Compositions comprising Sbi adjuvants and methods of use thereof

Abstract

The present application relates to compositions comprising Sbi adjuvants and methods of use thereof. Disclosed herein are fusion polypeptides comprising: (i) a fragment antigen comprising an epitope of a target protein antigen; and (ii) a complement binding polypeptide. The disclosure also provides fusion polynucleotides (e.g., mRNA) encoding the fusion polypeptides. Also disclosed herein are methods of making and using the fusion polypeptides and fusion polynucleotides of the present disclosure.

Description

[0001] This application is a divisional application of the invention patent application with application date of March 3, 2022, application number 202280016257.9, and name “Compositions containing Sbi adjuvant and methods of use thereof”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 63 / 156,860, filed on March 4, 2021, the entire contents of which are hereby incorporated by reference. Technical Field

[0004] The present invention relates to vaccine adjuvants. Background Art

[0005] In recent years, progress has been made in the development of vaccines for various diseases and conditions. However, these efforts have failed to produce broadly neutralizing vaccines. Summary of the invention

[0006] The present disclosure provides compositions for stimulating an immune response to an antigen and / or for enhancing the immunogenicity of an antigen. In some embodiments, the compositions disclosed herein comprise an immunogenic composition comprising: (1) an antigen fragment or antigen variant fused to (2) an adjuvant comprising a complement C3d binding region. In some embodiments, the immunogenic compositions disclosed herein can enhance the titer of the resulting antibody response and / or result in a measurable T cell response. In some embodiments, the adjuvant is or comprises a complement C3d binding region (e.g., Sbi III and / or Sbi IV) of a Sbi protein from Staphylococcus aureus. Also provided herein are pharmaceutical compositions and methods of using the pharmaceutical compositions to stimulate an immune response to an antigen and / or enhance the immunogenicity of an antigen.

[0007] The present disclosure provides a polypeptide comprising a complement C3d binding polypeptide from an immunoglobulin binding protein (Sbi) of Staphylococcus aureus. In some embodiments, the polypeptide is a fusion polypeptide comprising: (i) a fragment antigen comprising an epitope of a target protein antigen; and (ii) a complement C3d binding polypeptide from an immunoglobulin binding protein (Sbi) of Staphylococcus aureus.

[0008] In some embodiments, the complement C3d binding polypeptide is or comprises one or both of domain III and domain IV of Sbi of Staphylococcus aureus, or a functional fragment or variant thereof. In some embodiments, the complement C3d binding polypeptide is or comprises domain III of Sbi of Staphylococcus aureus, or a functional fragment or variant thereof. In some embodiments, the complement C3d binding polypeptide is or comprises domain IV of Sbi of Staphylococcus aureus, or a functional fragment or variant thereof. In some embodiments, the complement C3d binding polypeptide is or comprises domain III and domain IV of Sbi of Staphylococcus aureus, or a functional fragment or variant thereof.

[0009] In some embodiments, the fragment antigen has an amino acid sequence length of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the amino acid sequence length of the target protein antigen. In some embodiments, the fragment antigen has an amino acid sequence length of no more than 50% of the amino acid sequence length of the target protein antigen. In some embodiments, the fragment antigen has an amino acid sequence length of no more than 40%, 30%, 20%, 10% or 5% of the amino acid sequence length of the target protein antigen.

[0010] In some embodiments, the fragment antigen has a length of about 10-300 amino acid residues. In some embodiments, the fragment antigen has a length of at least 10 amino acid residues. In some embodiments, the fragment antigen has a length of less than about 300 amino acid residues. In some embodiments, the fragment antigen has a length of about 10-300, 10-250, 10-200, 10-150, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-300, 30-300, 40-300, 50-300, 60-300, 70-300, 80-300, 90-100, 100-300, 150-300, 200-300, 250-300, 20-250, 30-200, 40-150, 50-100, 60-90, or 70-80 amino acid residues. In some embodiments, the fragment antigen has a length of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 amino acid residues.

[0011] Also provided herein are fusion polypeptides comprising: (i) an antigenic variant or fragment antigenic variant comprising an epitope of a target protein antigen; and (ii) a complement C3d binding polypeptide from an immunoglobulin binding protein (Sbi) of Staphylococcus aureus.

[0012] In some embodiments, the complement C3d binding polypeptide is or comprises one or both of domain III and domain IV of Sbi of Staphylococcus aureus, or a functional fragment or variant thereof. In some embodiments, the complement C3d binding polypeptide is or comprises domain III of Sbi of Staphylococcus aureus, or a functional fragment or variant thereof. In some embodiments, the complement C3d binding polypeptide is or comprises domain IV of Sbi of Staphylococcus aureus, or a functional fragment or variant thereof. In some embodiments, the complement C3d binding polypeptide is or comprises domain III and domain IV of Sbi of Staphylococcus aureus, or a functional fragment or variant thereof.

[0013] In some embodiments, the amino acid sequence length of the antigen variant or fragment antigen variant is identical to the amino acid sequence length of the target protein antigen.

[0014] In some embodiments, antigen variants or fragment antigen variants include at least one modified amino acid compared to the target protein antigen. In some embodiments, the modified amino acid includes N-linked glycosylation. In some embodiments, antigen variants or fragment antigen variants include at least one amino acid mutation compared to the target protein antigen. In some embodiments, antigen variants or fragment antigen variants include 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 or 50 amino acid mutations compared to the target protein antigen. In some embodiments, the mutation introduces serine, threonine, alanine or an amino acid different from the amino acid present at the position in the target protein antigen at a specific position. In some embodiments, the mutation prevents the formation of disulfide bonds.

[0015] In some embodiments, the fragment antigen is characterized in that when expressed in vivo, the fragment antigen binds to a major histocompatibility complex (MHC) molecule. In some embodiments, the MHC molecule is or comprises an MHC I molecule or an MHC II molecule.

[0016] In some embodiments, the fragment antigen is characterized in that when expressed in vivo, the fragment antigen does not bind to an MHC molecule.

[0017] In some embodiments, a fragment antigen is characterized in that when expressed in vivo, the fragment antigen folds into a three-dimensional conformation that is substantially identical to the three-dimensional conformation of the fragment antigen when in its native position in the target protein antigen.

[0018] In some embodiments, the fragment antigen, antigen variant, or fragment antigen variant further comprises an amino acid sequence from a second target protein antigen.

[0019] In some embodiments, the target protein antigen is or comprises an infectious disease antigen. In some embodiments, the infectious disease antigen is or comprises a viral antigen, a bacterial antigen, a fungal antigen, or a combination thereof. In some embodiments, the viral antigen is or comprises an influenza antigen. In some embodiments, the viral antigen is or comprises a coronavirus polypeptide. In some embodiments, the coronavirus polypeptide is or comprises a SARS-CoV-2 protein. In some embodiments, the SARS-CoV-2 protein is or comprises a spike protein (SARS-CoV-2S) or a fragment thereof; an envelope protein (SARS-CoV-2E) or a fragment thereof; a membrane protein (SARS-CoV-2M) or a fragment thereof; a nucleocapsid protein (SARS-CoV-2N) or a fragment thereof; an auxiliary factor polypeptide or a fragment thereof; or any combination thereof. In some embodiments, the SARS-CoV-2 protein comprises a spike protein or a fragment thereof (e.g., RBD).

[0020] In some embodiments, the target protein antigen is or comprises a cancer antigen.

[0021] In some embodiments, the fragment antigen, antigen variant, or fragment antigen variant has an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:3.

[0022] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:4.

[0023] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:5.

[0024] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:6.

[0025] In some embodiments, the fragment antigen, antigen variant, or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:7.

[0026] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:20.

[0027] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:21.

[0028] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:22.

[0029] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:23.

[0030] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:24.

[0031] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:25.

[0032] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:26.

[0033] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:27.

[0034] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:28.

[0035] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:29.

[0036] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:30.

[0037] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:31.

[0038] In some embodiments, the fragment antigen, antigen variant, or fragment antigen variant has at least 80%, 85%, 90% or 100% identity to the amino acid sequence of SEQ ID NO:8.

[0039] In some embodiments, the complement C3d binding polypeptide comprises an Sbi domain III that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identical to the amino acid sequence of SEQ ID NO:9.

[0040] In some embodiments, the complement C3d binding polypeptide comprises an Sbi domain IV that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identical to the amino acid sequence of SEQ ID NO:10.

[0041] In some embodiments, Sbi domain III and Sbi domain IV are contiguous.

[0042] In some embodiments, Sbi domain III and Sbi domain IV are separated by a linker.

[0043] In some embodiments, (a) and (b) are encoded by nucleotide sequences and are located in the same nucleotide sequence or different nucleotide sequences. In some embodiments, (a) is located at the N-terminus of (b). In some embodiments, (a) is located at the C-terminus of (b).

[0044] In some embodiments, (a) and (b) are continuous or separated by a joint. In some embodiments, the joint is a peptidyl joint. In some embodiments, the peptidyl joint comprises at least 60% glycine and / or serine. In some embodiments, the joint is selected from a Gly-Gly-Gly-Gly-Ser (Gly4-Ser) joint or a histidine joint. In some embodiments, the joint is a Gly4-Ser joint. In some embodiments, the joint comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 repeats of a Gly4-Ser joint. In some embodiments, the joint comprises 3 repeats of a Gly4-Ser joint. In some embodiments, the joint comprises the sequence of SEQ ID NO: 11.

[0045] In some embodiments, the fusion polypeptide further comprises a secretory peptide. In some embodiments, the secretory peptide is about 10-30 amino acids in length. In some embodiments, the secretory peptide is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length. In some embodiments, the secretory peptide comprises an amino acid sequence with SEQ ID NO: 1 having at least 80%, 85%, 90% or 100% identity. In some embodiments, the secretory peptide comprises an amino acid sequence with SEQ ID NO: 2 having at least 80%, 85%, 90% or 100% identity.

[0046] In some embodiments, the fusion polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:12.

[0047] In some embodiments, the fusion polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:13.

[0048] In some embodiments, the fusion polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:14.

[0049] In some embodiments, the fusion polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:15.

[0050] In some embodiments, the fusion polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:16.

[0051] In some embodiments, the fusion polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:17.

[0052] In some embodiments, the fusion polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:18.

[0053] In some embodiments, the fusion polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:19.

[0054] In some embodiments, the fusion polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:32.

[0055] In some embodiments, the fusion polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:33.

[0056] In some embodiments, the fusion polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:34.

[0057] In some embodiments, the fusion polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:35.

[0058] In some embodiments, the fusion polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:36.

[0059] In some embodiments, the fusion polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:37.

[0060] In some embodiments, the fusion polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:38.

[0061] In some embodiments, the fusion polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:39.

[0062] In some embodiments, the fusion polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:40.

[0063] In some embodiments, the fusion polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:41.

[0064] In some embodiments, the fusion polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:42.

[0065] In some embodiments, the fusion polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:43.

[0066] In some embodiments, the fusion polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:44.

[0067] In some embodiments, the fusion polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:45.

[0068] In some embodiments, the fusion polypeptide is encoded by a polynucleotide that is or comprises RNA.In some embodiments, the fusion polynucleotide is or comprises messenger RNA.

[0069] In some embodiments, the fusion polypeptide is encoded by a polynucleotide that is or comprises DNA.

[0070] Disclosed herein are fusion polynucleotides encoding any of the fusion polypeptides disclosed herein.

[0071] Also disclosed herein is a fusion polynucleotide comprising a nucleotide sequence encoding: (i) a fragment antigen comprising an epitope of a target protein antigen; and (ii) a complement C3d binding polypeptide from an immunoglobulin binding protein (Sbi) of Staphylococcus aureus.

[0072] Disclosed herein are polynucleotides comprising a nucleotide sequence encoding: (i) an antigenic variant or a fragment antigenic variant comprising an antigenic epitope of a target protein; and (ii) a complement C3d binding polypeptide from an immunoglobulin binding protein (Sbi) of Staphylococcus aureus.

[0073] In some embodiments, the fusion polynucleotide is or comprises RNA.In some embodiments, the polynucleotide is or comprises messenger RNA.

[0074] In some embodiments, the fusion polynucleotide is or comprises DNA.

[0075] In some embodiments, the complement C3d binding polypeptide encoded by the polynucleotide is or comprises one or both of domain III and domain IV of Sbi of Staphylococcus aureus, or a functional fragment or variant thereof. In some embodiments, the complement C3d binding polypeptide encoded by the polynucleotide is or comprises domain III of Sbi of Staphylococcus aureus, or a functional fragment or variant thereof. In some embodiments, the complement C3d binding polypeptide encoded by the polynucleotide is or comprises domain IV of Sbi of Staphylococcus aureus, or a functional fragment or variant thereof. In some embodiments, the complement C3d binding polypeptide encoded by the polynucleotide is or comprises domain III and domain IV of Sbi of Staphylococcus aureus, or a functional fragment or variant thereof. In some embodiments, the complement C3d binding polypeptide encoded by the polynucleotide is or comprises domain III and domain IV of Sbi of Staphylococcus aureus, or a functional fragment or variant thereof.

[0076] In some embodiments, the fusion polynucleotide encodes a fragment antigen having an amino acid sequence length of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the amino acid sequence length of the target protein antigen. In some embodiments, the fusion polynucleotide encodes a fragment antigen having an amino acid sequence length of no more than 50% of the amino acid sequence length of the target protein antigen. In some embodiments, the fusion polynucleotide encodes a fragment antigen having an amino acid sequence length of no more than 40%, 30%, 20%, 10% or 5% of the amino acid sequence length of the target protein antigen.

[0077] In some embodiments, the fusion polynucleotide encodes an antigen variant or fragment antigen variant having an amino acid sequence length that is identical to the amino acid sequence length of the target protein antigen.

[0078] In some embodiments, the fusion polynucleotide encodes an antigen variant or fragment antigen variant comprising at least one modified amino acid compared to the target protein antigen.

[0079] In some embodiments, the fusion polynucleotide encodes a fragment antigen characterized in that when expressed in vivo, the fragment antigen binds to a major histocompatibility complex (MHC) molecule. In some embodiments, the MHC molecule is or comprises an MHC I molecule or an MHC II molecule.

[0080] In some embodiments, the fusion polynucleotide encodes a fragment antigen characterized in that when expressed in vivo, the fragment antigen does not bind to an MHC molecule.

[0081] In some embodiments, the fusion polynucleotide encodes a fragment antigen characterized in that when expressed in vivo, the fragment antigen folds into a three-dimensional conformation that is substantially identical to the three-dimensional conformation of the fragment antigen when in its native position in the target protein antigen.

[0082] In some embodiments, the fusion polynucleotide encodes a fragment antigen, antigen variant, or fragment antigen variant that also comprises an amino acid sequence from a second target protein antigen.

[0083] In some embodiments, the target protein antigen is or comprises an infectious disease antigen. In some embodiments, the infectious disease antigen is or comprises a viral antigen, a bacterial antigen, a fungal antigen, or a combination thereof. In some embodiments, the antigen is or comprises a viral antigen. In some embodiments, the viral antigen is or comprises an influenza antigen. In some embodiments, the viral antigen is or comprises a coronavirus polypeptide. In some embodiments, the coronavirus polypeptide is or comprises a SARS-CoV-2 protein. In some embodiments, the SARS-CoV-2 protein is or comprises a spike protein (SARS-CoV-2S) or a fragment thereof; an envelope protein (SARS-CoV-2E) or a fragment thereof; a membrane protein (SARS-CoV-2M) or a fragment thereof; a nucleocapsid protein (SARS-CoV-2N) or a fragment thereof; an auxiliary factor polypeptide or a fragment thereof; or any combination thereof. In some embodiments, the SARS-CoV-2 protein comprises a spike protein or a fragment thereof (e.g., RBD).

[0084] In some embodiments, the target protein antigen is or comprises a cancer antigen.

[0085] In some embodiments, (a) is located at the C-terminus of (b). In some embodiments, (a) and (b) are continuous or separated by a nucleotide sequence encoding a joint. In some embodiments, the joint is a peptidyl joint. In some embodiments, the peptidyl joint comprises at least 60% glycine and / or serine. In some embodiments, the joint is selected from a Gly-Gly-Gly-Gly-Ser (Gly4-Ser) joint or a histidine joint.

[0086] In some embodiments, the fusion polynucleotide further comprises a nucleotide sequence encoding a secretory peptide.

[0087] In some embodiments, the fusion polynucleotide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:46.

[0088] In some embodiments, the fusion polynucleotide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:47.

[0089] In some embodiments, the fusion polynucleotide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:48.

[0090] In some embodiments, the fusion polynucleotide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:49.

[0091] In some embodiments, the fusion polynucleotide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:50.

[0092] In some embodiments, the fusion polynucleotide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:51.

[0093] In some embodiments, the fusion polynucleotide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:52.

[0094] In some embodiments, the fusion polynucleotide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:53.

[0095] In some embodiments, the fusion polynucleotide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:54.

[0096] Disclosed herein are expression vectors comprising any of the fusion polynucleotides disclosed herein.

[0097] In some embodiments, the expression vector comprises a viral vector. In some embodiments, the viral vector comprises a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector or an RNA vector.

[0098] Also disclosed herein are compositions for delivering any of the fusion polypeptides disclosed herein.

[0099] Disclosed herein are compositions for delivering any of the fusion polynucleotides disclosed herein.

[0100] The present disclosure provides pharmaceutical compositions for delivering a fusion polypeptide disclosed herein, a fusion polynucleotide disclosed herein, or an expression vector comprising a fusion polynucleotide disclosed herein.

[0101] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, diluent, or a combination thereof.

[0102] Disclosed herein is a preparation method, which comprises: recombinantly linking a first nucleotide sequence encoding a fragment antigen comprising an epitope of a target protein antigen and a second nucleotide sequence encoding a complement C3d binding polypeptide of an immunoglobulin binding protein (Sbi) from Staphylococcus aureus to form a polynucleotide comprising the first nucleotide sequence and the second nucleotide sequence.

[0103] In some embodiments, the method further comprises expressing the polynucleotide in a cell to produce a fusion polypeptide encoded by the polynucleotide.

[0104] Disclosed herein is a cell comprising a fusion polypeptide disclosed herein, a fusion polynucleotide disclosed herein, or an expression vector comprising a fusion polynucleotide disclosed herein.

[0105] In some embodiments, the cell is contacted with the fusion polynucleotide, fusion polypeptide or expression vector. In some embodiments, contacting occurs in vivo, in vitro or ex vivo.

[0106] Disclosed herein is a kit comprising a fusion polypeptide disclosed herein, a fusion polynucleotide disclosed herein, or an expression vector comprising a fusion polynucleotide disclosed herein, and instructions for use.

[0107] In some embodiments, the kit further comprises: a polynucleotide comprising a nucleotide sequence encoding a target protein antigen; or a target protein antigen encoded by the polynucleotide.

[0108] The present disclosure provides a method comprising administering to a subject in need thereof at least one dose of a pharmaceutical composition comprising a fusion polypeptide disclosed herein, a fusion polynucleotide disclosed herein, or an expression vector comprising a fusion polynucleotide disclosed herein.

[0109] In some embodiments, the at least one dose is administered in an amount effective for inducing an immune response to the fragment antigen in the subject.

[0110] In some embodiments, the immune response includes the generation of neutralizing antibody titers against the fragment antigen.

[0111] In some embodiments, the neutralizing antibody titer is increased by at least 50% compared to the neutralizing antibody titer induced by the fragment antigen in the absence of the complement C3d binding polypeptide from the immunoglobulin binding protein (Sbi) of Staphylococcus aureus.

[0112] In some embodiments, the generation of neutralizing antibody titers has been established in mouse models using a dose of at least 0.1 μg.

[0113] In some embodiments, the at least one dose is administered in an amount effective for stimulating B cells while reducing induction of T cell responses.

[0114] Disclosed herein is a method comprising administering to a subject: a first dose of a pharmaceutical composition disclosed herein; and a second dose of a pharmaceutical composition disclosed herein. In some embodiments, the pharmaceutical composition comprises a fusion polypeptide disclosed herein, a fusion polynucleotide disclosed herein, or an expression vector comprising a fusion polynucleotide disclosed herein.

[0115] In some embodiments, the first dose and the second dose are administered at least 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, or 4 weeks apart.

[0116] In some embodiments, the first dose and the second dose are the same amount.

[0117] In some embodiments, the first dose and the second dose are different amounts.

[0118] Further disclosed herein is a method comprising: administering a dose of a pharmaceutical composition disclosed herein to a subject in need thereof, such that the subject receives: a first dose of a pharmaceutical composition that delivers a polynucleotide comprising a nucleotide sequence encoding a target protein antigen or a target protein antigen encoded by the polynucleotide; and a second dose of the pharmaceutical composition disclosed herein.

[0119] In some embodiments, (a) the polynucleotide comprising a nucleotide sequence encoding a target protein antigen further comprises a nucleotide sequence encoding a complement C3d binding polypeptide from an immunoglobulin binding protein (Sbi) of Staphylococcus aureus; or (b) the target protein antigen encoded by the polynucleotide further comprises a complement C3d binding polypeptide from an immunoglobulin binding protein (Sbi) of Staphylococcus aureus.

[0120] In some embodiments, the method further comprises, prior to the administering step, administering to the subject a first dose of a pharmaceutical composition that delivers a polynucleotide comprising a nucleotide sequence encoding a target protein antigen or a target protein antigen encoded by the polynucleotide.

[0121] The present disclosure provides a method comprising: administering a dose of a pharmaceutical composition disclosed herein to a subject in need thereof, so that the subject receives: a first dose of the pharmaceutical composition of the claims disclosed herein, and a second dose of a pharmaceutical composition delivering a polynucleotide comprising a nucleotide sequence encoding a target protein antigen or a target protein antigen encoded by the polynucleotide.

[0122] In some embodiments, (a) the polynucleotide comprising a nucleotide sequence encoding a target protein antigen further comprises a nucleotide sequence encoding a complement C3d binding polypeptide from an immunoglobulin binding protein (Sbi) of Staphylococcus aureus; or (b) the target protein antigen encoded by the polynucleotide further comprises a complement C3d binding polypeptide from an immunoglobulin binding protein (Sbi) of Staphylococcus aureus.

[0123] Disclosed herein is a method comprising: administering a dose of a pharmaceutical composition disclosed herein to a subject in need thereof, such that the subject receives: a first dose of a pharmaceutical composition comprising a polynucleotide encoding a nucleotide sequence of a fusion polypeptide comprising (i) a target protein antigen and (ii) a complement C3d binding polypeptide from an immunoglobulin binding protein (Sbi) of Staphylococcus aureus, or a fusion polypeptide encoded by the polynucleotide; and a second dose of the pharmaceutical composition disclosed herein.

[0124] In some embodiments, the method further comprises, prior to the administering step, administering to the subject a first dose of a polynucleotide comprising a nucleotide sequence encoding a fusion polypeptide comprising (i) a target protein antigen and (ii) a complement C3d binding polypeptide from an immunoglobulin binding protein (Sbi) of Staphylococcus aureus, or a pharmaceutical composition encoded by the polynucleotide for delivery.

[0125] In some embodiments, the first dose and the second dose are administered at least 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, or 4 weeks apart.

[0126] In some embodiments of any of the methods disclosed herein, the subject in need thereof is a subject suffering from or susceptible to a disease, disorder, or condition induced by a target protein antigen.

[0127] In some embodiments, the subject is a mammalian subject.

[0128] In some embodiments, the subject is a human subject.

[0129] In some embodiments, administration can be performed by intramuscular administration, intradermal administration, intravenous administration, subcutaneous administration, or a combination thereof.

[0130] Disclosed herein is a composition comprising a fusion polypeptide disclosed herein, or a fusion polynucleotide disclosed herein, or a pharmaceutical composition disclosed herein, which is used to enhance the immunogenicity of an antigen.

[0131] Also disclosed herein is a composition comprising the fusion polypeptide disclosed herein, or the fusion polynucleotide disclosed herein, or the pharmaceutical composition disclosed herein, which is used to stimulate an immune response against an antigen.

[0132] Further disclosed herein is a composition comprising a fusion polypeptide disclosed herein, or a fusion polynucleotide disclosed herein, or a pharmaceutical composition disclosed herein, which is used in a method for treating a disease or ameliorating a symptom of a disease.

[0133] The present disclosure provides a method for enhancing the immunogenicity of an antigen, comprising administering a fusion polypeptide disclosed herein, or a fusion polynucleotide disclosed herein, or a pharmaceutical composition disclosed herein to a subject in need thereof.

[0134] The present disclosure also provides a method for stimulating an immune response against an antigen, comprising administering a fusion polypeptide disclosed herein, or a fusion polynucleotide disclosed herein, or a pharmaceutical composition disclosed herein to a subject in need thereof.

[0135] Further provided herein is a method of treating a disease or ameliorating symptoms of a disease, comprising administering a fusion polypeptide disclosed herein, or a fusion polynucleotide disclosed herein, or a pharmaceutical composition disclosed herein to a subject in need thereof.

[0136] The present disclosure provides use of a composition comprising a fusion polypeptide disclosed herein, or a fusion polynucleotide disclosed herein, or a pharmaceutical composition disclosed herein in the preparation of a medicament for enhancing the immunogenicity of a fragment antigen.

[0137] The present disclosure also provides use of a composition comprising a fusion polypeptide disclosed herein, or a fusion polynucleotide disclosed herein, or a pharmaceutical composition disclosed herein in the preparation of a medicament for stimulating an immune response against an antigen.

[0138] Also provided herein is the use of a composition comprising the fusion polypeptide disclosed herein, or the fusion polynucleotide disclosed herein, or the pharmaceutical composition disclosed herein in the preparation of a medicament for treating a disease or improving the symptoms of a disease.

[0139] Provided herein is the use of a composition comprising a fusion polypeptide disclosed herein, or a fusion polynucleotide disclosed herein, or a pharmaceutical composition disclosed herein for enhancing the immunogenicity of a fragment antigen.

[0140] Also provided herein is the use of a composition comprising a fusion polypeptide disclosed herein, or a fusion polynucleotide disclosed herein, or a pharmaceutical composition disclosed herein for stimulating an immune response against an antigen.

[0141] The present disclosure provides use of a composition comprising a fusion polypeptide disclosed herein, or a fusion polynucleotide disclosed herein, or a pharmaceutical composition disclosed herein for treating a disease or improving symptoms of a disease.

[0142] In some embodiments of any of the methods or uses disclosed herein, the subject is a mammal.

[0143] In some embodiments of any of the methods or uses disclosed herein, the subject is a human.

[0144] In some embodiments of any of the methods or uses disclosed herein, a single dose of the fusion polypeptide, fusion polynucleotide, or pharmaceutical composition is administered.

[0145] In some embodiments of any of the methods or uses disclosed herein, multiple doses of the fusion polypeptide, fusion polynucleotide, or pharmaceutical composition are administered.

[0146] In some embodiments of any of the methods or uses disclosed herein, the fusion polypeptide, fusion polynucleotide, or pharmaceutical composition is administered at a dose of about 0.5 micrograms to 10 micrograms.

[0147] In some embodiments of any of the methods or uses disclosed herein, administration of the fusion polynucleotide, fusion polypeptide, or pharmaceutical composition results in a humoral response. In some embodiments, the humoral response is an antibody response.

[0148] In some embodiments of any of the methods or uses disclosed herein, administration of the fusion polynucleotide, fusion polypeptide, or pharmaceutical composition results in an increase in the titer of an antibody response. In some embodiments, the increase in titer is an increase of about 10-fold to about 500-fold. In some embodiments, the increase in titer of the antibody response is compared to the administration of an otherwise similar fusion polynucleotide that does not comprise Sbi domain III, or a fragment or variant thereof; and Sbi domain IV, or a fragment or variant thereof. In some embodiments, the increase in titer of the antibody response is compared to the administration of an otherwise similar fusion polypeptide that does not comprise Sbi domain III, or a fragment or variant thereof; and Sbi domain IV, or a fragment or variant thereof. In some embodiments, the increase in titer of the antibody response is compared to the administration of an otherwise similar pharmaceutical composition that does not comprise a nucleotide sequence encoding Sbi domain III, or a fragment or variant thereof; and Sbi domain IV, or a fragment or variant thereof.

[0149] In some embodiments of any of the methods or uses disclosed herein, the composition is administered via any of the following routes of administration: intramuscular, intravenous, subcutaneous, intrathecal, intradermal, ocular, intranasal, sublingual, or oral.

[0150] The present disclosure provides an in vitro method for selecting fragment antigens by identifying polypeptide fragments that fold into a three-dimensional conformation that is substantially identical to the three-dimensional conformation of the target protein antigen from which the fragment is derived.

[0151] In some embodiments, the method comprises the following steps: providing a mammalian cell display system that displays a library of fragment antigen candidates on the cell surface; exposing the mammalian cell display system to a composition comprising antibodies that bind to a target protein antigen from which the fragment antigen candidates are derived; detecting fragment antigen candidates that bind to the target protein antigen-binding antibodies, wherein binding of the fragment antigen candidate that binds to at least one of the target protein antigen-binding antibodies indicates a likelihood that the fragment antigen candidate folds into a three-dimensional conformation that is substantially identical to its three-dimensional conformation when in the target protein antigen.

[0152] In some embodiments, the method further comprises the step of generating antibodies that bind to the target protein antigen.

[0153] In some embodiments, the generation of antibodies comprises (i) immunizing an animal with a target protein antigen from which the fragment antigen candidate is derived or a polynucleotide encoding the target protein antigen; and (ii) identifying antibodies that bind to the target protein antigen.

[0154] In some embodiments, the mammalian cell display system comprises HEK293T cells, HeLa cells, or CHO cells.

[0155] In some embodiments, polypeptide fragments are identified based on binding affinity to antibodies in a serum binding assay or similar assay.

[0156] Also provided herein is a recombinant polynucleotide comprising a nucleotide sequence encoding an immunoglobulin binding protein (Sbi) of Staphylococcus aureus or a fragment thereof.

[0157] In some embodiments, the polynucleotide comprises a sequence encoding a complement C3d binding polypeptide from Sbi.

[0158] In some embodiments, the complement C3d-binding polypeptide is or comprises one or both of domain III and domain IV of Sbi of Staphylococcus aureus, or a functional fragment or variant thereof.

[0159] In some embodiments, the polynucleotide is or comprises RNA.In some embodiments, the polynucleotide is or comprises messenger RNA.

[0160] In some embodiments, the polynucleotide is or comprises DNA.

[0161] In some embodiments, the polynucleotide encodes a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the nucleotide sequence of SEQ ID NO:9.

[0162] In some embodiments, the polynucleotide encodes a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the nucleotide sequence of SEQ ID NO:10.

[0163] The present disclosure provides isolated polypeptides comprising an immunoglobulin binding protein (Sbi) of Staphylococcus aureus.

[0164] In some embodiments, the polypeptide comprises a complement C3d binding polypeptide from Sbi.

[0165] In some embodiments, the complement C3d-binding polypeptide is or comprises one or both of domain III and domain IV of Sbi of Staphylococcus aureus, or a functional fragment or variant thereof.

[0166] In some embodiments, the polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identical to the amino acid sequence of SEQ ID NO:9.

[0167] In some embodiments, the polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identical to the amino acid sequence of SEQ ID NO:10.

[0168] Also provided herein is an expression vector comprising a recombinant polynucleotide comprising a nucleotide sequence encoding an immunoglobulin binding protein (Sbi) of Staphylococcus aureus or a fragment thereof.

[0169] Also provided herein are compositions for the delivery of an isolated polypeptide comprising an immunoglobulin binding protein of Staphylococcus aureus (Sbi).

[0170] The present disclosure also provides a composition for delivering a recombinant polynucleotide comprising a nucleotide sequence encoding an immunoglobulin binding protein (Sbi) of Staphylococcus aureus or a fragment thereof.

[0171] Provided herein are pharmaceutical compositions for delivering: a recombinant polynucleotide comprising a nucleotide sequence encoding an immunoglobulin binding protein (Sbi) of Staphylococcus aureus or a fragment thereof; an isolated polypeptide comprising an immunoglobulin binding protein (Sbi) of Staphylococcus aureus; or an expression vector comprising a recombinant polynucleotide comprising a nucleotide sequence encoding an immunoglobulin binding protein (Sbi) of Staphylococcus aureus or a fragment thereof.

[0172] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, diluent, or a combination thereof.

[0173] The present disclosure provides a method comprising administering to a subject in need at least one dose of a pharmaceutical composition comprising: a recombinant polynucleotide comprising a nucleotide sequence encoding an immunoglobulin binding protein (Sbi) of Staphylococcus aureus or a fragment thereof; an isolated polypeptide comprising an immunoglobulin binding protein (Sbi) of Staphylococcus aureus; or an expression vector comprising a recombinant polynucleotide comprising a nucleotide sequence encoding an immunoglobulin binding protein (Sbi) of Staphylococcus aureus or a fragment thereof.

[0174] Also disclosed herein is a method for enhancing the immunogenicity of an antigen, comprising administering to a subject in need thereof a recombinant polynucleotide comprising a nucleotide sequence encoding an immunoglobulin binding protein (Sbi) of Staphylococcus aureus or a fragment thereof; an isolated polypeptide comprising an immunoglobulin binding protein (Sbi) of Staphylococcus aureus, or a pharmaceutical composition comprising the same.

[0175] The present disclosure also provides a method for stimulating an immune response against an antigen, comprising administering to a subject in need thereof a recombinant polynucleotide comprising a nucleotide sequence encoding an immunoglobulin binding protein (Sbi) of Staphylococcus aureus or a fragment thereof; an isolated polypeptide comprising an immunoglobulin binding protein (Sbi) of Staphylococcus aureus, or a pharmaceutical composition comprising the same.

[0176] Also provided herein is the use of an immunoglobulin binding protein of Staphylococcus aureus (Sbi) as an adjuvant in an immunogenic composition.

[0177] The present disclosure provides an immunoglobulin binding protein of Staphylococcus aureus (Sbi) for use as an adjuvant in an immunogenic composition.

[0178] These and other aspects encompassed by the present disclosure are described in more detail below and in the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0179] Figure 1A - Figure 1D depicts the improvement in antigenicity of the fusion of the isolated SARS-CoV-2 S protein domain with Sbi(III-IV). Figure 1AIt is a schematic diagram showing the possible mechanism of action of Sbi (III-IV) fusion. Sbi (III-IV) binds to C3d, which can in turn drive B cell activation by binding to CR2. C3d-driven B cell co-stimulation is orthogonal to helper T cell activation, which usually occurs through TCR recognition of homologous MHCII peptide complexes and CD40 signaling. Figure 1B is a diagram showing that the fusion of the RBD domain of SARS-CoV-2S protein with Sbi (III-IV) leads to an increase in IgG titers in BALB / c mice on the 21st day after 10 μg dose of IM delivered mRNA. Co-delivery of RBD fused to the transmembrane domain of S protein (total mRNA dose of 10 μg) leads to a further increase in titer. The top and bottom horizontal lines represent the detection limits of the dilution series used in titer measurements. Serum with titers falling outside this range is assigned values ​​from the extreme endpoints of the dilution series. The value is calculated as the geometric mean, and the error bars correspond to the geometric standard deviation. Figure 1C is an ELISPOT count of IFNγ / IL2 double-stained colony numbers. For each case, the spleens of 5 mice were pooled and plated in 4 wells at 500,000 cells / well. Each of the two wells was stimulated by an N-terminal or C-terminal S protein peptide library (PepMix SARS-CoV-2 spike glycoprotein mixture). The ratio was calculated by pooling colony counts across 4 wells, and the error bar was calculated as the Poisson error of the pooled colony counts. Figure 1D is a diagram depicting that the fusion fragment of the RBD domain of the S protein can result in a detectable humoral response. The data shown are as described in Figure 1B.

[0180] Figure 2A-2B depicts a high-throughput protein folding screen. Figure 2A is a schematic diagram of a screen for protein folding. Antisera are produced by vaccinating mice with mRNA vaccines encoding the full-length protein of interest. The collected antisera are then used to detect correctly folded fragments presented in a mammalian display system background in a protein fragment library. Figure 2B is a diagram showing positive and negative controls for validating the screening strategy. The RBD from SARS-CoV-2 is presented on the surface of HEK293T cells and stained with antisera collected from mice vaccinated with the RBD vaccine.

[0181] Figure 3 Depicted is the increase in virus neutralization when using a fusion of Sbi(III-IV) with the RBD domain of the SARS-CoV-2 S protein.

[0182] Figure 4A-4B Depicted is the stimulation of B cells by a fusion of Sbi(III-IV) with the RBD domain of the SARS-CoV-2 S protein.

[0183] Figure 5A-Figure 5B Results of a vaccination regimen consisting of a priming dose and a booster dose are depicted.

[0184] Figure 6 is a graph showing antibody titers generated with a vaccination regimen including a prime dose and a boost dose.

[0185] Figure 7A-7B Conserved hemagglutinin HA1 residues are depicted. Fig. 7A The context of the Y98, S / K136, W153, H183 and L / I194 receptor contacting residues within the structure of the HA1 glycoprotein trimer is shown. Figure 7B Depicted is the conservation of receptor contact residues between strains spanning 92 years of seasonal influenza.

[0186] Some definitions

[0187] About or approximately: As used herein, the terms "about" and "approximately" when used herein when referring to a value refer to a value similar to the value mentioned in the context. Generally speaking, those skilled in the art who are familiar with the context will understand the relevant degree of difference covered by "about" or "approximately" in this context. For example, in some embodiments, the term "about" or "approximately" may be covered in 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or a range of values ​​within a smaller range of the reference value.

[0188] Administration: As used herein, the term "administering" or "administration" generally refers to applying a composition to a subject to achieve delivery as a composition or an agent contained in a composition. One of ordinary skill in the art will appreciate the various routes that can be used for administration to a subject (e.g., a human) where appropriate. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, and the like. In some specific embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or include, for example, one or more of topical, intradermal, interdermal, transdermal, and the like), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intra-organ specific (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreous administration, and the like. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve applying a fixed number of doses. In some embodiments, administration may involve administration that is intermittent (e.g., multiple doses separated in time) and / or periodic (e.g., individual doses separated by the same time period) administration. In some embodiments, administration may involve continuous administration (e.g., perfusion) for at least a selected time period.

[0189] Adjuvant: As used herein, the term "adjuvant" refers to an agent that regulates and / or enhances an immune response to an agent that elicits an immune response. In some embodiments, the adjuvant is administered before, simultaneously with, or after the administration of the agent that elicits an immune response. In some embodiments, the adjuvant and the agent that elicits an immune response are in one composition. In some embodiments, the adjuvant and the agent that elicits an immune response are in different compositions. In some embodiments, the adjuvant is or comprises a nucleic acid, a polypeptide, a polysaccharide, or a small molecule. In some embodiments, the adjuvant is or comprises a complement binding domain. In some embodiments, the adjuvant is or comprises a C3d binding domain. In some embodiments, the adjuvant is or comprises domain III of the Sbi immunoglobulin binding protein of Staphylococcus aureus, or a functional fragment or variant thereof. In some embodiments, the adjuvant is or comprises domain IV of the Sbi immunoglobulin binding protein of Staphylococcus aureus, or a functional fragment or variant thereof. In some embodiments, the adjuvant comprises domain III and domain IV of Sbi of Staphylococcus aureus, or a functional fragment or variant thereof.

[0190] Antigen: As used herein, the term "antigen" refers to an agent that triggers an immune response; and / or (ii) an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or to an antibody. In some embodiments, an antigen triggers a humoral response (e.g., including the production of antigen-specific antibodies); in some embodiments, an antigen triggers a cellular response (e.g., involving a T cell that specifically interacts with a receptor). In some embodiments, an antigen comprises at least one epitope of a target protein. In some embodiments, an epitope may be a linear epitope. In some embodiments, an epitope may be a conformational epitope. In some embodiments, an antigen binds to an antibody and may or may not induce a specific physiological response in an organism. Generally speaking, an antigen may be or include any chemical entity, such as, for example, a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, a polymer (in some embodiments, except for a biopolymer [e.g., except for a nucleic acid or an amino acid polymer]), etc. In some embodiments, an antigen is or includes a polypeptide. In some embodiments, an antigen is or includes a polysaccharide. Those of ordinary skill in the art will appreciate that, in general, antigens can be provided in an isolated or pure form, or alternatively can be provided in a crude form (e.g., with other materials, e.g., in an extract containing an antigenic source such as a cell extract or other relatively crude preparation). In some embodiments, the antigen used according to the present invention is provided in a crude form. In some embodiments, the antigen is a recombinant antigen.

[0191] Antigenic variant: As used herein, the term "antigenic variant" refers to an antigen that shows significant structural identity with a target protein antigen, but is structurally different from the target protein antigen structure in terms of the presence or level of one or more chemical parts compared to the target protein antigen. In some embodiments, the antigenic variant is functionally different from the target protein antigen. In some embodiments, the antigenic variant is not functionally different from the target protein antigen. In some embodiments, the antigen comprises an epitope of the target protein antigen. In some embodiments, the antigenic variant is different from the target protein antigen due to one or more differences in the amino acid sequence and / or one or more differences in the chemical part (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone.

[0192] Delivery / contacting: As used interchangeably herein, the terms "delivery," "delivering," or "contacting" refer to the introduction of a fusion polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) into a target cell. The target cell may be cultured in vitro or ex vivo or present in a subject (in vivo). The method of introducing a fusion polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) into a target cell may vary with in vitro, ex vivo, or in vivo applications. In some embodiments, a fusion polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) may be introduced into a target cell in cell culture by in vitro transfection. In some embodiments, a fusion polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) may be introduced into a target cell via a delivery vehicle (e.g., nanoparticles, liposomes, and / or complexes with cell permeabilizing agents). In some embodiments, a fusion polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) can be introduced into a target cell in a subject by administering the fusion polynucleotide (e.g., as described herein) or fusion polypeptide (e.g., as described herein) to the subject.

[0193] Functional: As used herein, the term "functional" is used to refer to a form or fragment of an entity that exhibits a specific property and / or activity.

[0194] Fragment: A "fragment" of a material or entity as described herein has a structure that includes discrete portions of a whole but lacks one or more portions found in the whole. In some embodiments, a fragment consists of such discrete portions. In some embodiments, a fragment consists of or contains characteristic structural elements or portions found in the whole. In some embodiments, a fragment comprises a polynucleotide fragment. In some embodiments, a fragment comprises a polypeptide fragment. 300, 325, 350, 375, 400, 425, 450, 475, 500, 500, 600, 650, 700, 750, 800, 850, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, 6000, 6500, 7500, 8500, 9000, 9500 In some embodiments, a polynucleotide fragment or polypeptide fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomeric units (e.g., residues) found in the entire polynucleotide or entire polypeptide. The entire polypeptide or entire polynucleotide may be referred to as the "parent" of a polynucleotide fragment or polypeptide fragment in some embodiments.

[0195] Fragment antigen: "Fragment antigen" is used herein to refer to a fragment comprising an epitope of a target protein antigen. In some embodiments, the epitope is or comprises an epitope presented by MHC class I. In some embodiments, the epitope is or comprises an epitope presented by MHC class II. In some embodiments, the fragment antigen is a polypeptide fragment antigen. In some embodiments, the fragment antigen is encoded by a polynucleotide encoding a fragment antigen. In some embodiments, the polypeptide fragment antigen comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300 or more monomer units (e.g., residues) as found in a target protein antigen polypeptide. In some embodiments, the polypeptide fragment antigen comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomeric units (e.g., residues) found in the target protein antigen polypeptide. In some embodiments, the polypeptide fragment antigen comprises or consists of no more than about 50%, 40%, 30%, 20%, 10% or 5% of the monomeric units (e.g., residues) found in the target protein antigen polypeptide.

[0196] Fragment antigen variant: As used herein, the term "fragment antigen variant" refers to a fragment antigen that exhibits significant sequence and / or structural identity to a fragment antigen, but differs from the fragment antigen in sequence and / or structure in terms of the presence or level of one or more chemical moieties compared to the fragment antigen. In some embodiments, the fragment antigen variant is functionally different from the fragment antigen. In some embodiments, the fragment antigen variant is not functionally different from the fragment antigen. In some embodiments, the fragment antigen variant is different from the fragment antigen due to one or more differences in the amino acid sequence and / or one or more differences in the chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone.

[0197] Nucleic acid / oligonucleotide / polynucleotide: As used herein, the terms "nucleic acid" and "polynucleotide" and "oligonucleotide" can be used interchangeably and refer to polymers of 3 or more nucleotides. In some embodiments, nucleic acids include DNA. In some embodiments, nucleic acids include RNA. In some embodiments, nucleic acids include messenger RNA (mRNA). In some embodiments, nucleic acids are single-stranded. In some embodiments, nucleic acids are double-stranded. In some embodiments, nucleic acids include both single-stranded and double-stranded portions. In some embodiments, nucleic acids include a backbone containing one or more phosphodiester linkages. In some embodiments, nucleic acids include a backbone containing phosphodiester linkages and non-phosphodiester linkages. For example, in some embodiments, nucleic acids may include a backbone containing one or more phosphorothioate or 5'-N-phosphoramidite linkages and / or one or more peptide bonds, for example, as in "peptide nucleic acids". In some embodiments, nucleic acids include one or more or all natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, nucleic acid comprises one or more or all non-natural residues. In some embodiments, non-natural residues comprise nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, inserted bases, and combinations thereof). In some embodiments, non-natural residues comprise one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to the sugars in natural residues. In some embodiments, nucleic acid has a nucleotide sequence encoding a functional gene product such as an RNA or polypeptide. In some embodiments, nucleic acid has a nucleotide sequence comprising one or more introns. In some embodiments, nucleic acid can be prepared by separation from natural sources, enzymatic synthesis (e.g., by polymerization based on complementary templates, such as in vivo or in vitro), replication in a recombinant cell or system, or chemical synthesis.In some embodiments, the length of the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides. When the number of nucleotides is used as an indication of size (eg, the size of a fusion polynucleotide), a certain number of nucleotides refers to the number of nucleotides on a single strand (eg, a single strand of a fusion polynucleotide).

[0198] Polypeptide: As used herein, the term "polypeptide" generally has its art-recognized meaning of a polymer of at least three or more amino acids. One of ordinary skill in the art will appreciate that the term "polypeptide" is intended to be sufficiently general so as to encompass not only polypeptides having the complete sequences described herein, but also polypeptides representing functional, biologically active or characteristic fragments, portions or domains of such complete polypeptides (e.g., fragments, portions or domains that retain at least one activity). The polypeptide may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, and the like. In some embodiments, the polypeptide may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof.

[0199] RNA oligonucleotide: As used herein, the term "RNA oligonucleotide" refers to an oligonucleotide of a ribonucleotide. In some embodiments, the RNA oligonucleotide is single-stranded. In some embodiments, the RNA oligonucleotide is double-stranded. In some embodiments, the RNA oligonucleotide comprises a single-stranded and double-stranded portion. In some embodiments, the RNA oligonucleotide may comprise a main chain structure as described in the definition of "nucleic acid / oligonucleotide" above. The RNA oligonucleotide may be a regulatory RNA (e.g., siRNA, microRNA, etc.) or a messenger RNA (mRNA) oligonucleotide. In some embodiments where the RNA oligonucleotide is an mRNA oligonucleotide, the RNA oligonucleotide generally comprises a poly (A) (poly (A)) region at its 3' end. In some embodiments where the RNA oligonucleotide is an mRNA oligonucleotide, the RNA oligonucleotide generally comprises a cap structure recognized in the art for example for identifying mRNA and attaching mRNA to a ribosome to initiate translation at its 5' end. In some embodiments, a polynucleotide (e.g., a fusion polynucleotide) comprises an RNA oligonucleotide. When the number of ribonucleotides is used as an indication of size (eg, the size of a fusion polynucleotide), a certain number of nucleotides refers to the number of ribonucleotides on a single strand (eg, a single strand of a fusion polynucleotide).

[0200] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human). In some embodiments, the subject suffers from a disease, disorder, or illness. In some embodiments, the subject is susceptible to a disease, disorder, or illness. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or illness. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or illness. In some embodiments, the subject is a person having one or more characteristics that are characteristics of susceptibility or risk for a disease, disorder, or illness. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom diagnosis and / or therapy is administered and / or has been administered.

[0201] Variant: As used herein, the term "variant" refers to an entity that exhibits significant structural identity with a reference entity but is structurally different from the reference entity in the presence or level of one or more chemical moieties compared to the reference entity. In many embodiments, the variant is also functionally different from its reference entity. Generally, whether a particular entity is appropriately considered to be a "variant" of a reference entity is based on the degree of structural identity of the particular entity to the reference entity. For example, due to one or more differences in the amino acid sequence and / or one or more differences in the chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone, the variant polypeptide may be different from the reference polypeptide. Alternatively or additionally, in some embodiments, the variant polypeptide does not share at least one characteristic sequence element with the reference polypeptide. In some embodiments, the reference polypeptide has one or more biological activities. In some embodiments, the variant polypeptide shares one or more of the biological activities of the reference polypeptide. In some embodiments, the variant polypeptide lacks one or more of the biological activities of the reference polypeptide. In some embodiments, the variant polypeptide exhibits one or more biological activity levels reduced compared to the reference polypeptide.

[0202] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis (e.g., mRNA synthesis), and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions or as commonly accomplished in the art or as described herein. The aforementioned techniques and procedures can generally be performed according to conventional methods well known in the art, and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference for any purpose. DETAILED DESCRIPTION

[0203] Traditional vaccine adjuvants, such as alum and poly-I:C (poly-I:C), generally work by activating one or more Toll-like receptors (TLRs) and thereby causing local tissues to enter an antiviral state. In contrast, achieving high levels of antigen production with RNA vaccines (e.g., mRNA vaccines) requires minimal immunogenicity because localized innate immune responses can significantly reduce expression levels. The present disclosure provides adjuvant technologies native to RNA, such as adjuvant technologies native to mRNA, which can provide improvements in the intensity of immune responses without offsetting antigen expression. This can be achieved, for example, by using protein-based fusions to make RNA-encoded antigens (e.g., mRNA-encoded antigens) more immunogenic in a manner that is decoupled from the immunogenicity of the vaccine itself. In one embodiment, the fusion protein will directly stimulate and / or drive the uptake of B cells while minimizing nonspecific inflammation at sites where the RNA (e.g., mRNA) is expressed. A large group of candidate fusion domains were screened, and it was observed that the Sbi fragments described herein increased antibody titers across a range of antigen expression levels. This represents a first-in-class solution for preparing adjuvants that work well with RNA-based (e.g., mRNA-based) vaccines. Since viral and DNA vaccine vectors face the same immunogenicity-antigen expression optimization challenges, the Sbi fragments (e.g., SbiIII-IV) described herein can be used to improve other classes of nucleic acid-based vaccines.

[0204] Without wishing to be bound by theory, it is believed that considering the known information about the mechanism of action of SbiIII-IV, the fusion method described herein has the additional benefit of expanding the design space of effective antigens. The production of antibodies is driven by 1) binding antigens via B cell receptors (BCR) and 2) B cells activated by second signals. The most common activation signal is provided by helper T cells, which means that effective antigens need to be combined with BCR and need to contain peptides that are effectively presented on MHC and combined with T cell receptors (TCR). In contrast, SbiIII-IV is combined with complement fragments that can directly license B cells, thereby bypassing the need to produce a strong reaction in helper T cells. This can be possible to expand the design space of effective antigens by making it possible to use fragments that lack good T cell epitopes, for example. In some embodiments, one of the applications of the fusion architecture based on SbiIII-IV is to prepare a minimal antigen containing only a portion of a naturally occurring protein. As an example, the present disclosure provides compositions and uses of SbiIII-IV-based fusions comprising a portion of the SARS-CoV-2 spike protein (e.g., RBD and sub-RBD portions) as an exemplary antigen fused to an Sbi fragment (SbiIII-IV), which fusion has enhanced antigenicity (see Example 1) and / or stimulates a productive humoral immune response (see Examples 2-4).

[0205] The present disclosure recognizes for the first time that vaccination with a fragment antigen fused to an adjuvant comprising a complement C3d binding region can enhance the titer of the resulting antibody response and / or result in a measurable T cell response. In some embodiments, the adjuvant is or comprises the complement C3d binding region of the Sbi protein from Staphylococcus aureus. In some embodiments, the fragment antigen fused to an adjuvant comprising a complement C3d binding region acts as a synthetic immune synapse, thereby simulating a natural viral infection to drive a strong and appropriate immune response. In some embodiments, the fragment antigen fused to an adjuvant comprising a complement C3d binding region allows a small antigen lacking an MHC presenting peptide to elicit a meaningful humoral response.

[0206] The present disclosure also recognizes that vaccination with fragment antigens or variant antigens (rather than full-length antigens) can be used to develop vaccines that are less resistant to viral mutations and have broad neutralizing effects. The vaccination methods described herein can focus the immune response on conserved regions and / or functional regions to increase the breadth and efficacy of the vaccine. Without wishing to be bound by theory, it is believed that in some embodiments, immune focusing can occur by using fragment antigens that: (1) minimally contain epitopes of the target protein antigen that can be presented on MHC I and / or MHC II; and / or (2) have a conformation (confirmation) similar to the full-length or native target protein antigen.

[0207] Fragment or variant antigen

[0208] The present disclosure provides fragment antigens or variant antigens comprising epitopes of target protein antigens and compositions comprising the same. In some embodiments, the fragment antigen or variant antigen is fused with an adjuvant (eg, a C3d binding polypeptide) for use in an immunogenic composition.

[0209] In some embodiments, the fragment antigen or variant antigen comprises an epitope (e.g., a T cell epitope) of the target protein antigen. In some embodiments, the fragment antigen or variant antigen comprises a portion of an epitope (e.g., a T cell epitope) of the target protein antigen.

[0210] In some embodiments, the fragment antigen or variant antigen does not comprise an epitope (eg, a T cell epitope) of the target protein antigen.

[0211] In some embodiments, the fragment antigen has an amino acid sequence length of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the amino acid sequence length of the target protein antigen. In some embodiments, the fragment antigen has an amino acid sequence length of no more than 50% of the amino acid sequence length of the target protein antigen. In some embodiments, the fragment antigen has an amino acid sequence length of no more than 40%, 30%, 20%, 10% or 5% of the amino acid sequence length of the target protein antigen.

[0212] In some embodiments, the fragment antigen has a length of about 10-300 amino acid residues. In some embodiments, the fragment antigen has a length of at least 10 amino acid residues. In some embodiments, the fragment antigen has a length of less than about 300 amino acid residues. In some embodiments, the fragment antigen has a length of about 10-300, 10-250, 10-200, 10-150, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-300, 30-300, 40-300, 50-300, 60-300, 70-300, 80-300, 90-100, 100-300, 150-300, 200-300, 250-300, 20-250, 30-200, 40-150, 50-100, 60-90, or 70-80 amino acid residues. In some embodiments, the fragment antigen has a length of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 amino acid residues.

[0213] In some embodiments, the amino acid sequence length of the antigen variant or fragment antigen variant is identical to the amino acid sequence length of the target protein antigen.

[0214] In some embodiments, antigen variants or fragment antigen variants include at least one modified amino acid compared to the target protein antigen. In some embodiments, the modified amino acid includes N-linked glycosylation. In some embodiments, antigen variants or fragment antigen variants include at least one amino acid mutation compared to the target protein antigen. In some embodiments, antigen variants or fragment antigen variants include 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 or 50 amino acid mutations compared to the target protein antigen. In some embodiments, the mutation introduces serine, threonine, alanine or an amino acid different from the amino acid present at the position in the target protein antigen at a specific position. In some embodiments, the mutation prevents the formation of disulfide bonds.

[0215] In some embodiments, the fragment antigen is characterized in that when expressed in vivo, the fragment antigen binds to a major histocompatibility complex (MHC) molecule.

[0216] In some embodiments, the fragment antigen is characterized in that when expressed in vivo, the fragment antigen does not bind to an MHC molecule.

[0217] In some embodiments, the MHC molecule is or comprises an MHC I molecule or an MHC II molecule.

[0218] In some embodiments, a fragment antigen is characterized in that when expressed in vivo, the fragment antigen folds into a three-dimensional conformation that is substantially identical to the three-dimensional conformation of the fragment antigen when in its native position in the target protein antigen.

[0219] In some embodiments, the fragment antigen, antigen variant, or fragment antigen variant further comprises an amino acid sequence from a second target protein antigen.

[0220] Target protein antigen

[0221] Among other things, the present invention provides a fusion polypeptide comprising a fragment antigen or a variant antigen, which comprises an epitope of a target protein antigen. The present invention also provides a fusion polynucleotide encoding a fusion polypeptide comprising a fragment antigen or a variant antigen, which comprises an epitope of a target protein antigen.

[0222] In some embodiments, the target protein antigen is or comprises an infectious disease antigen. In some embodiments, the infectious disease antigen is or comprises a viral antigen, a bacterial antigen, a fungal antigen, or a combination thereof. In some embodiments, the viral antigen is or comprises an influenza antigen. In some embodiments, the viral antigen is or comprises a coronavirus polypeptide. In some embodiments, the coronavirus polypeptide is or comprises a SARS-CoV-2 protein, for example, as described herein.

[0223] In some embodiments, the target protein antigen is or comprises a cancer antigen.

[0224] SARS-CoV-2 Antigen

[0225] Exemplary antigens that may be included in any fusion polypeptide, fusion polynucleotide, composition, method, or use disclosed herein include one or more SARS-CoV-2 polypeptides. In some embodiments, the target protein antigen disclosed herein is or comprises a SARS-CoV-2 antigen. In some embodiments, the SARS-CoV-2 antigen is selected from: a spike glycoprotein (SARS-CoV-2S) polypeptide or an antigenic fragment thereof; an envelope protein (SARS-CoV-2E) polypeptide or an antigenic fragment thereof; a membrane protein (SARS-CoV-2M) polypeptide or an antigenic fragment thereof; a nucleocapsid protein (SARS-CoV-2N) polypeptide or an antigenic fragment thereof; an auxiliary factor polypeptide or an antigenic fragment thereof; or any combination thereof.

[0226] In some embodiments, the antigen (e.g., a fragment antigen or a variant antigen) comprises an epitope from a target protein antigen that is or comprises a SARS-CoV-2 polypeptide. In some embodiments, the antigen (e.g., a fragment antigen or a variant antigen) comprises an epitope from a SARS-CoV-2 spike glycoprotein (SARS-CoV-2S) polypeptide or a fragment thereof (e.g., RBD).

[0227] The SARS-CoV-2 S polypeptide is referenced by Gene ID: 43740568 and / or NCBI RefNC_045512.2. The amino acid sequence of the SARS-CoV-2 S polypeptide is provided by SEQ ID NO:58 below:

[0228] MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFR

[0229] SSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIR

[0230] GWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEF

[0231] RVY

[0232] SSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDL

[0233] PQ

[0234] GFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFL

[0235] LKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITN

[0236] LCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDL

[0237] CF

[0238] TNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGG

[0239] NYN

[0240] YLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQ

[0241] PY

[0242] RVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQ

[0243] QFG

[0244] RDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAI

[0245] HADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNS

[0246] PR

[0247] RARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTM

[0248] YICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKD

[0249] FG

[0250] GFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFN

[0251] GLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVT

[0252] QN

[0253] VLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSN

[0254] FGA

[0255] ISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMS

[0256] ECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDG

[0257] KAH

[0258] FPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPE

[0259] LD

[0260] SFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELG

[0261] KYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDED

[0262] DSE

[0263] PVLKGVKLHYT

[0264] The polynucleotide sequence of the SARS-CoV-2 S polypeptide is provided by SEQ ID NO:59 below:

[0265] 1atgtttgttt ttcttgtttt attgccacta gtctctagtc agtgtgttaa tcttacaacc

[0266] 61agaactcaat taccccctgc atacactaat tctttcacac gtggtgttta ttaccctgac

[0267] 121aaagttttca gatcctcagt tttacattca actcaggact tgttcttacc tttcttttcc

[0268] 181aatgttactt ggttccatgc tatacatgtc tctgggacca atggtactaa gaggtttgat

[0269] 241aaccctgtcc taccatttaa tgatggtgtt tattttgctt ccactgagaa gtctaacata

[0270] 301ataagaggct ggattttttgg tactacttta gattcgaaga cccagtccct acttattgtt

[0271] 361aataacgcta ctaatgttgt tattaaagtc tgtgaatttc aatttgtaa tgatccattt

[0272] 421ttgggtgttt attack aaacaacaaa agttggatgg aaagtgagtt cagagtttat

[0273] 481tctagtgcga ataattgcac ttttgaat gtctctcagc cttttcttat ggaccttgaa

[0274] 541ggaaaacagg gtaatttcaa aaatcttagg gaatttgtgt ttaagaat tgatggttat

[0275] 601tttaaaatat attctaagca cacgcctatt aatttagtgc gtgatctccc tcagggtttt

[0276] 661tcggctttag aaccattggt agatttgcca attacktatta acatcactag gtttcaaact

[0277] 721ttacttgctt tacatagaag ttatttgact cctggtgatt cttcttcagg ttggacagct

[0278] 781ggtgctgcag cttattatgt gggttatctt caacctagga cttttcttatt aaaatataat

[0279] 841gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctctc agaaacaaag

[0280] 901tgtacgttga aatccttcac tgtagaaaaa ggaatctatc aaacttctaa ctttagagtc

[0281] 961caaccaacag aatctattgt tagatttcct aatattacaa acttgtgccc ttttggtgaa

[0282] 1021gtttttaacg ccaccagatt tgcatctgtt tatgcttgga acaggaagag aatcagcaac

[0283] 1081tgtgttgctg attattctgt cctatataat tccgcatcat tttccacttt taaggtttat

[0284] 1141ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc agattcattt

[0285] 1201gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaaa gattgctgat

[0286] 1261 tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaattctaacaat

[0287] 1321 cttgattcta aggttggtgg taattataat tacctgtata gattgtttaggaagtctaat

[0288] 1381 ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtagcacaccttgt

[0289] 1441 aatggtgttg aaggttttaa ttgttacttt cctttacaat catatggtttccaacccact

[0290] 1501 aatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaacttctacatgca

[0291] 1561 ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaaatgtgtcaat

[0292] 1621 ttcaacttca atggtttaac aggcacaggt gttcttactg agtctaacaaaaagtttctg

[0293] 1681 cctttccaac aatttggcag agacattgct gacactactg atgctgtccgtgatccacag

[0294] 1741 acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtgttataacacca

[0295] 1801 ggaacaaata cttctaacca ggttgctgtt ctttatcagg atgttaactgcacagaagtc

[0296] 1861 cctgttgcta ttcatgcaga tcaacttact cctacttggc gtgtttattctacaggttct

[0297] 1921 aatgtttttc aaacacgtgc aggctgttta ataggggctg aacatgtcaacaactcatat

[0298] 1981 gagtgtgaca tacccattgg tgcaggtata tgcgctagtt atcagactcagactaattct

[0299] 2041 cctcggcggg cacgtagtgt agctagtcaa tccatcattg cctacactatgtcacttggt

[0300] 2101 gcagaaaatt cagttgctta ctctaataac tctattgcca tacccacaaattttactatt

[0301] 2161 agtgttacca cagaaattct accagtgtct atgaccaaga catcagtagattgtacaatg

[0302] 2221 tacatttgtg gtgattcaac tgaatgcagc aatcttttgt tgcaatatggcagtttttgt

[0303] 2281 acacaattaa accgtgcttt aactggaata gctgttgaac aagacaaaaacacccaagaa

[0304] 2341 gtttttgcac aagtcaaaca aatttacaaa acaccaccaa ttaaagattttggtggtttt

[0305] 2401 aatttttcac aaatattacc agatccatca aaaccaagca agaggtcatttattgaagat

[0306] 2461 ctacttttca acaaagtgac acttgcagat gctggcttca tcaaacaatatggtgattgc

[0307] 2521 cttggtgata ttgctgctag agacctcatt tgtgcacaaa agtttaacggccttactgtt

[0308] 2581 ttgccacctt tgctcacaga tgaaatgatt gctcaataca cttctgcactgttagcgggt

[0309] 2641 acaatcactt ctggttggac ctttggtgca ggtgctgcat tacaaataccatttgctatg

[0310] 2701 caaatggctt ataggtttaa tggtattgga gttacacaga atgttctctatgagaaccaa

[0311] 2761 aaattgattg ccaaccaatt taatagtgct attggcaaaa ttcaagactcactttcttcc

[0312] 2821 acagcaagtg cacttggaaa acttcaagat gtggtcaacc aaaatgcacaagctttaaac

[0313] 2881 acgcttgtta aacaacttag ctccaatttt ggtgcaattt caagtgttttaaatgatatc

[0314] 2941 ctttcacgtc ttgacaaagt tgaggctgaa gtgcaaattg ataggttgatcacaggcaga

[0315] 3001 cttcaaagtt tgcagacata tgtgactcaa caattaatta gagctgcagaaatcagagct

[0316] 3061 tctgctaatc ttgctgctac taaaatgtca gagtgtgtac ttggacaatcaaaaagagtt

[0317] 3121 gatttttgtg gaaagggcta tcatcttatg tccttccctc agtcagcacctcatggtgta

[0318] 3181 gtcttcttgc atgtgactta tgtccctgca caagaaaaga acttcacaactgctcctgcc

[0319] 3241 atttgtcatg atggaaaagc acactttcct cgtgaaggtg tctttgtttcaaatggcaca

[0320] 3301 cactggtttg taacacaaag gaatttttat gaaccacaaa tcattactacagacaacaca

[0321] 3361tttgtgtctg gtaactgtga tgttgtaata ggaattgtca acaacacagt ttatgatcct

[0322] 3421ttgcaacctg aattagactc attcaaggag gagttagata aatattttaa gaatcataca

[0323] 3481tcaccagatg ttgatttagg tgacatctct ggcattaatg cttcagttgt aaacattcaa

[0324] 3541aaagaaattg accgcctcaa tgaggttgcc aagaatttaa atgaatctct catcgatctc

[0325] 3601caagaacttg gaaagtatga gcagtatata aaatggccat ggtacatttg gctaggtttt

[0326] 3661atagctggct tgattgccat agtaatggtg acaattatgc tttgctgtat gaccagttgc

[0327] 3721tgtagttgtc tcaagggctg ttgttcttgt ggatcctgct gcaaatttga tgaagacgac

[0328] 3781tctgagccag tgctcaaagg agtcaaatta cattacacat aa

[0329] In some embodiments, the target protein antigen is or comprises a SARS-CoV-2S polypeptide. In some embodiments, the target protein antigen comprises a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of a SARS-CoV-2S polypeptide or a fragment thereof as described herein. In some embodiments, the target protein antigen comprises a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 58.

[0330] In some embodiments, the fragment antigen comprises an epitope of a SARS-CoV-2S polypeptide. In some embodiments, the fragment antigen comprises a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of a SARS-CoV-2S polypeptide or a fragment thereof as described herein. In some embodiments, the fragment antigen comprises a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 58.

[0331] In some embodiments, the fragment antigen comprises an epitope of a SARS-CoV-2S polypeptide. In some embodiments, the fragment antigen comprises a polynucleotide encoding a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of a SARS-CoV-2S polypeptide or a fragment thereof as described herein. In some embodiments, the fragment antigen comprises a polynucleotide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the nucleic acid sequence of SEQ ID NO: 59. Due to the degeneracy of the genetic code, one of ordinary skill in the art will understand that other DNA sequences (including codon-optimized sequences) can encode these polypeptides, as well as other polypeptides disclosed herein.

[0332] Adjuvant

[0333] In some embodiments, the adjuvants disclosed herein can be used to induce and / or modulate an immune response induced by an antigen described herein (e.g., a fragment antigen or an antigen variant). In some embodiments, the adjuvants disclosed herein comprise a complement binding polypeptide. In some embodiments, the complement binding polypeptide comprises a complement C3d binding polypeptide. An exemplary C3d binding polypeptide is an immunoglobulin binding protein (Sbi) of Staphylococcus aureus.

[0334] In some embodiments of any fusion polypeptide, fusion nucleotide, composition, method or use disclosed herein, the adjuvant disclosed herein can be used alone or in combination with an antigen disclosed herein to regulate and / or enhance an immune response. In one embodiment, the adjuvant disclosed herein may comprise a fusion polypeptide comprising a fragment antigen or a variant antigen and an adjuvant (e.g., a C3d binding polypeptide). In some embodiments, a fusion polynucleotide encoding a fusion polypeptide is also disclosed herein, comprising a fragment antigen or a variant antigen and an adjuvant (e.g., a C3d binding polypeptide). In some embodiments, the fusion polynucleotide comprises DNA or RNA. In some embodiments, the fusion polynucleotide comprises RNA, such as messenger RNA. In some embodiments, the fusion polynucleotide comprising RNA (e.g., messenger RNA) is characterized in that, when administered to a subject, it reduces the immunogenicity of the antigen contained in the fusion polynucleotide.

[0335] Staphylococcus aureus (S. aureus)

[0336] As disclosed herein, the immunoglobulin Staphylococcus aureus binding agent (Sbi) is an exemplary polypeptide that can bind complement C3d (as described in Clark et al. (2011) Mol Immunol. 48(4):452–462, the entire contents of which are incorporated herein by reference). Sbi comprises two immunoglobulin binding domains (domains I and II) and two complement C3d binding domains (domains III and IV). Sbi domains III and IV can bind C3d (in native C3, iC3b, and C3dg) and can lead to fluid phase depletion of C3 via activation of the alternative pathway (see Clark et al. 2011). It has also been shown that Sbi can be secreted and participate in S. aureus immune evasion (Burman et al., 2008 J. Biol. Chem; 283: 17579–17593).

[0337] Without wishing to be bound by theory, it is believed that in some embodiments, complement C3d binding polypeptides from S. aureus Sbi can be used as adjuvants for enhancing and / or modulating immune responses from antigens described herein. In some embodiments, the immune response is elicited by a fragment antigen or antigen variant disclosed herein. In some embodiments, the immune response is elicited by a component of Sbi of S. aureus.

[0338] Staphylococcus aureus Sbi is referred to by gene ID 3919725 and NCBI reference number NC_007795.1. The polynucleotide sequence of Sbi is provided herein as SEQ ID NO:56 below:

[0339] 1atgaaaaata aatatatctc gaagttgcta gttggggcag caacaattac gttagctaca

[0340] 61atgatttcaa atggggaagc aaaagcgagt gaaaacacgc aacaaacttc aactaagcac

[0341] 121caaacaactc aaaacaacta cgtaacagat caacaaaaag ctttttatca agtattacat

[0342] 181ctaaaaggta tcacagaaga acaacgtaac caatacatca aaacattacg cgaacaccca

[0343] 241gaacgtgcac aagaagtatt ctctgaatca cttaaagaca gcaagaaccc agaccgacgt

[0344] 301gttgcacaac aaaacgcttt ttacaatgtt cttaaaaatg ataacttaac tgaacaagaa

[0345] 361aaaaataatt acattgcaca aattaaagaa aaccctgata gaagccaaca agtttgggta

[0346] 421gaatcagtac aatcttctaa agctaaagaa cgtcaaaata ttgaaaatgc ggataaagca

[0347] 481attaaagatt tccaagataa caaagcacca cacgataaat cagcagcata tgaagctaac

[0348] 541tcaaaattac ctaaagattt acgtgataaa aacaaccgct ttgtagaaaa agtttcaatt

[0349] 601gaaaaagcaa tcgttcgtca tgatgagcgt gtgaaatcag caaatgatgc aatctcaaaa

[0350] 661ttaaatgaaa aagattcaat tgaaaacaga cgtttagcac aacgtgaagt taacaaagca

[0351] 721cctatggatg taaaagagca tttacagaaa caattagacg cattagttgc tcaaaaagat

[0352] 781gctgaaaaga aagtggcgcc aaaagttgag gctcctcaaa ttcaatcacc acaaattgaa

[0353] 841

[0354] 901gttcctcaat ctaaattat aggttactac caatcattaa aagattcatt taactatggt

[0355] 961tacaagtatt taacagatac ttataaaagc tataaagaaa aatatgatac agcaaagtac

[0356] 1021tactataata cgtactataa atacaaaggt gcgattgatc aaacagtatt aacagtacta

[0357] 1081ggtagtggtt ctaaatctta catccaacca ttgaaagttg atgataaaaa cggctactta

[0358] 1141gctaaatcat atgcacaagt aagaaactat gtaactgagt caatcaatac tggtaaagta

[0359] 1201ttatatactt tctaccaaaa cccaacatta gtaaaaacag ctattaaagc tcaagaaact

[0360] 1261gcatcatcaa tcaaaaatac attaagtaat ttattatcat tctggaaata a

[0361] The Sbi polypeptide sequence is provided herein as SEQ ID NO:57 below:

[0362] MKNKYISKLLVGAATITLATMISNGEAKASENTQQTSTKHQTTQNNYVTDQQKAFYQVLHLKGITEEQRNQYIKTLREHPERAQEVFSESLKDSKNPDRRVAQQNAFYNVLKNDNLTEQEKNNYIAQIKENPDRSQQVWVESVQSSKAKERQNIENADKAIKDFQDNKAPHDKSAAYEANSKLPKDLRDKNNRFVEKVSIEKAIVRHDERVKSANDAISKLNEKDSIENRRLAQREVNKAPMDVKEHLQKQLDALVAQKDAEKKVAPKVEAPQIQSPQIEKPKVESPKVEVPQIQSPKVEVPQSKLLGYYQSLKDSFNYGYKYLTDTYKSYKEKYDTAKYYYNTYYKYKGAIDQTVLTVLGSGSKSYIQPLKVDDKNGYLAKSYAQVRNYVTESINTGKVLYTFYQNPTLVKTAIKAQETASSIKNTLSNLLSFWK

[0363] In some embodiments, any fusion polypeptide, fusion nucleotide, composition, method or use disclosed herein comprises a complement C3d binding polypeptide of Sbi of Staphylococcus aureus. In some embodiments, the Sbi complement C3d binding polypeptide comprises one or both of domain III and domain IV of Sbi, or a functional fragment or variant thereof.

[0364] In some embodiments, the Sbi complement C3d binding polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identical to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the Sbi complement C3d binding polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identical to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the Sbi complement C3d binding polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 57.

[0365] In some embodiments, the Sbi complement C3d binding polypeptide is encoded by a polynucleotide encoding an amino acid having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO: 9. In some embodiments, the Sbi complement C3d binding polypeptide is encoded by a polynucleotide encoding an amino acid having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO: 10. In some embodiments, the Sbi complement C3d binding polypeptide is encoded by a polynucleotide encoding an amino acid having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO: 56. Due to the degeneracy of the genetic code, those of ordinary skill in the art will appreciate that other DNA sequences, including codon-optimized sequences, can encode these polypeptides, as well as other polypeptides disclosed herein.

[0366] Fusion peptide

[0367] The present disclosure provides fusion polypeptides comprising (i) a fragment antigen or antigen variant comprising an epitope of a target protein antigen; and (ii) a complement C3d binding polypeptide from an immunoglobulin binding protein (Sbi) of Staphylococcus aureus. Exemplary fusion polypeptides disclosed herein are provided in Tables 1 and 2.

[0368] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:3.

[0369] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:4.

[0370] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:5.

[0371] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:6.

[0372] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:7.

[0373] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90% or 100% identity to the amino acid sequence of SEQ ID NO:8.

[0374] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:20.

[0375] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:21.

[0376] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:22.

[0377] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:23.

[0378] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:24.

[0379] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:25.

[0380] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:26.

[0381] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:27.

[0382] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:28.

[0383] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:29.

[0384] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:30.

[0385] In some embodiments, the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:31.

[0386] In some embodiments, (a) and (b) are continuous or separated by a joint. In some embodiments, the joint is a peptidyl joint. In some embodiments, the peptidyl joint comprises at least 60% glycine and / or serine. In some embodiments, the joint is selected from a Gly-Gly-Gly-Gly-Ser (Gly4-Ser) joint or a histidine joint. In some embodiments, the joint is a Gly4-Ser joint. In some embodiments, the joint comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 repeats of a Gly4-Ser joint. In some embodiments, the joint comprises 3 repeats of a Gly4-Ser joint. In some embodiments, the joint comprises the sequence of SEQ ID NO: 11.

[0387] In some embodiments, the polypeptide further comprises a secretory peptide. In some embodiments, the secretory peptide is about 10-30 amino acids in length. In some embodiments, the secretory peptide is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length. In some embodiments, the secretory peptide comprises an amino acid sequence having at least 80%, 85%, 90% or 100% identity with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the secretory peptide comprises an amino acid sequence having at least 80%, 85%, 90% or 100% identity with the amino acid sequence of SEQ ID NO: 2.

[0388] In some embodiments, the polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:12.

[0389] In some embodiments, the polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:13.

[0390] In some embodiments, the polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:14.

[0391] In some embodiments, the polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:15.

[0392] In some embodiments, the polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:16.

[0393] In some embodiments, the polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:17.

[0394] In some embodiments, the polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:18.

[0395] In some embodiments, the polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:19.

[0396] In some embodiments, the polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:32.

[0397] In some embodiments, the polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:33.

[0398] In some embodiments, the polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:34.

[0399] In some embodiments, the polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:35.

[0400] In some embodiments, the polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:36.

[0401] In some embodiments, the polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:37.

[0402] In some embodiments, the polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:38.

[0403] In some embodiments, the polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:39.

[0404] In some embodiments, the polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:40.

[0405] In some embodiments, the polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:41.

[0406] In some embodiments, the polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:42.

[0407] In some embodiments, the polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:43.

[0408] In some embodiments, the polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:44.

[0409] In some embodiments, the polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:45.

[0410] Table 1: Exemplary amino acid sequences of components of polypeptide fusions disclosed herein

[0411]

[0412]

[0413]

[0414]

[0415]

[0416] Table 2: Exemplary amino acid sequences of polypeptide fusions encoded by the polynucleotide sequences disclosed herein

[0417]

[0418]

[0419]

[0420]

[0421]

[0422] Fusion polynucleotide Among other things, the present disclosure provides fusion polynucleotides encoding fusion polypeptides comprising (i) a fragment antigen or antigen variant comprising an epitope of a target protein antigen; and (ii) a complement C3d binding polypeptide from an immunoglobulin binding protein (Sbi) of Staphylococcus aureus. Exemplary fusion polynucleotide sequences are provided in Table 3.

[0423] In some embodiments, (a) and (b) are encoded by nucleotide sequences and are located in the same nucleotide sequence or different nucleotide sequences. In some embodiments, (a) is located at the N-terminus of (b). In some embodiments, (a) is located at the C-terminus of (b).

[0424] In some embodiments, (a) is located at the C-terminus of (b). In some embodiments, (a) and (b) are continuous or separated by a nucleotide sequence encoding a joint. In some embodiments, the joint is a peptidyl joint. In some embodiments, the peptidyl joint comprises at least 60% glycine and / or serine. In some embodiments, the joint is selected from a Gly-Gly-Gly-Gly-Ser (Gly4-Ser) joint or a histidine joint.

[0425] In some embodiments, the fusion polynucleotide further comprises a nucleotide sequence encoding a secretory peptide.

[0426] In some embodiments, the fusion polynucleotide is or comprises RNA.In some embodiments, the fusion polynucleotide is or comprises messenger RNA.

[0427] In some embodiments, the fusion polynucleotide is or comprises DNA.

[0428] In some embodiments, the fusion polynucleotide encodes a fragment antigen characterized in that when expressed in vivo, the fragment antigen binds to a major histocompatibility complex (MHC) molecule. In some embodiments, the MHC molecule is or comprises an MHC I molecule or an MHC II molecule.

[0429] In some embodiments, the fusion polynucleotide encodes a fragment antigen characterized in that when expressed in vivo, the fragment antigen does not bind to an MHC molecule.

[0430] In some embodiments, the fusion polynucleotide encodes a fragment antigen characterized in that when expressed in vivo, the fragment antigen folds into a three-dimensional conformation that is substantially identical to the three-dimensional conformation of the fragment antigen when in its native position in the target protein antigen.

[0431] In some embodiments, the fusion polynucleotide encodes a fragment antigen, antigen variant, or fragment antigen variant that also comprises an amino acid sequence from a second target protein antigen.

[0432] In some embodiments, the fusion polynucleotide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:46.

[0433] In some embodiments, the fusion polynucleotide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:47.

[0434] In some embodiments, the fusion polynucleotide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:48.

[0435] In some embodiments, the fusion polynucleotide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:49.

[0436] In some embodiments, the fusion polynucleotide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:50.

[0437] In some embodiments, the fusion polynucleotide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:51.

[0438] In some embodiments, the fusion polynucleotide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:52.

[0439] In some embodiments, the fusion polynucleotide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:53.

[0440] In some embodiments, the fusion polynucleotide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO:54.

[0441] Table 3: Exemplary polynucleotide sequences disclosed herein

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

[0448]

[0449] mRNA encoding polypeptide and preparation thereof

[0450] Provided herein, among other things, are polypeptides and polynucleotides that can be used to stimulate an immune response against an antigen and / or enhance the immunogenicity of an antigen.

[0451] In some embodiments, the polypeptides disclosed herein are encoded by a polynucleotide comprising RNA. In some embodiments, the polynucleotide comprises messenger RNA.

[0452] In some embodiments, polynucleotides comprising RNA (eg, mRNA) are formulated into lipid nanoparticle (LNP) formulations.

[0453] In some embodiments, the present disclosure provides LNP formulations for use in immunogenic compositions comprising polynucleotides containing RNA (eg, mRNA).

[0454] In some embodiments, LNP formulations comprising polynucleotides containing RNA (e.g., mRNA) are administered to a subject to enhance and / or modulate an immune response. In some embodiments, the immune response is elicited by an antigen contained in the polynucleotide.

[0455] In some embodiments, an LNP formulation comprising a polynucleotide comprising RNA (e.g., mRNA) is characterized in that when administered to a subject, it has reduced immunogenicity to an antigen (e.g., an antigen contained in the polynucleotide or an antigen contained in a polypeptide).

[0456] In some embodiments, a LNP formulation comprising a polynucleotide containing RNA (eg, mRNA) is characterized in that when administered to a subject, it focuses the immune response to conserved and / or functional regions of an epitope of a target protein antigen.

[0457] In some embodiments, an LNP formulation comprising a polynucleotide comprising RNA (eg, mRNA) is characterized in that, when administered to a subject, it increases the breadth and / or efficacy of an immune response against a target protein antigen in the subject.

[0458] In some embodiments, an LNP formulation comprising a polynucleotide comprising RNA (e.g., mRNA) is characterized in that when administered to a subject, it results in a humoral response (e.g., a broadly neutralizing humoral response) and / or a T cell-mediated response.

[0459] In some embodiments, the polypeptide disclosed herein comprises a fusion polypeptide as described herein. In some embodiments, the polynucleotide disclosed herein comprises a fusion polynucleotide as described herein. In some embodiments, the fusion polypeptide is encoded by a fusion polynucleotide comprising RNA. In some embodiments, the fusion polynucleotide comprises a messenger RNA.

[0460] In some embodiments, a fusion polynucleotide comprising RNA (eg, mRNA) is formulated into a lipid nanoparticle (LNP) formulation.

[0461] In some embodiments, the present disclosure provides LNP formulations for use in immunogenic compositions comprising a fusion polynucleotide comprising RNA (eg, mRNA).

[0462] In some embodiments, a LNP formulation comprising a fusion polynucleotide containing RNA (e.g., mRNA) is administered to a subject to enhance and / or modulate an immune response. In some embodiments, the immune response is elicited by a fragment antigen contained in the fusion polynucleotide. In some embodiments, the immune response is enhanced by an adjuvant (e.g., a C3d binding polypeptide) contained in the fusion polynucleotide.

[0463] In some embodiments, an LNP formulation comprising a fusion polynucleotide comprising RNA (e.g., mRNA) is characterized in that when administered to a subject, it has reduced immunogenicity to an antigen (e.g., an antigen contained in the fusion polynucleotide or an antigen contained in the fusion polypeptide).

[0464] In some embodiments, a LNP formulation comprising a fusion polynucleotide comprising RNA (eg, mRNA) is characterized in that when administered to a subject, it focuses the immune response to conserved and / or functional regions of an epitope of a target protein antigen.

[0465] In some embodiments, an LNP formulation comprising a fusion polynucleotide comprising RNA (eg, mRNA) is characterized in that when administered to a subject, it increases the breadth and / or efficacy of an immune response against a target protein antigen in the subject.

[0466] In some embodiments, an LNP formulation comprising a fusion polynucleotide comprising RNA (e.g., mRNA) is characterized in that when administered to a subject, it results in a humoral response (e.g., a broadly neutralizing humoral response) and / or a T cell-mediated response.

[0467] Composition

[0468] Among other things, the present disclosure provides compositions. Compositions disclosed herein, such as compositions comprising polypeptides or polynucleotides disclosed herein, can focus immune responses on conserved and / or functional regions of antigens. In some embodiments, such immune focusing can increase the breadth and / or efficacy of immune responses against antigens.

[0469] In some embodiments, a composition disclosed herein is characterized in that when administered to a subject, it reduces immunogenicity to an antigen (eg, an antigen contained in a fusion polynucleotide or an antigen contained in a fusion polypeptide).

[0470] In some embodiments, a composition disclosed herein is characterized in that when administered to a subject, it focuses the immune response to the conserved and / or functional regions of the epitope of the target protein antigen.

[0471] In some embodiments, a composition disclosed herein is characterized in that, when administered to a subject, it increases the breadth and / or efficacy of an immune response against a target protein antigen in the subject.

[0472] In some embodiments, a composition disclosed herein is characterized in that when administered to a subject, it results in a humoral response (e.g., a broadly neutralizing humoral response) and / or a T cell-mediated response.

[0473] In some embodiments, the composition comprises a polypeptide as described herein, e.g., a fusion polypeptide as described herein. In some embodiments, the composition comprises a polynucleotide, e.g., a fusion polynucleotide as described herein.

[0474] In some embodiments, the composition is or comprises a pharmaceutical composition, eg, as described herein.

[0475] In some embodiments, the composition is or comprises an expression vector comprising a polynucleotide disclosed herein, such as a fusion polynucleotide disclosed herein.

[0476] In some embodiments, the composition is or comprises an immunogenic composition, eg, as described herein.

[0477] Immunogenic composition

[0478] Disclosed herein are immunogenic compositions comprising (1) an antigenic fragment or antigenic variant comprising an epitope of a target protein antigen; and / or (2) an adjuvant comprising a complement C3d binding region. In some embodiments, the immunogenic compositions disclosed herein can enhance the titer of the resulting antibody response and / or result in a measurable T cell response.

[0479] In some embodiments, the immunogenic composition comprises an immunoglobulin binding protein of Staphylococcus aureus (Sbi) used as an adjuvant. In some embodiments, the immunogenic composition comprising Sbi used as an adjuvant comprises a Sbi polypeptide or a fragment thereof. In some embodiments, the immunogenic composition comprising Sbi used as an adjuvant comprises a polynucleotide encoding a Sbi polypeptide or a fragment thereof.

[0480] In some embodiments, the immunogenic composition comprises an antigenic fragment or antigenic variant comprising an epitope of a target protein antigen and an adjuvant comprising a complement C3d binding polypeptide.

[0481] In some embodiments, the immunogenic composition comprises a fusion polypeptide comprising an antigenic fragment or antigenic variant comprising an epitope of a target protein antigen fused to a complement C3d binding polypeptide. In some embodiments, the C3d binding polypeptide comprises an immunoglobulin binding protein (Sbi) of Staphylococcus aureus. In some embodiments, the complement C3d binding polypeptide is or comprises one or both of domain III and domain IV of Sbi of Staphylococcus aureus, or a functional fragment or variant thereof.

[0482] In some embodiments, the immunogenic composition comprises a fusion polynucleotide encoding a fusion polypeptide comprising an antigenic fragment or antigenic variant comprising an epitope of a target protein antigen fused to a complement C3d binding polypeptide. In some embodiments, the fusion polynucleotide comprises a C3d binding polypeptide containing an immunoglobulin binding protein (Sbi) of Staphylococcus aureus. In some embodiments, the complement C3d binding polypeptide is or comprises one or both of domain III and domain IV of Sbi of Staphylococcus aureus, or a functional fragment or variant thereof.

[0483] In some embodiments, an immunogenic composition comprises an expression vector comprising a polynucleotide or fusion polynucleotide disclosed herein.

[0484] Pharmaceutical composition

[0485] The pharmaceutical composition of the present disclosure may include a polypeptide disclosed herein (e.g., a fusion polypeptide), a polynucleotide disclosed herein (e.g., a fusion polynucleotide), or an expression vector comprising a polynucleotide (e.g., a fusion polynucleotide). In some embodiments, the pharmaceutical composition may include a pharmaceutically acceptable excipient, a diluent, or a combination thereof. In some embodiments, the pharmaceutical composition may include a buffer such as neutral buffered saline, phosphate buffered saline, etc.; a carbohydrate such as glucose, mannose, sucrose, or dextran; mannitol; a protein; a polypeptide or amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; and a preservative.

[0486] In some embodiments, the pharmaceutical composition is formulated for administration according to any of the administration routes disclosed herein. In some embodiments, the pharmaceutical composition is formulated for intramuscular administration, intradermal administration, intravenous administration, or subcutaneous administration.

[0487] Methods of using the compositions disclosed herein

[0488] The present disclosure provides, among other things, methods of using the fusion polypeptides described herein, the fusion polynucleotides described herein, or compositions comprising the same to stimulate an immune response to an antigen (eg, as a vaccine) or to enhance the immunogenicity of an antigen.

[0489] Also disclosed herein are methods of using the fusion polypeptides described herein, the fusion polynucleotides described herein, or compositions comprising the same to treat a disease or improve symptoms of a disease, such as a disease associated with an antigen described herein.

[0490] The use of compositions disclosed herein (e.g., compositions comprising fusion polypeptides disclosed herein or fusion polynucleotides) can focus immune responses on conserved and / or functional regions of antigens. In some embodiments, such immune focusing can improve the breadth and / or efficacy of immune responses against antigens.

[0491] In some embodiments, methods comprising administering a composition disclosed herein result in reduced immunogenicity to an antigen (eg, an antigen contained in a fusion polynucleotide or an antigen contained in a fusion polypeptide).

[0492] In some embodiments, the methods comprising administering a composition disclosed herein result in an immune response focused on conserved and / or functional regions of an epitope of a target protein antigen.

[0493] In some embodiments, the methods comprising administering a composition disclosed herein result in an increase in the breadth and / or efficacy of an immune response to a target protein antigen in a subject.

[0494] In some embodiments, the methods comprising administering a composition disclosed herein result in a humoral response (e.g., a broadly neutralizing humoral response) and / or a T cell-mediated response.

[0495] The present disclosure provides a method comprising administering to a subject in need thereof at least one dose of a pharmaceutical composition comprising a fusion polypeptide disclosed herein, a fusion polynucleotide disclosed herein, or an expression vector comprising a fusion polynucleotide disclosed herein. In some embodiments, the at least one dose is administered in an effective amount for inducing an immune response to a fragment antigen in a subject.

[0496] Disclosed herein is a method comprising administering to a subject: a first dose of a pharmaceutical composition disclosed herein; and a second dose of a pharmaceutical composition disclosed herein. In some embodiments, the pharmaceutical composition comprises a fusion polypeptide disclosed herein, a fusion polynucleotide disclosed herein, or an expression vector comprising a fusion polynucleotide disclosed herein.

[0497] In some embodiments, the first dose and the second dose are administered at least 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, or 4 weeks apart.

[0498] In some embodiments, the first and second doses are the same amount. In some embodiments, the first and second doses are different amounts.

[0499] The present disclosure provides a method for enhancing the immunogenicity of an antigen, comprising administering a fusion polypeptide disclosed herein, or a fusion polynucleotide disclosed herein, or a pharmaceutical composition disclosed herein to a subject in need thereof.

[0500] The present disclosure also provides a method for stimulating an immune response against an antigen, comprising administering a fusion polypeptide disclosed herein, or a fusion polynucleotide disclosed herein, or a pharmaceutical composition disclosed herein to a subject in need thereof.

[0501] Further provided herein is a method of treating a disease or ameliorating symptoms of a disease, comprising administering a fusion polypeptide disclosed herein, or a fusion polynucleotide disclosed herein, or a pharmaceutical composition disclosed herein to a subject in need thereof.

[0502] In some embodiments of any of the methods disclosed herein, a subject in need is a subject suffering from or susceptible to a disease, disorder or condition induced by a target protein antigen. In some embodiments, the subject is a mammalian subject. In some embodiments, the subject is a human subject.

[0503] In some embodiments of any of the methods or uses disclosed herein, a single dose of the fusion polypeptide, fusion polynucleotide, or pharmaceutical composition is administered. In some embodiments of any of the methods or uses disclosed herein, multiple doses of the fusion polypeptide, fusion polynucleotide, or pharmaceutical composition are administered.

[0504] In some embodiments of any of the methods or uses disclosed herein, the fusion polypeptide, fusion polynucleotide, or pharmaceutical composition is administered at a dose of about 0.5 micrograms to 10 micrograms.

[0505] In some embodiments of any of the methods or uses disclosed herein, administration of the fusion polynucleotide, fusion polypeptide, or pharmaceutical composition results in a humoral response. In some embodiments, the humoral response is an antibody response.

[0506] In some embodiments of any of the methods or uses disclosed herein, administration of the fusion polynucleotide, fusion polypeptide, or pharmaceutical composition results in an increase in the titer of an antibody response. In some embodiments, the increase in titer is an increase of about 10-fold to about 500-fold. In some embodiments, the increase in titer of the antibody response is compared to the administration of an otherwise similar fusion polynucleotide that does not comprise Sbi domain III, or a fragment or variant thereof; and Sbi domain IV, or a fragment or variant thereof. In some embodiments, the increase in titer of the antibody response is compared to the administration of an otherwise similar fusion polypeptide that does not comprise Sbi domain III, or a fragment or variant thereof; and Sbi domain IV, or a fragment or variant thereof. In some embodiments, the increase in titer of the antibody response is compared to the administration of an otherwise similar pharmaceutical composition that does not comprise a nucleotide sequence encoding Sbi domain III, or a fragment or variant thereof; and Sbi domain IV, or a fragment or variant thereof.

[0507] In some embodiments of any of the methods or uses disclosed herein, the composition is administered via any of the following routes of administration: intramuscular, intravenous, subcutaneous, intrathecal, intradermal, ocular, intranasal, sublingual, or oral.

[0508] Method for screening fragment antigens

[0509] Also provided herein is an in vitro method for selecting a fragment antigen, which is achieved by identifying such a polypeptide fragment, which is folded into a three-dimensional conformation that is substantially identical to the three-dimensional conformation when it is in the target protein antigen from which the fragment is derived. In some embodiments, the screening methods disclosed herein can identify antigens or fragment antigens that expand the design space of effective antigens. In some embodiments, the screening methods disclosed herein can identify antigens or fragment antigens that lack T cell epitopes or have a portion of T cell epitopes. In some embodiments, the screening methods disclosed herein can identify fragment antigens that contain only a portion of an epitope of a target protein antigen.

[0510] In some embodiments, the method comprises the following steps: providing a mammalian cell display system that displays a library of fragment antigen candidates on the cell surface; exposing the mammalian cell display system to a composition comprising antibodies that bind to a target protein antigen from which the fragment antigen candidates are derived; detecting fragment antigen candidates that bind to the target protein antigen-binding antibodies, wherein binding of the fragment antigen candidate that binds to at least one of the target protein antigen-binding antibodies indicates a likelihood that the fragment antigen candidate folds into a three-dimensional conformation that is substantially identical to its three-dimensional conformation when in the target protein antigen.

[0511] In some embodiments, the method further comprises the step of generating an antibody that binds to the target protein antigen. In some embodiments, the generation of the antibody comprises (i) immunizing an animal with a target protein antigen from which the fragment antigen candidate is derived or a polynucleotide encoding the target protein antigen; and (ii) identifying an antibody that binds to the target protein antigen.

[0512] In some embodiments, the mammalian cell display system comprises HEK293T cells, HeLa cells, or CHO cells.

[0513] In some embodiments, polypeptide fragments are identified based on binding affinity to antibodies in a serum binding assay or similar assay.

[0514] Other features of the present invention will become apparent in the course of the following description of exemplary embodiments, which are given for the purpose of illustrating the invention and are not intended to be limiting thereof.

[0515] Example

[0516] Example 1: Enhancement of Antigenicity of Fusion of SARS-CoV-2 S Protein Domain and Sbi(III-IV)

[0517] This embodiment describes the development of vaccines comprising fragment antigens that focus the immune response on specific regions of the antigen (e.g., conserved regions and / or functional key regions). Immunofocusing in this way can improve the breadth and efficacy of the vaccine. Among other things, there are two technical challenges in vaccination with antigens (e.g., fragment antigens) that are smaller than proteins or protein subdomains. First, the fragment antigens need to contain peptides that are effectively presented on the MHC complex. Second, the fragment antigen must have an epitope that is folded into a conformation identical to the conformation in the full protein.

[0518] BALB / c mice were administered a 10 μg dose of IM delivered mRNA encoding: (1) the RBD domain (RBD) of the SARS-CoV-2 S protein; (2) RBD fused to the transmembrane domain of the S protein (RBD-TM); (3) a fusion protein of the RBD domain of the SARS-CoV-2 S protein and Sbi (III-IV); or (4) a fusion protein of the RBD domain of the SARS-CoV-2 S protein and Sbi (III-IV) and RBD fused to the transmembrane domain of the S protein (RBD-TM). A group of unvaccinated mice served as controls. IgG titers in blood were assessed using ELISA on day 21 after vaccination. To assess T cell responses, spleens from 5 mice were pooled and plated into 4 wells at 500,000 cells / well. Two wells were each stimulated with either an N-terminal or C-terminal S protein peptide pool (PepMix SARS-CoV-2 spike glycoprotein cocktail).

[0519] The results showed that vaccination with a minimal antigen fused to the complement C3d binding region of the Sbi protein from Staphylococcus aureus ( Figure 1A ) enhanced the titer of the resulting antibody response ( FIG. 1B ). By screening a large panel of antigen presentation architectures, this strategy was expanded by co-delivering antigens fused to Sbi (III-IV) with the same antigen fused to a transmembrane domain ( FIG. 1B ). In addition to eliciting humoral responses, these mRNA-based vaccines also elicited IFNγ+IL2+multifunctional CD4+ responses ( FIG. 1C ). It was also observed that fusing peptides as short as 25 residues in length to Sbi (III-IV) enabled them to elicit meaningful antibody responses ( FIG. 1D ). Without wishing to be bound by any particular theory, C3d can directly activate B cells through complement receptor 2 (CR2) and allow the induction of immune responses in the absence of CD4+T cell signaling. Therefore, this scaffold can be used as a synthetic immune synapse, simulating natural viral infections to drive strong and appropriate immune responses. Therefore, in some embodiments, the fusion architecture described herein allows small antigens lacking MHC-presenting peptides to elicit meaningful humoral responses.

[0520] In order to solve the challenge of structure prediction, high-throughput screening was performed to determine whether the fragment antigen is folded into the same three-dimensional conformation as the whole protein (Figure 2A). The screening includes two steps. First, antiserum against the whole protein is produced by inoculating mice with mRNA vaccines encoding the full-length protein of interest. The polyclonal antibodies in the mouse serum are then used as probes for the fragments presented in the context of the mammalian surface display system. For example, HEK293T cells expressing a library of antigen fragments can be used for this step. The fragments bound to the antibodies produced by the whole protein may share a conformational state with the whole protein. Correctly folded fragments are identified by increasing the binding to serum antibodies (Figure 2B). This system allows for screening fragments in vitro with a flux that is two orders of magnitude higher than the actual flux in vivo.

[0521] Example 2: Fusion of Sbi(III-IV) with the RBD domain of SARS-CoV-2S protein increases virus neutralization

[0522] In this example, the virus neutralizing activity of sera obtained from mice vaccinated with mRNA encoding the RBD domain of the SARS-CoV-2S protein, or mice vaccinated with mRNA encoding a fusion of Sbi(III-IV) and the RBD domain of the SARS-CoV-2S protein was tested.

[0523] For this experiment, BABL / C mice were vaccinated as described in the "Animal Vaccination" section of Example 6. Specifically, 50 μL of the mRNA-LNP formulation of the indicated mRNA construct (10 μg mRNA dose) was injected into the right quadriceps of BALB / C mice. On days 10-21 after vaccination, serum from vaccinated mice was collected and incubated with target cells in the presence of SARS-CoV-2 virus. Serum from unvaccinated mice was used as a control.

[0524] like Figure 3 As shown, fusion of Sbi(III-IV) to the RBD domain of the SARS-CoV-2 S protein increased pseudotype virus neutralization compared to the RBD domain of the SARS-CoV-2 S protein alone. This effect was observed in both serum dilutions. This data suggests that vaccination with mRNA encoding a fusion of Sbi(III-IV) to the RBD domain of the SARS-CoV-2 S protein generates an antibody response with SARS-CoV-2 virus neutralizing properties.

[0525] Example 3: Stimulation of B cells by the fusion of Sbi (III-IV) and the RBD domain of SARS-CoV-2 S protein

[0526] This example describes the stimulation of B cells in mice administered a low dose of mRNA encoding a fusion of Sbi(III-IV) and the RBD domain of the SARS-CoV-2 S protein.

[0527] For this experiment, BABL / C mice were vaccinated as described in the "Animal Vaccination" section of Example 6. Specifically, mice were immunized with 0.5ug, 2.5ug or 10ug IM doses of mRNA containing a construct having a fusion of the RBD domain of the IL2 secretory peptide and the SARS-CoV-2S protein with Sbi (III-IV), also referred to as IL2 (ss) -RBD-Sbi (III-IV). Blood and spleen were collected from mice 10-21 days after inoculation. To assess titers, the blood of the animals was analyzed using an ELISA assay. To assess T cell responses, ELISPOTs for detecting IFNg and / or IL-2 were used.

[0528] like Figure 4A As shown, high titers were observed when the RBD-Sbi(III-IV) dose was reduced from 10 μg per mouse to 0.5 μg per mouse. Reducing the dose of IL2(ss)-RBD-Sbi(III-IV) to below 10 μg reduced the cellular response ( Figure 4B). These data suggest that the antibody response elicited by vaccination with IL2(ss)-RBD-Sbi(III-IV) involves bypassing T cells through direct stimulation of B cells.

[0529] Example 4: Boosting after primary immunization increases the fusion of Sbi (III-IV) with the RBD domain of SARS-CoV-2 S protein Antibody response induced by fusion

[0530] In this example, the effect of a vaccination regimen consisting of a priming dose followed by a booster dose was evaluated.

[0531] For this experiment, as described in the "animal vaccination" section of Example 6, BABL / C mice were vaccinated. Specifically, 50 μL mRNA mRNA-LNP preparations (10 μg mRNA initial immunization dose) were injected into mice in 4 groups, and the mRNA contained a construct of a fusion of the RBD domain with IL2 secretory peptide and SARS-CoV-2S protein and Sbi (III-IV), also referred to as IL2 (ss)-RBD-Sbi (III-IV). One group of mice did not receive additional vaccine administration (no booster group). The other three groups of mice received 50 μL mRNA-LNP preparations (10 μg mRNA doses) of booster doses on the 3rd, 7th and 14th days, respectively. Blood and serum were collected from mice 10-21 days after inoculation.

[0532] Figure 5A It was shown that administration of the booster dose increased the titer produced by IL2(ss)-RBD-Sbi(III-IV) by ≥776-fold (when administered on the 3rd day after priming), by ≥588.1-fold (when administered on the 7th day after priming), and by ≥9.2-fold (when administered on the 14th day after priming). Figure 5B It was shown that both booster vaccination and priming-only vaccination were able to block ACE2:RBD interaction in vitro at a serum dilution of 1:20. This data suggests that the antibody response elicited by vaccination with IL2(ss)-RBD-Sbi(III-IV) can be enhanced with a vaccination regimen that includes a priming dose and a boosting dose.

[0533] In the next experiment, the effect of different priming doses was tested. BALB / C mice were injected with a priming dose of 1ug or 5ug of an mRNA-LNP formulation containing an mRNA construct (S(ss)-RBD-Sbi(III-IV)) with a spike protein secretion peptide and a fusion of the RBD domain of the SARS-CoV-2S protein with Sbi(III-IV), or mRNA containing a construct (S(ss)-RBD) with a spike protein secretion peptide and the RBD domain of the SARS-CoV-2S protein. Mice received a booster dose on day 4 after the priming. Blood and serum were collected from mice 10-21 days after inoculation.

[0534] Figure 6 It was shown that priming with 1ug or 5ug of mRNA containing S(ss)-RBD-Sbi(III-IV) resulted in similar antibody titers. This data suggests that lower priming doses are sufficient to induce antibody responses.

[0535] Example 5: Broad-spectrum influenza A (H1N1) subtype vaccine resistant to HA antigen drift Development

[0536] This example describes the use of the methods provided in this disclosure to develop a broad spectrum of viruses (e.g., influenza A subtype H1N1 vaccines) that are resistant to HA antigenic drift. It is expected that this strategy will be widely applicable. This approach can be expanded by constructing drift-resistant and shift-resistant vaccines containing precisely designed antigens for all eighteen influenza HA subtypes.

[0537] The high-throughput folding screen described in Example 2 was used to identify the minimal antigen within the ribosome binding site (RBS) of the head domain of hemagglutinin HA1 ( Fig. 7A The RBS is essential for receptor binding and is widely conserved among influenza A (H1N1) viruses ( Figure 7B ), however, the surrounding variable residues often limit the breadth of RBS-guided antibodies (Raymond et al. (2018) PNAS 115(1): 168-173). This screen was used to identify the minimal foldable RBS that can focus the immune response on the conserved residues.

[0538] Example 6: Materials and Methods Used in Examples Disclosed herein

[0539] gBlock Amplification: Each gBlock template was prepared using Herculase II polymerase (Agilent) using an annealing temperature of 50°C with T7-AGG_fwd (gaattTAATACGACTCACTATAAGGcttgttctttttgcagaagc) and 120pA_rev (TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTagaatgtgaagaaactttctttttattag) (SEQ ID NO: 5) amplification.

[0540] mRNA synthesis : PCR products were cleaned up using a 0.8-fold ratio of SPRISelect beads:PCR reaction volume. 19.9 μL transcription mixture consisting of 1xHiScribe T7 High Yield buffer (NEB), 7.5 mM of each NTP, 7.5 mM CleanCapAG (TriLink Biotech), 2 M betaine (ThermoSci), 20 mM MgCl2, and 0.1 μL / μL HiScribe T7 polymerase mixture was added to a 2.1 uL DNA solution consisting of 200 ng T7 template in nuclease-free H2O. Transcription was performed at 50 °C for 1 hour. The transcribed RNA was purified using a 500 μg capacity Monarch RNA Cleanup kit, which was treated with DNAse I and purified again using a 500 μg capacity Monarch column. The mRNA was then treated with alkaline phosphatase (Millipore) at 37 °C for 10 min and purified using a 500 μg Monarch column. Concentrations were determined using a nanodrop spectrophotometer.

[0541] LNP formulation:Use NanoAssemlr Ignite microfluidic mixer (Precision Nanosystems) to prepare the preparation (mRNA-LNP) of mRNA in lipid nanoparticles.Use the preparation parameters recommended by the manufacturer, the GenVoy-ILM lipid mixture (Precision Nanosystems) is diluted to 12.5mM in absolute ethanol, and combined with the mRNA aqueous solution (0.14mg / mL) in PNI buffer (Precision Nanosystems).Immediately the preparation is diluted with 30:1 in phosphate buffered saline (pH 7.4), and concentrated using Amicon centrifugal filter (MilliporeSigma UFC901008).The preparation is stored at 4°C and used for in vivo research within 14 days.

[0542] Animal vaccination All animal experiments were conducted according to the guidelines set by the Charles River Accelerator Development Lab (CRADL) and approved by the CRADL Institutional Animal Care and Use committee. Female BALB / C mice (7-9 weeks old) were purchased from Charles River Laboratories and housed in CRADL. Before the study began, mice were adapted for at least 3 days. On the 1st day, 50 μL mRNA-LNP preparations (unless otherwise specified, 10 μg mRNA dosages were used) were injected into the right quadriceps of mice. For experiments involving two vaccinations, 50 μL mRNA-LNP preparations were injected into the left quadriceps of mice for booster immunization. At the 10th-21st day (as indicated), mice were euthanized, blood was collected via intracardiac rods, and spleens were dissected and collected for processing. Serum was separated from blood in MiniCollect serum separator tubes (Greiner Bio-One 450472) by centrifugation at 1200 xg for 10 min at 4°C. Fresh serum was stored at 4°C and used for evaluation of immunogenicity by ELISA and neutralization assays, the remainder was aliquoted and frozen at -80°C.

[0543] Spike titer ELISA assay: The ELISA protocol was adapted from the protocol previously established by Amanat et al. (Nat Med 26:1033–1036, 2020). Briefly, 96-well Immulon 4HBX plates (Thermo Fisher Scientific) were coated with 50 μl of 2 μg / ml SARS-CoV-2 (2019-nCoV) spike S1+S2 ECD-His recombinant protein (Sino Biological #40589-V08B1) in PBS per well at 4°C overnight. The plates were washed three times with 300 μl of PBS containing 0.1% Tween20 (PBS-T) and then blocked with 100 μl of PBS-T containing 3% skim milk per well for 1 h. Serial dilutions of serum and antibody controls were prepared in PBS-T containing 1% skim milk, and 100 μl of each serial dilution was added to the plate at room temperature and kept for 2 h. The wells were then washed three times in PBS-T as before. The wells were then incubated in 100 μl of a 1:3,000 dilution of goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (Sigma) in 1% milk PBS-T for 1 hour at room temperature. The plates were washed again three times in PBS-T. Within 10 min at room temperature, 100 μl of SIGMAFAST OPD (Sigma–Aldrich) solution was added to each well and kept for 10 min. The reaction was stopped by adding 50 μl of 3 M hydrochloric acid per well. The optical density was measured at 490 nm using a GloMax Discover (Promega) plate reader. The endpoint titer was determined by taking the last dilution before the signal dropped below 1 standard deviation above the mean of the signal from untreated control sera at the same dilution. If the signal never dropped below this threshold, the last dilution was taken as the titer. If no signal above the threshold was detected, the value in the dilution series before the lowest dilution tested was used. A chimeric monoclonal antibody reactive against the RBD of both SARS-CoV-1 and SARS-CoV-2 and a mouse monoclonal antibody reactive against SARS-CoV-1 were used as positive and negative controls, respectively.

[0544] Pseudotype neutralization assay:Human ACE2 overexpressing HEK cells (Integral Molecular) for viral transduction experiments were maintained in high glucose glutamine-containing DMEM (ThermoFisher Scientific 10564) supplemented with 1 μg / mL puromycin, 10% heat-inactivated fetal bovine serum, and 100U / mL penicillin / streptomycin. 4 μl (for 1:37.5 dilution) or 1 μl (for 1:500 dilution) of each serum was mixed with 35 μl of spike pseudotyped GFP reporter virus particles constructed using a second-generation lentiviral system (Integral Molecular, lot number CG-113A) in a puromycin-free medium to a total volume of 100 μl. The virus and serum were incubated in a 96-well cell culture plate at 37 ° C for 1.5 h. Then 20,000 freshly harvested HEK cells were added with 50 μL of puromycin-free medium and transduction was allowed to proceed for 3 days. The number of GFP-positive cells was quantified using the Cytation 5 Cell Imaging Multi-Mode Reader and Gen5 software (BioTek). Images of each well were taken using a 4x PL ACH objective in the GFP (469,525) channel with an LED set to 10.0, an integration time of 100 milliseconds, and a gain set to 11.0. Images were taken at a fixed focus height of 345 μm below the plate. Six images were taken for each well with automatic superposition so that the software could stitch them together. Image preprocessing was applied using a dark background and background flattening with a 60 μm rolling ball diameter. For cell analysis, a primary mask was applied to processed images with a dark background and a GFP threshold of 2000. The minimum and maximum object sizes were set to 10 μm and 1000 μm, respectively. Segmentation of contact objects, filling holes in the mask, inclusion of edge objects, and analysis of the entire image were all set to ON. Using these parameters, the software calculates the cell count, giving the number of GFP-positive cells per well.

[0545] ACE2:RBD inhibition assay : The ability of vaccinated sera to inhibit ACE2:RBD binding was measured using the SARS-CoV-2 Surrogate Virus Neutralization Test Kit (GenScript) essentially as described by Nie et al. (Nat Biotech 15:3699-3715, 2020).

[0546] Equivalent solutions

[0547] Those skilled in the art will recognize or be able to determine many equivalents of the specific embodiments of the invention described herein using only routine experimentation. It should be understood that, unless otherwise indicated or unless it is obvious to one of ordinary skill in the art that a contradiction or inconsistency will occur, the present invention covers all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc. from one or more of the listed claims are introduced into another claim that is subordinate to the same base claim (or any other claim that is related). In addition, it should also be understood that any embodiment or aspect of the present invention may be explicitly excluded from the claims, regardless of whether a specific exclusion is described in the specification. The scope of the present invention is not intended to be limited to the above description, but is set forth in the following claims.

[0548] Exemplary embodiments

[0549] Embodiment 1. A fusion polypeptide comprising:

[0550] (i) a fragment antigen comprising an epitope of a target protein antigen; and

[0551] (ii) Complement C3d binding polypeptide from the immunoglobulin binding protein (Sbi) of Staphylococcus aureus.

[0552] Embodiment 2. A fusion polypeptide as described in embodiment 1, wherein the complement C3d binding polypeptide is or comprises one or both of domain III and domain IV of Sbi of Staphylococcus aureus, or a functional fragment or variant thereof.

[0553] Embodiment 3. A fusion polypeptide as described in embodiment 1 or 2, wherein the fragment antigen has an amino acid sequence length of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the amino acid sequence length of the target protein antigen.

[0554] Embodiment 4. A fusion polypeptide as described in embodiment 1 or 2, wherein the fragment antigen has an amino acid sequence length of no more than 50% of the amino acid sequence length of the target protein antigen.

[0555] Embodiment 5. A fusion polypeptide as described in embodiment 4, wherein the fragment antigen has an amino acid sequence length of no more than 40%, 30%, 20%, 10% or 5% of the amino acid sequence length of the target protein antigen.

[0556] Embodiment 6. A fusion polypeptide as described in any one of embodiments 1 to 5, wherein the fragment antigen has a length of about 10-300 amino acid residues.

[0557] Embodiment 7. A fusion polypeptide as described in embodiment 6, wherein the fragment antigen has a length of at least 10 amino acid residues.

[0558] Embodiment 8. A fusion polypeptide as described in embodiment 6, wherein the fragment antigen has a length of less than about 300 amino acid residues.

[0559] Embodiment 9. A fusion polypeptide as described in any of embodiments 1 to 5, wherein the fragment antigen has a length of about 10-300, 10-250, 10-200, 10-150, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-300, 30-300, 40-300, 50-300, 60-300, 70-300, 80-300, 90-100, 100-300, 150-300, 200-300, 250-300, 20-250, 30-200, 40-150, 50-100, 60-90 or 70-80 amino acid residues.

[0560] Embodiment 10. A fusion polypeptide as described in any of embodiments 1 to 5, wherein the fragment antigen has a length of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 amino acid residues.

[0561] Embodiment 11. A fusion polypeptide comprising:

[0562] (i) an antigenic variant or a fragment antigenic variant comprising an epitope of a target protein antigen; and

[0563] (ii) Complement C3d binding polypeptide from the immunoglobulin binding protein (Sbi) of Staphylococcus aureus.

[0564] Embodiment 12. A fusion polypeptide as described in embodiment 11, wherein the complement C3d binding polypeptide is or comprises one or both of domain III and domain IV of Sbi of Staphylococcus aureus, or a functional fragment or variant thereof.

[0565] Embodiment 13. A fusion polypeptide as described in embodiment 11 or 12, wherein the amino acid sequence length of the antigen variant or fragment antigen variant is the same as the amino acid sequence length of the target protein antigen.

[0566] Embodiment 14. A fusion polypeptide as described in any one of Embodiments 11 to 13, wherein the antigen variant or fragment antigen variant comprises at least one modified amino acid compared to the target protein antigen.

[0567] Embodiment 15. The fusion polypeptide of embodiment 14, wherein the modified amino acid comprises N-linked glycosylation.

[0568] Embodiment 16. A fusion polypeptide as described in any one of Embodiments 11 to 15, wherein the antigen variant or fragment antigen variant comprises at least one amino acid mutation compared to the target protein antigen.

[0569] Embodiment 17. A fusion polypeptide as described in embodiment 16, wherein the antigen variant or fragment antigen variant comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 or 50 amino acid mutations compared to the target protein antigen.

[0570] Embodiment 18. A fusion polypeptide as described in embodiment 16 or 17, wherein the mutation introduces serine, threonine, alanine or an amino acid different from the amino acid present at the position in the target protein antigen at a specific position.

[0571] Embodiment 19. The fusion polypeptide of any one of Embodiments 16 to 18, wherein the mutation prevents the formation of a disulfide bond.

[0572] Embodiment 20. A fusion polypeptide as described in any of the preceding embodiments, wherein the fragment antigen is characterized in that when expressed in vivo, the fragment antigen binds to a major histocompatibility complex (MHC) molecule.

[0573] Embodiment 21. The fusion polypeptide of any one of embodiments 1 to 19, wherein the fragment antigen is characterized in that when expressed in vivo, the fragment antigen does not bind to an MHC molecule.

[0574] Embodiment 22. The fusion polypeptide of embodiment 20 or 21, wherein the MHC molecule is or comprises an MHC I molecule or an MHC II molecule.

[0575] Embodiment 23. A fusion polypeptide as described in any of the preceding embodiments, wherein the fragment antigen is characterized in that when expressed in vivo, the fragment antigen folds into a three-dimensional conformation that is substantially identical to the three-dimensional conformation of the fragment antigen when it is in its native position in the target protein antigen.

[0576] Embodiment 24. A fusion polypeptide as described in any of the preceding embodiments, wherein the fragment antigen, antigen variant or fragment antigen variant further comprises an amino acid sequence from a second target protein antigen.

[0577] Embodiment 25. A fusion polypeptide as described in any of the preceding embodiments, wherein the target protein antigen is or comprises an infectious disease antigen.

[0578] Embodiment 26. The fusion polypeptide of embodiment 25, wherein the infectious disease antigen is or comprises a viral antigen, a bacterial antigen, a fungal antigen, or a combination thereof.

[0579] Embodiment 27. A fusion polypeptide as described in Embodiment 26, wherein the target protein antigen is or comprises a cancer antigen.

[0580] Embodiment 28. A fusion polypeptide as described in embodiment 27, wherein the antigen is or comprises a viral antigen.

[0581] Embodiment 29. The fusion polypeptide of embodiment 26, wherein the viral antigen is or comprises an influenza antigen.

[0582] Embodiment 30. A fusion polypeptide as described in Embodiment 29, wherein the viral antigen is or comprises a coronavirus polypeptide.

[0583] Embodiment 31. A fusion polypeptide as described in Embodiment 30, wherein the coronavirus polypeptide is or comprises a SARS-CoV-2 protein.

[0584] Embodiment 32. A fusion polypeptide as described in Embodiment 31, wherein the SARS-CoV-2 protein is or comprises a spike protein (SARS-CoV-2S) or a fragment thereof; an envelope protein (SARS-CoV-2E) or a fragment thereof; a membrane protein (SARS-CoV-2M) or a fragment thereof; a nucleocapsid protein (SARS-CoV-2N) or a fragment thereof; an auxiliary factor polypeptide or a fragment thereof; or any combination thereof.

[0585] Embodiment 33. A fusion polypeptide as described in Embodiment 31 or 32, wherein the SARS-CoV-2 protein comprises a spike protein or a fragment thereof (e.g., RBD).

[0586] Embodiment 34. A fusion polypeptide as described in any of embodiments 1 to 33, wherein the fragment antigen, antigen variant or fragment antigen variant has an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:3.

[0587] Embodiment 35. A fusion polypeptide as described in any of embodiments 1 to 33, wherein the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:4.

[0588] Embodiment 36. A fusion polypeptide as described in any of embodiments 1 to 33, wherein the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:5.

[0589] Embodiment 37. A fusion polypeptide as described in any of embodiments 1 to 33, wherein the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:6.

[0590] Embodiment 38. A fusion polypeptide as described in any of embodiments 1 to 33, wherein the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:7.

[0591] Embodiment 39. A fusion polypeptide as described in any of embodiments 1 to 33, wherein the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:20.

[0592] Embodiment 40. A fusion polypeptide as described in any of embodiments 1 to 33, wherein the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:21.

[0593] Embodiment 41. A fusion polypeptide as described in any of embodiments 1 to 3, wherein the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:22.

[0594] Embodiment 42. A fusion polypeptide as described in any of embodiments 1 to 33, wherein the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:23.

[0595] Embodiment 43. A fusion polypeptide as described in any of embodiments 1 to 33, wherein the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:24.

[0596] Embodiment 44. A fusion polypeptide as described in any of embodiments 1 to 33, wherein the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:25.

[0597] Embodiment 45. A fusion polypeptide as described in any of embodiments 1 to 33, wherein the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:26.

[0598] Embodiment 46. A fusion polypeptide as described in any of embodiments 1 to 33, wherein the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:27.

[0599] Embodiment 47. A fusion polypeptide as described in any of embodiments 1 to 33, wherein the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:28.

[0600] Embodiment 48. A fusion polypeptide as described in any of embodiments 1 to 33, wherein the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:29.

[0601] Embodiment 49. A fusion polypeptide as described in any of embodiments 1 to 33, wherein the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:30.

[0602] Embodiment 50. A fusion polypeptide as described in any of embodiments 1 to 33, wherein the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:31.

[0603] Embodiment 51. A fusion polypeptide as described in any one of embodiments 1 to 33, wherein the fragment antigen, antigen variant or fragment antigen variant has at least 80%, 85%, 90% or 100% identity with the amino acid sequence of SEQ ID NO:8.

[0604] Embodiment 52. A fusion polypeptide as described in any of the preceding embodiments, wherein the complement C3d binding polypeptide comprises an Sbi domain III having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:9.

[0605] Embodiment 53. A fusion polypeptide as described in any of the preceding embodiments, wherein the complement C3d binding polypeptide comprises an Sbi domain IV having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:10.

[0606] Embodiment 54. A fusion polypeptide as described in any of the preceding embodiments, wherein the Sbi domain III and the Sbi domain IV are consecutive.

[0607] Embodiment 55. A fusion polypeptide as described in any one of embodiments 2 to 54, wherein the Sbi domain III and the Sbi domain IV are separated by a linker.

[0608] Embodiment 56. A fusion polypeptide as described in any of the preceding embodiments, wherein (a) and (b) are encoded by nucleotide sequences and are located in the same nucleotide sequence or in different nucleotide sequences.

[0609] Embodiment 57. A fusion polypeptide as described in any of the preceding embodiments, wherein (a) is located at the N-terminus of (b).

[0610] Embodiment 58. A fusion polypeptide as described in any one of embodiments 1 to 56, wherein (a) is located at the C-terminus of (b).

[0611] Embodiment 59. A fusion polypeptide as described in any of the preceding embodiments, wherein (a) and (b) are continuous or separated by a linker.

[0612] Embodiment 60. A fusion polypeptide as described in embodiment 59, wherein the linker is a peptidyl linker.

[0613] Embodiment 61. The fusion polypeptide of embodiment 60, wherein the peptidyl linker comprises at least 60% glycine and / or serine.

[0614] Embodiment 62. A fusion polypeptide as described in embodiment 60, wherein the linker is selected from a Gly-Gly-Gly-Gly-Ser (Gly4-Ser) linker or a histidine linker.

[0615] Embodiment 63. A fusion polypeptide as described in embodiment 62, wherein the linker is a Gly4-Ser linker.

[0616] Embodiment 64. A fusion polypeptide as described in embodiment 63, wherein the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 repeats of the Gly4-Ser linker.

[0617] Embodiment 65. A fusion polypeptide as described in Embodiment 64, wherein the linker comprises 3 repeats of the Gly4-Ser linker.

[0618] Embodiment 66. The fusion polypeptide of any one of Embodiments 62 to 65, wherein the linker comprises the sequence of SEQ ID NO: 11.

[0619] Embodiment 67. The fusion polypeptide of any one of Embodiments 1 to 66, wherein the polypeptide further comprises a secretory peptide.

[0620] Embodiment 68. A fusion polypeptide as described in embodiment 67, wherein the secretory peptide is about 10-30 amino acids in length.

[0621] Embodiment 69. A fusion polypeptide as described in embodiment 67 or 68, wherein the secretory peptide is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length.

[0622] Embodiment 70. A fusion polypeptide as described in any one of embodiments 67 to 69, wherein the secretory peptide comprises amino acids that are at least 80%, 85%, 90% or 100% identical to the amino acid sequence of SEQ ID NO:1.

[0623] Embodiment 71. A fusion polypeptide as described in any one of embodiments 67 to 69, wherein the secretory peptide comprises amino acids that are at least 80%, 85%, 90% or 100% identical to the amino acid sequence of SEQ ID NO:2.

[0624] Embodiment 72. A fusion polypeptide as described in any of the preceding embodiments, wherein the polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:12.

[0625] Embodiment 73. A fusion polypeptide as described in any of embodiments 1 to 71, wherein the polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:13.

[0626] Embodiment 74. A fusion polypeptide as described in any of embodiments 1 to 71, wherein the polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:14.

[0627] Embodiment 75. A fusion polypeptide as described in any of embodiments 1 to 71, wherein the polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:15.

[0628] Embodiment 76. A fusion polypeptide as described in any of embodiments 1 to 71, wherein the polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:16.

[0629] Embodiment 77. A fusion polypeptide as described in any of embodiments 1 to 71, wherein the polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:17.

[0630] Embodiment 78. A fusion polypeptide as described in any of embodiments 1 to 71, wherein the polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:18.

[0631] Embodiment 79. A fusion polypeptide as described in any of embodiments 1 to 71, wherein the polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:19.

[0632] Embodiment 80. A fusion polypeptide as described in any of embodiments 1 to 71, wherein the polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:32.

[0633] Embodiment 81. A fusion polypeptide as described in any of embodiments 1 to 71, wherein the polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:33.

[0634] Embodiment 82. A fusion polypeptide as described in any of embodiments 1 to 71, wherein the polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:34.

[0635] Embodiment 83. A fusion polypeptide as described in any of embodiments 1 to 71, wherein the polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:35.

[0636] Embodiment 84. A fusion polypeptide as described in any of embodiments 1 to 71, wherein the polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:36.

[0637] Embodiment 85. A fusion polypeptide as described in any of embodiments 1 to 71, wherein the polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:37.

[0638] Embodiment 86. A fusion polypeptide as described in any of embodiments 1 to 71, wherein the polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:38.

[0639] Embodiment 87. A fusion polypeptide as described in any of embodiments 1 to 71, wherein the polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:39.

[0640] Embodiment 88. A fusion polypeptide as described in any of embodiments 1 to 71, wherein the polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:40.

[0641] Embodiment 89. A fusion polypeptide as described in any of embodiments 1 to 71, wherein the polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:41.

[0642] Embodiment 90. A fusion polypeptide as described in any of embodiments 1 to 71, wherein the polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:42.

[0643] Embodiment 91. A fusion polypeptide as described in any of embodiments 1 to 71, wherein the polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:43.

[0644] Embodiment 92. A fusion polypeptide as described in any of embodiments 1 to 71, wherein the polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:44.

[0645] Embodiment 93. A fusion polypeptide as described in any of embodiments 1 to 71, wherein the polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:45.

[0646] Embodiment 94. A fusion polypeptide as described in any of the preceding embodiments, wherein the polypeptide is encoded by a polynucleotide that is or comprises RNA.

[0647] Embodiment 95. A fusion polypeptide as described in Embodiment 94, wherein the polynucleotide is or comprises a messenger RNA.

[0648] Embodiment 96. A fusion polypeptide as described in any of the preceding embodiments, wherein the polypeptide is encoded by a polynucleotide that is or comprises DNA.

[0649] Embodiment 97. A fusion polynucleotide encoding the fusion polypeptide of any one of embodiments 1 to 96.

[0650] Embodiment 98. A fusion polynucleotide comprising a nucleotide sequence encoding:

[0651] (i) a fragment antigen comprising an epitope of a target protein antigen; and

[0652] (ii) Complement C3d binding polypeptide from the immunoglobulin binding protein (Sbi) of Staphylococcus aureus.

[0653] Embodiment 99. A fusion polynucleotide comprising a nucleotide sequence encoding:

[0654] (i) an antigenic variant or a fragment antigenic variant comprising an epitope of a target protein antigen; and

[0655] (ii) Complement C3d binding polypeptide from the immunoglobulin binding protein (Sbi) of Staphylococcus aureus.

[0656] Embodiment 100. A fusion polynucleotide as described in embodiment 98 or 99, wherein the polynucleotide is or comprises RNA.

[0657] Embodiment 101. The fusion polynucleotide of embodiment 100, wherein the polynucleotide is or comprises a messenger RNA.

[0658] Embodiment 102. The fusion polynucleotide of any one of Embodiments 98 to 101, wherein the polynucleotide is or comprises DNA.

[0659] Embodiment 103. The fusion polynucleotide of Embodiment 98, wherein the complement C3d binding polypeptide is or comprises one or both of domain III and domain IV of Sbi of Staphylococcus aureus, or a functional fragment or variant thereof.

[0660] Embodiment 104. A fusion polynucleotide as described in embodiment 98 or 99, wherein the polynucleotide encodes a fragment antigen having an amino acid sequence length of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the amino acid sequence length of the target protein antigen.

[0661] Embodiment 105. A fusion polynucleotide as described in embodiment 98 or 99, wherein the polynucleotide encodes a fragment antigen having an amino acid sequence length not exceeding 50% of the amino acid sequence length of the target protein antigen.

[0662] Embodiment 106. A fusion polynucleotide as described in embodiment 105, wherein the polynucleotide encodes a fragment antigen having an amino acid sequence length not exceeding 40%, 30%, 20%, 10% or 5% of the amino acid sequence length of the target protein antigen.

[0663] Embodiment 107. A fusion polynucleotide as described in Embodiment 99, wherein the polynucleotide encodes an antigen variant or a fragment antigen variant having an amino acid sequence length that is the same as the amino acid sequence length of the target protein antigen.

[0664] Embodiment 108. A fusion polynucleotide as described in embodiment 99 or 107, wherein the polynucleotide encodes an antigen variant or a fragment antigen variant, which comprises at least one modified amino acid compared to the target protein antigen.

[0665] Embodiment 109. The fusion polynucleotide of any one of embodiments 98 to 108, wherein the polynucleotide encodes a fragment antigen characterized in that when expressed in vivo, the fragment antigen binds to a major histocompatibility complex (MHC) molecule.

[0666] Embodiment 110. The fusion polynucleotide of any one of embodiments 98 to 109, wherein the polynucleotide encodes a fragment antigen characterized in that when expressed in vivo, the fragment antigen does not bind to an MHC molecule.

[0667] Embodiment 111. The fusion polynucleotide of embodiment 109 or 110, wherein the MHC molecule is or comprises an MHC I molecule or an MHC II molecule.

[0668] Embodiment 112. A fusion polynucleotide as described in any one of embodiments 98 to 111, wherein the polynucleotide encodes a fragment antigen, which is characterized in that when expressed in vivo, the fragment antigen folds into a three-dimensional conformation that is substantially identical to the three-dimensional conformation of the fragment antigen when it is in its native position in the target protein antigen.

[0669] Embodiment 113. A fusion polynucleotide as described in any one of Embodiments 98 to 112, wherein the polynucleotide encodes a fragment antigen, an antigen variant, or a fragment antigen variant, and the fragment antigen, antigen variant, or fragment antigen variant also comprises an amino acid sequence from a second target protein antigen.

[0670] Embodiment 114. The fusion polynucleotide of any of the preceding embodiments, wherein the target protein antigen is or comprises an infectious disease antigen.

[0671] Embodiment 115. The fusion polynucleotide of embodiment 114, wherein the infectious disease antigen is or comprises a viral antigen, a bacterial antigen, a fungal antigen, or a combination thereof.

[0672] Embodiment 116. The fusion polynucleotide of any one of embodiments 98 to 113, wherein the target protein antigen is or comprises a cancer antigen.

[0673] Embodiment 117. The fusion polynucleotide of embodiment 114, wherein the antigen is or comprises a viral antigen.

[0674] Embodiment 118. The fusion polynucleotide of embodiment 114, wherein the viral antigen is or comprises an influenza antigen.

[0675] Embodiment 119. A fusion polynucleotide as described in Embodiment 114, wherein the viral antigen is or comprises a coronavirus polypeptide.

[0676] Embodiment 120. The fusion polynucleotide of Embodiment 119, wherein the coronavirus polypeptide is or comprises a SARS-CoV-2 protein.

[0677] Embodiment 121. A fusion polynucleotide as described in Embodiment 120, wherein the SARS-CoV-2 protein is or comprises a spike protein (SARS-CoV-2S) or a fragment thereof; an envelope protein (SARS-CoV-2E) or a fragment thereof; a membrane protein (SARS-CoV-2M) or a fragment thereof; a nucleocapsid protein (SARS-CoV-2N) or a fragment thereof; an auxiliary factor polypeptide or a fragment thereof; or any combination thereof.

[0678] Embodiment 122. The fusion polynucleotide of Embodiment 120 or 121, wherein the SARS-CoV-2 protein comprises a spike protein or a fragment thereof (e.g., RBD).

[0679] Embodiment 123. The fusion polynucleotide of any one of Embodiments 98 to 122, wherein (a) is located at the C-terminus of (b).

[0680] Embodiment 124. The fusion polynucleotide of any one of embodiments 98 to 123, wherein (a) and (b) are consecutive or separated by a nucleotide sequence encoding a linker.

[0681] Embodiment 125. The fusion polynucleotide of embodiment 124, wherein the linker is a peptidyl linker.

[0682] Embodiment 126. The fusion polynucleotide of embodiment 125, wherein the peptidyl linker comprises at least 60% glycine and / or serine.

[0683] Embodiment 127. The fusion polynucleotide of embodiment 126, wherein the linker is selected from a Gly-Gly-Gly-Gly-Ser (Gly4-Ser) linker or a histidine linker.

[0684] Embodiment 128. The fusion polynucleotide of any one of Embodiments 98 to 127, wherein the polynucleotide further comprises a nucleotide sequence encoding a secretory peptide.

[0685] Embodiment 129. A fusion polynucleotide as described in any one of embodiments 98 to 128, wherein the polynucleotide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleotide sequence of SEQ ID NO:46.

[0686] Embodiment 130. A fusion polynucleotide as described in any one of embodiments 98 to 128, wherein the polynucleotide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleotide sequence of SEQ ID NO:47.

[0687] Embodiment 131. A fusion polynucleotide as described in any one of embodiments 98 to 128, wherein the polynucleotide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleotide sequence of SEQ ID NO:48.

[0688] Embodiment 132. A fusion polynucleotide as described in any one of embodiments 98 to 128, wherein the polynucleotide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleotide sequence of SEQ ID NO:49.

[0689] Embodiment 133. A fusion polynucleotide as described in any one of embodiments 98 to 128, wherein the polynucleotide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleotide sequence of SEQ ID NO:50.

[0690] Embodiment 134. A fusion polynucleotide as described in any one of embodiments 98 to 128, wherein the polynucleotide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleotide sequence of SEQ ID NO:51.

[0691] Embodiment 135. A fusion polynucleotide as described in any one of embodiments 98 to 128, wherein the polynucleotide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleotide sequence of SEQ ID NO:52.

[0692] Embodiment 136. A fusion polynucleotide as described in any one of embodiments 98 to 128, wherein the polynucleotide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleotide sequence of SEQ ID NO:53.

[0693] Embodiment 137. A fusion polynucleotide as described in any one of embodiments 98 to 128, wherein the polynucleotide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleotide sequence of SEQ ID NO:54.

[0694] Embodiment 138. An expression vector comprising the fusion polynucleotide of any one of embodiments 97 to 137.

[0695] Embodiment 139. An expression vector as described in Embodiment 138, wherein the expression vector comprises a viral vector.

[0696] Embodiment 140. An expression vector as described in Embodiment 138 or 139, wherein the viral vector comprises a retroviral vector, an adenoviral vector, an adeno-associated viral vector or a lentiviral vector or an RNA vector.

[0697] Embodiment 141. A composition for delivering the fusion polypeptide of any one of Embodiments 1 to 96.

[0698] Embodiment 142. A composition for delivering the fusion polynucleotide of any one of embodiments 97 to 137.

[0699] Embodiment 143. A pharmaceutical composition for delivering the fusion polypeptide of any one of embodiments 1 to 96, the fusion polynucleotide of any one of embodiments 97 to 137, or the expression vector of any one of embodiments 138 to 140.

[0700] Embodiment 144. The pharmaceutical composition of embodiment 143, further comprising a pharmaceutically acceptable excipient, diluent or a combination thereof.

[0701] Embodiment 145. A preparation method comprising:

[0702] A first nucleotide sequence encoding a fragment antigen comprising an epitope of a target protein antigen and a second nucleotide sequence encoding a complement C3d binding polypeptide of an immunoglobulin binding protein (Sbi) from Staphylococcus aureus are recombinantly linked to form a polynucleotide comprising the first nucleotide sequence and the second nucleotide sequence.

[0703] Embodiment 146. The method of embodiment 145, further comprising expressing the polynucleotide in a cell to produce a fusion polypeptide encoded by the polynucleotide.

[0704] Embodiment 147. A cell comprising the fusion polypeptide of any one of embodiments 1 to 96, the fusion polynucleotide of any one of embodiments 97 to 137, or the expression vector of any one of embodiments 138 to 140.

[0705] Embodiment 148. The cell of Embodiment 147, wherein the cell is contacted with the fusion polynucleotide, fusion polypeptide or expression vector.

[0706] Embodiment 149. The cell of embodiment 147 or 148, wherein the contacting occurs in vivo, in vitro, or ex vivo.

[0707] Embodiment 150. A kit comprising the fusion polypeptide of any one of embodiments 1 to 96, the fusion polynucleotide of any one of embodiments 97 to 137, the expression vector of any one of embodiments 138 to 140, or the pharmaceutical composition of embodiment 143 or 144, and instructions for use.

[0708] Embodiment 151. A kit as described in Embodiment 150, which also includes: a polynucleotide comprising a nucleotide sequence encoding a target protein antigen; or a target protein antigen encoded by the polynucleotide.

[0709] Embodiment 152. A method comprising administering at least one dose of the pharmaceutical composition of embodiment 143 or 144 to a subject in need thereof.

[0710] Embodiment 153. The method of Embodiment 152, wherein the at least one dose is administered in an amount effective for inducing an immune response against the fragment antigen in the subject.

[0711] Embodiment 154. A method as described in Embodiment 153, wherein the immune response includes generating neutralizing antibody titers against the fragment antigen.

[0712] Embodiment 155. A method as described in Embodiment 154, wherein the neutralizing antibody titer is increased by at least 50% compared to the neutralizing antibody titer induced by the fragment antigen in the absence of the complement C3d binding polypeptide of the immunoglobulin binding protein (Sbi) from Staphylococcus aureus.

[0713] Embodiment 156. A method as described in Embodiment 154 or 155, wherein the generation of neutralizing antibody titers has been established in a mouse model using a dose of at least 0.1 μg.

[0714] Embodiment 157. The method of any one of Embodiments 152 to 156, wherein the at least one dose is administered in an amount effective for stimulating B cells while reducing induction of T cell responses.

[0715] Embodiment 158. A method comprising administering to a subject:

[0716] a) a first dose of the pharmaceutical composition of embodiment 143 or 144; and

[0717] b) a second dose of the pharmaceutical composition of embodiment 143 or 144.

[0718] Embodiment 159. The method of embodiment 158, wherein the first dose and the second dose are administered at least 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, or 4 weeks apart.

[0719] Embodiment 160. The method of embodiment 158 ​​or 159, wherein the first dose and the second dose are the same amount.

[0720] Embodiment 161. The method of embodiment 158 ​​or 159, wherein the first dose and the second dose are different amounts.

[0721] Embodiment 162. A method comprising:

[0722] Administering a dose of the pharmaceutical composition of embodiment 143 or 144 to a subject in need thereof such that the subject receives:

[0723] Delivery of a first dose of a polynucleotide comprising a nucleotide sequence encoding a target protein antigen or a pharmaceutical composition encoding a target protein antigen encoded by said polynucleotide; and

[0724] A second dose of the pharmaceutical composition of embodiment 143 or 144.

[0725] Embodiment 163. The method of embodiment 162, wherein:

[0726] (a) the polynucleotide comprising a nucleotide sequence encoding a target protein antigen further comprises a nucleotide sequence encoding a complement C3d binding polypeptide of an immunoglobulin binding protein (Sbi) from Staphylococcus aureus; or

[0727] (b) The target protein antigen encoded by the polynucleotide further comprises a complement C3d binding polypeptide derived from the immunoglobulin binding protein (Sbi) of Staphylococcus aureus.

[0728] Embodiment 164. A method as described in Embodiment 162 or 163, further comprising administering to the subject, before the administering step, the first dose of a pharmaceutical composition delivering a polynucleotide comprising a nucleotide sequence encoding a target protein antigen or a target protein antigen encoded by the polynucleotide.

[0729] Embodiment 165. A method comprising:

[0730] Administering a dose of the pharmaceutical composition of embodiment 143 or 144 to a subject in need thereof such that the subject receives:

[0731] a first dose of the pharmaceutical composition of embodiment 143 or 144, and

[0732] Delivery of the second dose comprises a polynucleotide comprising a nucleotide sequence encoding a target protein antigen or a pharmaceutical composition encoding the target protein antigen encoded by the polynucleotide.

[0733] Embodiment 166. The method of embodiment 165, wherein:

[0734] (a) the polynucleotide comprising a nucleotide sequence encoding a target protein antigen further comprises a nucleotide sequence encoding a complement C3d binding polypeptide of an immunoglobulin binding protein (Sbi) from Staphylococcus aureus; or

[0735] (b) The target protein antigen encoded by the polynucleotide further comprises a complement C3d binding polypeptide derived from the immunoglobulin binding protein (Sbi) of Staphylococcus aureus.

[0736] Embodiment 167. A method comprising:

[0737] Administering a dose of the pharmaceutical composition of embodiment 143 or 144 to a subject in need thereof such that the subject receives:

[0738] Delivery of the first dose comprises a polynucleotide comprising a nucleotide sequence encoding a fusion polypeptide comprising (i) a target protein antigen and (ii) a complement C3d binding polypeptide from an immunoglobulin binding protein (Sbi) of Staphylococcus aureus, or a pharmaceutical composition of a fusion polypeptide encoded by the polynucleotide; and

[0739] A second dose of the pharmaceutical composition of embodiment 143 or 144.

[0740] Embodiment 168. A method as described in Embodiment 167, further comprising administering to the subject a first dose of a polynucleotide comprising a nucleotide sequence encoding a fusion polypeptide comprising (i) a target protein antigen and (ii) a complement C3d binding polypeptide from an immunoglobulin binding protein (Sbi) of Staphylococcus aureus, or a pharmaceutical composition encoded by the polynucleotide, prior to the administering step.

[0741] Embodiment 169. The method of any one of Embodiments 162 to 167, wherein the first dose and the second dose are administered at least 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, or 4 weeks apart.

[0742] Embodiment 170. The method of any one of Embodiments 152 to 159, wherein the subject in need thereof is a subject suffering from or susceptible to a disease, disorder or condition induced by the target protein antigen.

[0743] Embodiment 171. The method of any one of Embodiments 152 to 170, wherein the subject is a mammalian subject.

[0744] Embodiment 172. A method as described in Embodiment 171, wherein the subject is a human subject.

[0745] Embodiment 173. The method of any one of embodiments 152 to 172, wherein the administering can be performed by intramuscular administration, intradermal administration, intravenous administration, subcutaneous administration, or a combination thereof.

[0746] Embodiment 174. A composition comprising the fusion polypeptide of any one of embodiments 1 to 96, or the fusion polynucleotide of any one of embodiments 97 to 137, or the pharmaceutical composition of embodiment 143 or 144, for enhancing the immunogenicity of an antigen.

[0747] Embodiment 175. A composition comprising the fusion polypeptide of any one of embodiments 1 to 96, or the fusion polynucleotide of any one of embodiments 97 to 137, or the pharmaceutical composition of embodiment 143 or 144, for stimulating an immune response to an antigen.

[0748] Embodiment 176. A composition comprising the fusion polypeptide of any one of embodiments 1 to 96, or the fusion polynucleotide of any one of embodiments 97 to 137, or the pharmaceutical composition of embodiment 143 or 144, for use in a method of treating a disease or ameliorating a symptom of a disease.

[0749] Embodiment 177. A method for enhancing the immunogenicity of an antigen, comprising administering to a subject in need thereof the fusion polypeptide of any one of embodiments 1 to 96, or the fusion polynucleotide of any one of embodiments 97 to 137, or the pharmaceutical composition of embodiment 143 or 144.

[0750] Embodiment 178. A method for stimulating an immune response to an antigen, comprising administering to a subject in need thereof the fusion polypeptide of any one of embodiments 1 to 96, or the fusion polynucleotide of any one of embodiments 97 to 137, or the pharmaceutical composition of embodiment 143 or 144.

[0751] Embodiment 179. A method of treating a disease or ameliorating the symptoms of a disease, comprising administering to a subject in need thereof the fusion polypeptide of any one of embodiments 1 to 96, or the fusion polynucleotide of any one of embodiments 97 to 137, or the pharmaceutical composition of embodiment 143 or 144.

[0752] Embodiment 180. Use of a composition comprising the fusion polypeptide of any one of embodiments 1 to 96, or the fusion polynucleotide of any one of embodiments 97 to 137, or the pharmaceutical composition of embodiment 143 or 144 in the preparation of a medicament for enhancing the immunogenicity of a fragment antigen.

[0753] Embodiment 181. Use of a composition comprising the fusion polypeptide of any one of embodiments 1 to 96, or the fusion polynucleotide of any one of embodiments 97 to 137, or the pharmaceutical composition of embodiment 143 or 144 in the preparation of a medicament for stimulating an immune response to an antigen.

[0754] Embodiment 182. Use of the fusion polypeptide of any one of embodiments 1 to 96, or the fusion polynucleotide of any one of embodiments 97 to 137, or the pharmaceutical composition of embodiment 143 or 144 in the preparation of a medicament for treating a disease or ameliorating the symptoms of a disease.

[0755] Embodiment 183. Use of a composition comprising the fusion polypeptide of any one of embodiments 1 to 96, or the fusion polynucleotide of any one of embodiments 97 to 137, or the pharmaceutical composition of embodiment 143 or 144 for enhancing the immunogenicity of a fragment antigen.

[0756] Embodiment 184. Use of a composition comprising the fusion polypeptide of any one of embodiments 1 to 96, or the fusion polynucleotide of any one of embodiments 97 to 137, or the pharmaceutical composition of embodiment 143 or 144 for stimulating an immune response to an antigen.

[0757] Embodiment 185. Use of the fusion polypeptide of any one of embodiments 1 to 96, or the fusion polynucleotide of any one of embodiments 97 to 137, or the pharmaceutical composition of embodiment 143 or 144 for treating a disease or ameliorating the symptoms of a disease.

[0758] Embodiment 186. The composition for use of any one of embodiments 174 to 176, the method of any one of embodiments 177 to 179, or the use of any one of embodiments 180 to 186, wherein the subject is a mammal.

[0759] Embodiment 187. The composition for use, the method or the use according to embodiment 186, wherein the subject is a human.

[0760] Embodiment 188. The composition for use of any one of embodiments 174 to 176, the method of any one of embodiments 177 to 179, or the use of any one of embodiments 180 to 186, wherein a single dose of the fusion polypeptide, fusion polynucleotide or pharmaceutical composition is administered.

[0761] Embodiment 189. The composition for use of any one of embodiments 174 to 176, the method of any one of embodiments 177 to 179, or the use of any one of embodiments 180 to 186, wherein multiple doses of the fusion polypeptide, fusion polynucleotide or pharmaceutical composition are administered.

[0762] Embodiment 190. The composition for use of any one of embodiments 174 to 176, the method of any one of embodiments 177 to 179, or the use of any one of embodiments 180 to 186, wherein the fusion polypeptide, fusion polynucleotide or pharmaceutical composition is administered at a dose of about 0.5 micrograms to 10 micrograms.

[0763] Embodiment 191. The composition for use of any one of embodiments 174 to 176, the method of any one of embodiments 177 to 179, or the use of any one of embodiments 180 to 186, wherein administration of the fusion polynucleotide, fusion polypeptide or pharmaceutical composition results in a humoral response.

[0764] Embodiment 192. The composition for use, method or use of embodiment 190, wherein the humoral response is an antibody response.

[0765] Embodiment 193. The composition for use of any one of Embodiments 174 to 176, the method of any one of Embodiments 177 to 179, or the use of any one of Embodiments 180 to 186, wherein administration of the fusion polynucleotide, fusion polypeptide or pharmaceutical composition results in an increase in the titer of an antibody response.

[0766] Embodiment 194. The composition for use, method or use of embodiment 193, wherein the increase in titer is from about 10-fold to about 500-fold increase.

[0767] Embodiment 195. The composition for use, method or use of embodiment 193 or 194, wherein the increase in the titer of the antibody response is compared to administration of an otherwise similar fusion polynucleotide that does not comprise Sbi domain III, or a fragment or variant thereof; and Sbi domain IV, or a fragment or variant thereof.

[0768] Embodiment 196. The composition for use, method or use of embodiment 193 or 194, wherein the increase in the titer of the antibody response is compared to administration of an otherwise similar fusion polypeptide that does not comprise Sbi domain III, or a fragment or variant thereof; and Sbi domain IV, or a fragment or variant thereof.

[0769] Embodiment 197. A composition for use, a method or a use as described in Embodiment 193 or 194, wherein the increase in the titer of the antibody response is compared to administration of an otherwise similar pharmaceutical composition that does not comprise a nucleotide sequence encoding Sbi domain III, or a fragment or variant thereof; and Sbi domain IV, or a fragment or variant thereof.

[0770] Embodiment 198. The composition for use of any one of embodiments 174 to 176, the method of any one of embodiments 177 to 179, or the use of any one of embodiments 180 to 186, wherein the composition is administered via any one of the following routes of administration: intramuscular, intravenous, subcutaneous, intrathecal, intradermal, ocular, intranasal, sublingual, or oral.

[0771] Embodiment 199. An in vitro method for selecting fragment antigens by identifying polypeptide fragments that fold into a three-dimensional conformation that is substantially identical to the three-dimensional conformation of the target protein antigen from which the fragment was derived.

[0772] Embodiment 200. The method of embodiment 199, wherein the method comprises the following steps:

[0773] providing a mammalian cell display system that displays a library of fragment antigen candidates on the cell surface;

[0774] exposing the mammalian cell display system to a composition comprising an antibody that binds to a target protein antigen from which the fragment antigen candidate is derived;

[0775] Detecting fragment antigen candidates that bind to the target protein antigen-binding antibodies, wherein binding of the fragment antigen candidate that binds to at least one of the target protein antigen-binding antibodies indicates the likelihood that the fragment antigen candidate folds into a three-dimensional conformation that is substantially identical to the three-dimensional conformation when in the target protein antigen.

[0776] Embodiment 201. A method as described in Embodiment 198 or 199, wherein the method further comprises the step of producing an antibody that binds to the target protein antigen.

[0777] Embodiment 202. A method as described in Embodiment 201, wherein the production of the antibody comprises (i) immunizing an animal with a target protein antigen from which the fragment antigen candidate is derived or a polynucleotide encoding the target protein antigen; and (ii) identifying an antibody that binds to the target protein antigen.

[0778] Embodiment 203. A method as described in any one of Embodiments 200 to 202, wherein the mammalian cell display system comprises HEK293T cells, HeLa cells or CHO cells.

[0779] Embodiment 204. The method of any one of Embodiments 200 to 203, wherein the polypeptide fragment is identified based on binding affinity to the antibody in a serum binding assay or similar assay.

[0780] Embodiment 205. A recombinant polynucleotide comprising a nucleotide sequence encoding an immunoglobulin binding protein (Sbi) of Staphylococcus aureus or a fragment thereof.

[0781] Embodiment 206. The polynucleotide of embodiment 205, wherein the polynucleotide comprises a sequence encoding a complement C3d binding polypeptide from Sbi.

[0782] Embodiment 207. The polynucleotide of Embodiment 206, wherein the complement C3d binding polypeptide is or comprises one or both of domain III and domain IV of Sbi of said Staphylococcus aureus, or a functional fragment or variant thereof.

[0783] Embodiment 208. The polynucleotide of any one of embodiments 205 to 207, wherein the polynucleotide is or comprises RNA.

[0784] Embodiment 209. The polynucleotide of any one of embodiments 205 to 207, wherein the polynucleotide is or comprises a messenger RNA.

[0785] Embodiment 210. The polynucleotide of any one of Embodiments 205 to 207, wherein the polynucleotide is or comprises DNA.

[0786] Embodiment 211. A polynucleotide as described in any of embodiments 205 to 210, wherein the polynucleotide encodes a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleotide sequence of SEQ ID NO:9.

[0787] Embodiment 212. A polynucleotide as described in any of embodiments 205 to 210, wherein the polynucleotide encodes a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleotide sequence of SEQ ID NO:10.

[0788] Embodiment 213. An isolated polypeptide comprising an immunoglobulin binding protein of Staphylococcus aureus (Sbi).

[0789] Embodiment 214. A polypeptide as described in Embodiment 213, which comprises a complement C3d binding polypeptide from Sbi.

[0790] Embodiment 215. The polypeptide of Embodiment 214, wherein the complement C3d binding polypeptide is or comprises one or both of domain III and domain IV of Sbi of said Staphylococcus aureus, or a functional fragment or variant thereof.

[0791] Embodiment 216. A polypeptide as described in embodiment 214 or 215, wherein the polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:9.

[0792] Embodiment 217. A polypeptide as described in embodiment 214 or 215, wherein the polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 100% identity with the amino acid sequence of SEQ ID NO:10.

[0793] Embodiment 218. An expression vector comprising the polynucleotide of any one of embodiments 205 to 212.

[0794] Embodiment 219. A composition for delivering a polypeptide as described in any one of embodiments 213 to 217.

[0795] Embodiment 220. A composition for delivering the polynucleotide of any one of embodiments 205 to 212.

[0796] Embodiment 221. A pharmaceutical composition for delivering the polynucleotide of any one of embodiments 205 to 212, the polypeptide of any one of embodiments 213 to 217, or the expression vector of embodiment 218.

[0797] Embodiment 222. The pharmaceutical composition of embodiment 221, further comprising a pharmaceutically acceptable excipient, diluent, or a combination thereof.

[0798] Embodiment 223. A method comprising administering at least one dose of the pharmaceutical composition of embodiment 221 or 222 to a subject in need thereof.

[0799] Embodiment 224. A method for enhancing the immunogenicity of an antigen, comprising administering to a subject in need thereof a polynucleotide of any one of embodiments 205 to 212, a polypeptide of any one of embodiments 213 to 217, or a pharmaceutical composition of embodiment 221 or 222.

[0800] Embodiment 225. A method for stimulating an immune response to an antigen, comprising administering to a subject in need thereof a polynucleotide of any one of embodiments 205 to 212, a polypeptide of any one of embodiments 213 to 217, or a pharmaceutical composition of embodiment 221 or 222.

[0801] Embodiment 226. Use of an immunoglobulin binding protein of Staphylococcus aureus (Sbi) as an adjuvant in an immunogenic composition.

[0802] Embodiment 227. An immunoglobulin binding protein of Staphylococcus aureus (Sbi) for use as an adjuvant in an immunogenic composition.

Claims

1. A fusion polypeptide comprising: (i) a fragment antigen comprising an epitope of a target protein antigen; and (ii) Complement C3d binding polypeptide from the immunoglobulin binding protein (Sbi) of Staphylococcus aureus.

2. The fusion polypeptide according to claim 1, wherein the complement C3d binding polypeptide is or comprises one or both of domain III and domain IV of Sbi of the Staphylococcus aureus, or a functional fragment or variant thereof.

3. The fusion polypeptide according to claim 1 or 2, wherein the fragment antigen has the following amino acid sequence length: (i) at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the amino acid sequence length of the target protein antigen; (ii) not exceeding 50% of the amino acid sequence length of the target protein antigen; (iii) about 10-300 amino acid residues; or (iv) combinations thereof.

4. A fusion polypeptide comprising: (i) an antigenic variant or a fragment antigenic variant comprising an epitope of a target protein antigen; and (ii) Complement C3d binding polypeptide from the immunoglobulin binding protein (Sbi) of Staphylococcus aureus.

5. The fusion polypeptide according to claim 4, wherein the complement C3d-binding polypeptide is or comprises one or both of domain III and domain IV of Sbi of the Staphylococcus aureus, or a functional fragment or variant thereof.

6. The fusion polypeptide according to claim 4 or 5, wherein: (i) the amino acid sequence length of the antigen variant or fragment antigen variant is the same as the amino acid sequence length of the target protein antigen; (ii) the antigen variant or fragment antigen variant comprises at least one modified amino acid compared to the target protein antigen; (iii) the antigen variant or fragment antigen variant comprises at least one amino acid mutation compared to the target protein antigen; or (iv) combinations thereof.

7. The fusion polypeptide of claim 6, wherein the modified amino acid comprises N-linked glycosylation.

8. The fusion polypeptide of claim 6, wherein the mutation: (a) introduces serine, threonine, alanine, or an amino acid different from the amino acid present at the position in the target protein antigen at a specific position; (b) prevents the formation of a disulfide bond; or both (a) and (b).

9. The fusion polypeptide of any one of the preceding claims, wherein the fragment antigen is characterized in that when expressed in vivo, the fragment antigen binds to a major histocompatibility complex (MHC) molecule.

10. The fusion polypeptide of any one of claims 1 to 9, wherein the fragment antigen is characterized in that when expressed in vivo, the fragment antigen does not bind to an MHC molecule.

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