Design of universal h5 influenza virus vaccine candidates via antigen reorientation

Abstract

New vaccine compositions comprising a modified antigen H5 HA bound to the surface of an adjuvant or carrier by electrostatic interactions are disclosed. The antigen of the vaccine composition is presented in a defined orientation on an adjuvant surface such that epitope accessibility is altered, and an immune response is redirected toward specific epitopes. In some embodiments the vaccine composition comprises one or more recombinant antigen polypeptides adsorbed to an alum particle. In some embodiments, the recombinant antigen polypeptide comprises a Region of Repetitive Carboxylic Groups (RRC) or a Region of Repetitive Lysyl / Guanidino Groups (RRL). The modified antigen H5 HA comprises a isoleucine to phenylalanine mutation in its HA2 subunit.

Description

Kilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 DESIGN OF UNIVERSAL H5 INFLUENZA VIRUS VACCINE CANDIDATES VIA ANTIGEN REORIENTATION RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application No.63 / 636,619, filed on April 19, 2024. The entire content of the provisional application is here being incorporated by reference for all purposes. FIELD OF THE INVENTION

[0002] The invention finds application in the fields of human and veterinary medicine. STATEMENT OF GOVERNMENT SUPPORT

[0003] This invention was made with Government support under the contract AI158125 awarded by the National Institutes of Health. The Government has certain rights in the invention. BACKGROUND

[0004] Since 2022, many countries, including the US, are experiencing the deadliest outbreak of bird flu caused by highly pathogenic avian influenza strains (e.g., H5N1). Avian influenza infection is historically associated with high case fatality rates (40-60%), although human-to- human transmission has been very rare. There is global concern that viral mutations may allow efficient transmission among humans and lead to the next influenza pandemic, where existing vaccine-induced immunity is unlikely to confer protection. The current strategy for seasonal influenza vaccination, including manufacturing, testing and stockpiling keeps the population at least six-12 months behind the rapidly evolving avian influenza virus. In addition, because avian influenza strains are often lethal to domesticated birds (e.g., chicken), current vaccine manufacturing that relies on millions of eggs will face serious logistic challenges. Therefore, there is an urgent need for universal influenza vaccines, which can broadly and durably protect against existing and emerging influenza strains. BRIEF SUMMARY OF THE INVENTION

[0005] This application incorporates the entire content of WO2023064631 by reference.Kilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068

[0006] In some embodiments, provided herein is a recombinant antigen polypeptide comprises a Region of Repetitive Carboxylic Groups (RRC) or a Region of Repetitive Lysyl / Guanidino Groups (RRL) inserted after an amino acid residue in a polypeptide. The amino acid residue corresponds to any one of 150-160 of SEQ ID NO: 7, the polypeptide shares at least 70% sequence identity to SEQ ID NO: 7 over the entire length of SEQ ID NO: 7, the polypeptide comprises a subsequence that shares at least 90% amino acid sequence identity with the HA2 subunit of H5 HA (SEQ ID NO: 23) over the entire length of the HA2 subunit (SEQ ID NO: 23), and the polypeptide comprises a phenylalanine at a position corresponding to 371F of SEQ ID NO: 7.

[0007] In some embodiments, the amino acid residue corresponds to 154N of SEQ ID NO: 7. In some embodiments, the recombinant antigen polypeptide comprises from N terminus to C terminus continuously: a first antigen fragment having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 5 over the entire length of SEQ ID NO: 5, an RRC or an RRL, and a second antigen fragment having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 6 over the entire length of SEQ ID NO: 6. In some embodiments, the polypeptide comprises a sequence selected from the group consisting of SEQ ID NO: 7-14. In some embodiments, provided herein is an influenza antigen-adjuvant complex comprising the recombinant antigen polypeptide of any of the claims 1-34 and an adjuvant. In some embodiments, the influenza antigen-adjuvant complex is formed by an electrostatic interaction between the RRC or the RRL and an adjuvant. In some embodiments, the antigen polypeptide comprises the RRC and wherein the adjuvant is alum (aluminum hydroxide). In some embodiments, the antigen polypeptide comprises an RRL, wherein the influenza antigen-adjuvant complex comprises an aluminum-based adjuvant selected from aluminum phosphate and amorphous aluminum hydroxyphosphate sulfate (AAHS).

[0008] In some embodiments, the RRC comprises a) [D]N wherein N is 8-18; b) [E]N wherein N is 8-18; c) a D-E copolymer [(D)X, (Glu)Y] where X is 1-17, Y is 1-17, and X + Y = 8-18; or d) a region having a high density of D and / or E. In some embodiments, the RRC comprises 8 to 12 amino acids selected from aspartic acid and glutamic acid. In some embodiments, the RRC is D8, D9, D10, D11, or D12. In some embodiments, RRL comprises a) [K]N wherein N is 8-18; b) [R]N wherein N is 8-18; c) a K-R copolymer [(K)X, (R)Y] where X is 1-17, Y is 1-17, and X + Y = 8-18 or [(K)X, (R)Y] where X is 1-17, Y is 1-17, and X + Y = 8-18); or d) a region having aKilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 high density of K and / or R. In some embodiments, N is 11 or X + Y is 11. In some embodiments, the H5 HA is presented as a trimer adsorbed to alum.

[0009] In some embodiments, the influenza antigen-adjuvant complex comprises an alum particle and a plurality of copies of the recombinant antigen polypeptide, wherein the antigen polypeptide comprises an RRC, wherein the plurality of copies of the antigen polypeptide is associated with the alum particle by electrostatic interaction between the alum particle and the RRC.

[0010] In some embodiments, the recombinant antigen polypeptide comprises one or more auxiliary elements. In some embodiments, the one or more auxiliary elements are selected from the group consisting of a polyhistidine tag and a trimerization domain.

[0011] Also provided herein is a polynucleotide encoding a polypeptide comprising the recombinant antigen polypeptide described herein and a cell comprising said polynucleotide.

[0012] Also provided herein is a vaccine composition comprising a plurality of the influenza antigen-adjuvant complexes described above.

[0013] Also provided herein is a method for eliciting an immune response in a mammal comprising administering the vaccine composition to the mammal.

[0014] Also provided herein is a method of preparing a recombinant vaccine composition comprising (a) expressing a nucleic acid sequence encoding a recombinant antigen polypeptide, wherein the recombinant antigen polypeptide comprises a Region of Repetitive Carboxylic Groups (RRC), or a Region of Repetitive Lysyl / Guanidino Groups (RRL) inserted after an amino acid residue in a polypeptide, wherein the amino acid residue corresponds to any one of 150-160 of SEQ ID NO: 7, wherein the polypeptide shares at least 70% sequence identity to SEQ ID NO: 7 over the entire length of SEQ ID NO: 7, wherein the polypeptide comprises a subsequence that shares at least 90% amino acid sequence identity with the HA2 subunit of H5 HA (SEQ ID NO: 23) over the entire length of the HA2 subunit (SEQ ID NO: 23), and wherein the recombinant antigen polypeptide comprises a phenylalanine at a position corresponding to 371F of SEQ ID NO: 7, and (b) adsorbing the recombinant antigen polypeptide to alum. In some embodiments, the amino acid residue corresponds to 154N of SEQ ID NO: 7. In some embodiments, the polypeptide comprises a sequence selected from the group of SEQ ID NO: 7-14.Kilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068

[0015] Also provided herein is a method of preparing a recombinant vaccine composition comprising (a) introducing a nucleic acid sequence encoding a recombinant antigen polypeptide to a host cell, wherein the recombinant antigen polypeptide comprises from N terminus to C terminus continuously: a first antigen fragment having an amino acid sequence with at least 90% sequence identity to H5 HA (SEQ ID NO: 5), an RRC or an RRL, and a second antigen fragment having an amino acid sequence having at least 90% sequence identity to H5 HA (SEQ ID NO: 6), wherein the recombinant antigen polypeptide comprises a phenylalanine at a position that corresponds to amino acid residue 371 (phenylalanine) (“371F”) of SEQ ID NO: 7, thereby producing the recombinant antigen polypeptide; and (b) adsorbing the recombinant antigen polypeptide to alum. Also provided herein is a recombinant vaccine composition produced by the method above.

[0016] In some embodiments, the recombinant antigen polypeptide comprises one or more auxiliary elements. In some embodiments, the one or more auxiliary elements are selected from the group consisting of a polyhistidine tag and a trimerization domain. . BRIEF SUMMARY OF THE DRAWINGS

[0017] FIG. 1 is a schematic representation of the reorientation of H5 hemagglutinin (H5 HA) by oligoD (poly-Asp) insertion into the head region of H5 HA. H5 HA sequence is based on H5 HA (A / Vietnam / 1203 / 2004).

[0018] FIG. 2A and 2B show the results of the biochemical characterization of H5 HA and reoriented H5 HA (“reoH5HA”). FIG. 2A shows the results of size exclusion chromatography coupled with multi-angle light scattering analysis. FIG. 2B shows the results of protein gel electrophoresis.

[0019] FIG. 3A and 3B show the thermal melting profiles and the melting temperatures of H5 HA and reoH5HA in solution and in the presence of alum, respectively.

[0020] FIG.4A and 4B show the stem antigenicity of H5 HA and reoH5HA. FIG.4A shows the stem-directed antibody binding profiles of H5 HA or reoH5HA measured by biolayer interferometry. In FIG. 4B, shifts in nanometers of antibody binding to H5 HA were used as theKilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 maximal binding value (1.0), and values for reoH5HA are normalized as a fraction of the maximal binding value of H5 HA.

[0021] FIG. 5A-5C show the epitope accessibility of reoH5HA on alum by ELISA. FIG. 5A shows a panel of monoclonal antibodies (mAbs) used for epitope analysis and their epitopes on H5 HA (H5.3 – H5 HA head, FluA-20 – HA trimer interface, H5M9 - HA1 vestigial esterase subdomain, MEDI8852 and FI6v3 – HA stem). FIG. 5B-5C show ELISA results detecting the binding of various mAbs to the reoH5HA on streptavidin-coated plates. FIG.5C shows the binding of monoclonal antibodies to the reoH5HA on alum-coated plates.

[0022] FIG.6A-6C show the immunogenicity of H5 HA and reoH5HA in mice. FIG.6A shows the study design of a prime-boost immunization study with H5 HA or reoH5HA adjuvanted withalum / CpG in BALB / c mice. FIG. 6B shows serum H5 HA-specific IgG titers over time (n = 10mice per group). FIG. 6C shows serum H5 HA-specific stem-only protein IgG titers over time(n = 10 mice per group). Stem-specific IgG titers were measured with the H1-stabilized stem (H1-SS) protein. Arrows indicate prime and boost immunizations. Dashed lines indicate limit of quantification.

[0023] FIG.7 shows the cross-reactivity to different HAs induced by reoH5HA. Cross-reactive binding of group 1 (H1 NC / 99, H1 CA / 09 and H2 JP / 57) and group 2 (H3 VC / 75 and H7 SH / 13)HAs by week 12 antisera. Each circle represents a single mouse (n = 10 mice per group). Thedashed line indicates limit of quantification. Sequence identity and similarity to H5 HA (A / Vietnam / 1203 / 2004) are calculated with local alignment using the Smith–Waterman algorithm.

[0024] FIG.8 shows the cross-reactivity to avian H5 HAs induced by reoH5HA. Cross-reactive binding of two avian H5 HAs (by week 7 and 12 antisera. Each circle represents a single mouse(n = 10 mice per group). The dashed line indicates limit of quantification.

[0025] FIG.9 shows cross-reactivity to different HAs induced by H5 HAPheand reoH5HAPhein mouse immunization. The graph shows the cross-reactive binding of group 1 (H1 NC / 99, H1 CA / 09, H1-SS and H2 JP / 57), group 2 (H3 VC / 75 and H7 NT / 27) and avian H5 HAs (aH5 HB / 20and aH5 BD / 23) by week 7 antisera. Each circle represents a single mouse (n = 10 mice per group).The dashed line indicates limit of quantification. Sequence identity and similarity to H5 HA (A / Vietnam / 1203 / 2004) are calculated with local alignment using the Smith–Waterman algorithm.Kilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 DETAILED DESCRIPTION OF THE INVENTION I. DEFINITIONS

[0026] As will be apparent from context, “vaccine” or “vaccine polypeptide” refers to the antigen (polypeptide) portion of a vaccine preparation, and “vaccine composition” refers to the antigen (polypeptide) in combination with an adjuvant (alum) and optionally other excipients.

[0027] As used herein, a “recombinant subunit vaccine” or “recombinant subunit vaccine polypeptide” refers to a recombinantly produced polypeptide intended for administration to a subject to elicit a protective immune response. Other terms used interchangeably with recombinant subunit vaccine include “recombinant polypeptide vaccine” “biosynthetic polypeptide vaccine,” and “genetically engineered polypeptide vaccine.”

[0028] As used herein, an “antigen polypeptide” or “antigenic portion” is a polypeptide or portion of a polypeptide that encodes a pathogen protein or portion of a pathogen protein (“pathogen antigen polypeptide”), or encodes a disease antigen or portion of disease antigen (“disease antigen polypeptide”), and elicits a desired protective immune response against the pathogen or disease antigen.

[0029] As used herein, a “disease antigen” refers to an antigen that is a target of a therapeutic vaccine, such as a cancer antigen. See Tagliamonte et al., 2014, “Antigen-specific vaccines for cancer treatment” Hum Vaccin Immunother.10(11):3332-3346. doi:10.4161 / 21645515.

[0030] As used herein, a “subject” to which a vaccine is administered may be a human or may be a non-human animal (e.g., a pet, such as a cat or dog, livestock, such as cows, sheep, pigs, goats, fish, and poultry).

[0031] As used herein, “Region of Repetitive Carboxylic Groups” (RRC) has the meaning set forth hereinbelow.

[0032] As used herein, “RRC-encoding sequence” is a nucleic acid sequence that encodes an RRC.

[0033] As used herein, an “aspartate residue” is an amino acid residue in a polypeptide, having the side chain CH2COOH. Aspartate is an -amino-acid residue anion resulting from the deprotonation of the carboxy group of an acid residue and is generally the form found inKilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 physiological conditions. The terms “aspartic acid,” ”aspartate,” “aspartic acid residue,” and ”aspartate residue,” are often used interchangeably in the literature and are equivalent terms as used herein. Aspartate is represented as “D” or “Asp.”

[0034] As used herein, a “poly-Asp sequence” (or, equivalently, an “[Asp]N sequence”) refers to 6 or more contiguous aspartate residues in an RRC portion of a recombinant polypeptide vaccine made as disclosed herein.

[0035] As used herein, a “poly-Asp encoding sequence” is a nucleic acid sequence that encodes multiple contiguous aspartate residues. In most systems, aspartate is encoded by the codons GAT and GAC. In some embodiments, a poly-Asp encoding DNA sequence comprises [GAY]N where G is guanine, A is adenine, Y is pyrimidine (C or T), and N = 6-40 or 8 to 20.

[0036] As used herein, a “glutamate residue” is an amino acid residue in a polypeptide, having the side chain CH2CH2COOH. Glutamate is an -amino-acid residue anion resulting from the deprotonation of the carboxy group of a glutamic acid residue and is generally the form found in physiological conditions. The terms “glutamic acid,” “glutamate,” “glutamic acid residue,” and “glutamate residue,” are often used interchangeably in the literature and are equivalent terms as used herein. Glutamate is represented as “E” or “Glu.”

[0037] As used herein, a “poly-Glu sequence” (or, equivalently, a “[Glu]N sequence”) refers to 6 or more contiguous glutamate residues in an RRC portion of a recombinant polypeptide.

[0038] As used herein, a “poly-Glu encoding sequence” is a nucleic acid sequence that encodes multiple contiguous aspartate residues. In most systems, aspartate is encoded by the codons CAG and CAA. In some embodiments, a poly-Glu encoding DNA sequence comprises [CAR]N where G is cytidine, A is adenine, R is purine (A or G), and N = 6 to 40 or N = 8 to 20 or 8 to 18.

[0039] An “RRC-containing polypeptide” is an antigenic polypeptide that can be used as a component of a vaccine and contains an RRC.

[0040] As used herein, in the context of an RRC, “introduction,” “installation,” and “insertion” are used interchangeably to refer to addition to and / or modification of a nucleic acid sequence encoding an RRC, e.g., poly-Asp or poly-Glu, for expression of an RRC-containing polypeptide (antigen). A polypeptide expressed from such a nucleic acid (i.e., a polypeptide having an RRCKilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 inserted) can be referred to as a polypeptide or antigen having an “inserted,” “installed,” or “introduced” RRC. Introduction of RRC-encoding codons is carried out using any suitable method, including molecular cloning and de novo synthesis of a polynucleotide. For ease of references, an insertion can be characterized as a “terminal insertion” or an “intervening insertion.”

[0041] As used herein, in one aspect, a “terminal” poly-Asp / poly-Glu / RRC sequence refers to a sequence found at the amino- or carboxy-terminus of a recombinant protein vaccine polypeptide. However, a poly-Asp / poly-Glu / RRC sequence that is at the amino-terminal but for an immediately preceding a single methionine can be considered a terminal RRC. As used herein, in cases in which an RRC-containing polypeptide is processed (e.g., by removal of a signal peptide) the amino- or carboxy-terminus of a recombinant protein refers to a terminus of a mature or processed protein or protein fragment as combined with alum and incorporated into the vaccine composition. In cases in which a recombinant protein is translated as a pre-protein (including a signal peptide), the terminal RRC can be positioned at the terminus of the mature protein. It will be understood that in a polynucleotide encoding pre-proteins, the terminal RRC encoding sequence can be positioned between codon corresponding to the C-terminus of the signal peptide and the codon corresponding to the N-terminus of mature polypeptide. Similarly, in the case of a pro-protein antigen (a protein that undergoes post-translational processing) the terminal RRC may be positioned such that it is located at the N- or C- terminus of the processed mature protein. Similarly, in the case of a protein antigen that is processed (e.g., cleaved) ex vivo after isolation from cells, the terminal RRC may be positioned such that it is located at the N- or C- terminus of the processed (e.g., cleaved) protein. In short, a terminal RRC may be positioned at the terminus of the polypeptide product that is combined with alum.

[0042] As used herein, “in the form immobilized on alum” refers to the antigen polypeptide associated with alum in a vaccine composition. For example, the form immobilized on alum may refer to a mature polypeptide after removal of a signal peptide and cleavage. In some cases the antigen polypeptide presented on alum is member of a multimer (e.g., trimer). In embodiments, the multimer may be a homomultimer or a heteromultimer.

[0043] As used herein, an RRC “at” the amino- or carboxy-terminus of a polypeptide, means that in the form immobilized on alum the RRC is a terminal sequence. As used herein, an RRC “near” the amino- or carboxy-terminus of a polypeptide, means that in the form immobilized onKilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 alum the RRC within twenty-five (25) residues of a polypeptide terminus, i.e., the twenty-fifth residue from the polypeptide terminus is part of the RRC. In some embodiments the RRC near a terminus is within 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 residues of a polypeptide terminus.

[0044] As used herein, an “intervening” RRC sequence refers to an RRC that is not at the amino- or carboxy-terminus of an antigen polypeptide in the form immobilized on alum. An intervening RRC can be an insertion at a position within an antigen polypeptide (a “contiguous intervening” sequence) or may be a substitution that replaces residues of the unmodified antigen. In either case, additional amino acid flanking one or both ends of the RRC sequence may be included in the introduced sequence.

[0045] As used here, a “contiguous intervening” poly-Asp / poly-Glu / RRC sequence refers to a poly-Asp / poly-Glu / RRC sequence within a polypeptide, where the poly-Asp / poly-Glu / RRC separates and is contiguous with two sequences that are contiguous in the unmodified antigen (e.g., a pathogen protein found in nature).

[0046] As used herein, “sequence identity” in reference to similarity of two proteins (a target protein and a reference protein) or two nucleic acids (a target nucleic acid and a reference nucleic acid) is a quantification of identity of amino acids or nucleobases when the reference and target sequence are optimally aligned. Sequence identity can be determined manually by inspection, especially when the target and reference have greater than 90% identity. Alternatively, for nucleotide sequences, percent identity to a reference nucleic acid sequence can be determined using a BLAST or BLAST 2.0 comparison program (described in Altschul et al. (1990) J. Mol. Biol.215: 403-410 and Altschul et al. (1977) Nucleic Acids Res.25: 3389-3402, respectively) with default parameters. BLASTP with default parameters can be used to determine percent to a reference polypeptide sequence. The BLASTN program uses as default parameters a word size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)). Software for BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) website.Kilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068

[0047] As used herein, a promoters or other regulatory elements, such as enhancers, is "operably linked" to a nucleic acid sequence when they affect to the expression of RNA from the nucleic acid sequence.

[0048] As used herein, an RRC-containing antigenic polypeptide can be described as a “derivative” of a non-RRC polypeptide (e.g., a naturally occurring pathogen protein, candidate subunit in development) when the RRC-containing antigenic polypeptide (“parental polypeptide”) shares sequence identity with at least a portion of the non-RRC antigenic polypeptide and elicits an immune response specific for the non-RRC polypeptide. Exclusive of an RRC(s) and auxiliary elements a derivative of a polypeptide may have at least about 50% sequence identity, at least about 60% sequence identity, at least about 70% sequence identity, at least about 80% sequence identity, or at least about 90% sequence identity with a corresponding “parental” polypeptide.

[0049] As used herein, the term “substantially similar,” when referring to sequence similarity, refers to that an amino acid sequence shares at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the reference amino acid sequence.

[0050] The terms “corresponding to,” “determined with reference to,” or “numbered with reference to” when used in the context of the identification of a given amino acid residue in a polypeptide sequence, refers to the position of the residue of a specified reference sequence when the given amino acid sequence is maximally aligned and compared to the reference sequence. The polypeptide that is aligned to the reference sequence need not be the same length as the reference sequence. “As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim element. As used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the term “comprising”, particularly in a description of components of a composition, in a description of a sequence (an amino acid sequence or a nucleic acid sequence), in a description of a method, or in a description of elements of a device, is understood to encompass those compositions, methods, or devices essentially of and consisting of the recitedKilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 components or elements. The invention illustratively described herein suitably may be practiced in the absence of any element, elements, limitation, or limitations which is not specifically disclosed herein. For example, a statement that a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 7, includes the scenario where the polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 7.

[0051] The term “reoH5HA” or “reoriented H5 HA” refers to a modified H5 HA that contains an amino acid sequence rich in charged residues (e.g., poly(aspartic acid), poly(glutamic acid), poly(lysine), and poly(arginine) inserted into the head region of the wild-type H5 HA such that it can be anchored onto adjuvant substances (e.g., alum adjuvants) in a predefined or “upside- down” configuration and induces immune response that focuses on the stem region of HA (HA-stem).

[0052] The term “reoH5HAphe” refers to a reoH5HA that further comprises a I Fsubstitution at a position corresponding to 371F of SEQ ID NO: 7. Exemplary reoH5HApheproteins include SEQ ID NOs 3 and 4.

[0053] The term “H5HAphe” refers to a modified H5 HA protein that contains a I Fsubstitution at a position corresponding to 371F of SEQ ID NO: 7. Exemplary H5HApheproteins include SEQ ID NO: 1-14. II. INTRODUCTION

[0054] This disclosure describes new vaccine compositions comprising a modified H5 HA antigen (a group 1 HA) bound to the surface of an adjuvant or carrier by electrostatic interactions. The modified H5 HA is a new reoriented HA immunogen that induces an immune response that focuses on the stem region of HA (HA-stem), which is evolutionarily conserved. This reoriented H5 HA not only induced cross-reactive antisera to both group 1 and 2 influenza A subtypes but also highly pathogenic avian influenza strains. This new reoriented H5 HA immunogen has potential to translate into a universal influenza vaccine candidate to protect against existing influenza A subtypes and even emerging avian strains.Kilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068

[0055] The reoriented H5 HA immunogen is generated by introducing an amino acid sequence rich in charged residues (e.g., poly(aspartic acid), poly(glutamic acid), poly(lysine), and poly(arginine) at a defined location in the target protein, as further disclosed below. In some embodiments, the defined location is immediately after the amino acid sequence corresponding to of LIKKX (SEQ ID: 24) in SEQ ID NO: 7. In some embodiments, the defined location is immediately after an amino acid residue corresponding to any one of 150-160 (e.g., 154) of SEQ ID NO: 7.

[0056] In addition, relative to the wild type H5 HA sequences, the reoriented H5 HA comprisesan I F mutation in the HA2 subunit of the H5 HA. This mutation further increases the cross-reactivity of antibodies produced in subjects immunized with the vaccine as compared to a reoriented H5 HA vaccine without such a mutation. A reoriented H5 HA comprising this mutation is referred to as reoH5HAphe in this disclosure. In some embodiments, a reoH5HAphe comprises a phenylalanine at a position that corresponds to 371F of SEQ ID NO: 7. This phenylalanine is the residue 45 of the HA2 subunit of the H5 HA. III. INFLUENZA VACCINES AND ANTIGEN MODIFICATIONS

[0057] Influenza Hemagglutinin (HA) is a glycoprotein found on the surface of influenza viruses. Influenza A viruses are divided into subtypes based on two proteins on the surface of the virus: hemagglutinin (HA) and neuraminidase (NA). There are 18 known HA subtypes and 11 known NA subtypes. It is responsible for binding the virus to cell membranes, such as, cells in the upper respiratory or erythrocytes. HA is also responsible for the fusion of the viral envelope with the endosomal membrane, after the pH drops in the endosome. HA is a homotrimeric integral membrane glycoprotein. HA is expressed as a precursor protein (referred to as HA0) that trimerizes and then is cleaved into two smaller polypeptides — the HA1 and HA2 subunits, which remain complexed. The mature form of HA is thus a trimer of HA1-HA2 heterodimers. The HA1 subunit includes a globular head region containing the hemagglutinin receptor binding site that interacts with sialic acid on the surface of eukaryotic cells. The HA2 subunit includes a long, helical chain, a transmembrane region, and a cytoplasmic region. A portion of the HA1 subunit and the helical chain portion of the HA2 subunit are referred to as the stem region of the Hemagglutinin (HA) protein.Kilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068

[0058] The head region of HA appears to be immunodominant, meaning that during viral infection or during vaccination, subjects often produce antibodies predominantly against the head region. The head region, however, has significantly higher sequence variability when compared to the stem region, and antibodies against it are often not protective against challenges with other viral isolates. The HA stem domain is highly conserved and appears to contain broadly neutralizing epitopes. As such, antibodies directed against the HA stem domain may protect against many strains of the virus.

[0059] Disclosed herein are the methods and compositions for preparing influenza vaccines effective against multiple related H5 influenza viruses. According to the present disclosure, regions of a polypeptide with a high density of carboxylic groups, known as a Region of Repetitive Carboxylic Groups or “RRC”, further discussed below, are introduced into an H5 influenza virus antigen polypeptide to produce an “enhanced antigen.” H5 influenza virus antigen polypeptide isgenetically engineered to contain an I F mutation at the position that corresponds to position 371of the wild type H5 HA (A / Viet Nam / 1203 / 2004) (SEQ ID NO: 15) or position 45 of the HA2 subunit of the wild type H5 HA. When administered, for example, as an antigen-alum complex, enhanced antigens increase humoral antibody responses and increase the neutralization potency of the antibody response relative to administration of an unmodified antigen-alum complex. Moreover, the antigenic-alum complexes of the invention may be designed to present antigens in a predetermined orientation. In an aspect, the orientation directs the immune system to generate antibodies against a specific region, or epitope, of the antigen, a process referred to herein as “immunofocusing.” For example, the orientation can be used to elicit production of neutralizing antibodies.

[0060] Furthermore, the recombinant antigen H5 HA polypeptide contains an isoleucine to phenylalanine substitution at a position that corresponds to residue 371 of wild type H5 HA (SEQ ID NO: 15). And therefore, this one amino acid substitution on an HA immunogen alters the establishment and expansion of broadly cross-reactive B cells and can be introduced to the H5 HA and reoH5HA design to expand the cross-reactivity of vaccine-induced antibodies.

[0061] As described in the Examples, below, the recombinant antigen H5 HA polypeptides disclosed herein are produced by site-specific introduction of repeating units of aspartate residues (“poly-Asp” or “[Asp]N”) and the single amino acid substitution (from isoleucine toKilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 phenylalanine). In studies using influenza hemagglutinin (HA) administered with alum, we have demonstrated that introduction of poly-Asp and the phenylalanine into antigens enhanced humoral antibody responses in a mouse model, resulted in minimal in-group variations among immunized individuals, and significantly increased the neutralization potency of the antibody response and broadened the cross-reactivity.

[0062] In some embodiments, the insertion is a poly(aspartic acid) [D]N, wherein the N is 8-18. In some embodiments, the insertion is a poly(aspartic acid) [E]N, wherein the N is 8-18. In some embodiments, the insertion is a D-E copolymer [(D)X, (Glu)Y] where X is 1-17, Y is 1-17, and X + Y = 8-18. In some embodiments, a region has a high density of D and / or E. In some embodiments, the insertion is 12D. In some embodiments, the insertion is 11D. The introduced sequence (or “region”) forms an electrostatic association with the adjuvant such that the antigen is retained on the surface in an advantageous orientation.

[0063] For illustration and not limitation three categories of vaccine compositions can be described:

[0064] (a) Vaccine polypeptides modified by introduction of a Region of Repetitive Carboxylic Groups (RRC). In this approach the modification introduces a region rich in aspartate (D) and / or glutamate (E) causing the polypeptide to associate with a negatively charged region of an alum aggregate (comprising aluminum hydroxide).

[0065] (b)Vaccine polypeptides modified by introduction of a Region of Repetitive Lysyl / Guanidino Groups (RRL). In this approach the modification introduces a region rich in lysine (K) and / or arginine (R), causing the polypeptide to associate with a negatively charged region of an aluminum-based adjuvant, such as aluminum phosphate and amorphous aluminum hydroxyphosphate sulfate (AAHS). See Section XIV, below.

[0066] (c) Vaccine polypeptides modified by introduction of an RRC or RRL and adsorbed to lipid nanoparticles (LNPs) used as carriers or adjuvants (e.g., liposomal saponin, monophosphoryl lipid A). See US Pat. No. 10,434,167 (“Non-toxic adjuvant formulation comprising a monophosphoryl lipid A (MPLA)-containing liposome composition and a saponin”), Rao et al., 2021, “Liposome Formulations as Adjuvants for Vaccines” In Gill et al. (eds) Nanoparticles for Rational Vaccine Design. Current Topics in Microbiology and Immunology, vol 433, Springer;Kilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 Alving et al., 2020, “Army Liposome Formulation (ALF) family of vaccine adjuvants” Expert Review of Vaccines, 19:279-292. The surface charge of LNPs can be fine-tuned by the lipid composition using art-known means. RRC-modified antigens will adsorb onto positively-charged LNPs and RRL-modified antigens will adsorb onto negatively-charged LNPs.

[0067] Without intending to be limited to a particular mechanism of action, we believe introduction of repetitive carboxylic groups from RRC not only increases the electrostatic interaction between antigens and alum but also provides RRCs that act as chelating ligands for aluminum that further enhances alum-binding. Without intending to be limited to a particular mechanism of action, we believe the modified interaction with alum surprisingly results in the advantageous properties of the antigen-alum complex described herein. Exemplary H5 HA proteins and the residue after which poly-D can be inserted, and the residue that can be mutated to phenylalanine and are shown in Table 1 H5 HA proteins and their poly-Asp insertion sites and phenylalanine mutation sites. Accession numbers are full-length HAs with the native signal peptides. Insertion sites are counted from the beginning of the ectodomain of the HAs (mature proteins without the signal peptides). Table 1 H5 HA proteins and their poly-Asp insertion sites and phenylalanine mutation sites. H H H c H H 0 H C HP09345Kilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 H5 - A / Hata / 2004UniProt:N154 I375H[0id residue in an H5 HA polypeptide, where the position corresponds to any one of 150-160 (i.e., 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160) of SEQ ID NO: 7. In some embodiments, a poly- Asp disclosed herein can be inserted after an amino acid sequence corresponding to a sequence of LIKKX (SEQ ID NO: 24). For example, an insertion of poly-Asp, into H5 HA (A / Viet Nam / 1203 / 2004) can be made after residue N154. FIG. 2 shows the analysis results of reoH5 HA by size exclusion chromatography and protein gel electrophoresis. Further, the engineered H5 HA shows a configuration that is consistent with an upside-down orientation, such that the stem of the HA is more accessible to the immune system and can generate an immune response that is effective against multiple related pathogens. See Example 2 and FIG. 5. The types of RRC and insertion location of the RRC may vary and they are further disclosed below in sections below entitled “RRC TYPES AND PROPERTIES” and “SITE OF RRC INSERTIONS.”

[0069] In some embodiments, provided herein is a recombinant antigen polypeptide comprises a Region of Repetitive Carboxylic Groups (RRC) or a Region of Repetitive Lysyl / Guanidino Groups (RRL) inserted into a polypeptide that shares at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO: 7 over the entire length of SEQ ID NO: 7.

[0070] In some embodiments, the polypeptide comprises a subsequence that shares at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with the HA2 subunit of H5 HA (SEQ ID NO: 23) over the entire length of the HA2 subunit (SEQ ID NO: 23).

[0071] In some embodiments, the recombinant antigen polypeptide comprises from N terminus to C terminus continuously: a first antigen fragment having an amino acid sequence with at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%,Kilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 at least 99%, or 100% amino acid sequence identity to SEQ ID NO: 5 over the entire length of SEQ ID NO: 5, an RRC or an RRL, and a second antigen fragment having an amino acid sequence having at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO: 5 over the entire length of SEQ ID NO: 6. The term “continuously” refers to that there is no intervening amino acid residue between any two of the 1) the first antigen fragment, 2) the RRC or RRL, and 3) the second antigen fragment. IV. RRC TYPES AND PROPERTIES

[0072] As noted above in Section II entitled “INTRODUCTION,” antigenic vaccine polypeptides can be modified by introduction of a Region of Repetitive Lysyl / Guanidino Groups (“RRL”). In this approach, the modification introduces a region rich in lysine (K) and / or arginine (R), causing the polypeptide to associate with a negatively charged region of an adjuvant, such as an aluminum-based adjuvant. Exemplary aluminum-based adjuvants include aluminum phosphate and amorphous aluminum hydroxyphosphate sulfate (AAHS). Polypeptides modified by introduction of RRLs can also associate with lipid-based adjuvants. Suitable adjuvants that can be used in methods and compositions disclosed herein are further described below.

[0073] RRL’s generally share the features of RRC’s, except that RRLs comprise lysine (K) and / or arginine (R) while RRCs comprise aspartic acid (D, Asp) and / or glutamic acid (E, Glu) and RRLs comprise lysine (K, Lys) and / or arginine (R, Arg). The description of RRLs is embodied in this disclosure: The reader is instructed to replace (except in working examples or otherwise clear from context) every reference to “aspartic acid” may be replaced with “lysine,” and every reference to “glutamic acid” may be replaced with “arginine” just as if the text had been duplicated and rewritten with these changes. As noted, references to alum in the context of RRCs will be understood to refer to adjuvants with a positive surface charge such as, but not limited to, those specifically listed herein). R A eGlutamic Acid (E, Glu, polyGlu, 12E, etc. ) Arginine (R, Arg, Polyarg, 12R, etc.)Kilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068

[0074] RRCs are rich in glutamic acid and / or aspartic acid, both of which are acidic amino acids with a side chain containing a terminal carboxyl group. RRCs are sometimes categorized as TYPE 1, TYPE 2, TYPE 3, or TYPE 4 RRCs. Each type of RRC begins with a glutamic acid or aspartic acid residue and ends with a glutamic acid or aspartic acid residue. In some embodiments the RRC contains aspartic acid residues and does not contain glutamic acid residues. In some embodiments the RRC contains glutamic acid residues and does not contain aspartic acid residues. In a polypeptide, an RRC can be described as the region of contiguous amino acid residues having a D or E at the amino end of the RRC, a D or E at the carboxy end of the RRC and having the properties of a TYPE 1-4 RRC. It will be recognized that in some cases an RRC may be inserted adjacent to a D or E containing sequence present in the unmodified antigen.

[0075] A TYPE 1 RRC contains a poly-Asp sequence (“poly-Asp” or “[Asp]N” or “poly-D” or “oligoD”). Several RRCs described in Examples are TYPE 1 RRCs. In some embodiments N is 6- 40 or 8-20 or 8-18.

[0076] A TYPE 2 RRC contains a poly-Glu sequence (“poly-Glu” or “[Glu]N” or “poly-E”). In some embodiments N is 6-40 or 8-20 or 8-18.

[0077] A TYPE 3 RRC can be described as a copolymer of Asp and Glu comprising N contiguous residues each independently selected from D and E, where N is 6-40 (e.g., [(Asp)X, (Glu)Y] where X is 1-39, Y is 1-39, and X + Y = 6-40 or where N is 6-20 [e.g., (Asp)X, (Glu)y where X is 1-19, Y is 1-19, and X + Y = 6-20]. The term “TYPE 3 RRC” includes such copolymers, as well as TYPE 1 and TYPE 2 RRCs.Residues in a TYPE 3 RRC can be described, without limitation, as a copolymer, a block copolymer, an alternating copolymer, or a random copolymer, such as DEDEDEDEDE (alternating copolymer), DDDEEEDDDEEE (block copolymer) or, e.g., EEEDEDDDEDEEED (random copolymer).

[0078] A TYPE 4 RRC, has a high density of Asp and / or Glu, but may include other residues as well. Examples of TYPE 4 RRCs are the sequences DDDDDLEEEEE and DEDEDDLEGEED. “High density” means that at least 50% of residues in the RRC are Asp or Glu. See TABLES 1A and 1B. In other embodiments, “High density” means that at least 50%, at least 60%, at least 75% or at least 80% of residues in the RRC are Asp or Glu. It will be apparent that the first residue of a TYPE 4 RRC will be D or E and the last residue of a TYPE 4 RRC will be D or E. It will alsoKilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 be apparent that all TYPE 1-3 RRCs (each having 100% Asp or Glu) are also TYPE 4 RRCs. Table 2 and Table 3 list various types of RRCs. Table 2 Exemplary RRCs which at least 80% of residues in the RRC are Asp or Glu Length Maximum Number of non E / D if at least Example o 6 1 1 2Table 3 Exemplary RRCs in which at least 50% of residues in the RRC are Asp or Glu L R 9 1

[0079] In some cases, the non-D non-E residues in a TYPE 4 RRC are small, non-polar and neutral amino acids such as glycine, leucine or alanine. In some cases, the RRC does not contain lysine or arginine (positively charged residues).

[0080] In some cases, the antigen protein is modified by introduction of two or more RRCs. In some cases, the RRCs are positioned in different flexible loops, but are close in three-dimensional space. In some embodiments, the flexible loop comprises one or more or all of residues 150-160 (i.e., 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160) of SEQ ID NO: 7.

[0081] The number of amino acid residues in an RRC (i.e., the “length” of the RRC) is generally in the range of 6 to 40. Often the RRC has a length of 8 to 20 residues. Generally, an RRC contains at least six residues that are D or E, preferably at least eight residues that are D or E. In some embodiments, the length of the RRC insertion sequence) is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. However, in some embodiments the RRC sequence comprises a greater or smaller number of Asp residues, such as 2 to 30 or 3 to 20 Asp residues.Kilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068

[0082] In some cases the RRC is 8 residues (e.g., 8D). In some cases, the RRC is 12 residues (e.g., 12D). As shown below in Example 2 and FIG. 5A-5C, 12D insertion into H5 HA (SEQ ID NO: 15) resulted in complete binding of the engineered H5 HA to alum and the desired “upside down” configuration. In some cases, the RRC is 11 residues (e.g., 11D), when inserted immediately after a sequence of LIKKD (SEQ ID NO: 26) or a residue D154 as in SEQ ID NO: 20. V. ALUM

[0083] As used herein, “alum” refers to insoluble aluminum hydroxide (also called aluminum oxyhydroxide) suitable for use as an adjuvant in humans and nonhuman animals. See HogenEsch et al., 2018, “Optimizing the utilization of aluminum adjuvants in vaccines: you might just get what you want.” npj Vaccines 3, 51. doi.org / 10. 1038 / s41541-018-0089-x; also see Baylor et al, 2002, “Aluminum salts in vaccines--US perspective” Vaccine 20 Suppl 3:S18-23. doi: 10.1016 / s0264-410x(02)00166-4. Alum has been described as aggregates of aluminum hydroxide nanoparticles or microparticles. See Harris et al. 2012, Alhydrogel(R) adjuvant, ultrasonic dispersion and protein binding: a TEM and analytical study. Micron 43, 192–200; Li et al., 2017 “Aluminum (Oxy)Hydroxide Nanosticks Synthesized in Bicontinuous Reverse Microemulsion Have Potent Vaccine Adjuvant Activity” ACS Appl Mater Interfaces. 2017;9(27):22893-22901. doi:10.1021 / acsami.7b03965; Orr et al., 2019,” Reprogramming the adjuvant properties of aluminum oxyhydroxide with nanoparticle technology” npj Vaccines 4, 1. doi.org / 10.1038 / s41541-018-0094-0. As used herein, the term “alum particles” is used to describe alum of various sizes and shapes, provided the alum is suitable for use as an adjuvant. Alum is available from a variety of commercial sources. ALHYDROGEL® type adjuvant is commercially available (CRODA, Invivogen).

[0084] As discussed below, in some cases an aluminum-based material with a surface negative charge is used as an aluminum-based adjuvant. Examples include aluminum phosphate and amorphous aluminum hydroxyphosphate sulfate (AAHS). Vaccine antigen polypeptides modified by introduction of a Regions of Repetitive Lysyl / Guanidino Groups (“RRL”) may be combined with aluminum-based adjuvants to prepare vaccines of the invention.Kilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 VI. GENETIC ENGINEERING AND EXPRESSION OF RECOMBINANT SUBUNIT VACCINE POLYPEPTIDES

[0085] Manipulation and expression of RRC recombinant subunit vaccines according to the invention can be carried out as desired. For example, recombinant DNA methodology may be used to modify an antigen-encoding sequence by site-specific introduction of an RRC encoding sequence. See, e.g., Irwin et al., 2012, “In-Fusion® Cloning with Vaccinia Virus DNA Polymerase” In: Isaacs S. (eds) Vaccinia Virus and Poxvirology. Methods in Molecular Biology (Methods and Protocols), vol 890. Humana Press, Totowa, NJ. doi.org / 10.1007 / 978-1-61779-876- 4_2. As used herein, “introduction” of an RRC does not imply use of any particular methodology. Exemplary methods include insertion and ligation, homologous recombination, and introduction using a CRISPR / CAS system.

[0086] Recombinant subunit polypeptide vaccines can be produced using any suitable method, including expression as heterologous proteins in recombinant systems. Exemplary expression systems are well known and include bacteria, yeast, insect cell, mammalian cell, plant and transgenic animal platforms. See, e.g., Cid and Bolívar, 2021, “Platforms for Production of Protein-Based Vaccines: From Classical to Next-Generation Strategies” Biomolecules 11, 1072.doi.org / 10.3390 / biom11081072; also see Man Wang et al., 2016, “Recent advances in the production of recombinant subunit vaccines in Pichia pastoris, Bioengineered 7:3,155-165, DOI:10.1080 / 21655979.2016.1191707. VII. EXPRESSION OF RECOMBINANT SUBUNIT VACCINE POLYPEPTIDE

[0087] A recombinant subunit vaccine can be prepared by (a) obtaining a first polynucleotide comprising a sequence that encodes an antigen polypeptide; (b) introducing an RRC-encoding nucleic acid sequence into the sequence that encodes the antigen polypeptide, thereby producing a second polynucleotide encoding a chimeric protein sequence having (i) an RRC portion and (ii) an antigen polypeptide sequence portion(s). Typically, the nucleic acid sequence encoding the chimeric protein sequence linked to a promoter that drives transcription of the protein-encoding a sequence. The chimeric protein encoded by the second polynucleotide is expressed to produce an RRC containing vaccine antigen polypeptide. The polypeptide can be expressed using art-known methods, e.g., as discussed above, such as a cell-based or cell-free expression system. The vaccine polypeptide can be purified using routineKilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068

[0088] In an aspect, the invention provides vaccine polypeptides and vaccine compositions prepared using the methods described herein. In one approach, the method further includes the step of adsorbing the polypeptide to alum to produce a protein-alum complex. See Section XII below, titled “Adsorbing Antigen to Alum.” In one approach, the method further includes the step of combining the protein-alum complex with excipients. Components (e.g., protein, alum, excipient) can be combined in any order. VIII. SEQUENCE CHARACTERISTICS OF A RECOMBINANT SUBUNIT VACCINE POLYPEPTIDE

[0089] The recombinant subunit vaccine (or “antigen polypeptide”) comprises a sequence that elicits an immune response, such as an immune response against a pathogen. In one approach the antigen polypeptide has a sequence found in nature (e.g., a polypeptide expressed by the pathogen). Insertion of the RRC sequence results in a protein in which a pathogen sequence is close to or adjacent to an RRC in an arrangement not found in nature. In a related approach insertion of the RRC sequence results in a protein in which the RRC is adjacent to a pathogen sequence. A recombinant subunit vaccine containing an RRC or other RRC sequence can be recognized by reference to naturally occurring sequences identified in databases such as GenBank or UniProt. A hallmark of some vaccine polypeptides is an RRC adjacent to or near a known pathogen sequence. It will be understood that a characteristic of the recombinant subunit vaccine polypeptides is the presence of RRC near or adjacent to known or naturally occurring sequences (e.g., pathogen sequences), i.e., an arrangement not found in nature as can be readily deduced by reference to a sequence database.

[0090] In a related approach, the antigen polypeptide is a component of a vaccine that is approved or licensed by a regulatory agency, as is discussed in greater detail herein below. In this case the RRC is inserted to improve the properties of the known vaccine. In another related approach, the antigen polypeptide is a known (e.g., published) vaccine polypeptide candidate. In this case the RRC is inserted to improve the properties of the candidate vaccine. In general, the hallmarks of an RRC-containing recombinant subunit vaccine can be recognized by reference to a sequence database, having the hallmark of RRC adjacent to or close a known sequence of a licensed vaccine polypeptide or vaccine polypeptide candidate.Kilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068

[0091] For illustration and not limitation, the structure of the chimeric polypeptide produced by insertion of RRC can be described as shown in Table 4. Table 4 Exemplary chimeric polypeptides produced by insertion of RRC N-[A1]-[D]N-[A2]-C, where A1 and A2 are contiguous amino acid sequences of an H5 HA p r c N w d s s t s t A s s e c N N N s o N N y s E E n sKilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068

[0092] In some embodiments, a recombinant antigen polypeptide disclosed herein comprises a Region of Repetitive Carboxylic Groups (RRC) or a Region of Repetitive Lysyl / Guanidino Groups (RRL) inserted after an amino acid sequence corresponding to a sequence of LIKKX (SEQ ID NO: 24) in a polypeptide. In some embodiments, a recombinant antigen polypeptide disclosed herein comprises an RRC or RRL inserted after an amino acid residue in a polypeptide, wherein the amino acid residue corresponds to any one of 150-160 (i.e., 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160) of SEQ ID NO: 7. The polypeptide further has an amino acid sequence that shares at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO: 7 over the entire length of SEQ ID NO: 7. In some embodiments, the polypeptide comprises a subsequence that shares at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with the HA2 subunit of H5 HA (SEQ ID NO: 23) over the entire length of the HA2 subunit (SEQ ID NO: 23). In some embodiments, the recombinant antigen polypeptide comprises a phenylalanine at a position that corresponds to 371F of SEQ ID NO: 7.

[0093] In some embodiments, a recombinant antigen polypeptide disclosed herein comprises from N terminus to C terminus continuously: a first antigen fragment having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 5 over the entire length of SEQ ID NO: 5, an RRC or an RRL, and a second antigen fragment having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 6 over the entire length of SEQ ID NO: 6. The recombinant antigen polypeptide comprises a phenylalanine at a position amino acid that corresponds to 371F of SEQ ID NO: 7. IX. SITES OF PHE MUTATION

[0094] Wild-type H5 HAs share a conserved isoleucine residue in their HA2 subunits, and the isoleucine residue can be substituted with phenylalanine to increase the cross-reactivity of antibodies induced by the vaccines comprising the H5 HAs. The positions of the isoleucine in the various types of wild-type H5 HAs are shown in Table 1 H5 HA proteins and their poly-Asp insertion sites and phenylalanine mutation sites. Although the exact positions of the isoleucineKilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 may vary, they generally align with residue 371 of the mature protein of H5 - A / Viet Nam / 1203 / 2004, or the residue 371 of SEQ ID NO: 7. As compared to the wild-type H5 - A / Viet Nam / 1203 / 2004, SEQ ID NO 1 and SEQ ID NO: 7 both contain an isoleucine to phenylalanine at position 371. SEQ ID NO: 1 additionally comprises a Foldon trimerization domain and His-Tag at the C terminus. IX. SITE OF RRC INSERTION

[0095] Optimal sites of insertion were determined. The effects of RCC insertion were assessed by comparing thermal melting relative to a reference sequence. Retention of conformational epitopes was assessed by determining the effect of the insertion using panels of antibodies. Methods of determining the optimal sites of insertion are also disclosed in WO2023064631. The effect of alum binding by the RRC-containing antigen relative to the wild-type antigen were assessed in a variety of ways, including as described in Example 2, FIG. 6A-C and FIG. 7. The ability of adjuvated antigen to elicit an immune response can be determined using art-known methods such as measuring antibody response including specific IgG production including. See Example 4 and FIG. 5, 11, 14, and 18 of WO2023064631. In one approach, the ability of vaccination to generate neutralizing antibodies is assessed. See Example 1. Methods of assessing ability of vaccination to generate neutralizing antibodies is also described in WO2023064631, Example 4. X. IMMUNOFOCUSING

[0096] As noted above, the position of an inserted RRC can be used to control the orientation of the antigen polypeptide relative to the alum surface, providing methods for immunofocusing. The control of antigen orientation on alum by introducing RRC to different locations on antigen proteins has several advantages. For example, the RRC-containing antigenic proteins of this disclosure are useful as vaccine immunogens that can direct the immune system of a subject immunized with such vaccine immunogens to generate antibodies against a specific region, or epitope, of a protein that is known to be productive or neutralizing in the case of an infection See Weidenbacher and Kim, 2019, “Protect, Modify, Deprotect (PMD): A strategy for creating vaccines to elicit antibodies targeting a specific epitope,” PNAS 116 (20): 9947-9952 and Weidenbacher and Kim, 2019, WO 2019 / 222674, both incorporated by reference for all purposes, for a discussion.Kilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068

[0097] In one aspect the invention provides a vaccine composition comprising antigen protein molecules adsorbed to alum particles, wherein the antigen protein molecules comprise a Region of Repetitive Carboxylic Groups and wherein a majority of the antigen protein molecules in the composition that are adsorbed to alum have the same orientation relative to a surface of the alum particle to which it is adsorbed. Orientation can be determined as described above (in section captioned “Site of RRC Insertion”) and as described in Examples 2. In one approach, antigen proteins in antigen protein-alum complexes have the same orientation relative to the alum surface when a panel of 4, 5, or more monoclonal antibodies against the protein exhibit substantially similar binding patterns. XI. AUXILIARY ELEMENTS INCLUDING SPACERS AND TRIMERIZATION DOMAINS

[0098] As used herein, “auxiliary element” refers to a functional element in an RRC-containing polypeptide sequence that is not present in the antigen sequence that is modified by insertion of the RRC into, e.g., a naturally occurring sequence. Without limitation examples of auxiliary elements include tags for analysis or purification (e.g., a histidine tag or Avi-Tag, and the like), spacer elements (e.g., a glycine-serine spacer having the structure [N]3-5 where N is glycine or serine, e.g., GGS), and trimerization domains (e.g., foldon, GCN4, GCN4-pIQI). Exemplary auxiliary elements are shown in Table 5. Table 5 Exemplary auxiliary elements A [ T F n G GCN4-pIQI MKQIEDKIEEILSKQYHIENEIARIKKLIGER Trimerization domain

[0099] Gly-Ser spacers provide flexibility in the polypeptide that allows the RRC (or an auxiliary element) to adopt orientations to facility binding to alum.

[0100] Trimerization domains may be included in the vaccine preparation. Influenza Hemagglutinin (HA) proteins are found in nature as trimers. In some embodiments a trimerizationKilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 domain is included in the RRC-containing polypeptide stabilize the trimeric structure of the antigen complex. For example, the RRC-containing polypeptides set forth in SEQ ID NOs: 1-4. The trimerization domain foldon (or GCN4) in these polypeptides can be replaced with GCN4 (or foldon) or any other trimerization domain. It will be recognized that some antigen polypeptides will comprise a dimerization domain or other multimerization domain. XII. ADSORBING ANTIGEN TO ALUM

[0101] Methods for combining a vaccine protein and alum to make a protein-alum complex are well known. For a general description see HogenEsch et al., 2018, “Optimizing the utilization of aluminum adjuvants in vaccines: you might just get what you want.” npj Vaccines 3, 51. doi.org / 10. 1038 / s41541-018-0089-x. Also see Example 1, below (protein antigens with alum (protein:alum, 1:10, w / w) for 30 min PBS at room temperature, followed by addition of naïve mouse serum to a final concentration of 10% (v / v). XIII. Vaccine Compositions

[0102] In one aspect, the invention provides a first composition comprising (1) a polypeptide having a defined sequence and having an RRC insertion and (2) alum, wherein at least some of the polypeptides are adsorbed to alum particles.

[0103] In an embodiment, a majority of the polypeptides in the first composition that are associated with alum are associated in the same orientation.

[0104] In addition to protein and alum, the vaccine compositions may include one or more other vaccine reagents selected from citric acid monohydrate, trisodium citrate dihydrate, sugars (e.g.,2-hydroxypropyl- -cyclodextrin), sodium chloride, thiomersal, antibiotics, MgCl2 (for OPV),MgSO4, lactose-sorbitol and sorbitol-gelatine. Additional adjuvants

[0105] In general, the vaccine compositions may include other adjuvants. A list of approved adjuvants is included here: www.cdc.gov / vaccinesafety / concerns / adjuvants.html. In an embodiment, the composition comprises CpG oligonucleotides.Kilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 XV. VACCINATION TARGETS

[0106] The methods and vaccine compositions disclosure herein may be used for any therapeutic or prophylactic treatment responsive to vaccination. Exemplary diseases, pathogens, pathogen polypeptides, and disease-associated polypeptides are known and additional targets will be identified in the future. Exemplary targets, for illustration and not limitation, are described below. See Cid and Bolivar, 2021, “Platforms for Production of Protein-Based Vaccines: From Classical to Next-Generation Strategies” Biomolecules 11(8), 1072. XVI. EXEMPLARY EMBODIMENTS

[0107] This disclosure includes the following non-limiting exemplary embodiments.

[0108] Embodiment 1. A recombinant antigen polypeptide comprises a Region of Repetitive Carboxylic Groups (RRC) or a Region of Repetitive Lysyl / Guanidino Groups (RRL) inserted after an amino acid residue in a polypeptide, wherein the amino acid residue corresponds to any one of amino acid residues 150-160 of SEQ ID NO: 7, wherein the polypeptide shares at least 70% sequence identity to SEQ ID NO: 7 over the entire length of SEQ ID NO: 7, wherein the polypeptide comprises a subsequence that shares at least 90% amino acid sequence identity with the HA2 subunit of H5 HA (SEQ ID NO: 23) over the entire length of the HA2 subunit (SEQ ID NO: 23), and wherein the polypeptide comprises a phenylalanine at a position corresponding to amino acid residue 371 (phenylalanine) of SEQ ID NO: 7.

[0109] Embodiment 2. The recombinant antigen polypeptide of embodiment 1, wherein the amino acid residue corresponds to amino acid residue 154 (Asparagine) of SEQ ID NO: 7.

[0110] Embodiment 3. The recombinant antigen polypeptide of embodiment 1 or 2, wherein the recombinant antigen polypeptide comprises from N terminus to C terminus continuously: (i) a first antigen fragment having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 5 over the entire length of SEQ ID NO: 5, (ii) an RRC or an RRL, and (iii) a second antigen fragment having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 6 over the entire length of SEQ ID NO: 6.Kilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068

[0111] Embodiment 4. The recombinant antigen polypeptide of any one of embodiments 1- 3, wherein the polypeptide comprises a sequence selected from the group consisting of SEQ ID NO: 7-14.

[0112] Embodiment 5. An influenza antigen-adjuvant complex comprising the recombinant antigen polypeptide of any of the embodiments 1-4 and an adjuvant.

[0113] Embodiment 6. The influenza antigen-adjuvant complex of embodiment 5, wherein the influenza antigen-adjuvant complex is formed by an electrostatic interaction between the RRC or the RRL and an adjuvant.

[0114] Embodiment 7. The influenza antigen-adjuvant complex of embodiment 6, wherein the antigen polypeptide comprises the RRC and wherein the adjuvant is alum (aluminum hydroxide).

[0115] Embodiment 8. The influenza antigen-adjuvant complex of any one of embodiments 1-7, wherein the antigen polypeptide comprises an RRL, wherein the influenza antigen-adjuvant complex comprises an aluminum-based adjuvant selected from aluminum phosphate and amorphous aluminum hydroxyphosphate sulfate (AAHS).

[0116] Embodiment 9. The influenza antigen-adjuvant complex of any one of embodiments 1-8, wherein the RRC comprises a) [D]Nwherein N is 8-18; b) [E]Nwherein N is 8-18; c) a D-E copolymer [(D)X, (Glu)Y] where X is 1-17, Y is 1-17, and X + Y = 8-18; or d) a region having a high density of D and / or E.

[0117] Embodiment 10. The influenza antigen-adjuvant complex of any of embodiments 5- 9, wherein the RRC comprises 8 to 12 amino acids, wherein each amino acid residue in the RRC is aspartic acid or glutamic acid.

[0118] Embodiment 11. The influenza antigen-adjuvant complex of embodiment 10, wherein the RRC is D8, D9, D10, D11, or D12.Kilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068

[0119] Embodiment 12. The influenza antigen-adjuvant complex of embodiment 8 wherein RRL comprises a) [K]Nwherein N is 8-18; b) [R]N wherein N is 8-18; c) a K-R copolymer [(K)X, (R)Y] where X is 1-17, Y is 1-17, and X + Y = 8-18 or [(K)X, (R)Y] where X is 1-17, Y is 1-17, and X + Y = 8-18); or d) a region having a high density of K and / or R.

[0120] Embodiment 13. The influenza antigen-adjuvant complex of embodiment 9 or embodiment 10 wherein N is 11 or X + Y is 11.

[0121] Embodiment 14. The influenza antigen-adjuvant complex of any one of embodiments 5-13, wherein H5 HA is presented as a trimer adsorbed to alum.

[0122] Embodiment 15. The influenza antigen-adjuvant complex of any of embodiments 5- 14, wherein the complex comprises an alum particle and a plurality of copies of the recombinant antigen polypeptide, wherein the antigen polypeptide comprises an RRC, wherein the plurality of copies of the antigen polypeptide is associated with the alum particle by electrostatic interaction between the alum particle and the RRC.

[0123] Embodiment 16. The influenza antigen-adjuvant complex of any one of embodiments 5-15 in which the recombinant antigen polypeptide comprises one or more auxiliary elements.

[0124] Embodiment 17. The influenza antigen-adjuvant complex of embodiment 16, wherein the one or more auxiliary elements are selected from the group consisting of a polyhistidine tag and a trimerization domain.

[0125] Embodiment 18. A polynucleotide encoding a polypeptide comprising the recombinant antigen polypeptide described in any of embodiments 5-16.

[0126] Embodiment 19. A cell comprising the polynucleotide of embodiment 18.

[0127] Embodiment 20. A vaccine composition comprising a plurality of the influenza antigen-adjuvant complexes of any of embodiments 5-17.Kilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068

[0128] Embodiment 21. A method for eliciting an immune response in a mammal comprising administering the vaccine composition of embodiment 20 to the mammal.

[0129] Embodiment 22. A method of preparing a recombinant vaccine composition comprising (a) expressing a nucleic acid sequence encoding a recombinant antigen polypeptide, wherein the recombinant antigen polypeptide comprises a Region of Repetitive Carboxylic Groups (RRC), or a Region of Repetitive Lysyl / Guanidino Groups (RRL) inserted after an amino acid residue in a polypeptide, wherein the amino acid residue corresponds to any one of 150-160 of SEQ ID NO: 7, wherein the polypeptide shares at least 70% sequence identity to SEQ ID NO: 7 over the entire length of SEQ ID NO: 7, wherein the polypeptide comprises a subsequence that shares at least 90% amino acid sequence identity with the HA2 subunit of H5 HA (SEQ ID NO: 23) over the entire length of the HA2 subunit (SEQ ID NO: 23), and wherein the recombinant antigen polypeptide comprises a phenylalanine at a position corresponding to amino acid residue 371 (phenylalanine) of SEQ ID NO: 7; and (b) adsorbing the recombinant antigen polypeptide to alum.

[0130] Embodiment 23. The recombinant antigen polypeptide of embodiment 22, wherein the amino acid residue corresponds to amino acid residue 154 (Asparagine) of SEQ ID NO: 7.

[0131] Embodiment 24. The method of embodiment 22 or 23, wherein the polypeptide comprises a sequence selected from the group consisting of SEQ ID NO: 7-14.

[0132] Embodiment 25. A method of preparing a recombinant vaccine composition comprising (a) introducing a nucleic acid sequence encoding a recombinant antigen polypeptide to a host cell, wherein the recombinant antigen polypeptide comprises from N terminus to C terminus : a first antigen fragment having an amino acid sequence with at least 90% sequence identity to H5 HA (SEQ ID NO: 5), an RRC or an RRL, and a second antigen fragment having an amino acid sequence having at least 90% sequence identity H5 HA (SEQ ID NO: 6), wherein the RRC orKilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 the RRL is an intervening sequence, optionally, the RRC or the RRL is a contiguous intervening sequence, wherein the recombinant antigen polypeptide comprises a phenylalanine at a position that corresponds to amino acid residue 371 (phenylalanine) of SEQ ID NO: 7, thereby producing the recombinant antigen polypeptide; and (b) adsorbing the recombinant antigen polypeptide to alum.

[0133] Embodiment 26. The method of embodiment 22-25, where the recombinant antigen polypeptide comprises one or more auxiliary elements.

[0134] Embodiment 27. The method of embodiment 26, wherein the one or more auxiliary elements are selected from the group consisting of a polyhistidine tag and a trimerization domain.

[0135] Embodiment 28. A recombinant vaccine composition produced by the method of embodiments 22-27. XVII. INFORMAL SEQUENCES

[0136] In the following sequences, the double underlined is the Foldon trimerization domain (SEQ ID NO: 28) and His-Tag; OligoD insertion and Phe45HA2 are highlighted in bold with underlines; and MEKIVLLFAIVSLVKS(SEQ ID NO: 27) is a signal sequence. SEQ ID NO: 1 >H5 HA with Phe45HA2(H5 HAPhe) (without the signal peptide) DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGN PMCDEFINVPEWSYIVEKANPVNDLCFPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEAS LGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQ NPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKI VKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNS PQRETGGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAFDGVTNKVNSIIDK MNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVKilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 GSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH SEQ ID NO: 2 >H5 HA with Phe45HA2(H5 HAPhe) (with the signal peptide) MEKIVLLFAIVSLVKS DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGN PMCDEFINVPEWSYIVEKANPVNDLCFPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEAS LGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQ NPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKI VKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNS PQRETGGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAFDGVTNKVNSIIDK MNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKV RLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREE GSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH SEQ ID NO: 3 >reoH5HA with Phe45HA2(reoH5HAPhe) (without signal peptide) DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGN PMCDEFINVPEWSYIVEKANPVNDLCFPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEAS LGVSSACPYQGKSSFFRNVVWLIKKNDDDDDDDDDDDDSTYPTIKRSYNNTNQEDLLVLWGIHH PNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESN GNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKS NRLVLATGLRNSPQRETGGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAFDKilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 GVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDF HDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREE GSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH SEQ ID NO: 4 >reoH5HA with Phe45HA2(reoH5HAPhe) (with the signal peptide) MEKIVLLFAIVSLVKS DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGN PMCDEFINVPEWSYIVEKANPVNDLCFPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEAS LGVSSACPYQGKSSFFRNVVWLIKKNDDDDDDDDDDDDSTYPTIKRSYNNTNQEDLLVLWGIHH PNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESN GNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKS NRLVLATGLRNSPQRETGGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAFD GVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDF HDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEGS GYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH SEQ ID NO: 5 >part of reoH5HA with or without Phe45HA2 (reoH5HAPhe) before the insertion site (without signal peptide) DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGN PMCDEFINVPEWSYIVEKANPVNDLCFPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEAS LGVSSACPYQGKSSFFRNVVWLIKKNKilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 SEQ ID NO: 6 >part of reoH5HA with or without Phe45HA2 (reoH5HAPhe) after the insertion site (without signal peptide) STYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSK VNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPM GAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRETGGLFGAIAGFIEGGWQGhMVD GWYGYHHSNEQGSGYAADKESTQKAFDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKK MEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNE CMESVRNGTYDYPQYSEEARLKREE (SEQ ID NO: 7-14 are the H5 HAphesequences) SEQ ID NO: 7 H5 sequence referenced for generation of reoH5HA or reoH5HAPhe>A / Viet Nam / 1203 / 2004 (GenBank: AII30339.1) DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKKHNGKLCDLDGVKPLILRDCSVAGWLL GNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSS HEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAE QTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFI APEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNR LVLATGLRNSPQRETRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKA371FDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENER TLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARL KREE SEQ ID NO: 8 >A / Cygnus columbianus / Hubei / 50 / 2020 (A0A8E4ZAK5) Poly D insertion site is after N154 DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLNGVKPLILKDCSVAGWLL GNPMCDEFIRVPEWSYIVERANPANDLCYPGSLNDYEELKHLLSRINHFEKILIIPKSSWPN HETSLGVSAACPYQGAPSFFRNVVWLIKKNDAYPTIKISYNNTNREDLLILWGIHHSNNAEE QTNLYKNPTTYISVGTSTLNQRLVPKIATRSQVNGQRGRMDFFWTILKPDDAIHFESNGNFI KILPATRICK TOWNSEND 784170181Kilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 APEYAYKIVKKGDSTIMKSGVEYGHCNTKCQTPVGAINSSMPFHNIHPLTIGECPKYVKSNK LVLATGLRNSPLREKRRKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQK A374FDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLME NERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEE ARLKREE SEQ ID NO: 9 >A / duck / Bangladesh / 58330 / 2023 (GenBank: PP068332.1), Poly D insertion site is after D1541DHICIGYHANNSTKQVDTIMEKNVTVTHAQDILEKTHNGKLCDLNGVKPLILKDCSVAGWL LGNPMCDEFINVPEWSYIVEKANPANGLCYPGNFNDYEELKHLLSRINHFEKIQIIPKDSWS NHEASLGVSAACPYQGNSSFFRNVVWLIKKDNAYPTIKKSYNNTNQEDLLILWGIHHPNDEA EQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKINGQSGRIDFFWTILKPNDAIHFESNGNF IAPEYAYKIVKKGDSTIMKSEVEYGNCNTRCQTPIGAINSSMPFHNIHPLTIGECPKYVKSN KLVLATGLRNSPQKERRRKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQ KA374FDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLM ENERTLDFHDSNVRNLYDKVRLQLKDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSE EARLKREE SEQ ID NO: 10 >A / CanadaGoose / Michigan / 22-008506-005 / 2022 (GenBank: OQ957564.1), Poly D insertion site is after N154 DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLNGVKPLILKDCSVAGWLL GNPMCDEFIRVPEWSYIVERANPANDLCYPGSLNDYEELKHMLSRINHFEKILIIPKSSWPN HETSLGVSAACPYQGAPSFFRNVVWLIKKNDAYPTIKISYNNTNREDLLILWGIHHSNNAEE QTNLYKNPTTYISVGTSTLNQRLAPKIATRSQVNGQRGRMDFFWTILKPDDAIHFESNGNFI APEYAYKIVKKGDSTIMKSGVEYGHCNTKCQTPVGAINSSMPFHNIHPLTIGECPKYVKSNK LVLATGLRNSPLREKRRKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQK AFDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENE RTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEAR LKREE SEQ ID NO: 11 >A / American wigeon / South Carolina / AH0195145 / 2021 (GenBank: OQ789275.1), Poly D insertion site is after N1541DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLNGVKPLILKDCSVAGWL LGNPMCDEFIRVPEWSYIVERANPANDLCYPGSLNDYEELKHMLSRINHFEKILIIPKSSWP NHETSLGVSAACPYQGAPSFFRNVVWLIKKNDAYPTIKISYNNTNREDLLILWGIHHSNNAE EQTNLYKNPTTYISVGTSTLNQRLAPKIATRSQVNGQRGRMDFFWTILKPDDAIHFESNGNF IAPEYAYKIVKKGDSTIMKSGVEYGHCNTKCQTPVGAINSSMPFHNIHPLTIGECPKYVKSN KLVLATGLRNSPLREKRRKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQ KAFDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENKilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 ERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEA RLKREE SEQ ID NO: 12 >A / Chicken / Scotland / 1959 (P09345), Poly D insertion site is after D1541DQICIGYHANKSTKQVDTIMEKNVTVTHAQDILERTHNGKLCSLNGVKPLILRDCSVAGWL LGNPMCDEFLNVPEWSYIVEKDNPINSLCYPGDFNDYEELKHLLSSTNHFEKIQIIPRSSWS NHDASSGVSSACPYIGRSSFFRNVVWLIKKDNAYPTIKRSYNNTNQEDLLILWGIHHPNDAA EQTKLYQNPTTYVSVGTSTLNQRSIPEIATRPKVNGQSGRMEFFWTILKPNDAINFESNGNF IAPEYAYKIVKKGDSAIMKSGLAYGNCDTKCQTPVGAINSSMPFHNIHPHTIGECPKYVKSD RLVLATGLRNVPQRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAF DGITNKVNSIIDKMNTQFKAVGKEFNNLERRVENLNKKMEDGFLDVWTYNVELLVLMENERT LDFHDSNVKNLYDKVRLQLKDNARELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLN REE SEQ ID NO: 13 >A / Hatay / 2004 (Q5QQ29), Poly D insertion site is after N1541DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCALDGVKPLILRDCSVAGWL LGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWS SHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAA EQIKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNF IAPEYAYKLVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSN RLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKEST QKAFDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLME NERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRSGTYDYPQYSEE ARLKREE SEQ ID NO: 14 >A / Cambodia / 408008 / 2005 (F2Z918), Poly D insertion site is after N154, the I1DQICIGYHANNSTEQVDTIMERNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWL LGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWS SHEASLGVSAACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVMWGIHHPNDAA EQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNF IAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSN RLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKEST QKAFDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLME NERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEE ARLKREE SEQ ID NO: 15-22 are the wild-type sequencesKilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 SEQ ID NO: 15 H5 sequence referenced for generation of reoH5HA or reoH5HAPhe>A / Viet Nam / 1203 / 2004 (GenBank: AII30339.1) DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKKHNGKLCDLDGVKPLILRDCSVAGWLL GNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSS HEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAE QTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFI APEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNR LVLATGLRNSPQRETRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKA371IDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENER TLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARL KREE SEQ ID NO: 16 >A / Cygnus columbianus / Hubei / 50 / 2020 (A0A8E4ZAK5) Poly D insertion site is after N154 DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLNGVKPLILKDCSVAGWLL GNPMCDEFIRVPEWSYIVERANPANDLCYPGSLNDYEELKHLLSRINHFEKILIIPKSSWPN HETSLGVSAACPYQGAPSFFRNVVWLIKKNDAYPTIKISYNNTNREDLLILWGIHHSNNAEE QTNLYKNPTTYISVGTSTLNQRLVPKIATRSQVNGQRGRMDFFWTILKPDDAIHFESNGNFI APEYAYKIVKKGDSTIMKSGVEYGHCNTKCQTPVGAINSSMPFHNIHPLTIGECPKYVKSNK LVLATGLRNSPLREKRRKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQK AIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENE RTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEAR LKREE SEQ ID NO: 17 >A / duck / Bangladesh / 58330 / 2023 (GenBank: PP068332.1), Poly D insertion site is after D1541DHICIGYHANNSTKQVDTIMEKNVTVTHAQDILEKTHNGKLCDLNGVKPLILKDCSVAGWL LGNPMCDEFINVPEWSYIVEKANPANGLCYPGNFNDYEELKHLLSRINHFEKIQIIPKDSWS NHEASLGVSAACPYQGNSSFFRNVVWLIKKDNAYPTIKKSYNNTNQEDLLILWGIHHPNDEA EQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKINGQSGRIDFFWTILKPNDAIHFESNGNF IAPEYAYKIVKKGDSTIMKSEVEYGNCNTRCQTPIGAINSSMPFHNIHPLTIGECPKYVKSN KLVLATGLRNSPQKERRRKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQ KAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMEN ERTLDFHDSNVRNLYDKVRLQLKDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEA RLKREE SEQ ID NO: 18 KILPATRICK TOWNSEND 784170181Kilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 >A / CanadaGoose / Michigan / 22-008506-005 / 2022 (GenBank: OQ957564.1), Poly D insertion site is after N154 DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLNGVKPLILKDCSVAGWLL GNPMCDEFIRVPEWSYIVERANPANDLCYPGSLNDYEELKHMLSRINHFEKILIIPKSSWPN HETSLGVSAACPYQGAPSFFRNVVWLIKKNDAYPTIKISYNNTNREDLLILWGIHHSNNAEE QTNLYKNPTTYISVGTSTLNQRLAPKIATRSQVNGQRGRMDFFWTILKPDDAIHFESNGNFI APEYAYKIVKKGDSTIMKSGVEYGHCNTKCQTPVGAINSSMPFHNIHPLTIGECPKYVKSNK LVLATGLRNSPLREKRRKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQK AIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENE RTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEAR LKREE SEQ ID NO: 19 >A / American wigeon / South Carolina / AH0195145 / 2021 (GenBank: OQ789275.1), Poly D insertion site is after N1541DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLNGVKPLILKDCSVAGWL LGNPMCDEFIRVPEWSYIVERANPANDLCYPGSLNDYEELKHMLSRINHFEKILIIPKSSWP NHETSLGVSAACPYQGAPSFFRNVVWLIKKNDAYPTIKISYNNTNREDLLILWGIHHSNNAE EQTNLYKNPTTYISVGTSTLNQRLAPKIATRSQVNGQRGRMDFFWTILKPDDAIHFESNGNF IAPEYAYKIVKKGDSTIMKSGVEYGHCNTKCQTPVGAINSSMPFHNIHPLTIGECPKYVKSN KLVLATGLRNSPLREKRRKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQ KAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMEN ERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEA RLKREE SEQ ID NO: 20 >A / Chicken / Scotland / 1959 (P09345), Poly D insertion site is after D154 DQICIGYHANKSTKQVDTIMEKNVTVTHAQDILERTHNGKLCSLNGVKPLILRDCSVAGWLL GNPMCDEFLNVPEWSYIVEKDNPINSLCYPGDFNDYEELKHLLSSTNHFEKIQIIPRSSWSN HDASSGVSSACPYIGRSSFFRNVVWLIKKDNAYPTIKRSYNNTNQEDLLILWGIHHPNDAAE QTKLYQNPTTYVSVGTSTLNQRSIPEIATRPKVNGQSGRMEFFWTILKPNDAINFESNGNFI APEYAYKIVKKGDSAIMKSGLAYGNCDTKCQTPVGAINSSMPFHNIHPHTIGECPKYVKSDR LVLATGLRNVPQRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAID GITNKVNSIIDKMNTQFKAVGKEFNNLERRVENLNKKMEDGFLDVWTYNVELLVLMENERTL DFHDSNVKNLYDKVRLQLKDNARELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLNR EE SEQ ID NO: 21 >A / Hatay / 2004 (Q5QQ29), Poly D insertion site is after N1541DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCALDGVKPLILRDCSVAGWL LGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSKilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 EQIKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNF IAPEYAYKLVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSN RLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKEST QKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLME NERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRSGTYDYPQYSEE ARLKREE SEQ ID NO: 22 >A / Cambodia / 408008 / 2005 (F2Z918), Poly D insertion site is after N154, the I1DQICIGYHANNSTEQVDTIMERNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWL LGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWS SHEASLGVSAACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVMWGIHHPNDAA EQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNF IAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSN RLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKEST QKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLME NERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEE ARLKREE SEQ ID NO: 23 (HA2 subunit sequence of SEQ ID NO: 1 or 7) GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAFDGVTNKVNSIIDKMNTQFE AVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRD NAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREE SEQ ID NO: 24 LIKKX, where X can be N or D SEQ ID NO: 25 LIKKN SEQ ID NO: 26 LIKKD SEQ ID NO: 27 MEKIVLLFAIVSLVKS SEQ ID NO: 28 GSGYIPEAPRDGQAYVRKDGEWVLLSTFLGKilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 XVII. EXAMPLES Example 1 Methods

[0137] Antigen cloning. DNA encoding influenza hemagglutinins (HAs of H5 - A / Viet Nam / 1203 / 2004) were cloned into the pADD2 vector with a foldon trimerization domain followed by an Avi-Tag and a hexahistidine tag on the C-terminus. OligoD was also inserted into the flexible loop regions on H5 HA in pADD2 expression plasmids. All plasmid sequences were confirmed by Sanger sequencing (Sequetech). For transfection purposes, plasmids were transformed into StellarTMcells, isolated by Maxiprep kits (NucleoBond Xtra Maxi kit, Macherey Nagel), filtered through a sterile 0.45-μm membrane in a biosafety cabinet and stored at -20 C.

[0138] Protein expression and purification. All antigens were expressed in Expi-293F cells. Expi-293F cells were cultured at 37 C under constant shaking (120 rpm) in a humidified CO2(8%) incubator. Expi-293F cells were transfected at a density of 3-4 × 106cells / mL. For 200 mL transfection of antigen proteins, the transfection mixture was made by adding 120 μg plasmid DNA (from Maxiprep) to 20 mL expression media, followed by the dropwise addition of 260 μL FectoPro transfection reagent (Polyplus) with vigorous mixing. Transfection mixtures were incubated at room temperature for 10 minutes before being transferred to Expi-293F cells. D- glucose (4 g / L, Sigma-Aldrich) and valproic acid (3 mM, Acros Organics) were added to the cells immediately post-transfection to increase recombinant protein production. Cells were boosted again with D-glucose three days post-transfection and harvested on day four by centrifugation at 7000 ×g for five min. The supernatant was filtered through a 0.22-μm membrane for subsequent purification processes.

[0139] Antigen proteins with hexahistidine tags were purified with HisPurTMNi-NTA resin (Thermo Fisher). Briefly, the filtered supernatant from Expi-293F cells was mixed with Ni-NTA resin (1 mL resin per liter supernatant) and incubated at 4 C overnight. The mixture was then passed through a gravity-flow column, washed with 20 mM imidazole in HEPES buffer saline (HBS, 20 mM HEPES, pH 7.4, 150 mM NaCl), and then eluted with 250 mM imidazole in HBS. Elution was concentrated with centrifugal filters (30 or 50 kDa MWCO, Millipore Sigma) and buffer-exchanged to HBS for size-exclusion chromatography using a SuperoseTM6 (IncreaseKilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 10 / 300 GL, Cytiva) on an ÄKTA Protein Purification System (Cytiva). Peak fractions were pooled, concentrated, buffer-exchanged to HBS with 10% glycerol and filtered through a 0.22-μm membrane.

[0140] The concentration of all proteins was determined by absorbance at 280 nm (A280), and the purity was assessed by protein gel electrophoresis. Protein samples were flash-frozen in liquid nitrogen and stored at -20 C.

[0141] Size-exclusion chromatography–multi-angle light scattering (SEC-MALS) analysis. SEC-MALS analysis of wild-type and oligoD-modified proteins was performed on a 1260 Infinity II high-performance liquid chromatography system (Agilent) coupled with a miniDAWN and Optilab detectors (Wyatt Technologies) for light scattering and refractive index analysis. Purified protein samples were loaded onto a SuperdexTM200 column (Increase 3.2 / 300, Cytiva) sequentially for analysis. ASTRA software (Wyatt Technologies) was used for data analysis.

[0142] Differential scanning fluorimetry. Thermal melting profiles of proteins were measured by differential scanning fluorimetry on a Prometheus NT.48 instrument (NanoTemper). Protein samples (0.1 mg / mL) were loaded into glass capillaries (NanoTemper) and then subjected to a temperature gradient from 20 to 95 C at a heating rate of 1 C per min. Alternatively, protein samples (0.1 mg / mL) were pre-mixed with alum (10 mg / mL, Alhydrogel®, InvivoGen) at a ratio of 1:10 (protein: alum, w / w) for 30 minutes at room temperature before loading into glass capillaries. HBS and alum (diluted to 1 mg / mL in HBS) were also loaded into glass capillaries and measured as controls. Intrinsic fluorescence (350 nm and 330 nm) was recorded as a function of temperature. Thermal melting curves were plotted using the first derivative of the ratio (350 nm / 330 nm). Melting temperatures were calculated automatically by the instrument (PR.ThermControl software, version 2.3.1) and represented peaks in the thermal melting curves.

[0143] Streptavidin- and alum-based ELISA. To investigate antigen orientation on alum, we measured mAb binding in streptavidin- or alum-based ELISAs. For streptavidin-based ELISAs, Nunc 96-well MaxiSorp plates were coated with streptavidin (4 μg / mL in DPBS, 60 μL per well) for one hour at room temperature. These plates were washed three times with Milli-Q H2O and then blocked with ChonBlock (120 μL per well) overnight at 4 C. For subsequent steps, all dilutions were made in DPBS with 0.05% Tween-20 and 0.1% BSA, and ELISA plates were rinsed with PBST in between steps. Biotinylated HA HA (2 μg / mL) were added to the platesKilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 and incubated for one hour at room temperature. mAbs were serially diluted (10-fold dilution starting from 20 nM) and then added to the ELISA plates for one-hour incubation at room temperature. Rabbit anti-human IgG, HRP-conjugated (1:4,000) was added for one-hour incubation before rinsing with PBST six times.

[0144] For alum-based ELISA, Nunc 96-well MaxiSorp plates were coated with ZsGreen-Avi- His-12D (4 μg / mL in DPBS, 60 μL per well) for one hour at room temperature. These plates were washed three times with Milli-Q H2O and then blocked with ChonBlock overnight at 4 C. For subsequent steps, all dilutions were made in DPBS with 0.05% Tween-20 and 0.1% BSA unless otherwise noted, and ELISA plates were rinsed with PBST in between steps. Alum (100 μg / mL in HBS) was added to the plates and incubated for one hour at room temperature. After rinsing, HA or reoriented HA (2 μg / mL) was added to the plates and incubated for one hour at room temperature. mAbs were serially diluted (10-fold dilution starting from 20 nM) and then added to the ELISA plates for one-hour incubation at room temperature. Rabbit anti-human IgG, HRP- conjugated (1:4,000) was added for one-hour incubation before rinsing with PBST six times. ELISA plates were developed with the TMB substrate for five minutes and terminated with sulfuric acid. Absorbance at 450 nm was recorded on a microplate reader.

[0145] Bio-layer interferometry (BLI). BLI experiments are performed on an OctetRed 96 system. All samples are diluted with Octet buffer (DPBS with 0.02% Tween-20 and 0.1% BSA), and assays are performed under agitation (1000 rpm). Monoclonal antibodies (200 nM) are loaded onto anti-human Fc sensors and then dipped into antigen solutions (150 nM HA) for binding analysis, followed by dissociation into Octet buffer. Data are processed by Data Analysis software and then plotted.

[0146] Statistical analyses. Statistics are analyzed using GraphPad Prism software. Non- transformed data are presented as arithmetic mean ± s.d. Log-transformed data (ELISA titers and NT50) are presented as geometric mean ± s.d. P values of 0.05 or less are considered significant. EXAMPLE 2 ALUM-BINDING H5 HA

[0147] We chose an H5 HA (A / Vietnam / 1203 / 2004), which shares about 92% sequence identity to avian H5 HAs (for example, A / Cygnus columbianus / Hubei / 50 / 2020). To create a reoriented version of H5 HA (reoH5HA), we aligned the structure of H2 and H5 HAs and identified an oligoDKilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 insertion site (inserting after the amino acid sequence of LIKKN (SEQ ID NO: 25) or N154 using SEQ ID NO: 7 as a reference) within the head region of H5 HA (FIG. 1). Both H5 HA and reoH5HA were successfully expressed and purified from Expi293F cells via transient transfection. They appeared as homogenous and monodisperse populations on size exclusion chromatography coupled with multi-angle light scattering (FIG. 2A) and protein gel electrophoresis (FIG. 2B). ReoH5HA maintained the thermal melting profile and melting temperature similar to H5 HA (FIG.3A), including in the presence of alum (FIG.3B). OligoD insertion into the head of H5 HA did not appreciably affect the binding profiles of stem-directed monoclonal antibodies (mAbs) against the protein (FIG.4).

[0148] To validate the “upside down” configuration of reoH5HA on alum, we measured binding of five mAbs to reoH5HA on streptavidin- or alum-coated ELISA plates (FIG.5A). All antibodies bound reoH5HA with high affinity when its C-terminus was anchored on streptavidin-coated ELISA plates (FIG.5B). By contrast, when reoH5HA was adsorbed on alum-coated ELISA plates, only mAbs targeting non-head epitopes (MEDI8852, FI6v3 and H5M9) showed binding (FIG. 5C), suggesting that reoH5HA adopted an “upside down” configuration where head epitopes were no longer accessible.

[0149] We then immunized mice with H5 HA or reoH5HA adjuvanted with alum (150 μg) and CpG oligodeoxynucleotide (5 μg) (FIG. 6A). After prime-boost immunizations on day 0 and 21, mice from both groups developed robust H5 HA-specific IgG responses (FIG. 6B). Compared with H5 HA, reoH5HA elicited significantly higher antibody titers against HA-stem, as measured by binding of antisera to the H1-stabilized stem (H1-SS) protein (FIG.6C).

[0150] After two doses, mice immunized with H5 HA mostly cross-reacted with group 1 HAs (H1 NC / 99, H1 CA / 09 and H2 JP / 57) while cross-reactivity to group 2 HAs (H3 VC / 75 and H7 / SH / 13) was barely detectable (FIG. 7). By contrast, mice immunized with reoH5HA cross- reacted to all HAs we tested except H3 VC / 75 with much higher titers. Cross-reactive antibody titers also correlated with the degree of sequence identity and similarity to H5 HA. We further examined cross-reactivity to two avian H5 HAs (A / Cygnus columbianus / Hubei / 50 / 2020 [HB / 20] and A / duck / Bangladesh / 58330 / 2023 [BD / 23]) and found that reoH5HA elicited significantly higher cross-reactive titers to both avian H5 HAs than did H5 HA (FIG.8).Kilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068

[0151] In addition, we introduced a point mutation (isoleucine to phenylalanine on HA2) to the stem region of H5 HA and reoH5HA, because the bulky phenylalanine residue (Phe) on their stem region has been shown to expand the breadth of B cell responses upon vaccination [Andrews et al., 2022, PMID: 35115707]. We then immunized mice with H5 HAPhe or reoH5HAPhe adjuvanted with alum (150 μg) and CpG oligodeoxynucleotide (5 μg). After prime-boost immunizations on day 0 and 21, mice from both groups developed robust H5 HA-specific IgG responses (FIG. 8). Compared with H5 HAPhe, reoH5HAPheelicited significantly higher cross-reactive antibody titers against group 1 (H1 NC / 99, H1 CA / 09, H1-SS and H2 JP / 57), group 2 (H3 VC / 75 and H7 NT / 27) and avian H5 HAs (aH5 HB / 20 and aH5 BD / 23). The cross-reactive antibody titers correlated with the degree of sequence identity and similarity. ***

[0152] It is understood that the examples and embodiments described in the present disclosure are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited in the present disclosure are hereby incorporated by reference in their entirety for all purposes.

Claims

Kilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 CLAIMS 1. A recombinant antigen polypeptide comprises a Region of Repetitive Carboxylic Groups (RRC) or a Region of Repetitive Lysyl / Guanidino Groups (RRL) inserted after an amino acid residue in a polypeptide, wherein the amino acid residue corresponds to any one of amino acid residues 150-160 of SEQ ID NO: 7, wherein the polypeptide shares at least 70% sequence identity to SEQ ID NO: 7 over the entire length of SEQ ID NO: 7, wherein the polypeptide comprises a subsequence that shares at least 90% amino acid sequence identity with the HA2 subunit of H5 HA (SEQ ID NO: 23) over the entire length of the HA2 subunit (SEQ ID NO: 23), and wherein the polypeptide comprises a phenylalanine at a position corresponding to amino acid residue 371 (phenylalanine) of SEQ ID NO:

7.

2. The recombinant antigen polypeptide of claim 1, wherein the amino acid residue corresponds to amino acid residue 154 (Asparagine ) of SEQ ID NO:

7.

3. The recombinant antigen polypeptide of claim 1, wherein the recombinant antigen polypeptide comprises from N terminus to C terminus continuously: (i) a first antigen fragment having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 5 over the entire length of SEQ ID NO: 5, (ii) an RRC or an RRL, and (iii) a second antigen fragment having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 6 over the entire length of SEQ ID NO:

6.

4. The recombinant antigen polypeptide of claim 1, wherein the polypeptide comprises a sequence selected from the group consisting of SEQ ID NO: 7-14.

5. An influenza antigen-adjuvant complex comprising the recombinant antigen polypeptide of any of the claims 1-4 and an adjuvant.Kilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 6. The influenza antigen-adjuvant complex of claim 5, wherein the influenza antigen- adjuvant complex is formed by an electrostatic interaction between the RRC or the RRL and an adjuvant.

7. The influenza antigen-adjuvant complex of claim 6, wherein the antigen polypeptide comprises the RRC and wherein the adjuvant is alum (aluminum hydroxide).

8. The influenza antigen-adjuvant complex of claim 6, wherein the antigen polypeptide comprises an RRL, wherein the influenza antigen-adjuvant complex comprises an aluminum- based adjuvant selected from aluminum phosphate and amorphous aluminum hydroxyphosphate sulfate (AAHS).

9. The influenza antigen-adjuvant complex of claim 5, wherein the RRC comprises a) [D]N wherein N is 8-18; b) [E]N wherein N is 8-18; c) a D-E copolymer [(D)X, (Glu)Y] where X is 1-17, Y is 1-17, and X + Y = 8-18; or d) a region having a high density of D and / or E.

10. The influenza antigen-adjuvant complex of any of claims 5-9, wherein the RRC comprises 8 to 12 amino acids, wherein each amino acid residue in the RRC is aspartic acid or glutamic acid.

11. The influenza antigen-adjuvant complex of claim 10, wherein the RRC is D8, D9, D10, D11, or D12.

12. The influenza antigen-adjuvant complex of claim 8 wherein RRL comprises a) [K]Nwherein N is 8-18; b) [R]Nwherein N is 8-18; c) a K-R copolymer [(K)X, (R)Y] where X is 1-17, Y is 1-17, and X + Y = 8-18 or [(K)X, (R)Y] where X is 1-17, Y is 1-17, and X + Y = 8-18); or d) a region having a high density of K and / or R.Kilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 13. The influenza antigen-adjuvant complex of claim 9 or claim 10 wherein N is 11 or X + Y is 11.

14. The influenza antigen-adjuvant complex of any one of claims 5-13, wherein H5 HA is presented as a trimer adsorbed to alum.

15. The influenza antigen-adjuvant complex of any of claims 5-14, wherein the complex comprises an alum particle and a plurality of copies of the recombinant antigen polypeptide, wherein the antigen polypeptide comprises an RRC, wherein the plurality of copies of the antigen polypeptide is associated with the alum particle by electrostatic interaction between the alum particle and the RRC.

16. The influenza antigen-adjuvant complex of any one of claims 5-15 in which the recombinant antigen polypeptide comprises one or more auxiliary elements.

17. The influenza antigen-adjuvant complex of claim 16, wherein the one or more auxiliary elements are selected from the group consisting of a polyhistidine tag and a trimerization domain.

18. A polynucleotide encoding a polypeptide comprising the recombinant antigen polypeptide described in any of claims 5-16.

19. A cell comprising the polynucleotide of claim 18.

20. A vaccine composition comprising a plurality of the influenza antigen-adjuvant complexes of any of claims 5-17.

21. A method for eliciting an immune response in a mammal comprising administering the vaccine composition of claim 20 to the mammal.

22. A method of preparing a recombinant vaccine composition comprisingKilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 (a) expressing a nucleic acid sequence encoding a recombinant antigen polypeptide, wherein the recombinant antigen polypeptide comprises a Region of Repetitive Carboxylic Groups (RRC), or a Region of Repetitive Lysyl / Guanidino Groups (RRL) inserted after an amino acid residue in a polypeptide, wherein the amino acid residue corresponds to any one of 150-160 of SEQ ID NO: 7, wherein the polypeptide shares at least 70% sequence identity to SEQ ID NO: 7 over the entire length of SEQ ID NO: 7, wherein the polypeptide comprises a subsequence that shares at least 90% amino acid sequence identity with the HA2 subunit of H5 HA (SEQ ID NO: 23) over the entire length of the HA2 subunit (SEQ ID NO: 23), and wherein the recombinant antigen polypeptide comprises a phenylalanine at a position corresponding to amino acid residue 371 (phenylalanine) of SEQ ID NO: 7; and (b) adsorbing the recombinant antigen polypeptide to alum.

23. The recombinant antigen polypeptide of claim 22, wherein the amino acid residue corresponds to amino acid residue 154 (Asparagine ) of SEQ ID NO:

7.

24. The method of claim 22, wherein the polypeptide comprises a sequence selected from the group consisting of SEQ ID NO: 7-14.

25. A method of preparing a recombinant vaccine composition comprising (a) introducing a nucleic acid sequence encoding a recombinant antigen polypeptide to a host cell, wherein the recombinant antigen polypeptide comprises from N terminus to C terminus : a first antigen fragment having an amino acid sequence with at least 90% sequence identity to H5 HA (SEQ ID NO: 5), an RRC or an RRL, and a second antigen fragment having an amino acid sequence having at least 90% sequence identity to H5 HA (SEQ ID NO: 6), wherein the RRC or the RRL is an intervening sequence, optionally, the RRC or the RRL is a contiguous intervening sequence, wherein the recombinant antigen polypeptide comprises a phenylalanine at a position that corresponds to amino acid residue 371 of SEQ ID NO: 7,Kilpatrick Docket 110221-1488799-011410WO Biohub Docket: CZB-306S-PC / S24-068 thereby producing the recombinant antigen polypeptide; and (b) adsorbing the recombinant antigen polypeptide to alum.

26. The method of claim 22-25, where the recombinant antigen polypeptide comprises one or more auxiliary elements.

27. The method of claim 26, wherein the one or more auxiliary elements are selected from the group consisting of a polyhistidine tag and a trimerization domain.

28. A recombinant vaccine composition produced by the method of claims 22-27.

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