweaning diarrhea, bovine, equine and / or kitten upper respiratory disease syndrome, feline upper respiratory infection (URI), feline upper respiratory tract disease (FeURTD), feline herpesvirus (FHV), canine parvovirus (CPV).

[0252] The present invention is also characterized by the following aspects. 1. A method for inducing an immune response in a subject by administering an antigen and an adjuvant, wherein the antigen is associated with a first population of polymersomes and the adjuvant is associated with a second population of polymersomes, and the two polymersome populations are administered to the subject. 2. The method of embodiment 1, wherein the antigen is associated with the first population of polymersomes by encapsulation of the antigen within the first population of polymersomes, by incorporation of the antigen into the surrounding membrane of the polymersomes of the first population of polymersomes, by conjugation of the antigen to the outer surface of the polymersomes by covalent bonds, and / or by conjugation of the antigen to the outer surface of the polymersomes by non-covalent bonds. 3. The method of embodiment 1 or embodiment 2, wherein the adjuvant is associated with the second population of polymersomes by encapsulation of the adjuvant within the second population of polymersomes, by incorporation of the adjuvant into the surrounding membrane of the polymersomes of the second population of polymersomes, by conjugation of the adjuvant to the outer surface of the polymersomes by covalent bonds, and / or by conjugation of the adjuvant to the outer surface of the polymersomes by non-covalent bonds. 4. The method of embodiment 2 or embodiment 3, wherein a first population of polymersomes has an antigen encapsulated within the polymersomes and a second population of polymersomes has an adjuvant encapsulated within the polymersomes. 5. The method of embodiment 2 or embodiment 3, wherein a first population of polymersomes has an antigen covalently or non-covalently conjugated to the outer surface of the polymersomes, and a second population of polymersomes has an adjuvant covalently or non-covalently conjugated to the outer surface of the polymersomes. 6. The method of embodiment 2 or embodiment 3, wherein a first population of polymersomes has an antigen incorporated into the surrounding membrane of the polymersomes and a second population of polymersomes has an adjuvant incorporated into the surrounding membrane of the polymer. 7. The method of embodiment 2 or embodiment 3, wherein a first population of polymersomes has an antigen encapsulated within the polymersomes and a second population of polymersomes has an adjuvant covalently conjugated to the outer surface of the polymersomes. 8. The method of embodiment 2 or embodiment 3, wherein a first population of polymersomes has an antigen covalently conjugated to the outer surface of the polymersomes, and a second population of polymersomes has an adjuvant encapsulated within the polymersomes. 9. The method of any one of the preceding embodiments, wherein the first population of polymersomes and the second population of polymersomes are administered simultaneously (at the same time) or at different times. 10. The method of embodiment 9, wherein the step of simultaneously administering the first population of polymersomes and the second population of polymersomes comprises administering the two polymersome populations together (co-administering) or administering each of the two polymersome populations separately. 11. The method of any one of the preceding embodiments, wherein the two polymersome populations are prepared separately. 12. The method of embodiment 11, wherein the two polymersome populations are mixed together prior to administration. 13. The method of any one of embodiments 1-12, wherein the two polymersome populations are administered by a route of administration selected from the group consisting of oral administration, intranasal administration, administration to a mucosal surface, inhalation, intradermal administration, intraperitoneal administration, subcutaneous administration, intravenous administration, and intramuscular administration. 14. The method of any one of the preceding embodiments, wherein the subject is a mammal, including a human, or a non-mammal. 15. The method of embodiment 14, wherein the subject is a mammal and is vaccinated against a disease selected from the group consisting of cancer, viral infections, and bacterial infections. 16. The method of embodiment 15, wherein the subject is a human and is vaccinated against a coronavirus infection. 17. The method of embodiment 16, wherein the coronavirus is pathogenic to humans. 18. The method of embodiment 16 or embodiment 17, wherein the coronavirus is a betacoronavirus. 19. The method of any one of embodiments 16 to 18, wherein the coronavirus is a sarbecovirus or a merbecovirus. 20. The method of embodiment 19, wherein the coronavirus is MERS-CoV, SARS-CoV-2, or SARS-CoV-1. 21. The method of embodiment 14, wherein the subject is a non-mammalian animal and is to be vaccinated against a disease selected from the group consisting of a viral infection and a bacterial infection. 22. The method of embodiment 21, wherein the non-mammalian animal is a bird (e.g., a poultry such as a chicken, duck, goose, or turkey), a fish, or a crustacean. 23. The method of embodiment 21, wherein the bird is a chicken, duck, goose, or turkey. 24. The method of embodiment 21, wherein the fish is salmon, trout, or tilapia. 25. The method of embodiment 1621, wherein the crustacean is a shrimp, prawn, or crab. 26. The method of embodiment 14, wherein the mammal is a goat, sheep, cow, or pig. 27. The method of embodiment 26, wherein the animal is a pig and is vaccinated against porcine epidemic diarrhea virus. 28. The method of embodiment 26, wherein the animal is an ungulate and is vaccinated against foot-and-mouth disease virus. 29. The method of any one of the previous embodiments, wherein the encapsulated antigen is a soluble or solubilized antigen. 30. The antigen, preferably a soluble or solubilized encapsulated antigen, (i) a polypeptide, (ii) carbohydrates; (iii) a polynucleotide that is not an antisense oligonucleotide and is preferably a DNA molecule or an mRNA molecule; (iv) a combination of (i) and / or (ii) and / or (iii) 3. The method of any one of the preceding aspects, selected from the group consisting of: 31. The method of embodiment 14, wherein the first and / or second population of polymersomes are oxidation-stable polymersomes comprising a soluble encapsulated antigen or adjuvant, or wherein the first and / or second population of polymersomes are oxidation-sensitive polymersomes comprising a soluble encapsulated antigen or adjuvant. 32. An amide moiety, and / or (ii) a secondary amine moiety, and / or (iii) a 1,2,3-triazole moiety, which is preferably a 1,4-disubstituted[1,2,3]triazole moiety or a 1,5-disubstituted[1,2,3]triazole moiety, and / or (iv) a pyrazoline moiety, and / or (vi) an ester moiety, and / or (vii) a carbamate moiety and / or a carbonate moiety, in the method of any one of the preceding embodiments. 33. The method of embodiment 32, wherein the covalent bond conjugating the antigen or adjuvant to the outer surface of the first polymersome population and / or the second polymersome population is formed by reacting a reactive group present on the outer surface of the polymersome with a reactive group on the antigen or adjuvant. 34. The method of embodiment 33, wherein the covalent bond is selected from the group consisting of: (i) a carboxamide bond, (ii) a 1,4-disubstituted[1,2,3]triazole or 1,5-disubstituted[1,2,3]triazole bond, and (iii) a substituted pyrazoline bond. 35. The method of embodiment 34, wherein (i) the reactive groups present on the outer surface of the polymersome are aldehyde groups and the reactive groups on the antigen or adjuvant are amine groups, thereby forming a carboxamide group, or (ii) the reactive groups present on the outer surface of the polymersome are alkyne groups and the reactive groups on the antigen or adjuvant are azide groups, thereby forming a 1,2,3-triazole group, more preferably a 1,4-disubstituted or 1,5-disubstituted 1,2,3-triazole, preferably by copper- or ruthenium-catalyzed azide-alkyne cycloaddition, or (iii) the reactive groups present on the outer surface of the polymersome are methacrylate and / or hydroxyl groups and the reactive groups on the antigen or adjuvant are tetrazole groups, thereby forming a pyrazoline group, preferably wherein formation of the pyrazoline group comprises a nitrilimine intermediate. 36. The method of embodiment 35, wherein the carboxamide bond is further reacted with a reducing agent to form a secondary amine. 37. The method of any one of embodiments 32-36, wherein the covalent bond is formed by a linker moiety. 38. The method of embodiment 37, wherein the linker moiety L is a peptide linker or a straight-chain or branched-chain hydrocarbon-based linker. 39. The method of embodiment 37 or embodiment 38, wherein the linker moiety comprises 1 to about 550 main chain atoms, 1 to about 500 main chain atoms, 1 to about 450 main chain atoms, 1 to about 350 main chain atoms, 1 to about 300 main chain atoms, 1 to about 250 main chain atoms, 1 to about 200 main chain atoms, 1 to about 150 main chain atoms, 1 to about 100 main chain atoms, 1 to about 50 main chain atoms, 1 to about 30 main chain atoms, 1 to about 20 main chain atoms, 1 to about 15 main chain atoms, or 1 to about 12 main chain atoms, or 1 to about 10 main chain atoms, wherein a main chain atom is a carbon atom optionally replaced with one or more heteroatoms selected from the group consisting of N, O, P, and S. 40. The method of any one of embodiments 37 to 39, wherein the linker moiety comprises a membrane anchor domain that incorporates the linker moiety into the membrane of the polymersome. 41. The method of embodiment 40, wherein the membrane anchor domain comprises a lipid. 42. The method of embodiment 41, wherein the lipid is a phospholipid or a glycolipid. 43. The method of embodiment 42, wherein the glycolipid comprises glycophosphatidylinositol (GPI). 44. The method of embodiment 42, wherein the phospholipid is a sphingophospholipid or a glycerophospholipid. 45. The method of embodiment 44, wherein the sphingophospholipid comprises distearoylphosphatidylethanolamine [DSPE] conjugated to polyethylene glycol (PEG) (DSPE-PEG) or a cholesterol-based conjugate. 46. ​​The method of embodiment 45, wherein the DSPE-PEG contains from 2 to about 500 ethylene oxide units. 47. The method of any one of embodiments 32 to 46, wherein the linker is non-hydrolyzable and / or non-oxidizable under physiological conditions. 48. The method of any one of embodiments 2 to 47, wherein the antigen incorporated into the surrounding membrane of the polymersome is a membrane-bound protein or lipid antigen. 49. The method of embodiment 48, wherein the membrane-bound protein comprises an extracellular fragment or extracellular domain of a transmembrane protein. 50. The method of embodiment 48 or embodiment 49, wherein the membrane-bound protein is a transmembrane protein, a G protein-coupled receptor, a neurotransmitter receptor, a kinase, a porin, an ABC transporter, an ion transporter, an acetylcholine receptor, or a cell adhesion receptor. 51. The method of any one of embodiments 48 to 50, wherein the lipid antigen is a synthetic lipid or a naturally occurring lipid. 52. The method of any one of embodiments 2 to 51, wherein the non-covalent bond for conjugating the antigen and / or adjuvant to the outer surface of the polymersomes of the first polymersome population and / or the second polymersome population comprises a binding pair selected from the group consisting of streptavidin and biotin, avidin and biotin, streptavidin and a streptavidin-binding peptide, and avidin and an avidin-binding peptide, or is an electrostatic interaction. 53. The method of any one of embodiments 1 to 52, wherein the first population of polymersomes and the second population of polymersomes comprise or are formed from the same at least one amphiphilic polymer. 54. The method of any one of embodiments 1 to 52, wherein the first population of polymersomes and the second population of polymersomes comprise or are formed from at least one different amphiphilic polymer. 55. The method of any one of the preceding embodiments, wherein the first and / or second population of polymersomes are oxidatively stable. 56. Administration of the first and / or second population of polymersomes induces CD8 (+) 2. The method of any one of the previous aspects, wherein the method is capable of eliciting a T cell mediated immune response, preferably wherein the elicitation is in vivo, ex vivo or in vitro elicitation. 57. The encapsulated antigen comprises a soluble portion of a membrane protein (MP) or a membrane-associated peptide (MAP), preferably the antigen comprises a soluble portion of influenza hemagglutinin, swine influenza hemagglutinin, a spike protein such as porcine epidemic diarrhea virus spike protein, a spike protein of a human pathogenic coronavirus such as MERS-CoV spike protein, SARS-CoV-2 spike protein, or SARS-CoV-1 spike protein, ovalbumin (OVA), B16 peptide or MC38 peptide, more preferably the antigen is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO: 10. The method of any one of the preceding embodiments, comprising a polypeptide that is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to a polypeptide sequence selected from the group consisting of SEQ ID NOs:43-46, SEQ ID NOs:34-41, SEQ ID NOs:48-51, and SEQ ID NO:65. 58. The method of any one of the preceding embodiments, wherein the encapsulated antigen comprises a fragment of a viral spike protein, wherein the fragment comprises, consists essentially of, or consists of: the S1 portion of the spike protein, the S2 portion of the spike protein, a combination of the S1 and S2 portions of the spike protein, the receptor binding domain (RBD) of the spike protein, or a combination thereof. 59. The method of any one of the preceding embodiments, wherein the first population and / or second population of polymersomes are oxidatively stable in the presence of serum components, preferably wherein the oxidative stability is in vivo, ex vivo, or in vitro oxidative stability. 60. The method of any one of the preceding embodiments, wherein the first and / or second population of polymersomes are stable within endosomes, preferably said stability being in vivo, ex vivo, or in vitro. 61. The method of any one of the preceding embodiments, wherein the first population and / or second population of polymersomes have improved oxidative stability compared to the oxidative stability of the corresponding liposomes, preferably wherein the improved stability is improved stability in vivo, ex vivo, or in vitro. 62. The first and / or second population of polymersomes release their contents, including soluble encapsulated antigens, in an oxidation-independent manner to target CD8 (+) The method of any one of the preceding aspects, wherein the antibody is capable of triggering a T cell mediated immune response, preferably wherein the release is in vivo, ex vivo or in vitro release. 63. The first and / or second population of polymersomes are CD8 (+) The method of any one of the preceding aspects, wherein the antibody is capable of eliciting a cellular immune response, including a T cell mediated immune response, preferably wherein the immune response is an in vivo, ex vivo or in vitro immune response. 64. The first and / or second population polymersomes are (+) The method of any one of the preceding aspects, wherein the method is capable of eliciting a cellular and / or humoral immune response, including a T cell mediated immune response, preferably wherein the immune response is an in vivo, ex vivo or in vitro immune response. 65. The method of embodiment 64, wherein the humoral immune response comprises the production of specific antibodies, and more preferably, the immune response is an in vivo, ex vivo, or in vitro immune response. 66. The first and / or second population of polymersomes comprises effector CD4 (+) The method of any one of the previous aspects, wherein the method is capable of enhancing the frequency of T cells, preferably wherein the enhancement is in vivo, ex vivo or in vitro enhancement. 67. The method of embodiment 64, wherein the cellular immune response comprises a T cell-mediated immune response, preferably an in vivo, ex vivo, or in vitro immune response. 68. The first and / or second population of polymersomes induces antigen-specific CD8+ binding compared to free antigen. (+) The method of any one of the preceding aspects, wherein the method is capable of enhancing clonal expansion of T cells, preferably wherein the expansion is in vivo, ex vivo or in vitro expansion. 69. The first and / or second population of polymersomes are capable of binding antigen-specific effector CD8 (+) The method of any one of the preceding aspects, wherein the method is capable of inducing T cells, preferably wherein the induction is in vivo, ex vivo or in vitro induction. 70. The first and / or second population of polymersomes are antigen-specific CD8 (+) 2. The method of any one of the preceding aspects, wherein the method is capable of enhancing the cytotoxic phenotype of the T cells, preferably wherein the enhancement is in vivo, ex vivo or in vitro enhancement. 71. The method of any one of the preceding embodiments, wherein the first and / or second population of polymersomes are capable of targeting lymph node-resident macrophages and / or B cells, preferably wherein the targeting is in vivo, ex vivo, or in vitro targeting. 72. The method of any one of the preceding embodiments, wherein the first and / or second population of polymersomes are reduction-stable, preferably the first and / or second population of polymersomes are reduction-stable in the presence of serum components, and more preferably the reduction-stability is in vivo, ex vivo, or in vitro reduction-stable. 73. The method of any one of the preceding embodiments, wherein the first and / or second population of polymersomes have reduced permeability, preferably the reduced permeability is relative to the permeability of the corresponding liposomes, and more preferably the permeability is in vivo, ex vivo, or in vitro permeability. 74. The method of any one of the preceding embodiments, wherein the first population and / or the second population of polymersomes are capable of releasing their contents within endosomes, preferably the endosomes are late endosomes, and more preferably the release is in vivo, ex vivo, or in vitro release. 75. The method of any one of the preceding embodiments, wherein the first and / or second population of polymersomes have one or more of the following capabilities: (i) the ability to induce a cellular immune response; preferably, the cellular immune response is CD8 (+) More preferably, the cellular immune response comprises a CD8 T cell-mediated immune response. (+) and most preferably the cellular immune response is a cellular immune response to a soluble portion of influenza hemagglutinin, swine influenza hemagglutinin, a spike protein such as a porcine epidemic diarrhea virus spike protein, a spike protein of a human pathogenic coronavirus such as a MERS-CoV spike protein, a SARS-CoV-2 spike protein, or a SARS-CoV-1 spike protein, ovalbumin (OVA), a B16 peptide, or an MC38 peptide, and even most preferably the cellular immune response is a cellular immune response to a soluble portion of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NOs:12-14, SEQ ID NOs:43-46, SEQ ID NOs:34-41, SEQ ID NOs: a cellular immune response to a polypeptide that is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to a polypeptide sequence selected from the group consisting of SEQ ID NOs:48-51, and SEQ ID NO:65; (ii) the ability to release the contents of the polymersome within an endosome; preferably the endosome is a late endosome, and more preferably the contents comprise a soluble portion of influenza hemagglutinin, swine influenza hemagglutinin, a spike protein, such as a porcine epidemic diarrhea virus spike protein, a spike protein of a human pathogenic coronavirus, such as a MERS-CoV spike protein, a SARS-CoV-2 spike protein, or a SARS-CoV-1 spike protein, ovalbumin (OVA), a B16 peptide, or an MC38 peptide, and most preferably the contents are SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NOs:12-14, SEQ ID NOs:43-46, SEQ ID NOs: SEQ ID NOs:34-41, SEQ ID NOs:48-51, and SEQ ID NO:65; (iii) Oxidation-independent release of polymersome contents to CD8 (+)the ability to trigger a T cell-mediated immune response; preferably, the contents comprise a soluble portion of influenza hemagglutinin, swine influenza hemagglutinin, a spike protein, such as a porcine epidemic diarrhea virus spike protein, a spike protein of a human pathogenic coronavirus, such as a MERS-CoV spike protein, a SARS-CoV-2 spike protein, or a SARS-CoV-1 spike protein, ovalbumin (OVA), a B16 peptide, or an MC38 peptide, and more preferably, the contents comprise a soluble portion of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NOs:12-14, SEQ ID NOs:43-46, SEQ ID NOs:34-41, SEQ ID NOs:48-51, and SEQ ID NOs: including a polypeptide that is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to a polypeptide sequence selected from the group consisting of ID NO:65; (iv) the ability to stimulate an immune response against the antigen; preferably the antigen comprises a soluble portion of influenza hemagglutinin, swine influenza hemagglutinin, a spike protein, such as a porcine epidemic diarrhea virus spike protein, a spike protein of a human pathogenic coronavirus, such as a MERS-CoV spike protein, a SARS-CoV-2 spike protein, or a SARS-CoV-1 spike protein, ovalbumin (OVA), a B16 peptide, or an MC38 peptide; more preferably the antigen is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NOs:12-14, SEQ ID NOs:43-46, SEQ ID NOs:34-41, SEQ ID NOs: SEQ ID NOs:48-51, and SEQ ID NO:65; (v) CD8 (+)the ability to trigger cross-protection induced by a T cell-mediated immune response; preferably the response is a response to a soluble portion of influenza hemagglutinin, swine influenza hemagglutinin, a spike protein, such as a porcine epidemic diarrhea virus spike protein, ovalbumin (OVA), a B16 peptide or an MC38 peptide, and more preferably the response is a response to a soluble portion of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, 13 and 14, SEQ ID NOs:43 to 46, SEQ ID NOs:34 to 41, SEQ ID NOs:48 to 51, and SEQ ID NOs: is a response to a polypeptide that is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to a polypeptide sequence selected from the group consisting of NO:65; (vi) the ability to deliver a peptide or protein to antigen-presenting cells (APCs); preferably the peptide or protein comprises or is derived from influenza hemagglutinin, swine influenza hemagglutinin, a spike protein, such as a porcine epidemic diarrhea virus spike protein, a spike protein of a human pathogenic coronavirus, such as a MERS-CoV spike protein, a SARS-CoV-2 spike protein, or a SARS-CoV-1 spike protein, an ovalbumin (OVA), a B16 peptide, or a soluble portion of an MC38 peptide, and more preferably the peptide or protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NOs:12, 13, and 14, SEQ ID NOs:43 to 46, SEQ ID NOs:47 to 48, SEQ ID NOs:49 to 50, SEQ ID NOs:51 to 52, SEQ ID NOs:53 to 54, SEQ ID NOs:55 to 56, SEQ ID NOs:57 to 58, SEQ ID NOs:59 to 60, SEQ ID NOs:61 to 62, SEQ ID NOs:63 to 64, SEQ ID NOs:65 to 66, SEQ ID NOs:67 to 68, SEQ ID NOs:69 to 70, SEQ ID NOs:69 to 71, SEQ ID NOs:69 to 72, SEQ ID NOs:69 to 73, SEQ ID NOs:69 to 74, SEQ ID NOs:69 to 75, SEQ ID NOs:69 to 76, SEQ ID NOs:69 to 7 comprising or derived from a polypeptide that is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to a polypeptide sequence selected from the group consisting of SEQ ID NOs:34-41, SEQ ID NOs:48-51, and SEQ ID NO:65; (vii) CD8 (+) T cell-mediated immune response and / or CD4 (+)the ability to trigger an immune response, including a T cell-mediated immune response; preferably the response is a response to a soluble portion of influenza hemagglutinin, swine influenza hemagglutinin, ovalbumin (OVA), a spike protein, such as a porcine epidemic diarrhea virus spike protein, a spike protein of a human pathogenic coronavirus, such as a MERS-CoV spike protein, a SARS-CoV-2 spike protein, or a SARS-CoV-1 spike protein, a B16 peptide, or an MC38 peptide, and more preferably the response is a response to a soluble portion of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NOs:12, 13 and 14, SEQ ID NOs:43 to 46, SEQ ID NOs:34 to 41, SEQ ID NOs: a response to a polypeptide that is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to a polypeptide sequence selected from the group consisting of SEQ ID NOs:48-51, and SEQ ID NO:65; (viii) the ability to stimulate an immune response in a subject; preferably the response is a response to a soluble portion of influenza hemagglutinin, swine influenza hemagglutinin, a spike protein, such as a porcine epidemic diarrhea virus spike protein, a spike protein of a human pathogenic coronavirus, such as a MERS-CoV spike protein, a SARS-CoV-2 spike protein, or a SARS-CoV-1 spike protein, ovalbumin (OVA), a B16 peptide, or an MC38 peptide, and more preferably the response is a response to a soluble portion of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NOs:12, 13, and 14, SEQ ID NOs:43 to 46, SEQ ID NOs:34 to 41, SEQ ID NOs: a response to a polypeptide that is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to a polypeptide sequence selected from the group consisting of SEQ ID NOs:48-51, and SEQ ID NO:65; (ix) the ability to immunize a non-human animal; preferably, the immunization is against influenza hemagglutinin, swine influenza hemagglutinin, a spike protein, such as a porcine epidemic diarrhea virus spike protein, a spike protein of a human pathogenic coronavirus, such as a MERS-CoV spike protein, a SARS-CoV-2 spike protein, or a SARS-CoV-1 spike protein, ovalbumin (OVA), a B16 peptide, or a soluble portion of an MC38 peptide, and more preferably, the immunization is against SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NOs:43-46, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:4 immunization against a polypeptide that is at least 60% or more (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to a polypeptide sequence selected from the group consisting of SEQ ID NOs:34-41, SEQ ID NOs:48-51, and SEQ ID NO:65; (x) the first population and / or second population of polymersomes have altered antigenicity compared to the corresponding antigenicity of...

Claims

1. an adjuvant associated with one or more polymersome populations, in a method for inducing an immune response in a subject by administration of the adjuvant alone, The adjuvant, wherein the sole administration is characterized in that an antigen is not administered to the subject in combination with the adjuvant.

2. The single administration comprises administering (i) prophylactic administration, preferably for antiviral and / or immunomodulatory prophylaxis; (ii) therapeutic administration, preferably for antiviral and / or immunomodulatory treatment; (iii) administration to reduce stress levels in a subject (e.g., to reduce stress levels during transportation, during cattle transport, and / or when the cattle are mixed with other animals); (iv) Any combination of (i) to (iii).

2. The adjuvant of claim 1, which is one or more of:

3. The adjuvant of any one of claims 1 to 2, wherein the adjuvants are independently associated with the same or different polymersome populations, preferably the adjuvants are CpG oligonucleotides of class A, B or C.

4. The adjuvant of any one of claims 1 to 3, wherein the adjuvant is (i) a CpG oligonucleotide of class A, B or C, or (ii) a mixture comprising at least one CpG oligonucleotide of class A, B or C.

5. The adjuvant of claim 4, wherein the CpG oligonucleotide is selected from the group of oligonucleotides having sequences shown in SEQ ID NOs: 18, 62-64 and 67-77.

6. The adjuvant comprises one or more polymersome populations and: (i) encapsulating the adjuvant within the one or more polymersome populations; (ii) incorporating and / or embedding the adjuvant in the surrounding membrane of the one or more polymersome populations; (iii) covalently conjugating the adjuvant to the outer surface of the one or more polymersome populations; (iv) non-covalently conjugating the adjuvant to the outer surface of the one or more polymersome populations; and / or (v) any combination of (i) to (iv) 6. The adjuvant of any one of the preceding claims, wherein the adjuvant is independently associated with one or more of the following:

7. The adjuvant is (i) CpG oligodeoxynucleotides (i.e., CpG ODNs), preferably CpG oligodeoxynucleotides selected from the group consisting of CpG classes A, B and C, more preferably CpG oligodeoxynucleotides selected from the group consisting of CpG oligodeoxynucleotides having SEQ ID NOs: 18, 62-64 and 67-77; (ii) non-antigenic components derived from bacterial and mycobacterial cell walls and proteins 20. The adjuvant of any one of the preceding claims, independently selected from the group consisting of:

8. 2. The adjuvant of any one of the preceding claims, wherein the polymersome is an oxidatively stable polymersome.

9. 20. The adjuvant according to any one of the preceding claims, wherein the immune response comprises stimulating the production and / or secretion of one or more cytokines, preferably wherein the immune response is an innate immune response.

10. 10. The adjuvant of any one of the preceding claims, wherein the immune response comprises stimulating the production and / or secretion of one or more cytokines including stimulating the production and / or secretion of interleukin-6 (IL-6), preferably wherein the production and / or secretion of interleukin-6 (IL-6) predominates over the production and / or secretion of interleukin-12 (IL-12), and more preferably wherein the production and / or secretion of interleukin-6 (IL-6) is not accompanied by the production and / or secretion of interleukin-12 (IL-12).

11. 20. The adjuvant of any one of the preceding claims, wherein the route of single administration is selected from the group consisting of oral administration, intranasal administration, administration to a mucosal surface, inhalation, topical administration, intradermal administration, intraperitoneal administration, subcutaneous administration, intravenous administration and intramuscular administration.

12. The object is (i) a mammal, preferably a human, cat, dog, cow, goat, sheep, cow, horse or pig; and (ii) non-mammalian animals; 20. The adjuvant according to any one of the preceding claims, selected from the group consisting of:

13. The method is for treating a variety of diseases, including cancer (e.g., sarcoma, fibrosarcoma), atopic dermatitis, African swine fever, avian influenza, bovine spongiform encephalopathy, brucellosis, Cattle Fever Tick, chronic wasting disease, swine fever, contagious equine metritis, equine herpesvirus, equine infectious anemia, equine piroplasmosis, equine viral arteritis, foot and mouth disease, Johne's disease, Mycoplasma ovipneumoniae, porcine epidemic diarrhea virus, pseudorabies, rabbit hemorrhagic disease virus, Schmallenberg virus, scrapie, spring viremia of carp, influenza A virus in swine, tuberculosis, vesicular stomatitis, West Nile virus, stress-related diseases (e.g., pasteurellosis, Mannheimia haemolytica, 5. The adjuvant of any one of the preceding claims, which is a method of treatment and / or prevention against a disease selected from the group consisting of: rabies, rabies haemolytica and coccidiosis), viral diseases (e.g. antiviral treatment or prevention), immune diseases (e.g. immunomodulatory treatment or prevention), feline calicivirus, coronavirus, herpes virus, canine parvovirus, porcine postweaning diarrhea, bovine, equine and / or kitten upper respiratory disease syndrome, feline upper respiratory tract infection (URI), feline upper respiratory tract disease (FeURTD), feline herpes virus (FHV), canine parvovirus (CPV).

14. Polymersomes have the following properties: i) containing an oxidation-stabilized film; ii) is synthetic; iii) free of encapsulated and associated antigens; iv) comprising a membrane of an amphiphilic polymer; v) containing an amphiphilic synthetic block copolymer that forms a vesicle membrane; vi) having a diameter of more than 70 nm, preferably in the range of about 100 nm to about 1 μm, or about 100 nm to about 750 nm, or about 100 nm to about 500 nm, or about 125 nm to about 250 nm, about 140 nm to about 240 nm, about 150 nm to about 235 nm, about 170 nm to about 230 nm, or about 220 nm to about 180 nm, or about 190 nm to about 210 nm, and most preferably the diameter is about 200 nm; vii) having a vesicular morphology; viii) being self-assembling; and / or ix) essentially non-immunogenic or essentially non-antigenic, preferably the block copolymer or amphiphilic polymer is non-immunogenic or non-antigenic. The adjuvant of any of the preceding claims, comprising one or more of:

15. (i) the one or more polymersome populations comprise or are formed from amphiphilic polymers, including or consisting of diblock or triblock (ABA or ABC) copolymers; and / or (ii) the one or more polymersome populations comprise a lipid polymer; 10. The adjuvant according to any one of the preceding claims.

16. (a) the amphiphilic polymer comprises a copolymer poly(N-vinylpyrrolidone)-b-PLA; (b) the amphiphilic polymer is a poly(butadiene)-poly(ethylene oxide) (PB-PEO) diblock copolymer, or the amphiphilic polymer is a poly(dimethylsiloxane)-poly(ethylene oxide) (PDMS-PEO) diblock copolymer or a poly(dimethylsiloxane)-poly(acrylic acid) (PDMS-PAA), the PB-PEO diblock copolymer preferably comprising 5-50 blocks PB and 5-50 blocks PEO, or the PB-PDMS diblock copolymer preferably comprising 5-100 blocks PDMS and 5-100 blocks PEO; (c) the amphiphilic polymer is a poly(lactide)-poly(ethylene oxide) / 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (PLA-PEO / POPC) copolymer, preferably having a ratio of PLA-PEO to POPC (e.g., PLA-PEO / POPC) of 75 to 25 (e.g., 75 / 25); (d) the amphiphilic polymer is a poly(caprolactone)-poly(ethylene oxide) / 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (PCL-PEO / POPC) copolymer, preferably having a ratio of PCL-PEO to POPC (e.g., PCL-PEO / POPC) of 75 to 25 (e.g., 75 / 25); (e) the amphiphilic polymer is polybutadiene-polyethylene oxide (BD); and / or (f) the first and / or second population of polymersomes comprises a diblock copolymer PBD 21 -PEO 14 (BD21) and / or triblock copolymer PMOXA 12 -PDMS 55 -PMOXA 12 and / or (g) the first and / or second population of polymersomes comprises a diblock copolymer PBD 21 -PEO 14 (BD21) and DOTAP (e.g., 85 mol % BD21 and 15 mol % DOTAP), 10. The adjuvant according to any one of the preceding claims.

17. 2. The adjuvant of any one of the preceding claims, wherein the single administration comprises one or more administrations of the first and / or second population of polymersomes encapsulating the adjuvant, wherein the adjuvant is a CpG oligonucleotide, and wherein a single dose of the CpG oligonucleotide is from about 7.5 μg to about 12.5 μg of CpG per kg of subject body weight.

18. An adjuvant for use in eliciting an immune response in a subject, said use comprising administration of said adjuvant alone, said adjuvant being associated with one or more polymersome populations.

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