Stable hemagglutinin (HA) trimers as influenza vaccine antigens

Abstract

The present invention provides novel engineered influenza hemagglutinin (HA) proteins, related polynucleotide sequences, and vaccine compositions comprising nanoparticle compositions. The engineered HA proteins are stabilized relative to wild type HA proteins by replacing one or more conserved residues in the extracellular domain of HA2 with hydrophobic residues. The invention also provides methods of using such vaccine compositions in a variety of therapeutic applications, such as for the prevention or treatment of influenza virus infection.

Description

[0001] Cross-references to related applications

[0002] This patent application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 503,725 (filed May 23, 2023; currently pending). The entire disclosure of that priority application is incorporated herein by reference in its entirety and for all purposes. Background Technology

[0003] Influenza is an acute respiratory illness in humans with a short incubation period followed by a spectrum of clinical symptoms ranging from asymptomatic to severe. Influenza viruses cause epidemics and pandemics, causing 250,000 to 500,000 deaths worldwide each year and contributing to the global health burden. Influenza viruses belong to the Orthomyxoviridae family and can be classified into four types: types A, B, C, and D. Influenza A and B viruses (IAV and IBV) comprise eight negative-sense single-stranded viral RNA gene segments that encode essential viral proteins such as hemagglutinin (HA), neuraminidase (NA), and matrix 2 (M2) proteins. In contrast, influenza C and D viruses (ICV and IDV) have seven viral RNA gene segments because the hemagglutinin-esterase fusion (HEF) protein viral RNA replaces the HA and NA viral RNAs. IAV is the primary cause of influenza pandemics and is the main contributor to annual human influenza epidemics. IAV utilizes wild aquatic birds as the natural reservoir and can infect other species besides humans, such as pigs. IAV is more diverse than IBV due to the ability to adapt and infect multiple species. Both IAV and IBV have a host-derived lipid membrane that is decorated on the surface with HA, NA, and M2. IAV can be divided into subtypes based on the genetic and antigenic properties of the surface antigens HA and NA (which mediate viral entry and release, respectively). To date, 18 HA (H1 to 18) and 9 NA subtypes (N1 to 11) have been identified, which make up the circulating IAV strains. IAV can be divided into two phylogenetically distinct groups. Group 1 comprises H1, H2, H5, H6, H8, H9, H11, H13, H16, H17, and H18, and group 2 consists of H3, H4, H7, H10, H14, and H15. HA within a subtype has up to 15% sequence diversity, while HA subtypes within a group and between the two groups have 40% and 60% sequence diversity, respectively. H1N1 (group 1) and H3N2 (group 2) and two antigenically distinct IBV lineages (Victoria and Yamagata) are used in most seasonal influenza vaccines, with the strain backbone determined by the World Health Organization (WHO) on an annual basis.

[0004] A major challenge to influenza vaccine development is that the surface antigens (HA and NA) evolve in response to pressure from the host immune system, a process known as antigenic drift and antigenic shift. Antigenic drift is evident in circulating seasonal IAV, where mutations in the surface antigens allow for reinfection of the same host, while antigenic shift is caused by less frequent reassortment events and is the cause of pandemics. The HA protein is the main target of the humoral response. Hundreds of neutralizing antibodies (NAbs) have been isolated from infected or vaccinated individuals. Some broadly neutralizing antibodies (bNAbs) can block IAV of multiple HA subtypes or react with both IAV and IBV. Despite the low sequence identity of the HA proteins of IAV and IBV, they adopt the same protein fold. Structural analysis of HA in complex with NAbs and bNAbs has identified a number of neutralizing epitopes at the head, at the stem, and at the interface thereof. In general, the stem contains more conserved epitopes than the head. But the head does contain some epitopes that are conserved within one HA subtype or across different strains of multiple HA subtypes. Head epitopes are located in or outside the receptor-binding site (RBS), which is the main target of the antibody response induced by vaccines. Currently, all seasonal influenza vaccines and recombinant HA-based vaccines in preclinical development use wild-type HA.

[0005] Despite substantial progress in influenza vaccine design, there remains a need in the medical field for more effective and potent vaccine immunogens, e.g., for preventing influenza virus infection. The present invention addresses such unmet needs in the art. SUMMARY

[0006] In one aspect, the present invention provides engineered influenza virus hemagglutinin (HA) proteins. These proteins comprise a modified HA2 ectodomain having a substitution of the amino acid residue at position 95 or 106 with a hydrophobic residue. The numbering of the amino acids in HA2 is based on influenza A H3 subtype. In addition to the modified HA2 ectodomain, some engineered influenza virus HA proteins comprise an intact HA1 protein, while others do not comprise the HA head domain. Some engineered influenza HA proteins are based on influenza A virus, and the residue that is substituted in HA2 is N95. Some engineered influenza HA proteins are based on influenza B virus, and the residue that is substituted in HA2 is Q95. Some engineered influenza HA proteins are based on influenza C virus, and the residue that is substituted in HA2 is E95.

[0007] In various embodiments, the conserved residue at position 95 or 106 in HA2 can be replaced with Ala, Val, Leu, He, Met, Phe, Tyr, or Trp. Some engineered influenza HA proteins of the application comprise a modified HA2 ectodomain having a substitution of both amino acid residues at positions 95 and 106 with a hydrophobic residue. In some of these embodiments, the substitution at each position is independently replaced with Ala, Val, Leu, He, Met, Phe, Tyr, or Trp. Some of these engineered influenza HA proteins are based on a Group 1 influenza A virus, and the residue replaced at position 106 is R106. Some of these engineered influenza HA proteins are based on a Group 2 influenza A virus, and the residue replaced at position 106 is H106. Some of these engineered influenza HA proteins are based on an influenza B virus, and the residue replaced at position 106 is G106. In these embodiments, the substitution at each position is independently replaced with Ala, Val, Leu, He, Met, Phe, Tyr, or Trp.

[0008] Some engineered influenza HA proteins of the application comprise a modified HA2 ectodomain having (1) a substitution at position 95 with a hydrophobic residue, and (2) an additional amino acid substitution at one or both of positions 51 and 103 with a hydrophobic residue. In some of these embodiments, the HA protein is based on an influenza A virus, and the additional amino acid residues that are replaced are K51 and E103.

[0009] In some embodiments, the engineered influenza virus HA proteins of the application can additionally comprise a subunit sequence of a self-assembling nanoparticle fused to the C-terminus of the engineered HA protein.

[0010] In some related aspects, the application provides (1) pharmaceutical compositions comprising an engineered influenza HA protein described herein and a pharmaceutically acceptable carrier, (2) polynucleotides encoding an engineered influenza HA protein described herein, (3) vectors comprising one of the polynucleotides encoding an engineered influenza HA protein described herein; and (4) host cells comprising one polynucleotide encoding an engineered influenza HA protein described herein or a vector containing the polynucleotide. In some pharmaceutical compositions of the application, the engineered HA protein is displayed on the surface of a self-assembling nanoparticle.

[0011] In another aspect, the present application provides an influenza virus vaccine composition comprising the engineered HA protein described herein displayed on the surface of a self-assembling nanoparticle. In some embodiments, the self-assembling nanoparticle is composed of a trimeric sequence, and the C-terminus of the engineered HA protein is fused to the N-terminus of the subunit sequence of the nanoparticle. In some embodiments, the self-assembling nanoparticle scaffold sequence is I3-01, E2p, or ferritin. In some embodiments, the scaffold vaccine additionally comprises a T-cell epitope and / or a lock domain fused to the C-terminus of the nanoparticle subunit sequence.

[0012] In yet another aspect, the present application provides a method of treatment or use for treating or preventing an influenza virus infection in a subject. The method or use entails administering to the subject a pharmaceutical composition or nanoparticle vaccine composition comprising an engineered influenza HA protein described herein.

[0013] A further understanding of the nature and advantages of the application can be realized by reference to the remaining portions of the specification and the claims. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figures 1A-1C Results from hemagglutinin (HA) metastability analysis are shown.

[0015] Figures 2A-2B Results from negative stain EM analysis of H5N1 and H7N9 HA proteins are shown.

[0016] Figures 3A-3D Determination of hemagglutinin metastability causes is shown. Figure 3A An alignment of HA2 ectodomain sequences from 10 HAs from four IAV subtypes (H1, H3, H5, and H7) and two IBV lineages (Victoria and Yamagata) is shown in Table 1.

[0017] Figures 4A-4B Stabilizing effect of a single amino acid mutation at N95 on H1N1 hemagglutinin (HA) trimeric protein is shown. DETAILED DESCRIPTION

[0018] I SUMMARY

[0019] The present invention is based, in part, on research by the inventors into the cause of metastability of influenza virus hemagglutinin (HA). As a class I viral fusion protein, HA has inherent metastability that leads to substandard immune responses to vaccination. As detailed herein, the inventors first discovered that HA trimers can disassemble or show high levels of flexibility in the head or stem. The inventors then identified key residues in HA2 as potential causes of HA metastability. Based on the H3 numbering system, residue 95 is highly conserved across different influenza types and subtypes, which is Asn (N) in IAV HA (e.g., A / California / 04 / 2009 H1N1, GenBank Accession No. FJ966082 and ACP41105), Gln (Q) in IBV HA (e.g., B / Brisbane / 60 / 2008, GenBank Accession No. FJ766840 and ACN29380), and Glu (E) in ICV HA (e.g., C / Johannesburg / 1 / 1966, GenBank Accession No. AM410041 and CAL69520). The inventors also showed that a single mutation at position 95 of HA2 (e.g., N95L for IAV) can stabilize the HA trimer of A / California / 07 / 2009 H1N1 (a pandemic strain with unstable HA) in a closed pre-fusion trimer conformation, as shown by negative stain EM analysis. This mutation enables the H1N1-CA-2009 HA trimer to be purified by head NAbs and by stem bNAbs (the latter preferentially bind the trimer conformation). For IAV, this mutation can be combined with a hydrophobic mutation at R106 (IAV, A / California / 04 / 2009 H1N1, GenBank: FJ966082) or K51 and E103 (putative pH sensor, according to Milder et al., Proc. Natl. Acad. Sci. USA 2022, 119: e2115379119 numbering) to further stabilize the HA trimer. Regardless of the delivery system, HA containing the N95 / Q95 mutation provides an excellent antigen for development of influenza A / B vaccine. Moreover, these mutations can be used to stabilize headless or stem-only HA antigens for vaccine development.

[0020] Based on these studies, the present application provides engineered and stabilized influenza HA trimer sequences in the form of proteins or nucleic acids (DNA / mRNA) or carried by viral vectors, which can be used as influenza vaccines. In some related embodiments, the present application provides VLP-type influenza vaccines comprising nanoparticles presenting stabilized HA trimers and stem antigens. The influenza vaccines based on the present application have multiple technical advantages. The hydrophobic mutation at position 95 can stabilize HA trimers from different influenza types / subtypes / strains, providing a universal solution for HA stabilization. As shown for A / California / 04 / 09 HA, which readily dissociates into monomers in soluble form, a single N95L mutation is sufficient to stabilize the HA trimer, regardless of the purification method used. Considering the low pH conditions (pH = 5.5) used in the negative stain EM analysis, the observed structural stability indicates that the N95L-containing HA protein is also resistant to low pH. In some related embodiments, the vaccines can have engineered HA proteins that combine the hydrophobic mutation at position 95 with other mutations in the HA2 ectodomain, such as (1) a hydrophobic mutation at position 106 or (2) hydrophobic mutations at K51 and E103 (Milder et al., Proc. Natl. Acad. Sci. USA 2022, 119: e2115379119). This can result in further improved stability and reduced pH sensitivity (senility) of different HA trimers.

[0021] The vaccines based on engineered HA sequences can be delivered as influenza vaccines through different platforms (DNA, mRNA, vectors, proteins, and nanoparticles). The stabilized HA trimers and stem antigens (both in soluble and particulate forms) can be readily produced in ExpiCHO cells at reasonable yields. Since CHO is one of the major mammalian cell lines used for industrial production of protein therapeutics and vaccines, and ExpiCHO is a transient expression version of this CHO cell line, it is expected that the influenza vaccines obtained from ExpiCHO cells have the same properties as those from industrial CHO cell lines. This will enable GMP production of stabilized HA trimers, stem antigens, and their nanoparticles for human use.

[0022] Unless otherwise stated herein, the engineered influenza virus HA proteins or immunogens, polynucleotides encoding, expression vectors and host cells, and related therapeutic applications of the present application can be produced or performed according to the procedures exemplified herein or conventional practices well known in the art. See, e.g., Methods in Enzymology, Vol. 289: Solid Phase Peptide Synthesis, J. N. Abelson, M. I. Simon, G. B. Fields (Eds.), Academic Press; 1st edition (1997) (ISBN-13: 978-0121821906); U.S. Patent Nos. 4,965,343 and 5,849,954; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., (3rd edition, 2000); Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (supplemented through 2003); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986); or Methods in Enzymology: Guide to Molecular Cloning Techniques Vol. 152, S.L. Berger and A. R. Kimmerl, Eds., Academic Press Inc., San Diego, USA (1987); Current Protocols in Protein Science (CPPS) (John E. Coligan et al., Eds., John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et al., Eds., John Wiley and Sons, Inc.), and Culture of Animal Cells: A Manual of Basic Technique by R.Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barnes, eds., Academic Press, 1st edition, 1998. The following sections provide additional guidance in practicing the compositions and methods of the present application.

[0023] II. DEFINITIONS

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The following references provide one of ordinary skill with a general definition of many of the terms used in this application: Academic Press Dictionary of Science and Technology, Morris (ed.), Academic Press (1st edition, 1992); Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (eds.), Oxford University Press (revised edition, 2000); Encyclopaedic Dictionary of Chemistry, Kumar (ed.), Anmol Publications Pvt. Ltd. (2002); Dictionary of Microbiology and Molecular Biology, Singleton et al. (eds.), John Wiley & Sons (3rd edition, 2002); Dictionary of Chemistry, Hunt (ed.), Routledge (1st edition, 1999); Dictionary of Pharmaceutical Medicine, Nahler (ed.), Springer-Verlag Telos (1994); Dictionary of Organic Chemistry, Kumar and Anand and (eds.), Anmol Publications Pvt. Ltd. (2002); and A Dictionary of Biology (Oxford Paperback Reference), Martin and Hine (eds.), Oxford University Press (4th edition, 2000). Further definitions for some of these terms are provided herein when they are specifically applied to the present application.

[0025] As used herein, the singular form "a", "an", and "the" include plural references unless the context clearly dictates otherwise. For example, "a trimer of Env origin" can refer to either a single or multiple trimer molecules of Env origin, and can be considered equivalent to the phrase "at least one trimer of Env origin".

[0026] The terms "antigen" or "immunogen" as used herein are used interchangeably to refer to a substance, typically a protein, capable of inducing an immune response in a subject. The term also refers to a protein that is immunologically active in the sense that it is capable of eliciting an immune response of the humoral and / or cellular type against the protein once administered to a subject (either directly or by administering to the subject a nucleotide sequence or vector encoding the protein). Unless otherwise indicated, the term "vaccine immunogen" is used interchangeably with "protein antigen" or "immunogen polypeptide."

[0027] The term "conservatively modified variant" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refers to those nucleic acids that encode identical or essentially identical amino acid sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For a polypeptide sequence, "conservatively modified variants" refers to those variants in which at least one amino acid residue is replaced by a different residue having similar properties. Families of amino acid residues having similar properties have been defined in the art. These families include amino acids that are basic, acidic, uncharged polar, nonpolar, and beta-branched. Examples of families are: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0028] Epitope refers to an antigenic determinant. These are specific chemical groups or peptide sequences on a molecule that are antigenic such that they elicit a specific immune response, e.g., an epitope is a region of an antigen to which B and / or T cells respond. Epitopes can be formed both by contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of the protein.

[0029] An effective amount of a vaccine or other agent is sufficient to produce a desired response, e.g., to reduce or eliminate a sign or symptom of a disorder or disease (e.g., seasonal influenza). For example, this can be the amount required to inhibit viral replication or measurably alter an outward symptom of viral infection (e.g., an increase in T cell count in the case of influenza infection). Typically, the amount is sufficient to measurably inhibit replication or infectivity of the virus. When administered to a subject, a dose is typically used that reaches a target tissue concentration (e.g., in lymphocytes) that has been shown to achieve in vitro inhibition of viral replication. In some embodiments, an “effective amount” is an amount that treats (including preventing) one or more symptoms and / or underlying cause of any disorder or disease (e.g., for treating an influenza infection). In some embodiments, an effective amount is a therapeutically effective amount. In some embodiments, an effective amount is an amount that prevents the occurrence of one or more signs or symptoms of a particular disease or disorder (e.g., one or more signs or symptoms associated with the disease).

[0030] A fusion protein, as used herein, is a recombinant protein comprising amino acid sequences from at least two unrelated proteins that have been joined together by peptide bonds to form a single protein. The unrelated amino acid sequences can be joined directly to one another, or they can be joined using a linker sequence. Proteins, as used herein, are unrelated if their amino acid sequences are not typically found joined together by peptide bonds in their natural environment (e.g., within a cell). For example, the amino acid sequence of a bacterial enzyme (e.g., B. stearothermophilus dihydrolipoyl acyltransferase (E2p)) and the amino acid sequence of an influenza HA protein are not typically found joined together by peptide bonds.

[0031] An immunogen is a protein or portion thereof that is capable of inducing an immune response in a mammal (e.g., a mammal infected or at risk of being infected by a pathogen). Administration of an immunogen can result in protective immunity and / or active immunity against a pathogen of interest.

[0032] An immune response refers to the response of cells of the immune system (e.g., B cells, T cells, or monocytes) to a stimulus. In some embodiments, the response is specific to a particular antigen (“antigen-specific response”). In some embodiments, the immune response is a T cell response, e.g., a CD4+ response or a CD8+ response. In some further embodiments, the response is a B cell response and results in the production of specific antibodies.

[0033] An immunogenic composition refers to a composition comprising an immunogenic polypeptide that induces a measurable CTL response against a virus expressing the immunogenic polypeptide, or that induces a measurable B cell response (e.g., production of antibodies) against the immunogenic polypeptide.

[0034] The amino acid numbering or amino acid numbering system used herein refers to the numbering or linear position of an amino acid residue in an HA protein (or HA1 or HA2 protein) from a prototype influenza strain. Through standard sequence alignment, it allows for comparison of sequences of different HA proteins (or HA1 or HA2 proteins) from other influenza strains or engineered HA proteins described herein to the sequence of the prototype sequence. With such standard or normalized amino acid numbering, it is easy to identify and name conserved amino acid residues in HA proteins from a variety of viral strains or engineered HA proteins. For example, unless otherwise specified herein, the amino acid renumbering of the HA2 protein can be based on the HA2 sequence of an IAV H3 subtype virus (e.g., the A / Victoria / 361 / 2011 H3N2 strain exemplified herein). In some embodiments, the numbering of the HA protein can be based on the sequence of any well-known viral strain, the sequence of the A / California / 04 / 2009 H1N1 strain that caused the pandemic, or other influenza strains described herein.

[0035] Sequence identity or similarity between two or more nucleic acid sequences or between two or more amino acid sequences is represented as the identity or similarity between sequences. Sequence identity can be measured in percentage identity; the higher the percentage, the more identical the sequences. When two sequences are compared and aligned for maximum correspondence over a comparison window, or designated region, as measured using one of the following sequence comparison algorithms, or by manual alignment and visual inspection, and if two sequences have a specified percentage of identical amino acid residues or nucleotides (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a designated region, or when not designated, over the entire sequence), then the two sequences are“substantially identical.” Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0036] Homologs or orthologs of nucleic acid or amino acid sequences have a relatively high degree of sequence identity / similarity when aligned using standard methods. Sequence alignment methods for comparison are well known in the art. Various programs and alignment algorithms are described in Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al., Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, which shows detailed considerations of sequence alignment methods and homology calculations.

[0037] The term "subject" refers to any animal classified as a mammal, for example, humans and non-human mammals. Examples of non-human animals include dogs, cats, cows, horses, sheep, pigs, goats, rabbits, and the like. Unless otherwise stated, the term "patient" or "subject" are used interchangeably herein. Preferably, the subject is a human.

[0038] The terms "treat" or "alleviate" include administering a compound or agent to a subject to arrest or slow the development of symptoms, complications, or biochemical indicia of a disease (e.g., an influenza infection), to alleviate symptoms either preclinically or postclinically, or to arrest or slow the development of a disease, condition, or disorder. Those in need of treatment include those already with the disease or condition as well as those in which the disease or condition is to be prevented. Treatment can be prophylactic (to prevent or slow the onset of a disease, or to prevent the manifestation of clinical or subclinical symptoms of a disease) or therapeutic (to arrest or slow the progression of symptoms after their appearance).

[0039] A vaccine refers to a pharmaceutical composition that elicits a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, a vaccine elicits an antigen-specific immune response against an antigen of a pathogen, such as a viral pathogen, or against a cellular component associated with a pathological condition. A vaccine can include a polynucleotide (e.g., a nucleic acid encoding a disclosed antigen), a peptide or polypeptide (e.g., a disclosed antigen), a virus, a cell, or one or more cellular components. In some embodiments of the application, a vaccine or vaccine immunogen or vaccine composition is expressed from a fusion construct and self-assembles into a nanoparticle that displays an immunogen polypeptide or protein on the surface.

[0040] An influenza vaccine refers to an immunogenic composition capable of stimulating an immune response that is administered to prevent, ameliorate, or treat an influenza virus infection. An influenza vaccine can include, for example, attenuated or killed (e.g., split) influenza viruses, virus-like particles (VLPs), and / or antigenic polypeptides (e.g., engineered hemagglutinins described herein) or DNA derived therefrom, or any recombinant form of such immunogenic materials.

[0041] A virus-like particle (VLP) refers to a non-replicating viral shell derived from any of several viruses. VLPs are typically composed of one or more viral proteins, such as, but not limited to, those proteins known as capsid proteins, coat proteins, shell proteins, surface proteins, and / or envelope proteins or particle-forming polypeptides derived from these proteins. Upon recombinant expression of the proteins in an appropriate expression system, VLPs can self-assemble. Methods for producing particular VLPs are known in the art. The presence of VLPs following recombinant expression of viral proteins can be detected using routine techniques known in the art, such as by electron microscopy, biophysical characterization, and the like. See, e.g., Baker et al. (1991) Biophys. J. 60: 1445-1456; and Hagensee et al. (1994) J. Virol. 68:4503-4505. For example, VLPs can be isolated by density gradient centrifugation and / or identified by a characteristic density band. Alternatively, freeze-etch electron microscopy can be performed on a vitreous-hydrated sample of the VLP preparation, and images recorded under appropriate exposure conditions.

[0042] Self-assembling nanoparticles refer to spherical protein shells with diameters of tens of nanometers and well-defined surface geometry, formed from identical copies of non-viral proteins that are able to self-assemble into nanoparticles with similar appearance to VLPs. Some examples known include ferritin (FR), which is conserved across species and forms 24-mer, and B. Stearothermophilus dihydroxy-acid dehydratase (E2p), Aquifex aeolicus lumazine synthase (LS), and Thermotoga maritima encapsulin, which all form 60-mer. Self-assembling nanoparticles can form spontaneously upon recombinant expression of the proteins in appropriate expression systems. Methods for production, detection, and characterization of nanoparticles can be performed using the same techniques developed for VLPs.

[0043] III. Engineered influenza hemagglutinin (HA) proteins

[0044] The present invention provides engineered or redesigned influenza virus hemagglutinin (HA) proteins or polypeptides (and polynucleotide sequences) for use in generating influenza virus vaccines. As a class I viral fusion protein, HA plays an important role in viral entry: by binding to host receptors (sialylated glycans on respiratory endothelial cells), and promoting membrane fusion in the low pH environment of the endosomal compartment after entry into the cell by endocytosis. During viral replication, HA is first expressed as a precursor protein, a non-covalent homotrimer called HA0. Uncleaved HA0 is cleaved by a cellular protease into HA1 and HA2, which form the functional HA spike on the virion surface. Each monomer of the HA protein is composed of a globular head domain and a membrane proximal stem or handle domain. The head domain is composed of only a portion of HA1 (including the receptor binding domain and a vestigial esterase domain), while the stem domain contains portions of both HA1 and HA2.

[0045] The engineered HA proteins or vaccine antigens of the application bear one or more specific modifications in HA2 relative to wild-type HA proteins, as detailed herein. They can be derived from the HA protein of any influenza virus. The HA sequence in which HA2 is modified can be a complete HA protein, a sequence substantially identical to a wild-type HA protein, or a variant or fragment of a complete HA protein (e.g., a headless HA). Numerous HA sequences from influenza isolates are known in the art. For example, the National Center for Biotechnology Information (NCBI) maintains a database containing thousands to tens of thousands of complete HA polypeptide sequences for each influenza type or subtype. Using this database, the skilled artisan can readily identify sequences suitable for practicing the application. Thus, suitable HA sequences can include, for example, wild-type or variant HA polypeptide or polynucleotide sequences of IAV (H1-H18), IBV, ICV, and IDV. In various embodiments, they can be derived from the HA of influenza viruses that mediate infection of different hosts (e.g., humans as well as non-human species).

[0046] Generally, the engineered HA proteins of the application comprise stabilizing mutations in HA2. In some embodiments, the engineered HA proteins comprise a complete HA1 protein and a modified HA2 ectodomain. In other embodiments, the engineered HA proteins can be headless or stem-only HA proteins that comprise a modified HA2 ectodomain but do not comprise a complete HA1 protein. Typically, the stabilizing mutations in the modified HA2 proteins are one or more single amino acid substitutions. In some preferred embodiments, the mutations comprise replacement of (1) the amino acid residue at position 95 (or equivalent position in different HA sequences) of HA2 and / or (2) the amino acid residue at position 106 (or equivalent position in different HA sequences) of HA2 with a hydrophobic residue. Unless otherwise specified, the amino acid numbering for a given HA2 protein is based on influenza A virus H3 subtype. One example of an H3 HA protein structure is PDB 4O5N, which is the hemagglutinin crystal structure of influenza virus strain A / Victoria / 361 / 2011 (H3N2) (Accession Code AGB08328; SEQ ID NO: 2). See, e.g., Lee et al., Nat. Commun. 5: 3614, 2014. The HA2 portion of this HA sequence is shown in SEQ ID NO: 6. Because the atomic structure of the H3N2 HA protein was first determined by crystallography, historically H3N2 HA has been used as a template to determine residue numbering for other HA protein structures. Thus, according to this H3-based amino acid numbering system, corresponding amino acid residues that are conserved across multiple domains (e.g., HA2 ectodomain), regions (e.g., head and stem), and structural motifs and boundaries in HA proteins from any other influenza virus can be readily determined (e.g., by sequence alignment) or otherwise known in the art. It should be noted that there is significant sequence conservation between HA2 proteins from different influenza virus types and different strains of the same virus type. Thus, the H3 numbering system is particularly accurate for identifying conserved amino acid positions in HA2 proteins.

[0047] In particular, the sequence and structure of HA2 is conserved across all viruses (especially influenza A, B, and C, and possibly D, although there is little structural information reported for D). As well characterized in the literature, the HA2 protein is highly conserved across influenza strains, and any conserved residues in the protein structure of different influenza viruses (e.g., N95) will be at the same position based on the H3 numbering system. See, e.g., Heiny et al., PLoS ONE 2007, 2(11):e1190; Fouchier et al., J. Virol. 2005;79:2814-2822; Nobusawa et al., Virology 1991;182:475-485; Okuno et al., J. Virol. 1993;67:2552-2558; and Chun et al., Vaccine 2008, 26:6068-6076. Thus, the amino acid numbering in H3 subtype HA2 can be applied to any other viral type and subtype. For example, the residue at position 95 in HA2 is conserved across all IAV, IBV, and ICV. In particular, the residue at position 95 is N95 for IAV, Q95 for IBV, and E95 for ICV. In addition to the HA2 sequence of the IAV H3N2 strain described above (SEQ ID NO:6), the highly conserved nature of position 95 is evident in the amino acid sequences of the HA2 portion of the HA protein from 3 other exemplary viral strains, IAV strain A / California / 04 / 2009 H1N1, IBV strain B / Brisbane / 60 / 2008, and ICV strain C / Johannesburg / 1 / 1966 (SEQ ID NOs:5, 7, and 8, respectively). Similarly to position 95, other HA2 amino acid positions in the engineered HA proteins of the application for substitution are also highly conserved across different influenza viruses. For example, the residue at position 106 is R106 for group 1 IAV, H106 for group 2 IAV, and G106 for IBV.

[0048] As an alternative to the H3-based HA2 numbering described above, the conserved residues in the extracellular domain of HA2 to be modified can also be numbered based on other prototypical viral sequences. For example, the two HA2 positions 95 and 106 described above are positions 439 and 450, respectively, in the full-length HA protein sequence of prototypical IAV strain A / California / 04 / 2009 H1N1 (accession number ACP41105; SEQ ID NO:1). As used herein Figure 3AAs exemplified herein, these highly conserved residues at corresponding positions in HA proteins of other IAVs, as well as in HA proteins of other types of influenza viruses (e.g., IBV and ICV), can be readily determined by sequence alignment. As an example, residue N439 in the HA protein of this prototype H1N1 strain (i.e., N95 based on H3-based HA2 numbering) corresponds to residue N440 in the HA protein of the above-mentioned IAV H3N2 strain A / Victoria / 361 / 2011, residue Q457 in the HA protein of the prototype IBV strain B / Brisbane / 60 / 2008 (Accession No. ACN29380; SEQ ID NO: 3), and residue E464 in the HA protein of the ICV strain C / Johannesburg / 1 / 1966 (Accession No. CAL69520; SEQ ID NO: 4).

[0049] As noted above, some engineered HA proteins of the application comprise only the HA2 ectodomain, rather than the full HA2 protein. Sequences for the HA2 ectodomain from influenza viruses can be readily determined by, e.g., sequence alignment. For example, the wild-type HA2 ectodomain sequences for the exemplary IAV H1N1 strain A / California / 04 / 2009 and IAV H3N2 strain A / Victoria / 361 / 2011 are shown in SEQ ID NOs: 9 and 10, respectively. The boundaries of the HA2 ectodomain in several other prototype influenza virus strains described herein can also be readily determined, including the HA2 ectodomain boundaries for IAV A / Hong Kong / 1 / 1968 H3N2 (GenBank: CY044261), IAV A / Viet Nam / 1203 / 2004 H5N1 (GenBank: AY818135), and IAV A / Guangdong / HP001 / 2017 H7N9 (GenBank: KY643843).

[0050] In some embodiments, the engineered HA proteins of the application comprise an HA2 ectodomain with a substitution at position 95. The residue at this position in the HA2 ectodomain (e.g., N95, Q95, or E95) can be replaced with any hydrophobic residue. One particular engineered HA protein exemplified herein is derived from IAV H1N1 strain A / California / 04 / 2009, which comprises a HA1 and a HA2 ectodomain with an N95L substitution (see Example 3). As demonstrated herein, this single substitution is sufficient to effectively stabilize the HA in a closed trimeric conformation. In other embodiments, the engineered HA proteins of the application can have a substitution at position 106. The residue at this position in the HA2 ectodomain (e.g., R106, H106, or G106) can also be replaced with any hydrophobic residue. Still other engineered HA proteins of the application have substitutions at both position 95 and position 106. In these embodiments, each residue at these two positions can be replaced with any hydrophobic residue. In various embodiments, the replacement amino acid residue at each of position 95 and position 106 can independently be Ala, Val, Leu, He, Met, Phe, Tyr, or Trp.

[0051] In addition to the amino acid substitution at position 95 and / or position 106, some engineered HA proteins of the application can have substitutions at additional positions in HA2. In some of these embodiments, the additional substitutions include at least one substitution at position 51 and position 103 with a hydrophobic residue. In some embodiments, the engineered HA protein comprises additional substitutions at residues 51 and 103 with a hydrophobic residue. As demonstrated herein, these additional modifications of the HA2 ectodomain further improve the stability of the HA trimer and reduce its pH sensitivity. As one specific example, the engineered HA protein can comprise substitutions at N95, K51, and E103, each with a hydrophobic amino acid residue.

[0052] SEQ ID NO: 1: HA of IAV A / California / 04 / 2009 H1N1 sequence (Accession No. ACP41105; Garten et al., Science 325 (5937), 197-201, 2009):

[0053]

[0054] SEQ ID NO: 5: HA2 portion of SEQ ID NO: 1:

[0055]

[0056] SEQ ID NO: 9: HA2 ectodomain portion of SEQ ID NO: 1:

[0057]

[0058] SEQ ID NO: 2: HA of IAV A / Victoria / 361 / 2011 (H3N2) (Accession No. AGB08328):

[0059]

[0060]

[0061] SEQ ID NO: 6: HA2 portion of SEQ ID NO: 2

[0062]

[0063] SEQ ID NO: 10: HA2 ectodomain portion of SEQ ID NO: 2

[0064]

[0065] SEQ ID NO: 3: HA of IBV B / Brisbane / 60 / 2008 (Accession No. ACN29380)

[0066]

[0067] SEQ ID NO: 7: HA2 portion of SEQ ID NO: 3

[0068]

[0069]

[0070] SEQ ID NO: 4: HA of ICV C / Johannesburg / 1 / 1966 (Accession No. CAL69520; Crescenzo-Chaigne et al., J. Virol. 81, 11282-11289, 2007)

[0071]

[0072] SEQ ID NO: 8: HA2 portion of SEQ ID NO: 4

[0073]

[0074] In addition to the exemplary HA proteins from which some of the engineered HA proteins of the application are derived, some other engineered influenza HA proteins of the application can be derived from an HA protein sequence that is substantially identical to one of these exemplary sequences, including conservatively modified variant sequences. In addition to the specific amino acid substitutions described above, one can readily determine other residues in a wild-type HA sequence that can tolerate alteration. Again, this can be accomplished by sequence alignment and also taking into account conserved influenza HA motifs and residues known in the art.

[0075] The various engineered influenza HA proteins of the application can be obtained or produced according to the protocols exemplified herein or methods well known in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., (3rded., 2000); and Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (Supplement 49, 2003). Following recombinant expression (e.g., in ExpiCHO cells as detailed herein), the proteins can be purified by any conventional procedure of implementation. See, e.g., Guide to Protein Purification, Ed. Deutscher, Meth. Enzymol. 185, Academic Press; San Diego, 1990; and Scopes, Protein Purification: Principles and Practice, Springer Verlag, New York, 1982. Substantial purification indicates purification from other proteins or cellular components. A substantially purified protein is at least 60%, 70%, 80%, 90%, 95%, or 98% pure. Once purified, the antigenicity and other properties of the engineered influenza HA proteins can also be readily assayed using standard methods, e.g., antigenic profile analysis using known bNAb and non-Nab, differential scanning calorimetry (DSC), electron microscopy, binding analysis by ELISA, Biolayer Interferometry (BLI), Surface Plasmon Resonance (SPR), and co-crystallography analysis, as exemplified herein.

[0076] IV. Scaffolded influenza HA protein compositions

[0077] The present application provides influenza HA-based vaccine compositions comprising a heterologous scaffold presenting or incorporating the engineered influenza HA proteins described herein. In the construction of the vaccines of the present application, any heterologous scaffold can be used to present the engineered influenza HA proteins. These include nanoparticles, virus-like particles, protein carriers (e.g., immunoglobulin chains or domains such as Fc, KLH, BSA, tetanus toxoid, and diphtheria toxoid), and various chemical scaffolds. In some embodiments, virus-like particles (VLPs) can be used, such as bacteriophage Q β VLPs and nanoparticles. In some preferred embodiments, the heterologous scaffold used to present or display the engineered influenza HA proteins is a nanoparticle. Various nanoparticle platforms can be used to generate the vaccine compositions of the present application. Generally, the nanoparticles used in the present application need to be formed from multiple copies of a single subunit.

[0078] In some preferred embodiments, the engineered influenza HA protein is presented on a self-assembling nanoparticle, such as a self-assembling nanoparticle derived from E2p, I3-01v9, and ferritin (FR). E2p is a redesigned variant of dihydroxysphingosyl acyltransferase from Bacillus stearothermophilus that has been shown to self-assemble into a heat-stable 60-mer nanoparticle. See, e.g., He et al., Nat. Commun. 7: 12041, 2016. Similarly, I3-01 is an engineered protein that can self-assemble into a super-stable nanoparticle. See, e.g., Hsia et al., Nature 535, 136-139, 2016. Ferritin is a globular protein found in all animals, bacteria, and plants. The globular form of ferritin is composed of monomeric subunit proteins (also referred to as monomeric ferritin subunits), which are polypeptides with a molecular weight of about 17 to 20 kDa. The amino acid sequences of the E2p, I3-01v9, and ferritin nanoparticle subunits, as well as some other suitable nanoparticle sequences, are known in the art. See, e.g., WO2017 / 192434, WO2019 / 089817, and WO19 / 241483. Other nanoparticles or VLPs known in the art that can also be used in the practice of the present application include, e.g., PdxJ from Pseudomonas aeruginosa, CsgA from Thermotoga maritima, CsgG from Myxococcus xanthus, bacteriophage Qbeta virus particles, Flock House Virus (FHV) particles, ORSAY virus particles, and infectious bursal disease virus (IBDV) particles. In yet other embodiments, the scaffold for presenting the engineered influenza HA protein of the present application can also include molecules having the following PDB IDs: 1JIG (12-mer Dlp-2 from Bacillus anthracis), 1UVH (12-mer DPS from Mycrobacterium smegmatis), 2YGD (24-mer ocular lens chaperone protein alphaB-crystallin), 3CS0 (24-mer DegP24), 3MH6 and 3MH7 (24-mer HtrA protease), 3PV2 (12-mer HtrA homolog DegQ WT), 4A8C (12-mer DegQ from E. Coli.), 4A9G (24-mer DegQ from E. Coli.), 4EVE (12-mer HP-NAP from Helicobacter pylori strain YS29), and 4GQU (24-mer HisB from Mycobacterium tuberculosis).In various embodiments, the nanoparticle vaccines of the present application can use any of these known scaffolds, conservatively modified variants thereof, or variants having a substantially identical (e.g., at least 90%, 95%, or 99% identical) sequence.

[0079] In some embodiments, the influenza HA proteins displayed on the nanoparticle platform can optionally comprise a trimerization motif, e.g., SHP or foldon, as exemplified herein. Some influenza nanoparticle vaccine compositions can additionally comprise other structural components that serve to further enhance the stability and antigenicity of the displayed immunogen. In some embodiments, a lock protein domain can be inserted into the nanoparticle construct, e.g., by covalent fusion to the C-terminus of the nanoparticle subunit. The lock domain can be any dimeric protein capable of forming an interface through specific interactions, e.g., hydrophobic (van der Waals) contacts, hydrogen bonds, and / or salt bridges. General guidance and specific examples for selecting lock domains are described in the art, e.g., PCT 2019 / 036917.

[0080] In some embodiments, the scaffold influenza vaccine of the present application can also comprise a T cell epitope to promote a robust T cell response and direct B cell development towards bNAb. The T cell epitope can be located in any position relative to other structural components, so long as it does not affect the presentation of the engineered HA protein on the nanoparticle surface. Any T cell epitope sequence or peptide known in the art can be used in the practice of the present application. This includes any polypeptide sequence comprising an MHC class II epitope and which can effectively activate CD4+ and CD8+ T cells after immunization, e.g., a T-helper epitope that activates CD4+ T helper cells. See, e.g., Alexander et al., Immunity 1, 751-761, 1994; Ahlers et al., J. Clin. Invest. 108: 1677-1685, 2001; Fraser et al., Vaccine 32, 2896-2903, 2014; De Groot et al., Immunol. Cell Biol. 8:255-269, 2002; and Gene Ther. 21: 225-232, 2014. In some embodiments, the T cell epitope inserted into the nanoparticle vaccine construct is a universal pan DR epitope peptide (PADRE). See, e.g., Hung et al., Mole. Ther. 15: 1211-19, 2007; Wu et al., J. Biomed. Sci. 17: 88, 2010; and Bissati et al., npj Vaccines 2: 24, 2017. Additional examples of suitable T cell epitopes are also described in the art, e.g., D and TpD epitopes (Fraser et al., Vaccine 32, 2896-2903, 2014).

[0081] The scaffold influenza vaccine compositions of the present application can be constructed according to standard recombinant techniques and other methods described in the art, e.g., He et al., Nat. Comm. 7, 12041, 2016; Kong et al., Nat. Comm. 7, 12040, 2016; He et al., Sci Adv. 4(11): eaau6769, 2018; and PCT publications WO2017 / 192434, WO2019 / 089817, and WO19 / 241483. In various embodiments, the nanoparticle displaying any engineered influenza HA protein can be constructed by fusing the influenza HA protein to a subunit of the nanoparticle, e.g., the E2p subunit. Preferably, the C-terminus of the influenza HA protein sequence is fused to the N-terminus of the nanoparticle subunit sequence. In some embodiments, a short peptide spacer can be used to connect the influenza HA protein sequence and the nanoparticle subunit sequence.

[0082] V. Polynucleotides and expression constructs

[0083] The engineered influenza HA proteins and scaffold vaccine compositions of the application are generally produced by first producing an expression construct (i.e., expression vector) comprising operably linked coding sequences for the various structural components described herein. Thus, in some related aspects, the application provides polynucleotides (e.g., DNA or RNA) encoding engineered influenza HA proteins or polypeptides, and polynucleotides (e.g., DNA or RNA) encoding subunit sequences of nanoparticles displaying influenza HA proteins, as well as expression vectors comprising such polynucleotides and host cells (e.g., ExpiCHO cells as exemplified herein) for producing engineered influenza HA proteins and vaccine compositions. Fusion polypeptides encoded by such polynucleotides or expressed by such vectors are also encompassed by the application.

[0084] Polynucleotides and related vectors can be readily produced by standard molecular biology techniques or the protocols exemplified herein. For example, general protocols for cloning, transfection, transient gene expression, and obtaining stable transfectant cell lines are described in the art, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., (3rded., 2000); and Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou edition, 2003). Introduction of mutations into polynucleotide sequences by PCR can be performed as described in, e.g., PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (eds.), Academic Press, San Diego, CA, 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Applications 1 : 17, 1991.

[0085] The choice of particular vector depends on the intended use of the fusion polypeptide. For example, the vector selected must be capable of driving expression of the fusion polypeptide in the desired cell type, whether that cell type is prokaryotic or eukaryotic. Many vectors contain sequences that allow both eukaryotic expression of operably linked gene sequences and prokaryotic vector replication. Vectors useful in the present application can replicate autonomously, i.e., the vector exists extrachromosomally, and its replication is not necessarily linked to the replication of the host genome. Alternatively, the replication of the vector can be linked to the replication of the host chromosomal DNA, e.g., the vector can integrate into the chromosome of the host cell, as is the case with retroviral vectors and as is achieved in stably transfected cell lines. Both viral-based and non-viral based expression vectors can be used to produce immunogens in mammalian host cells. Non-viral vectors and systems include plasmids, episomal vectors (typically with an expression cassette for expressing a protein or RNA), and artificial chromosomes (see, e.g., Harrington et al., Nat. Genet. 15:345, 1997). Useful viral vectors include those based on lentivirus or other retroviruses, adenovirus, adeno-associated virus, cytomegalovirus, herpes virus, vectors based on SV40, papillomavirus, Epstein Barr virus, vaccinia virus vectors, and Semliki Forest virus (SFV). See Brent et al., supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68:143, 1992.

[0086] A variety of known cells or cell lines can be used in the practice of the present application, depending on the particular vector used to express the fusion polypeptide. The host cell can be any cell into which a recombinant vector carrying a fusion of the present application can be introduced and in which the vector can drive expression of the fusion polypeptide. It can be prokaryotic, such as any of a number of bacterial strains, or it can be eukaryotic, such as a yeast or other fungal cell, an insect or amphibian cell, or a mammalian cell, including, for example, a rodent, simian, or human cell. The cell expressing a fusion polypeptide of the present application can be a primary culture cell or can be an established cell line. Thus, in addition to the cell lines exemplified herein (e.g., CHO cells), a number of other host cell lines known in the art can be used in the practice of the present application. These include, for example, a variety of Cos cell lines, HeLa cells, HEK293, AtT20, BV2, and N18 cells, myeloma cell lines, transformed B cells, and hybridomas.

[0087] Expression of polypeptides using mammalian tissue cell cultures is discussed generally in, for example, Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y., 1987. The fusion polypeptide expression vector can be introduced into the selected host cell by any of a number of suitable methods known to those of skill in the art. In order to introduce the fusion polypeptide-encoding vector into mammalian cells, the method used will depend on the form of the vector. For plasmid vectors, the DNA encoding the fusion polypeptide sequence can be introduced into mammalian cells by any of a number of transfection procedures, including, for example, lipid-mediated transfection ("lipofection"), DEAE-dextran-mediated transfection, electroporation, or calcium phosphate precipitation. These methods are described in detail in Brent et al., supra. Lipofection reagents and methods suitable for transient transfection of a wide variety of transformed and untransformed or primary cells are widely available, making lipofection an attractive method for introducing constructs into eukaryotic, and especially cultured mammalian cells. For example, LipofectAMINE TM (Life Technologies) or LipoTaxi TM (Stratagene) kits are available. Other companies that provide reagents and methods for lipofection include Bio-Rad Laboratories, Clontech, Glen Research, Life Technologies, JBL Scientific, MBI Fermentas, PanVera, Promega, Quantum Biotechnologies, Sigma-Aldrich, and Wako Chemicals USA.

[0088] For long-term, high-yield production of recombinant fusion polypeptides, stable expression is preferred. Rather than using expression vectors that contain viral origins of replication, host cells can be transformed with fusion polypeptide-encoding sequences and selection markers controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) that regulate their own expression to allow for stable integration and maintenance of the sequences in the chromosomes of the host cells. Selection markers in the recombinant vectors confer resistance to the selection reagent and allow cells to maintain the vector stable integrated into their chromosomes. Commonly used selection markers include neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al., J. Mol. Biol., 150: 1, 1981), and hygro, which confers resistance to hygromycin (Santerre et al., Gene, 30: 147, 1984). Through appropriate selection, the transfected cells can contain integrated copies of the fusion polypeptide-encoding sequences.

[0089] VI. Pharmaceutical compositions and therapeutic applications

[0090] This invention provides pharmaceutical compositions or immunogenic compositions and related methods for preventing and treating influenza infections (e.g., influenza) using modified influenza HA proteins and nanoparticles displaying the modified HA proteins as described herein. In some embodiments, the modified influenza HA sequence (peptide or polynucleotide sequence) or nanoparticles displaying the modified protein are included in the pharmaceutical composition. The pharmaceutical composition may be a therapeutic or prophylactic formulation. Typically, the composition additionally contains one or more pharmaceutically acceptable carriers, and optionally other therapeutic ingredients (e.g., antibiotics or antiviral drugs). Various pharmaceutically acceptable additives may also be used in the composition.

[0091] Some of the pharmaceutical compositions of the present invention are vaccines. For vaccine compositions, suitable adjuvants may additionally be included. Some examples of suitable adjuvants include, for example, aluminum hydroxide, lecithin, Freund's adjuvant, and MPL. TM And IL-12. In some embodiments, the modified influenza HA protein and associated vaccines, as disclosed herein, can be formulated into controlled-release or timed-release formulations. This can be achieved in compositions comprising sustained-release polymers or via microencapsulation delivery systems or bioadhesive gels. A variety of pharmaceutical compositions can be prepared according to standard procedures known in the art. See, for example, Remington's Pharmaceutical Sciences, 19th Edition, Supplement, Mack Publishing Company, Easton, Pa., 1995; Sustained and Controlled Release Drug Delivery Systems, edited by JR Robinson, Marcel Dekker, Inc., New York, 1978); U.S. Patent Nos. 4,652,441 and 4,917,893; U.S. Patent Nos. 4,677,191 and 4,728,721; and U.S. Patent No. 4,675,189.

[0092] The therapeutic methods of the application involve administering a modified influenza HA molecule (protein or nucleic acid) of the application or a pharmaceutical composition comprising the molecule to a subject having an influenza infection (e.g., influenza) or at risk of developing an influenza infection (e.g., influenza). Generally, the immunogenic compositions of the application are administered in an amount sufficient to induce an immune response against an influenza virus or group of viruses. For prophylactic applications, the immunogenic compositions are provided prior to any symptoms (e.g., prior to infection). The prophylactic administration of the immunogenic compositions serves to prevent or ameliorate any subsequent infection. Thus, in some embodiments, the subject to be treated is a subject having or at risk of developing an influenza virus infection, e.g., due to exposure or potential exposure to the virus. Following administration of a therapeutically effective amount of the disclosed therapeutic compositions, the subject can be monitored for viral infection, symptoms associated with influenza virus infection, or both. For therapeutic applications, the immunogenic compositions are provided at or following the onset of symptoms of disease or infection (e.g., following the onset of symptoms of influenza, or following the diagnosis of an influenza virus infection). Thus, the immunogenic compositions can be provided prior to an anticipated exposure to the virus to attenuate the severity, duration, or extent of the anticipated infection and / or associated disease symptoms, following an exposure or suspected exposure to the virus, or following the onset of actual infection. The appropriate amount of the modified HA protein or related nanoparticle composition can be determined based on the particular disease or condition to be treated or prevented, the severity, the age of the subject, and other personal attributes of the particular subject (e.g., the overall state of health of the subject and the robustness of the subject's immune system). Determination of effective doses is guided by animal model studies followed by human clinical trials, and by administration regimens that significantly reduce the occurrence or severity of the targeted disease symptoms or conditions in the subject.

[0093] The pharmaceutical compositions of the application can be combined with other agents known in the art for the treatment or prevention of influenza virus infection. Administration of the pharmaceutical compositions and known antiviral agents can be simultaneous or sequential. Pharmaceutical compositions comprising the modified influenza HA proteins or nanoparticle vaccines of the application can be provided as components of a kit. Optionally, such kits comprise additional components including packaging, instructions, and a variety of other reagents such as buffers, substrates, antibodies or ligands (e.g., control antibodies or ligands), and detection reagents. Optional instructions can also be provided in the kits.

[0094] Examples

[0095] The following examples are provided to illustrate, but not limit, the application.

[0096] Example 1 Analysis of hemagglutinin (HA) metastability

[0097] In this study, we designed six soluble constructs against HAs from four IAV subtypes (H1, H3, H5, and H7) and two IBVs. Each HA construct encoded HA1, HA2 ectodomain, a restriction site (AS), foldon, and His6 tag. The HA2 ectodomain was terminated at D518 of IAV A / California / 04 / 2009 H1N1 and K519 of IAV A / Hong Kong / 1 / 1968 H3N2 (GenBank: CY044261), respectively. These HA constructs were transiently expressed in ExpiCHO cells and purified using nickel and antibody columns, followed by size-exclusion chromatography (SEC) on a Superdex 200 10 / 300 column Figure 1A ). Overall, a trimer peak was observed for all tested HAs, regardless of the purification method. Notably, all HAs from Group 1 IAV and IBV Victoria lineage showed a trimer peak at 10.7 to 10.9 ml, while all HAs from Group 2 IAV and IBV Yamagata lineage showed a trimer peak at 11.1 to 11.7 ml, indicating a difference in molecular size. Negative stain EM was performed to assess H1N1 Figure 1B ) and H3N2 Figure 1C ) HAs. Indeed, the H1N1 HA showed an open trimer, while the H3N2 HA was a closed trimer, which is in very good agreement with the crystal structure (PDB ID: 4FNK). Thus, the “open trimer” display is the main form of H1 HA metastability.

[0098] We further analyzed H5N1 and H7N9 HAs by SEC and negative stain EM. Similarly, the HA constructs encoded HA1, HA2 ectodomain, AS, foldon, and His6 tag. Notably, the HA2 ectodomain was terminated at S520 of A / Viet Nam / 1203 / 2004 H5N1 (GenBank: AY818135) and D513 of IAV A / Guangdong / HP001 / 2017 H7N9 (GenBank: KY643843), respectively. These HA constructs were transiently expressed in ExpiCHO cells and purified using nickel or antibody columns, followed by SEC using a Superdex 200 10 / 300 column, as shown in Figure 1A . As mentioned above, the H5N1 HA and H7N9 HA showed a trimer peak at 10.9 and 11.7 ml, respectively. Then, negative stain EM was performed to assess H5N1-VN-2004 Figure 2A) and H7N9-GD-2017 ( Figure 2B ) HA. For H5N1 HA, we observed a higher flexibility of the HA2 region in 2D class images ( Figure 2A , middle). Thus, applying the C3 symmetry did not lead to a reasonable 3D reconstruction of the HA trimer. Even when the Cl symmetry was enforced, the 3D model showed a lower resolution density at the bottom of the HA trimer ( Figure 2A , right). A small fraction of the HA proteins showed as monomers ( Figure 2A , right, bottom). For H7N9 HA, we observed a clear pattern of dissociation of the head domain ( Figure 2B , middle). Less than 50% of the HA proteins were closed trimers and allowed a 3D reconstruction of the intact trimer ( Figure 2B , right, upper). In contrast, more than 50% of the HA proteins could only be analyzed by enforcing the Cl symmetry, resulting in a 3D model with two HA protomers ( Figure 2B , right, bottom). In summary, our EM analysis revealed other forms of HA metastability for H5 and H7.

[0099] Example 2 Determination of the cause of hemagglutinin (HA) metastability

[0100] To determine the cause of HA metastability, we aligned the HA2 ectodomain from 10 HA from four IAV subtypes (H1, H3, H5, and H7) and two IBV lineages (Victoria and Yamagata) ( Figure 3A ). We identified several positions that can contribute to HA metastability according to the widely used H3 numbering system. First, we hypothesized that the polar / charged residue at position 95 is the underlying cause of HA metastability because it is located at the C-terminus of the central C-helix of the HA trimer and forms repulsive polar-polar interactions ( Figure 3B ). We analyzed 15,821, 12,493, and 7,081 non-redundant HA sequences from human, avian, and swine (swain) origins from the NCBI Influenza Virus Resource website and found that the presence of the polar / charged residue is conserved throughout the database. Second, we hypothesized that position 106, R106 (group 1 IAV) / H106 (group 2 IAV) / G106 (IBV), in different HA sequences can be another cause of HA metastability because it is located at the first residue of the connecting segment between the C-helix and the D-helix and defines the subtype / type-specific properties ( Figure 3C). For group 1 IAV, R106 forms a repulsive interaction, forcing the HA trimer to open, while for group 2 IAV, the side chain of H106 tightly packs with the side chains of K51 and Q105 from the adjacent protomers and acts as a pH sensor to enable the HA trimer to conditionally open. For IBV, G106 increases the flexibility of the D-helix. Third, we hypothesized that the K51 / E103 pair (Milder et al., Proc. Natl. Acad. Sci. USA 2022, 119: e2115379119) can be mutated to hydrophobic residues to stabilize the packing between the A-helix and the C-helix ( Figure 3D ). This salt bridge was recently proposed as a pH sensor and mutated to stabilize the HA. However, Midler et al. reported that the H1N1 HA-K51I / E103I mutant showed an open conformation in negative stain EM analysis. Notably, this proposed pH sensor only exists in IAV HA. In summary, position 95 is the most critical for HA metastability, and can be mutated to hydrophobic residues (e.g., Ala, Val, Leu, lie, Met, Phe, Tyr, and Trp), which can be used alone or together with hydrophobic mutations at position 106 or at K51 / E103 to stabilize the HA trimer. All residues in this section are numbered according to the alignment with H3 HA or the H3 numbering system.

[0101] Example 3 Stabilization of H1N1 hemagglutinin (HA) trimers by a single N95L mutation

[0102] Given the importance of position 95 for HA metastability, we created the HA-N95L mutant against the H1N1 pandemic strain A / California / 04 / 09. Following the same protocol, we transiently (and at small scale) expressed this HA protein in ExpiCHO cells and purified it using a nickel column and two antibody columns. The purified protein was analyzed by SEC on a Superdex 200 10 / 300 column and the SEC profile was compared to that of the wild-type HA ( Figure 4A ). In the case of the N95L mutation, the HA trimer peak shifted to the right from 10.6 to 10.7 ml, as observed for the wild-type HA, to 11.6 to 11.9 ml ( Figure 4A ). The structure of the HA-N95L mutant purified by 2D1 (head NAb) and CR6261 (stem NAb) columns was evaluated by negative stain EM ( Figure 4B ). In both cases, the single mutation at N95L was found to effectively stabilize the HA in the closed trimer conformation, which is in excellent agreement with the crystal structure (PDB ID: 3LZG) Figure 4B). Thus, the results confirmed our HA metastability hypothesis and demonstrated the utility of hydrophobic mutations at N95L for trimer stabilization.

[0103]

[0104] Accordingly, the present application has been disclosed with the broadest possible scope that the Federal Laws will allow. It should be understood to those skilled in the art that various modifications, alternative constructions, and equivalents can be used without departing from the spirit and scope of the present application.

[0105] It should also be noted that all publications, sequence listings, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes and to the same extent as if each were individually so denoted. To the extent the definitions in the text conflict with those in the incorporated references, the text definitions control.

Claims

1. A modified influenza virus hemagglutinin (HA) protein comprising a modified HA2 extracellular domain, wherein the modified HA2 extracellular domain comprises a substitution of an amino acid residue at position 95 or 106 with a hydrophobic residue; wherein the amino acid numbering of HA2 is based on the influenza A H3 subtype.

2. The modified influenza virus hemagglutinin (HA) protein of claim 1, further comprising the complete HA1.

3. The modified influenza virus hemagglutinin (HA) protein of claim 1, which does not contain the HA head domain.

4. The modified influenza virus hemagglutinin (HA) protein of claim 1, wherein the HA protein is derived from influenza A virus and the replaced amino acid residue is N95.

5. The modified influenza virus hemagglutinin (HA) protein of claim 1, wherein the HA protein is derived from influenza B virus and the replaced amino acid residue is Q95.

6. The modified influenza virus hemagglutinin (HA) protein of claim 1, wherein the HA protein is derived from influenza C virus and the replaced amino acid residue is E95.

7. The modified influenza virus hemagglutinin (HA) protein of claim 1, wherein the substituted residues are Ala, Val, Leu, Ile, Met, Phe, Tyr, or Trp.

8. The modified influenza virus hemagglutinin (HA) protein of claim 1, wherein the modified HA2 extracellular domain comprises the substitution of both amino acid residues at positions 95 and 106 with hydrophobic residues.

9. The modified influenza virus hemagglutinin (HA) protein of claim 8, wherein (1) the HA protein is derived from group 1 influenza A virus and the residue at position 106 is R106, (2) the HA protein is derived from group 2 influenza A virus and the residue at position 106 is H106, or (3) the HA protein is derived from influenza B virus and the residue at position 106 is G106.

10. The modified influenza virus hemagglutinin (HA) protein of claim 8, wherein each substitution is independently replaced by Ala, Val, Leu, Ile, Met, Phe, Tyr, or Trp.

11. The modified influenza virus hemagglutinin (HA) protein of claim 1, comprising (1) a substitution at position 95 and (2) an additional amino acid substitution at one or both positions 51 and 103 with a hydrophobic residue.

12. The modified influenza virus hemagglutinin (HA) protein of claim 11, wherein the HA protein is derived from influenza A virus, and the additional substituted amino acid residues are K51 and E103.

13. The modified influenza virus hemagglutinin (HA) protein of claim 1, further comprising a subunit sequence of a self-assembled nanoparticle fused to the C-terminus of the modified HA protein.

14. A pharmaceutical composition comprising the modified influenza virus hemagglutinin (HA) protein of claim 1 and a pharmaceutically acceptable carrier.

15. The pharmaceutical composition of claim 14, wherein the modified HA protein is displayed on the surface of the self-assembled nanoparticles.

16. A polynucleotide encoding the modified influenza virus hemagglutinin (HA) protein of claim 1.

17. A vector comprising the polynucleotide of claim 14.

18. A host cell comprising the polynucleotide of claim 14.

19. An influenza virus vaccine composition comprising the modified HA protein of claim 1 displayed on the surface of self-assembled nanoparticles.

20. The vaccine composition of claim 19, wherein the self-assembled nanoparticles comprise a trimer sequence, and wherein the C-terminus of the modified HA protein is fused to the N-terminus of a subunit sequence of the nanoparticles.

21. The vaccine composition of claim 19, wherein the self-assembled nanoparticles comprise I3-01, E2p, or ferritin.

22. The vaccine composition of claim 19, further comprising a locking domain and / or a T-cell epitope fused to the C-terminus of the nanoparticle subunit sequence.

23. A method of treating or preventing influenza virus infection in a subject, comprising administering the pharmaceutical composition of claim 14 to the subject to treat or prevent influenza virus infection in the subject.

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