WO2021231729A1 - Adjuvanted stabilized stem hemagglutinin nanoparticles and methods of using the same to induce broadly neutralizing antibodies against influenza - Google Patents

Adjuvanted stabilized stem hemagglutinin nanoparticles and methods of using the same to induce broadly neutralizing antibodies against influenza Download PDF

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WO2021231729A1
WO2021231729A1 PCT/US2021/032252 US2021032252W WO2021231729A1 WO 2021231729 A1 WO2021231729 A1 WO 2021231729A1 US 2021032252 W US2021032252 W US 2021032252W WO 2021231729 A1 WO2021231729 A1 WO 2021231729A1
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influenza
vaccine composition
sequence
protein
helix
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PCT/US2021/032252
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French (fr)
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Gary J. Nabel
Chih-Jen Wei
Nicole DARRICARRERE
Masaru Kanekiyo
John R. Mascola
Barney S. Graham
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Sanofi
The United States Of America, As Represented By The Secretary, Dept. Of Health And Human Services
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • Influenza is caused by a virus that attacks mainly the upper respiratory tract - the nose, throat and bronchi and rarely also the lungs.
  • the infection usually lasts for about a week. It is characterized by sudden onset of high fever, myalgia, headache and severe malaise, nonproductive cough, sore throat, and rhinitis. Most people recover within one to two weeks without requiring any medical treatment. However, in the very young, the elderly and people suffering from medical conditions, such as lung diseases, diabetes, cancer, kidney or heart problems, influenza poses a serious risk. In these people, the infection may lead to severe complications of underlying diseases, pneumonia and death. Annual influenza epidemics are thought to result in between three and five million cases of severe illness and between 250,000 and 500,000 deaths every year around the world.
  • Influenza virus is a member of Orthomyxoviridae family. There are three main subtypes of influenza viruses, designated influenza A, influenza B, and influenza C.
  • the influenza virion contains a segmented negative-sense RNA genome, which encodes the following proteins: hemagglutinin (HA), neuraminidase (NA), matrix (Ml), proton ion-channel protein (M2), nucleoprotein (NP), polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), polymerase acidic protein (PA), and nonstructural protein 2 (NS2).
  • HA hemagglutinin
  • NA neuraminidase
  • Ml matrix
  • M2 proton ion-channel protein
  • NP nucleoprotein
  • PB1 polymerase basic protein 1
  • PB2 polymerase basic protein 2
  • PA polymerase acidic protein
  • NS2 nonstructural protein 2
  • the HA, NA, Ml, and M2 are membrane associated, whereas NP, PB1, PB2, PA, and NS2 are nucleocapsid associated proteins.
  • the Ml protein is the most abundant protein in influenza particles.
  • the HA and NA proteins are envelope glycoproteins, responsible for virus attachment and penetration of the viral particles into the cell. Specifically, HA binds the influenza virus to host cells with sialic acid-containing on surface structures on their membranes.
  • HA and NA proteins are the sources of the major immunodominant epitopes for virus neutralization and protective immunity, making them important components for prophylactic influenza vaccines.
  • the genetic makeup of influenza viruses allows frequent minor genetic changes, known as antigenic drift.
  • the amino acid sequence of the major antigens of influenza, particularly HA is highly variable across groups, subtypes and strains. For this reason, current seasonal influenza vaccines must be administered every year and require yearly updates to account for mutations in HA and NA proteins (antigenic drift) and to match rapidly-evolving viral strains.
  • HI and H3 subtypes account for about 75% of the confirmed influenza infections (M.
  • H3 subtypes are most frequently revised in seasonal vaccines as they harbor the highest sequence variability (J. D. Allen et al., Human Vaccines & Immunotherapeutics, 2018, 14:1840-1847).
  • Seasonal influenza vaccines confer protection against specific viral strains but have restricted breadth that limit their protective efficacy.
  • the influenza strains used in the vaccine are selected by the WHO/CDC based on the agencies’ best guess as to the prevalent influenza strains for the upcoming flu season. Often times, the guess is not accurate and the vaccine strains do not match the seasonal influenza strains, limiting the effectiveness of the seasonal vaccines.
  • Seasonal vaccines are also not designed to provide protection against pandemic strains that can result from antigen shift.
  • Pandemic outbreaks of influenza are caused by the emergence of a pathogenic and transmissible virus to which the human population is immunologically naive. Because the virus is new, the human population has little to no immunity against it. The virus spreads quickly from person-to-person worldwide. Three times in the last century, the influenza A viruses have undergone major genetic changes mainly in their HA-component, resulting in global pandemics and large tolls in terms of both disease and deaths. HI and H3 are the major seasonal subtypes that have caused pandemics (E. D. Kilboume et al., Emerging infectious diseases, 2006, 12:9-14).
  • pandemic The most infamous pandemic was the “Spanish Flu” which affected large parts of the world population and is thought to have killed at least 40 million people in 1918-1919. More recently, two other influenza A pandemics occurred in 1957 (“Asian influenza”) and 1968 (“Hong Kong influenza”) and caused significant morbidity and mortality globally. In contrast to current influenza epidemics, these pandemics were associated with severe outcomes also among healthy younger persons, albeit not on such a dramatic scale as the “Spanish flu” where the death rate was highest among healthy young adults.
  • H1N1 pandemic which infected over 60 million people in one year (www.cdc.gov/flu/pandemic-resources/2009-hlnl- pandemic.html).
  • Swine influenza has also been associated with influenza A subtypes H1N2, H2N3, H3N1, and H3N2.
  • pandemic influenza antigens e.g., the HA amino acid sequence of the pandemic strain
  • vaccines have traditionally been unavailable until the later stages of a pandemic.
  • recombinant protein engineering is required to present these epitopes in a favorable manner to the immune system. For example, when full-length HA is used as an immunogen, the antibodies elicited are predominantly those that bind to the highly-variable, immunodominant head domain.
  • a strategy has been devised to induce antibodies to the subdominant highly-conserved stem region and involves removing the head region of HA, stabilizing the remaining HA stem regions through protein engineering, and fusing the stabilized stem region (HA-ss) to a multimerizing protein, such as ferritin.
  • This approach was first used to generate group 1 HA stem immunogens. See e.g., PCT International Publication No. WO2015/183969; and Yassine et al., 2015, Nat. Med. 21:1065-70. It was also used to generate group 2 HA stem immunogens. See e.g., PCT International Publication No. WO2018/045308; and Corbeh et al., Therapeutics and Prevention, 2019, 10(1).
  • stabilizing group 2 HA stem was unexpectedly more difficult than stabilizing group 1 HA stem and required the use of different strategies other than the simpler hydrophobic repacking used to stabilize group 1 HA stem nanoparticles.
  • influenza vaccines that can better address the current problems of antigenic drift, antigenic shift, and virus mismatch by providing broader protection against multiple influenza strains, including the HI and/or H3 subtypes that account for the majority of seasonal infections and pandemic outbreaks.
  • a vaccine that could provide broad protection against these viruses would provide a significant public health benefit, extending protection to drifted seasonal strains and/or pandemic strains and reducing or eliminating the need for annual vaccine reformulations.
  • a broadly protective vaccine is desired to effectively control seasonal influenza infection and to avert pandemic outbreaks. The elicitation of broadly neutralizing antibodies that prevent infection would be a transformative solution to prevent influenza.
  • HA hemagglutinin
  • NHPs non-human primates
  • bnAbs neutralized diverse influenza viruses and shared a mode of recognition similar to human bnAbs indicating that this vaccine composition can be used to protect against diverse influenza viruses, including influenza viruses generated through antigenic drift and antigen shift that give rise to seasonal infections and pandemic outbreaks.
  • a first aspect is directed to a vaccine composition
  • a vaccine composition comprising stem-stabilized influenza hemagglutinin-multimerizing protein (e.g., ferritin) nanoparticles and a squalene-based oil-inwater adjuvant emulsion
  • the stem-stabilized influenza hemagglutinin-multimerizing protein nanoparticles comprise a protein with self-assembling multimerization properties (e.g., ferritin) joined to a modified, group 2 influenza hemagglutinin (HA) protein (e.g., modified HA protein from H3 influenza virus) to form a protein construct and wherein the protein construct forms stem-stabilized influenza hemagglutinin nanoparticles when expressed in cells
  • the modified influenza virus HA protein lacks an antigenic head region and comprises a modified stem region from a group 2 influenza HA protein wherein the modified stem region comprises at least one immunogenic epitope, and wherein the vaccine composition elicits broadly neutralizing influenza antibodies when administered to a subject
  • a second aspect is directed to a method of vaccinating against influenza virus, the method comprising administering to a subject in need thereof an immunologically effective dose of the vaccine composition, as described herein, wherein administration of the vaccine composition elicits broadly neutralizing influenza antibodies in the human subject.
  • Figures 1A-C show antibody responses after group 2 stem nanoparticle vaccination (H3-SS-np + AF03) in cynomolgus macaques.
  • H3-SS-np + AF03 group 2 stem nanoparticle vaccination
  • A Anti-H3 and anti-HIO IgG responses determined by ELISA after three immunizations administered at weeks 0, 4 and 10 (see vertical arrows) with H3-SS-np with AF03 adjuvant.
  • B Left: Neutralization measured by a reporter- microneutralization (MN) assay for 2005 H3 (A/Wisconsin/67/05, H3N2) in NHP serum, in the presence of competitors, either 2005 HA (WT) or Astern (I45N, Q47T).
  • MN reporter- microneutralization
  • HA lentiviral vector reporter assay for 2013 H10 (A/Jiangxi/IPB13b/2013, H10N8) in NHP serum. Specificity was determined with protein competitors, either wildtype 2009 HA (WT) or Astern (D19N, G33E). The IC50 is the dilution factor of serum that achieves 50% neutralization.
  • C Microneutralization activity of macaque antisera was assayed 2 weeks after the third immunization using the following diverse H3N2, H10N8 or H7N9 strains:
  • H3N2 A/Singapore/INFIMH- 16-0019/2016 (H3N2), A/Hong Kong/4801/2014 (H3N2), A/Victoria/361/2011 (H3N2), A/Wisconsin/67/2005 (H3N2), A/Shangdong/9/1993 (H3N2), A/Port Chalmers/1/1973 (H3N2), A/Aichi/02/1968 (H3N2), A/Jiangxi-Donghu/346-2/2013 (H10N8) and A/Anhui/1/2013 (H7N9).
  • Figures 2A-B show isolation and repertoire analysis of monoclonal antibodies from monkeys immunized with a vaccine composition comprising H3-SS-np and AF03 using antigen-specific B cell sorting.
  • the bnAbs were sorted using a single H3 HA probe (H3+) derived from A/Perth/16/2009 (A) or counter sorted using two H3 HA probes (H3+/H3+) derived from divergent strains, A/Hong Kong/1/1968 and A/Victoria/361/2011 (B).
  • Figures 3A-B show repertoire analysis of lambda and kappa light chains from H3-SS- np-immunized monkeys.
  • Pie charts depict the frequency of usage of specific variable and joining germline genes, in lambda (A) and kappa (B) light chains sequenced from single-sorted B cells that were either H3+ (Perth 2009 probe) or H3+/H3+ (Hong Kong 1968 and Victoria 2011 probes). As indicated, some germline genes could not be assigned unambiguously despite >90% sequence match.
  • Figures 4A-B show the breadth and specificity of mAbs isolated from immune macaques, including (A) microneutralization (MN) activity of mAbs isolated from group 2 immune macaques in a reporter assay with H3N2, H7N9 and H10N8 strains (two previously reported human bnAbs, CR6261 and CR8020, were included as controls in the assay); and (B) binding of H3-3B10 mAb to wildtype 2009 H3 HA (WT) and a Astern mutant (D19N, G33E).
  • MN microneutralization
  • WT wildtype 2009 H3 HA
  • Astern mutant D19N, G33E
  • FIGS 5A-C show that vaccine-induced NHP Fabs bind to the conserved epitope on the influenza hemagglutinin stem.
  • Figure 6 shows the binding of antibodies isolated from H3-SS-np-immunized cynomolgus macaques to wildtype and AStem (D19N, G33E) H3 HA trimers.
  • ELISA plates were coated with either wildtype or mutant HA (A/Perth/16/2009) trimers and probed for binding with indicated monkey and human mAbs.
  • Figure 7A-D shows where the 3B10 antibody makes contacts with the HA1 and HA2 portions of the stabilized stem region of the fusion protein.
  • the CDR-H1 make contacts with the N- terminus of the fusion peptide through van der Waals and hydrogen-bonding interactions. Residues from CDRHl and the fusion peptide involved in interactions are shown as sticks.
  • the CDR-H2 make contacts with the N- and C-terminus of the fusion peptide through hydrogen-bonding interactions.
  • C The CDR-H3 make contacts with the C-terminus of the fusion peptide through van der Waals and hydrogen-bonding interactions.
  • Continuous Cryo-EM density was observed in the HA1-HA2 connection region. The model is shown as cartoon with relevant residue numbers labeled.
  • This application discloses vaccine compositions comprising a squalene-based oil-inwater adjuvant emulsion, such as AF03, and nanoparticles formed from a protein construct comprising a stem-stabilized group 2 influenza hemagglutinin (HA) joined to a protein with self-assembling multimerization properties (e.g., ferritin).
  • the stem-stabilized HA substantially lacks the head region.
  • the protein construct forms nanoparticles that are antigenic with respect to the stem region of the HA polypeptide.
  • the vaccine compositions induce broadly neutralizing antibodies (bnAbs) to the conserved stem region of the influenza HA.
  • HA can refer to influenza hemagglutinin protein, or a portion thereof.
  • HA-ss refers to a stabilized stem region, or a portion of the stem region, from an influenza HA protein.
  • the HA portion of such a designation can also refer to the subtype of the hemagglutinin protein.
  • a stabilized stem region from a subtype 3 hemagglutinin can be referred to as H3-ss.
  • a protein construct comprising HA-ss joined to a ferritin protein can be referred to as HA-ss-F, or H3-ss-F if the stabilized stem region is from a subtype 3 HA.
  • the HA-ss-F fusion protein forms antigenic nanoparticles, or HA-ss-np for short, H3-ss-np if the stabilized stem region is from a subtype 3 HA.
  • a Ty pe, or Group of influenza virus refers to influenza Type A, influenza Type B or influenza type C. It is understood by those skilled in the art that the designation of a virus as a specific Type relates to sequence difference in the respective Ml (matrix) protein or P (nucleoprotein).
  • Type A influenza viruses are further divided into group 1 and group 2. These groups are further divided into subtypes, which refers to classification of a virus based on the sequence of its HA protein.
  • strain refers to viruses within a subtype that differ from one another in that they have small, genetic variations in their genome.
  • Antigen refers to an agent that elicits an immune response; and/or (ii) an agent that is bound by a T cell receptor (e.g., when presented by an MHC molecule) or to an antibody (e.g., produced by a B cell) when exposed or administered to an organism.
  • an antigen elicits a humoral response (e.g., including production of antigen-specific antibodies) in an organism; alternatively or additionally, in some embodiments, an antigen elicits a cellular response (e.g., involving T-cells whose receptors specifically interact with the antigen) in an organism.
  • a particular antigen may elicit an immune response in one or several members of a target organism (e.g., mice, rabbits, primates, humans), but not in all members of the target organism species.
  • an antigen elicits an immune response in at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the members of a target organism species.
  • an antigen binds to an antibody and/or T cell receptor, and may or may not induce a particular physiological response in an organism.
  • an antigen may bind to an antibody and/or to a T cell receptor in vitro, whether or not such an interaction occurs in vivo.
  • an antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens.
  • Antigens include the stem-stabilized influenza hemagglutinin-ferritin protein constructs (HA-ss-F) or nanoparticles obtained therefrom (HA-ss-np) as described herein.
  • Broadly neutralizing antibodies are antibodies that neutralize more than one subtype and/or strain of influenza virus.
  • broadly neutralizing antibodies elicited against a vaccine composition comprising a modified influenza HA protein construct from one sub-type of virus may neutralize another sub-type of virus.
  • broadly neutralizing antibodies elicited against a vaccine composition comprising a modified influenza HA protein construct from an H3 influenza virus may neutralize viruses from two or more of the following sub-types: HI, H2, H4, H5, H6, H7, H8, H8, H10, Hll, H12, H13, H14, H15, H16, H17 or H18, or from two or more of the following group 2 sub-types: H3, H4, H7, H10, H14, or H15.
  • broadly neutralizing antibodies elicited against a vaccine composition comprising a modified influenza HA protein construct from one sub-type may neutralize other strains of the same sub-type of virus.
  • broadly neutralizing antibodies elicited against a vaccine composition comprising a modified influenza HA protein construct from an H3 influenza virus may neutralize viruses from other divergent group 2 strains, including, for example, one or more H3 and/or H10 strains.
  • Exemplary H3 strains include, but are not limited to, A/Singapore/INFIMH- 16-0019/2017, A/Hong Kong/4801/2014, A/Victoria/361/2011, A/Wisconsin/67/2005, A/Shangdong/9/1993, A/Port Chalmers/ 1/1973, and A/Aichi/02/1968.
  • Exemplary H10 strains include, but are not limited to, A/Jiangxi/IPB13b/2013.
  • Divergent group 2 strains may also have an HA2 polypeptide sharing at least 60% identity with the HA2 polypeptide from with the wild type HA2 polypeptide from A/Finland/486/2004 (SEQ ID NO: 4).
  • Carrier refers to a diluent, adjuvant, excipient, or vehicle with which a composition is administered.
  • carriers can include sterile liquids, such as, for example, water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as, for example, peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • carriers are or include one or more solid components.
  • epitope includes any moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component in whole or in part.
  • an epitope is comprised of a plurality of chemical atoms or groups on an antigen.
  • such chemical atoms or groups are surface- exposed when the antigen adopts a relevant three-dimensional conformation.
  • such chemical atoms or groups are physically near to each other in space when the antigen adopts such a conformation.
  • at least some such chemical atoms are groups are physically separated from one another when the antigen adopts an alternative conformation (e.g., is linearized).
  • Excipient refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example to provide or contribute to a desired consistency or stabilizing effect.
  • suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • Fusion protein refers to a protein encoded by a nucleic acid sequence engineered from nucleic acid sequences encoding at least a portion of two different (e.g., heterologous) proteins. To create a fusion protein nucleic acid sequences are joined such that the resulting reading does not contain an internal stop codon.
  • “Glycosylation,” as used herein, refers to the addition of a saccharide unit to a protein.
  • N-glycan refers to a saccharide chain attached to a protein at the amide nitrogen of an N (asparagine) residue of the protein. As such, an N-glycan is formed by the process ofN-glycosylation. This glycan may be a polysaccharide.
  • Immune response refers to a response of a cell of the immune system, such as a B cell, T cell, dendritic cell, macrophage or polymorphonucleocyte, to a stimulus such as an antigen, immunogen, or vaccine.
  • An immune response can include any cell of the body involved in a host defense response, including for example, an epithelial cell that secretes an interferon or a cytokine.
  • An immune response includes, but is not limited to, an innate and/or adaptive immune response.
  • a protective immune response refers to an immune response that protects a subject from infection (prevents infection or prevents the development of disease associated with infection).
  • lymphocytes such as B or T cells
  • cytokines or chemokines secretion of cytokines or chemokines
  • inflammation inflammation
  • antibody production and the like.
  • An antibody response or humoral response is an immune response in which antibodies are produced.
  • a “cellular immune response” is one mediated by T cells and/or other white blood cells.
  • Immunogen refers to a compound, composition, or substance which is capable, under appropriate conditions, of stimulating an immune response, such as the production of antibodies or a T cell response in an animal, including compositions that are injected or absorbed into an animal.
  • immunize means to render a subject protected from an infectious disease, such as by vaccination.
  • neutralizing antibodies are antibodies that prevent influenza virus from completing one round of replication.
  • one round of replication refers the life cycle of the virus, starting with attachment of the virus to a host cell and ending with budding of newly formed virus from the host cell. This life cycle includes, but is not limited to, the steps of attaching to a cell, entering a cell, cleavage and rearrangement of the HA protein, fusion of the viral membrane with the endosomal membrane, release of viral ribonucleoproteins into the cytoplasm, formation of new viral particles and budding of viral particles from the host cell membrane. Accordingly, a neutralizing antibody is one that inhibits one or more such steps.
  • Pandemic strain A “pandemic” influenza strain is one that has caused or has capacity to cause pandemic infection of human populations. In some embodiments, a pandemic strain has caused pandemic infection. In some embodiments, such pandemic infection involves epidemic infection across multiple territories; in some embodiments, pandemic infection involves infection across territories that are separated from one another (e.g., by mountains, bodies of water, as part of distinct continents, etc.) such that infections ordinarily do not pass between them.
  • prevention refers to prophylaxis, avoidance of disease manifestation, a delay of onset, and/or reduction in frequency and/or severity of one or more symptoms of a particular disease, disorder or condition (e.g., infection for example with influenza virus). In some embodiments, prevention is assessed on a population basis such that an agent is considered to “prevent” a particular disease, disorder or condition if a statistically significant decrease in the development, frequency, and/or intensity of one or more symptoms of the disease, disorder or condition is observed in a population susceptible to the disease, disorder, or condition.
  • Seasonal strain A “seasonal” influenza strain is one that has caused or has capacity to cause a seasonal infection (e.g., annual epidemic) of human populations. In some embodiments, a seasonal strain has caused seasonal infection.
  • Sequence identity The similarity between amino acid or nucleic acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. “Sequence identity” between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences. “Sequence identity” between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. Homologs or variants of a given gene or protein will possess a relatively high degree of sequence identity when aligned using standard methods.
  • % identical refers, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing said sequences, after optimal alignment, with respect to a segment or “window of comparison”, in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math.
  • Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100.
  • the degree of identity is given for a region which is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence.
  • the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments in continuous nucleotides.
  • the degree of identity is given for the entire length of the reference sequence.
  • Nucleic acid sequences or amino acid sequences having a particular degree of identity to a given nucleic acid sequence or amino acid sequence, respectively, may have at least one functional property of said given sequence, e.g., and in some instances, are functionally equivalent to said given sequence.
  • One important property includes the ability to act as a cytokine, in particular when administered to a subject.
  • a nucleic acid sequence or amino acid sequence having a particular degree of identity to a given nucleic acid sequence or amino acid sequence is functionally equivalent to said given sequence.
  • Stem-stabilized influenza hemagglutinin-ferritin protein construct As used herein, “stem-stabilized influenza hemagglutinin-ferritin protein construct” or the like is used herein to refer to a non-naturally occurring polypeptide comprising a ferritin polypeptide joined to a modified influenza hemagglutinin (HA) polypeptide, where the immunodominant head region of the HA is substantially removed, and the polypeptide forms nanoparticles that are antigenic with respect to the stem region of the HA polypeptide when expressed in cells.
  • Antigenicity may be a feature of the modified influenza HA polypeptide as part of the larger construct. That is, it is sufficient that the construct can serve as an antigen that generates antibodies against the modified influenza HA polypeptide, regardless of whether the modified influenza HA polypeptide without the ferritin could do so.
  • Subject means any member of the animal kingdom. In some embodiments, “subject” refers to humans. In some embodiments, “subject” refers to non-human animals. In some embodiments, subjects include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In certain embodiments, the nonhuman subject is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a ferret, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig).
  • a mammal e.g., a rodent, a mouse, a rat, a rabbit, a ferret, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig.
  • a subject may be a transgenic animal, genetically-engineered animal, and/or a clone.
  • the subject is an adult, an adolescent or an infant.
  • terms “individual” or “patient” are used and are intended to be interchangeable with “subject.”
  • Vaccination refers to the administration of a composition intended to generate an immune response, for example to a disease-causing agent such as influenza.
  • Vaccination can be administered before, during, and/or after exposure to a disease-causing agent, and/or to the development of one or more symptoms, and in some embodiments, before, during, and/or shortly after exposure to the agent.
  • Vaccines may elicit both prophylactic (preventative) and therapeutic responses.
  • Methods of administration vary according to the vaccine, but may include inoculation, ingestion, inhalation or other forms of administration.
  • Inoculations can be delivered by any of a number of routes, including parenteral, such as intravenous, subcutaneous, intraperitoneal, intradermal, or intramuscular.
  • Vaccines may be administered with an adjuvant to boost the immune response.
  • vaccination includes multiple administrations, appropriately spaced in time, of a vaccinating composition.
  • the vaccine compositions for immunization against influenza virus described in this application include a squalene-based adjuvant emulsion, like AF03, and a protein construct comprising a stem-stabilized group 2 influenza HA and a protein with self-assembling multimerization properties (e.g., ferritin), where the protein construct self assembles into immunogenic nanoparticles when expressed in cells.
  • a squalene-based adjuvant emulsion like AF03
  • a protein construct comprising a stem-stabilized group 2 influenza HA and a protein with self-assembling multimerization properties (e.g., ferritin), where the protein construct self assembles into immunogenic nanoparticles when expressed in cells.
  • protein constructs as described herein are capable of self-assembling into 24-mer nanoparticles (also called HA-ss-np) expressing eight trimers of HA stem protein on each vertex formed by three subunits coming together and displaying octahedral symmetry.
  • the vaccine composition When administered to a subject, the vaccine composition induces broadly neutralizing antibodies to the conserved stem region of the influenza HA.
  • HA-ss-F is formed by joining two proteins that normally do not exist in nature: a modified stem region of an influenza HA protein and a ferritin protein.
  • HA-ss-F protein constructs and the nanoparticles (HA-ss-np) formed by those protein constructs have been previously described.
  • PCT International Publication No. W02018/0453008 which is hereby incorporated by reference in its entirety, describes in detail the production of group 2 HA-ss- F. See also, Corbett et ak, Therapeutics and Prevention, 2019, 10(1), 1-18, which is hereby incorporated by reference in its entirety.
  • Hemagglutinin is the immunodominant surface influenza protein and is composed of two structurally distinct domains or regions: the globular head and the stem (also referred to as the stalk).
  • the HA protein is formed from a precursor protein called HAO, which is transported through the golgi complex and transported to the plasma membrane where it is cleaved by cellular proteases into HA1 and HA2, which are linked by a disulfide bond.
  • HAO precursor protein
  • the head region of the influenza HA is composed of the majority of the HA1 polypeptide, including the receptor-binding domain (about 145 amino acids) and the vestigial esterase domain (about 75 amino acids).
  • the stem region which anchors the HA protein into the viral lipid envelope, includes the HA2 polypeptide and a portion of the HA1 polypeptide.
  • a first portion of the stem region is located upstream of the N-terminus of the head region, while a second portion of the stem region is located downstream of the C-terminus of the head region.
  • a first stem region refers to the portion of the HA stem region that is located upstream of the N- terminus of the HA head region.
  • a second stem region refers to the portion of the HA stem region that is located downstream of the C-terminus of the HA head region.
  • the head region corresponds to amino acids 60-329 of the full length HA1 polypeptide of the H3N2 influenza virus A/Finland/486/2004 (SEQ ID NO: 2).
  • the first stem region located upstream of the N-terminus of the head region corresponds to amino acids 1-59 (or amino acids 17-59 when the signal sequence is removed) of the full length HA1 polypeptide of the H3N2 influenza virus A/Finland/486/2004 (SEQ ID NO: 2)
  • the second stem region located downstream of the C-terminus of the head region corresponds to 1) amino acids 330-345 of the full length HA1 polypeptide of the H3N2 influenza virus A/Finland/486/2004 (SEQ ID NO: 2) and 2) amino acids 1-221 of the HA2 polypeptide of the H3N2 influenza virus A/Finland/486/2004 (SEQ ID NO: 4).
  • HA proteins The amino acid numbering of the A/Finland/486/2004 H3N2 influenza is used merely by way of reference.
  • HA proteins it is understood by those skilled in the art that HA proteins from different influenza viruses may have different lengths due to sequence differences (insertions, deletions) in the protein.
  • reference to a corresponding region refers to a region of another protein that is identical, or nearly so (e.g., at least 90% identical, at least 95%, identical, at least 98% identical or at least 99% identical), in sequence, structure and/or function to the region being compared.
  • the corresponding region in another HA protein may not have the same residue numbers but will have a nearly identical sequence and will perform the same function.
  • numbering systems are used by those in the field, which relate amino acid positions to a reference sequence.
  • corresponding amino acid residues in HA proteins from different strains of influenza may not have the same residue number with respect to their distance from the N-terminal amino acid of the protein.
  • residue 100 in A/New Caledoma' ; 2Q/1999 (1999 NC, HI) does not mean it is the 100* residue from the N-terminal amino acid.
  • residue 100 of A/New Caledonia/20/1999 (1999 NC, HI) aligns with residue 100 of influenza H3N2 strain.
  • the use of such numbering systems is understood by those skilled in the art. While the H3 numbering system can be used to identify the location of ammo acids, unless otherwise noted, the location of amino acid residues in HA proteins will be identified by general reference to the position of a corresponding amino acid from a sequence disclosed herein.
  • the HA-ss portion of the HA-ss-F protein construct comprises at least one immunogenic epitope from the stem region (Embodiment HAla).
  • the HA-ss portion is constructed by removing the immunodominant head region of the influenza HA protein (Embodiment HAlb). This can be achieved by removing all five antigenic sites of the head region (e.g., antigenic sites A-E in the head region of group 2 influenza strains) (Embodiment HA1 c). The entire antigenic site may be removed or a sufficient number of amino acid residues of each antigenic site such that no immunogenic epitopes remain in the head region (Embodiment HAld).
  • the head region is “substantially removed” or “substantially replaced” meaning that all but up to 5 contiguous amino acids of the head region are removed (e.g., 5, 4, 3, 2, 1 or 0 contiguous amino acids of the head region are removed) (Embodiment HAle). In other embodiments, at least 95% of the head region is removed (Embodiment HA11). In certain embodiments, all amino acid residues of the head region are removed (Embodiment HAlg).
  • the first stem region and the second stem region can be joined together directly, or they can be joined with a linker sequence (Embodiment HA2a).
  • the head region is replaced with a linker sequence that connects the first and second stem regions (Embodiment HA2b).
  • Any linker sequence may be used so long as the first and second stem region sequences are able to form the desired structure (Embodiment HA2c). While any amino acids may be used to make the linker sequence, it is preferred to use amino acids lacking large or charged side chains (Embodiment HA2d).
  • Preferred amino acids include, but are not limited to, serine, glycine, valine and alanine (Embodiment HA2e).
  • the linker is made from serine and glycine residues (Embodiment HA21).
  • the length of the linker sequence may vary, but preferred embodiments use the shortest possible sequence in order to allow the stem sequences to form the desired structure (Embodiment HA2g).
  • the linker sequence is less than 10 amino acids in length (Embodiment HA2h).
  • the linker sequence is less than 5 amino acids in length (Embodiment HA2i).
  • the linker sequence is 6-7 amino acids in length (Embodiment HA2j).
  • the linker sequence consists of the amino acid sequence GSG (Embodiment HA2k). In one embodiment, the linker sequence forms a loop (Embodiment HA21). In certain embodiments, the linker sequence is comprised of the following amino acid residues: valine, isoleucine, phenylalanine, cysteine, proline, glycine, and asparagine (Embodiment HA2m).
  • the linker sequence consists of one of the following amino acid sequences: VFPGCGV (SEQ ID NO: 5), VFPCGV (SEQ ID NO: 6), VFPGCV (SEQ ID NO: 7), CFNGIC (SEQ ID NO: 8), and VFPNCGV (SEQ ID NO: 9), wherein optionally a first cysteine residue in the linker sequence forms a disulfide bond with a second cysteine residue in the second stem region downstream of the C-terminus of the head region (Embodiment HA2n).
  • the linker sequence comprises less than 5 contiguous amino acids from the head region of an HA protein (Embodiment HA2o).
  • the second stem region includes a small portion of the HA1 polypeptide and the entire the HA2 polypeptide.
  • the HA2 polypeptide comprises two central helices (helix A and helix C) that form the majority of the stem backbone that extends from the transmembrane portion of the HA2 polypeptide to the head region.
  • a helix A sequence refers to a sequence from an influenza A (e.g., group 2 influenza virus, such as H3) HA protein that forms helix A in an HA2 polypeptide
  • a helix C sequence refers to a sequence from an influenza A (e.g., group 2 influenza virus, such as H3) HA protein that forms helix C in an HA2 polypeptide.
  • the HA2 polypeptide also includes an interhelical region of about 32 amino acids that connects helix A and helix C (“HA2 interhelical region”).
  • the HA2 polypeptide and HA2 interhelical region of the H3N2 strain A/Finland/486/2004 correspond to SEQ ID NO: 4 and amino acids 60-92 of SEQ ID NO: 4, respectively.
  • the “HA2 interhelical region” refers to a portion of the HA2 influenza polypeptide that connects helix A to helix C in a wild type HA protein. i. HA2 Interhelical Region
  • the stem region can improve stability.
  • shortening or deleting the HA2 interhelical region can improve the stability of the protein construct (Embodiment HA4a).
  • the HA2 interhelical region that is shortened or removed comprises amino acids 60-92 of SEQ ID NO: 4 or an analogous amino acid sequence from an influenza A virus other than A/Finland/486/2004 (Embodiment HA4b).
  • helix A with or without a helix A extension sequence
  • helix C amino acid sequences flanking this region can be joined together directly, or they can be joined with a linker sequence (Embodiment HA4c). It is not necessary to delete the entire HA2 interhelical region, so long as helix A and helix C are able to be joined and form the desired structure (Embodiment HA4d).
  • the HA2 interhelical region is “substantially removed” or “substantially replaced,” meaning that up to 5 amino acids of the HA2 interhelical region remain, such as 5, 4, 3, 2, 1, or 0 amino acid(s) of the HA2 interhelical region (Embodiment HA4e). In other embodiments, at least 90% of the HA2 interhelical region is removed (Embodiment HA4I). In certain embodiments, all amino acid residues of the HA2 interhelical region are removed (Embodiment HA4g). Any linker sequence may be used so long as the helix A and helix C sequences are able to form the desired structure (Embodiment HA4h).
  • Embodiment HA4i While any amino acids may be used to make the linker sequence, it is preferred to use amino acids lacking large or charged side chains (Embodiment HA4i). Preferred amino acids include, but are not limited to, serine, glycine, valine and alanine (Embodiment HA4j).
  • the linker sequences that replaces the HA2 interhelical region is a glycine-rich loop (Embodiment HA4k).
  • the linker is made from serine and glycine residues (Embodiment HA41).
  • the length of the linker sequence may vary, but preferred embodiments use the shortest possible sequence in order to allow the stem sequences to form the desired structure (Embodiment HA4m).
  • the linker sequence is less than 10 amino acids in length (Embodiment HA4n). In one embodiment, the linker sequence is less than 5 amino acids in length (Embodiment HA4o). In one embodiment, the linker sequence is 3-6 amino acids in length (Embodiment HA4p). In one embodiment, the linker sequence is 3-6 amino acids in length and includes at least two glycine residues (Embodiment HA4q). In one embodiment, the linker sequence comprises or consists of the amino acid sequence GSG (Embodiment HA4r). In one embodiment, the linker sequence consists of the amino acid sequence GGP or GGPD (SEQ ID NO: 10) (Embodiment HA4s). ii. Helix A Extension
  • the outer helix A in certain H3 strains is approximately 5 amino acids shorter at its C- terminus than in HI HA.
  • the stability of the protein construct can be improved by extending the length of helix A at its C-terminus, particularly for group 2 HA-ss constructs or H3-ss constructs (Embodiment HA5a).
  • the helix A extension sequence comprises amino acid residues with helix-forming propensities (Embodiment HA5b).
  • the helix A extension sequence is less than 10 amino acids in length (Embodiment HA5c).
  • the helix A extension sequence is 5 or fewer amino acids in length (Embodiment HA5d).
  • the helix A extension sequence comprises, or consists of, X1LMX2Q, or helix-forming variants thereof, wherein the amino acids at positions Xiand X2 are acidic amino acids, such as glutamic acid or aspartic acid (SEQ ID NO: 11; Embodiment HA5e). It should be noted that Xi and X2 can, but need not, be the same amino acid residue.
  • the helix A extension sequence comprises, or consists of, X1LMX2Q, or helix-forming variants thereof, wherein the amino acids at positions Xiand X2 are independently selected from the group consisting of glutamine, glutamic acid, asparagine, aspartic acid, glycine, and proline (SEQ ID NO: 12; Embodiment HA5f).
  • the helix A extension sequence comprises or consists of ALMAQ (SEQ ID NO: 13) or ELMEQ (SEQ ID NO: 14), or helix-forming variants thereof (Embodiment HA5g).
  • the helix A extension sequence comprises or consists of the amino acid sequence of ALMAQ (SEQ ID NO: 13) or ELMEQ (SEQ ID NO: 14) (Embodiment HA5h). iii. Mutations to HA2 Polypeptide
  • the stability of the protein construct can be further stabilized by one or more mutations (Embodiment HA6a).
  • the second stem region comprises one or more mutations (Embodiment HA6b).
  • the second stem region may contain one or more mutations that improve side chain repacking and/or resurfacing (Embodiment HA6c).
  • Repacking mutations generally refer to mutations at residues on the core of the protein (inside) to reduce or eliminate atomic voids that would cause structural instability. See e.g., Liang et ak, Biophysical Journal, 2001, 81:751-66.
  • Resurfacing mutations generally refers to mutations on the protein surface (outside) to reduce or eliminate issues such as hydrophobic patches that can make the protein sticky or prone to aggregation and, therefore, unstable. See e.g., Chapman et ak, Cell Chem Biol, 2016, 23(5):543-53. For example, after removal of the HA head a new surface would be generated (analogous to a wound) and this new surface could include hydrophobic amino acids that would normally be shielded from water in the wild type HA. Such residues can be replaced by others with more hydrophilic properties while conserving the protein structure.
  • the one or more mutations that improve side chain repacking and/or resurfacing are at residues K51 and/or El 03, where the amino acid numbering, by way of reference, is based on the HA2 polypeptide of the A/Finland/486/2004 H3N2 influenza virus (SEQ ID NO: 4) (Embodiment HA6d).
  • the one or more mutations that improve side chain repacking and/or resurfacing further comprise mutations at one or more of amino acid residues L52, L55, N95, T107, and/or N116, where the amino acid numbering, by way of reference, is based on the HA2 polypeptide of the A/Finland/486/2004 H3N2 influenza virus (SEQ ID NO: 4) (Embodiment HA6e).
  • the one or more mutations that improve side chain repacking and/or resurfacing are at amino acid residues K51, L52, L55, N95, E103, T107, and N116, where the amino acid numbering, by way of reference, is based on the HA2 polypeptide of A/Finland/486/2004 (SEQ ID NO: 4) (Embodiment HA61).
  • the one or more mutations that improve side chain repacking and/or resurfacing are K51M and/or E103L (Embodiment HA6g).
  • the one or more mutations that improve side chain repacking and/or resurfacing further comprises one or more of L52V, L55V, N95L, T107V, and/or N116R (Embodiment HA6h).
  • the one or more mutations that improve side chain repacking and/or resurfacing are K51M, L52V, L55V, N95L, E103L, E103L, T107V, and N116R (Embodiment HA6i).
  • the second stem region may also contain one or more mutations that stabilize the N- terminus of helix C (Embodiment HA7a).
  • the one or more mutations that stabilize the N-terminus of helix C increase the helix-forming propensities of helix C (Embodiment HA7b).
  • the one or more mutations that stabilize the N- terminus of helix C are at residues W92 and/or S93, where the amino acid numbering, by way of reference, is based on the HA2 polypeptide of A/Finland/486/2004 (SEQ ID NO: 4) (Embodiment HA7c).
  • the one or more mutations that stabilize the N- terminus of helix C are W92D, S93A, and/or S93C (Embodiment HA7d).
  • the one or more mutations that stabilize the N-terminus of helix C comprise is at residue W92, including, for example, W92D (Embodiment HA7e). In certain embodiments, the one or more mutations that stabilize the N-terminus of helix C comprise W92D and S93C (Embodiment HA7f).
  • the cysteine residue corresponding to amino acid residue 93 forms a disulfide bond with the cysteine residue in the linker sequence that joins the first and second stem regions, optionally where the linker sequence joining the first and second stem regions consists of one of the following amino acid sequences: VFPGCGV (SEQ ID NO: 5), VFPCGV (SEQ ID NO: 6), VFPGCV (SEQ ID NO: 7), and VFPNCGV (SEQ ID NO: 9) (Embodiment HA7g).
  • the modified HA stem region is from a group 2 influenza HA (Embodiment HA8a).
  • the group 2 influenza HA is from one of the currently recognized group 2 subtypes: H3, H4, H7, H10, H14, and HI 5 (Embodiment HA8b).
  • the group 2 influenza HA is an H3 HA (Embodiment HA8c).
  • the modified stem region is from the HA of one of the following H3 influenza strains (where 3 sequences are provided, they correspond to the full length hemagglutinin, HA1, and HA2 in that order): A/Finland/486/2004 (SEQ ID NOs 1, 2, and 4), A/Singapore/INFIMH- 16-0019/2017 (SEQ ID NOs 15-17), A/Hong Kong/4801/2014 (SEQ ID NOs 18-20), A/Victoria/361/2011 (SEQ ID NOs 21-23), A/Wisconsin/67/2005 (SEQ ID NOs 24-26), A/Shangdong/9/1993 (SEQ ID NOs 27-29), A/Port Chalmers/ 1/1973 (SEQ ID NOs 30-32), or A/Aichi/02/1968 (SEQ ID NOs 80-82) (Embodiment HA8d).
  • H3 influenza strains where 3 sequences are provided, they correspond to the full length hemagglutinin, HA1, and
  • the modified stem region is from the HA of one of the following H3 influenza strains: A/Denmark/35/2005 (SEQ ID NO: 33), A/Bangladesh/558/2012 (SEQ ID NO: 34), A/Sao Paulo/89403/2010 (SEQ ID NO: 35), A/Bangladesh/541/2012 (SEQ ID NO: 36), A/Bangladesh/542/2012 (SEQ ID NO: 37), A/Tocantins/979/2010 (SEQ ID NO: 38), A/Tunisia/17332/2011 (SEQ ID NO: 39), A/Norway/88/2003 (SEQ ID NO: 40), A/Japan/ AF2844/2012 (SEQ ID NO: 41), A/Texas/2977/2012 (SEQ ID NO: 42), A/North Carolina/AF2716/2011 (SEQ ID NO: 43), or A/Norway /70/2005 (SEQ ID NO: 44) (Embod
  • the group 2 influenza HA is an H7 HA (Embodiment HA8g).
  • the modified stem region is from the HA of one of the following H7 influenza strains: A/duck/Chiba/24-203-44/2012 (SEQ ID NO: 45), A/chicken/ Germany/2003 (SEQ ID NO: 46), A/chicken/Italy /444/1999 (SEQ ID NO: 47), A/mallard/Italy/4810-7/2004 (SEQ ID NO: 48), A/Anhui/1/2013 (SEQ ID NOs 49-51), A/Anhui/DEWH72-03/2013 (SEQ ID NO: 52), A/Shanghai/JS01/2013 (SEQ ID NO: 53), A/Guangdong/02/2013 (SEQ ID NO: 54), A/Shenzhen/ S P44/ 2014 (SEQ ID NO: 55), or A/Beijing/3/2013 (SEQ ID NO:
  • the group 2 influenza HA is an H10 HA (Embodiment HA8j).
  • the modified stem region is from an H10 influenza strain, including, but not limited to A/Jiangxi/IPB13b/2013 (SEQ ID NOs 58-60) (Embodiment HA8k).
  • the H10 strain is an H10N8 strain (Embodiment HA81).
  • Exemplary group 2 HA-ss protein constructs that can be used also include those disclosed in PCT International Publication No. WO2018/045308 and Corbett et ak, Therapeutics and Prevention, 2019, 10(1), 1-18, each of which is hereby incorporated by reference in its entirety (Embodiment HA8m).
  • the group 2 influenza HA is an H3 HA and the H3-ss comprises a first linker sequence that substantially replaces the head region of the H3 HA and a modified stem region from the H3 HA, wherein the modified stem region comprises at least one immunogenic epitope (Embodiment HA8n).
  • the H3-ss comprises a first HA stem region covalently joined to a second HA stem region by a first linker sequence, wherein the head region of the H3 HA has been substantially replaced by the first linker sequence, wherein the second stem region comprises a first portion comprising a helix A sequence, a helix A extension sequence at the C-terminus of the helix A sequence, a second linker sequence, and a second portion comprising a helix C sequence, wherein the second linker sequence covalently joins the helix A extension sequence and the helix C sequence and substantially replaces the HA2 interhelical region, wherein the helix A extension sequence extends the length of the helix A sequence by up to 5 amino acid residues, and wherein the second stem region comprises at least one first mutation that improves side chain repacking and/or resurfacing and at least one second mutation at the N-terminus of the helix C sequence that increases the helix-forming propens
  • the at least one first mutation comprises a mutation at K51, L52, L55, N95, E103, T107, and N116, wherein the amino acid numbering, by way of reference, is based on the HA2 polypeptide of A/Finland/486/2004 H3N2 influenza virus (SEQ ID NO: 4) (Embodiment HA9b).
  • the at least one second mutation comprises a mutation at W92 and S93, wherein the amino acid numbering, by way of reference, is based on the HA2 polypeptide of A/Finland/486/2004 H3N2 influenza virus (SEQ ID NO: 4) (Embodiment HA9c).
  • the at least one first mutation comprises a mutation at K51, L52, L55, N95, E103, T107, and N116 and the at least one second mutation comprises a mutation at W92 and S93, wherein the amino acid numbering, by way of reference, is based on the HA2 polypeptide of the A/Finland/486/2004 H3N2 influenza virus (SEQ ID NO: 4) (Embodiment HA9d).
  • the at least one first mutation comprises K51M, L52V, L55V, N95L, E103L, T107V, and N116R (Embodiment HA9e).
  • the at least one second mutation comprises W92D and S93C (Embodiment HA9f).
  • the at least one first mutation comprises K51M, L52V, L55V, N95L, E103L, T107V, and N116R and the at least one second mutation comprises W92D and S93C (Embodiment HA9g).
  • the first linker forms a loop and contains a first cysteine residue that forms a disulfide bond with a second cysteine residue in the second stem region (Embodiment HAlOa). In certain embodiments, the first linker consists of 6-7 amino acids (Embodiment HAlOb).
  • the first linker sequence consists one of the following amino acid sequences: VFPGCGV (SEQ ID NO: 5), VFPCGV (SEQ ID NO: 6), VFPGCV (SEQ ID NO: 7), CFNGIC (SEQ ID NO: 8), and VFPNCGV (SEQ ID NO: 9), wherein optionally a first cysteine residue in the linker sequence forms a disulfide bond with a second cysteine residue in the N-terminus of the helix C sequence in the second stem region (Embodiment HAlOc).
  • the second linker consists of 3-6 amino acids including at least two glycine residues (Embodiment HAlla). In certain embodiments, the second linker consists of the amino acid sequence GGP (Embodiment HA1 lb).
  • the helix A extension sequence consists of ALMAQ (SEQ ID NO: 13) or ELMEQ (SEQ ID NO: 14) (Embodiment HA12).
  • the H3 HA comprises a first HA stem region covalently joined to a second HA stem region by a first linker sequence, wherein the head region of the H3 HA has been substantially replaced by the first linker sequence, wherein the second stem region comprises a first portion comprising a helix A sequence, a helix A extension sequence at the C-terminus of the helix A sequence, a second linker sequence, and a second portion comprising a helix C sequence, wherein the first linker sequence consists of the amino acid sequence VFPGCGV (SEQ ID NO: 5), wherein a first cysteine residue in the linker sequence forms a disulfide bond with a second cysteine residue in the N-terminus of the helix C sequence in the second stem region, wherein the second linker sequence consists of the amino acid sequence GGP and covalently joins the helix A extension sequence and the helix C sequence, and substantially replaces the HA2 interhelical region, where
  • the H3-ss is from the A/Finland/486/2004 H3N2 strain (SEQ ID NO: 1) (Embodiment HA14a).
  • the HA-ss comprises the amino acid sequence (signal sequence in bold italics):
  • the H3-ss comprises the amino acid sequence (no signal sequence):
  • the modified HA sequence is linked to a multimerizing protein or portion thereof.
  • a multimerizing protein refers to a protein monomer that is capable of binding to other monomeric subunit proteins such that the monomeric subunit proteins self-assemble into a nanoparticle. Any monomeric subunit protein can be used to produce the protein construct, so long as the protein construct is capable of forming a multimeric structure displaying HA protein on its surface.
  • the multimerizing protein is ferritin. a. Ferritin
  • Ferritin is an iron storage protein found in almost all living organisms. Ferritin has been extensively studied and engineered for a number of potential bioeliemical/biomedical purposes [Iwahori, K. U.S. Patent 2009/0233377 (2009); Mel drum, F.C. et al. Science 257, 522-523 (1992); Naitou, M. et al. U.S. Patent 2011/0038025 (2011); Yamashita, I. Biochim BiophysActa 1800, 846-857 (2010)], including its use as a vaccine platform for displaying exogenous epitope peptides [Carter, D.C. et al. U.S. Patent 2006/0251679 (2006); Li, C.Q. et al.
  • Ferritin ’ s use as a vaccine platform is particularly interesting because of its self-assembly and multivalent presentation of antigen which induces stronger B cell responses than monovalent form as well as induce T-cell independent antibody responses [Bachmann, M.F. et al. Annu Rev Immunol 15, 235-270 (1997); Dintzis, H.M. et al. Proc Natl Acad Sci USA 73, 3671-3675 (1976)]. Further, the molecular architecture of ferritin, which consists of 24 subunits assembling into an octahedral cage with 432 symmetry can display multimeric antigens on its surface.
  • Ferritin genes are found in many species and generally show a conserved highly alpha- helical structure despite sequence variation. As such, any ferritin can be used in the fusion proteins described herein, including bacterial, insect, and human ferritin, despite its sequence identity to any particularly described ferritin.
  • the ferritin is bacterial, insect, fungal, bird, or mammalian (Embodiment Fla). In some embodiments, the ferritin is human, optionally with one or more mutations described herein (Embodiment Fib). In some embodiments, the ferritin is bacterial (Embodiment Flc), optionally with one or more mutations described herein. In some embodiments, the ferritin is H. pylori ferritin ( see SEQ ID NO: 65 for exemplary H. pylori ferritin sequences), optionally with one or more mutations described herein (Embodiment Fid).
  • the ferritin is Pyrococcus furiosus ferritin (NCBI seq WP_011011871.1; SEQ ID NO: 70), optionally with one or more mutations described herein (Embodiment Fie).
  • NCBI seq WP_011011871.1; SEQ ID NO: 70 Pyrococcus furiosus ferritin
  • Embodiment Fie The lower sequence homology between H. pylori ferritin (or other bacterial ferritins) and human ferritin may decrease the potential for autoimmunity when used as a vaccine platform (see Kanekiyo et al., Cell 162, 1090-1100 (2015)).
  • the ferritin is a light chain and/or heavy chain ferritin (Embodiment F2a).
  • the ferritin is human heavy chain ferritin (FTH1, GENE ID No: 2495; SEQ ID NO: 71) or human light chain ferritin (FTL, GENE ID No: 2512; SEQ ID NO: 72), optionally with one or more modifications described herein (Embodiment F2b).
  • the ferritin is Trichoplusia ni heavy chain ferritin (GenBank: AY970291.1; SEQ ID NO: 73) or Trichoplusia ni light chain ferritin (GenBank: AY970292.1; SEQ ID NO: 74), optionally with one or more mutations described herein (Embodiment F2c).
  • HA-ss-F protein constructs need not comprise the full-length sequence of a ferritin protein.
  • Portions, or regions, of the ferritin protein can be used as long as the portion comprises an amino acid sequence that directs self-assembly of monomeric ferritin subunits into the globular form of the protein (Embodiment 3a).
  • a region comprising N-terminal amino acids of the ferritin protein are removed (Embodiment 3b).
  • amino acids 1-4 of the wild typ Q Helicobacter pylori ferritin protein may be removed (see e.g., SEQ ID NOs: 66-69) (Embodiment 3c). More specific regions are described in Zhang, Y. 2011, Int. J. Mol. Sci., 12, 5406-5421, which is incorporated herein by reference in its entirety (Embodiment 3d).
  • the ferritin comprises a sequence having greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 97%, greater than 98%, or greater than 99% identity to a wild-type ferritin, including, but not limited to H. pylori ferritin, P. furiosus ferritin or human ferritin (Embodiment F4).
  • a ferritin monomer is a single ferritin molecule (or, where applicable, a single ferritin heavy or light chain) that has not assembled with other ferritin molecules.
  • Ferritin protein self- assembles into a globular protein complex comprising multiple individual monomers.
  • the self- assembled ferritin complex may be referred to as a ferritin particle or nanoparticle.
  • the ferritin comprises one or more mutations (Embodiment F5a).
  • the one or more mutations comprise changes to the amino acid sequence of a wild-type ferritin and/or an insertion, e.g., at the N- or C-terminus (Embodiment F5b).
  • one, two, three, four, five, or more different amino acids are mutated in the ferritin as compared to wild-type ferritin (in some embodiments, in addition to any N- terminal insertion) (Embodiment F5c).
  • a mutation simply refers to a difference in the sequence (such as a substituted, added, or deleted amino acid residue or residues) relative to the corresponding wild-type ferritin.
  • the ferritin is a H. pylori ferritin (see e.g., SEQ ID NO: 65 or 66) with one or more mutations described herein (Embodiment F5d).
  • N-glycans can result from glycosylation of asparagine side chains and can differ in structure between humans and other organisms such as bacteria and yeast. Thus, it may be desirable to reduce or eliminate non-human glycosylation and/or N-glycan formation in ferritin according to the disclosure. In some embodiments, controlling glycosylation of ferritin improves the efficacy and/or safety of the composition, especially when used for human vaccination.
  • ferritin is mutated to inhibit formation of an N-glycan (Embodiment F6a).
  • a mutated ferritin has reduced glycosylation as compared to its corresponding wild type ferritin (Embodiment F6b).
  • the ferritin comprises a mutation at an asparagine residue that is glycosylated in the wild type ferritin (Embodiment F6c).
  • the asparagine is N19 of H. pylori ferritin or a position that corresponds to N19 of H. pylori ferritin as determined by pair-wise or structural alignment (Embodiment F6d).
  • mutating such an asparagine, e.g., N 19 of H. pylori ferritin decreases glycosylation of ferritin (Embodiment F6e).
  • the mutation replaces the asparagine with a glutamine (Embodiment F6f).
  • the ferritin is an H. pylori ferritin (e.g., SEQ ID NO: 65 or 66) comprising an N19Q mutation (Embodiment F6g).
  • SEQ ID NOs 68 and 69 are exemplary ferritin sequences comprising aN19Q mutation (Embodiment F6h).
  • a mammal exposed to a glycosylated protein produced in bacteria or yeast may generate an immune response to the glycosylated protein, because the pattern of glycosylation of a given protein in bacterial or yeast could be different from the pattern of glycosylation of the same protein in a mammal. Thus, some glycosylated therapeutic proteins may not be appropriate for production in bacteria or yeast.
  • decreased glycosylation of ferritin by amino acid mutation facilitates protein production in bacteria or yeast (Embodiment F7a).
  • decreased glycosylation of ferritin reduces the potential for adverse effects in mammals upon administration of mutated ferritin that is expressed in bacteria or yeast (Embodiment F7b).
  • the reactogenicity in a human subject of a mutated ferritin produced in bacteria or yeast is lower because glycosylation is decreased (Embodiment F7c).
  • the incidence of hypersensitivity responses in human subjects is lower following treatment with a mutated ferritin with reduced glycosylation compared to wildtype ferritin (Embodiment F7d).
  • degradation in a subject of a composition comprising a mutated ferritin with reduced glycosylation is slower compared with a composition comprising a wild- type ferritin, or a composition comprising a corresponding ferritin with wild-type glycosylation (Embodiment F7e).
  • a composition comprising a mutated ferritin with reduced glycosylation has reduced clearance in a subject compared with a composition comprising a wild-type ferritin, or a composition comprising a corresponding ferritin with wild- type glycosylation (Embodiment F7f).
  • a composition comprising a mutated ferritin with reduced glycosylation has a longer-serum half-life compared to wild-type ferritin, or a composition comprising a corresponding ferritin with wild-type glycosylation (Embodiment F7g).
  • the ferritin comprises a H. pylori ferritin comprising the amino acid sequence:
  • the multimerizing protein is lumazine synthase or a portion thereof (Embodiment F9b).
  • the influenza HA-ss is joined to at least 50, at least 100 or least 150 amino acids from lumazine synthase, wherein the protein construct is capable of forming a nanoparticle (Embodiment F9c).
  • influenza HA-ss is joined to a protein at least 85%, at least 90%, at least 95%, or at least 98% identical to lumazine synthase, wherein the protein construct is capable of forming a nanoparticle (Embodiment F9d).
  • positions of mutations corresponding to those described with respect to a given polypeptide can be identified by pairwise or structural alignment.
  • Structural alignment is relevant to large protein families such as ferritin where the proteins share similar structures despite considerable sequence variation and many members of the family have been structurally characterized.
  • Structural alignment involves identifying corresponding residues across two (or more) polypeptide sequences by (i) modeling the structure of a first sequence using the known structure of the second sequence or (h) comparing the structures of the first and second sequences where both are known and identifying the residue in the first sequence most similarly positioned to a residue of interest in the second sequence. Corresponding residues are identified in some algorithms based on distance minimization in the overlaid structures (e.g., what set of paired alpha carbons provides a minimized root-mean-square deviation for the alignment). Structural modeling and alignment is well known in the art, as discussed, for example, in U.S. Patent Nos 6,859,736 and 8,738,343; Aslam et al., Journal of Biotechnology, 2016, 20:9-13 and Bordoli et al., Nature Protocols, 2009, (4): 1-13.
  • HA-ss-F protein constructs have been previously described.
  • PCT International Publication No. WO2018/0453008 which is hereby incorporated by reference in its entirety, describes in detail the production of group 2 HA-ss-F protein constructs and H3-ss-np formed when the protein constructs are expressed in cells. See also, Corbett et al., Therapeutics and Prevention, 2019, 10(1), 1-18, which is hereby incorporated by reference in its entirety. Any of these previously described group 2 HA-ss-F constructs can be used to produce the HA-ss-np used in the vaccine compositions described herein.
  • the HA-ss- np component of the vaccine composition can also be formed using any of the protein constructs described in this application, including any of the HA-ss-F protein constructs.
  • the modified stem region of the influenza HA and ferritin are genetically fused as a fusion protein.
  • a linker sequence connects the C-terminus of the second stem region to the N-terminus of the ferritin protein. Any linker sequence may be used so long as the HA-ss and ferritin are able to form the desired immunogenic nanoparticles. While any amino acids may be used to make the linker sequence, it is preferred to use amino acids lacking large or charged side chains. Preferred amino acids include, but are not limited to, serine, glycine, valine and alanine. In one embodiment, the linker is made from serine and glycine residues.
  • the glycine-serine linker is GS, SGG, GGGS (SEQ ID NO: 75), 2XGGGS (SEQ ID NO: 76), 3XGGGS (SEQ ID NO: 77), 4XGGGS (SEQ ID NO: 78), or 5XGGGS (SEQ ID NO: 79).
  • the length of the linker sequence may vary, but preferred embodiments use the shortest possible sequence in order to allow the stem sequences to form the desired structure.
  • the linker sequence is less than 10 amino acids in length.
  • the linker sequence is less than 5 amino acids in length.
  • the linker sequence is 3 amino acids in length.
  • the linker sequence consists of the amino acid sequence SGG.
  • the modified stem region of the influenza HA and ferritin may also be joined by other mechanisms, including non- genetically linked, for example, by chemical conjugation.
  • HA-ss Embodiments can be combined with any of the ferritin embodiments described herein or otherwise known in the art (“F Embodiments”) to generate a desired HA-ss-F protein construct that assemble into nanoparticles (HA-ss-np) expressing trimers of HA stem protein on their surface when expressed in cells.
  • F Embodiments ferritin embodiments described herein or otherwise known in the art
  • the HA-ss component of the HA-ss-F construct may be represented by any one of the HA-ss Embodiments described herein (e.g., HAla-g, HA2a-o, HA3, HA4a-s, HA5a-h, HA6a-i, HA7a-g, HA8a-n, HA9a-g, HAlOa-c, HAlla-b, HA12, HA13, and HA14a-c) or any combination of two or more of those HA Embodiments.
  • HA-ss Embodiments described herein e.g., HAla-g, HA2a-o, HA3, HA4a-s, HA5a-h, HA6a-i, HA7a-g, HA8a-n, HA9a-g, HAlOa-c, HAlla-b, HA12, HA13, and HA14a-c
  • ferritin component of the HA-ss-F construct may be represented by any one of the described F Embodiments described herein (e.g., Fla-c, F2a-c, F3a-d, F4, F5a-d, F6a-h, F7a-g, F8, F9a-d) or any combination of two or more of those F Embodiments.
  • any of the HA Embodiments or combinations thereof may be combined with any of the F Embodiments or combinations thereof.
  • Embodiment HA8n is combined with any of the F Embodiments described herein.
  • Embodiment HA8n can be combined with Embodiment Fid, F5d, or F8.
  • Embodiment HA9a is combined with any of the F Embodiments described herein.
  • Embodiment HA9a can be combined with Embodiment Fid, F5d, or F8.
  • any of Embodiments HA9b to HA9g, HAlOa to HAlOc, HAlla to HAllb, and/or HA12 or Embodiment HA9a further comprising any of Embodiments HA9b to HA9g , HAlOato HAlOc, HAlla to HAllb, and/or HA12 is combined with any of the F Embodiments described herein.
  • any of Embodiments HA9b to HA9g or Embodiment HA9a further comprising any of Embodiments HA9b to HA9g, HA12a to HA12c, HAlla to HAllb, and/or HA12 can be combined with Embodiment Fid, F5d, or F8.
  • Embodiment HA13 is combined with any of the F Embodiments described herein.
  • Embodiment HA13 can be combined with Embodiment Fid, F5d, or F8.
  • Embodiment HA14a is combined with any of the
  • Embodiment HA14a can be combined with Embodiment Fid, F5d, or F8.
  • the Embodiment HA14b or HA14c is combined with any of the F Embodiments described herein.
  • Embodiment HA14b or HA14c can be combined with Embodiment Fid, F5d, or F8.
  • the FG-ss-F comprises the amino acid sequence (signal sequence in bold italics):
  • the FG-ss-F comprises the amino acid sequence (no signal sequence):
  • HA-ss-F nanoparticles described herein are administered with a squalene- based adjuvant emulsion, as described in PCT International Publication No. W02007/006939 and U.S. Patent No. 8,703,095, which are hereby incorporated by reference in their entireties.
  • a squalene- based adjuvant emulsion as described in PCT International Publication No. W02007/006939 and U.S. Patent No. 8,703,095, which are hereby incorporated by reference in their entireties.
  • such an emulsion can be obtained by means of a phase inversion temperature process, which permits the production of a monodisperse emulsion, the droplet size of which is very small, thus permitting the emulsion to be filtered with sterilizing filters having a cutoff threshold of 200 nm.
  • the squalene-based adjuvant comprises an oil-in-water adjuvant emulsion comprising at least: squalene, an aqueous solvent, a polyoxyethylene alkyl ether hydrophilic nonionic surfactant, and a hydrophobic nonionic surfactant.
  • the emulsion is thermoreversible, optionally wherein 90% of the population by volume of the oil drops has a size less than 200 nm.
  • the polyoxyethylene alkyl ether is of formula CFE- (CH 2 ) x -(0-CH 2 -CH 2 )n-0H, in which n is an integer from 10 to 60, and x is an integer from 11 to 17.
  • the polyoxyethylene alkyl ether surfactant is polyoxyethylene(12) cetostearyl ether.
  • 90% of the population by volume of the oil drops has a size less than 160 nm. In certain embodiments, 90% of the population by volume of the oil drops has a size less than 150 nm. In certain embodiments, 50% of the population by volume of the oil drops has a size less than 100 nm. In certain embodiments, 50% of the population by volume of the oil drops has a size less than 90 nm.
  • the adjuvant further comprises at least one alditol, including, but not limited to, glycerol, erythritol, xylitol, sorbitol and mannitol.
  • the hydrophilic/lipophilic balance (HLB) of the hydrophilic nonionic surfactant is greater than or equal to 10. In certain embodiments, the HLB of the hydrophobic nonionic surfactant is less than 9. In certain embodiments, the HLB of the hydrophilic nonionic surfactant is greater than or equal to 10 and the HLB of the hydrophobic nonionic surfactant is less than 9.
  • the hydrophobic nonionic surfactant is a sorbitan ester, such as sorbitan monooleate, or a mannide ester surfactant.
  • the amount of squalene is between 5 and 45%.
  • the amount of polyoxyethylene alkyl ether surfactant is between 0.9 and 9%.
  • the amount of hydrophobic nonionic surfactant is between 0.7 and 7%.
  • the adjuvant comprises: i) 32.5% of squalene, ii) 6.18% of polyoxyethylene(12) cetostearyl ether, iii) 4.82% of sorbitan monooleate, and iv) 6% of mannitol.
  • the adjuvant further comprises an alkylpolyglycoside and/or a cryoprotective agent, such as a sugar, in particular dodecylmaltoside and/or sucrose.
  • a cryoprotective agent such as a sugar, in particular dodecylmaltoside and/or sucrose.
  • the adjuvant comprises AF03, as described in Klucker et ak, J. Pharm. Sci. 2012, 101(12):4490-500, which is hereby incorporated by reference in its entirety.
  • the present disclosure provides vaccine compositions comprising HA-ss-np, as described herein, in combination with a squalene-based oil-in-water adjuvant emulsion, such as AF03, as described herein.
  • administration of the vaccine composition induces broadly neutralizing antibodies in non-human primates.
  • the vaccine composition may also include other pharmaceutically acceptable excipients.
  • excipients in general, the nature of the excipient will depend on the particular mode of administration being employed.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • solid compositions for example, powder, pill, tablet, or capsule forms
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • vaccine compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pharmaceutically acceptable salts to adjust the osmotic pressure, preservatives, stabilizers, buffers, sugars, amino acids, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • auxiliary substances such as wetting or emulsifying agents, pharmaceutically acceptable salts to adjust the osmotic pressure, preservatives, stabilizers, buffers, sugars, amino acids, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • the vaccine composition is a sterile, liquid solution formulated for parenteral administration, such as intravenous, subcutaneous, intraperitoneal, intradermal, or intramuscular.
  • parenteral administration such as intravenous, subcutaneous, intraperitoneal, intradermal, or intramuscular.
  • the vaccine composition may also be formulated for intranasal or inhalation administration.
  • the vaccine composition can also be formulated for any other intended route of administration.
  • a vaccine composition is formulated for intradermal injection, intranasal administration or intramuscular injection.
  • injectables are prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • injection solutions and suspensions are prepared from sterile powders or granules. General considerations in the formulation and manufacture of pharmaceutical agents for administration by these routes may be found, for example, in Remington ’s Pharmaceutical Sciences, 19 th ed., Mack Publishing Co., Easton, PA, 1995; incorporated herein by reference. At present the oral or nasal spray or aerosol route ( e.g .
  • the vaccine composition is administered using a device that delivers a metered dosage of the vaccine composition.
  • Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices such as those described in U.S. Patent No. 4,886,499, U.S. Patent No. 5,190,521, U.S. Patent No. 5,328,483, U.S. Patent No. 5,527,288, U.S. Patent No. 4,270,537, U.S. Patent No. 5,015,235, U.S. Patent No. 5,141,496, U.S. Patent No. 5,417,662 (all of which are incorporated herein by reference).
  • Intradermal compositions may also be administered by devices which limit the effective penetration length of a needle into the skin, such as those described in WO1999/34850, incorporated herein by reference, and functional equivalents thereof.
  • jet injection devices which deliver liquid vaccines to the dermis via a liquid jet injector or via a needle which pierces the stratum comeum and produces a jet which reaches the dermis. Jet injection devices are described for example in U.S. Patent No. 5,480,381, U.S. Patent No. 5,599,302, U.S. Patent No. 5,334,144, U.S. Patent No. 5,993,412, U.S. Patent No. 5,649,912, U.S. Patent No. 5,569,189, U.S. Patent No.
  • Preparations for parenteral administration typically include sterile aqueous or nonaqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti- oxidants, chelating agents, and inert gases and the like.
  • the present disclosure provides methods of administering the vaccine compositions described herein to a subject.
  • the methods may be used to vaccinate a subject against an influenza virus.
  • the vaccination method comprises administering to a subject in need thereof an immunologically effective dose of a vaccine composition comprising HA-ss-np, as described herein, and a squalene-based oil-in-water adjuvant emulsion, such as AF03, as described herein, wherein administration of the vaccine composition elicits broadly neutralizing influenza virus antibodies in the subject.
  • the present disclosure provides a vaccine composition comprising HA-ss-np, as described herein, and a squalene-based oil-in-water adjuvant emulsion, such as AF03, as described herein, for use in vaccinating a subject against an influenza virus.
  • a vaccine composition comprising HA-ss-np, as described herein, and a squalene-based oil-in-water adjuvant emulsion, such as AF03, as described herein, for use in vaccinating a subject against an influenza virus.
  • the bnAbs bind the stem region and neutralize influenza viruses from two or more of the following sub-types: HI, H2, H4, H5, H6, H7, H8, H8, H10, Hll, H12, H13, H14, H15, H16, H17 or H18.
  • the bnAbs bind the stem region and neutralize influenza viruses from two or more of the following group 2 sub-types: H3, H4, H7, H10, H14, or H15.
  • the bnAbs bind the stem region and neutralize influenza viruses from one or more of the following group 1 sub-types: HI, H2, H5, H6, H8, H9, Hll, H12, H13, H16, H17 or H18 and one or more ofthe following group 2 sub-types: H3, H4, H7, H10, H14, or H15.
  • the bnAbs bind the stem region and neutralize H3 influenza strains and one or more group 2 influenza strains selected from one or more of H4, H7, H10, H14, and HI 5 influenza strains.
  • the bnAbs bind the stem region and neutralize at least one H3 influenza strain and at least one H7 strain (e.g., AJ Anhui/1/2013) or H10 strain (e.g., A/Jiangxi/IPB13b/2013). In certain embodiments, the bnAbs bind the stem region and neutralize at least one H3, H7 (e.g., A/Anhui/1/2013), and H10 (e.g., A/Jiangxi/IPB13b/2013) influenza strain.
  • H7 strain e.g., AJ Anhui/1/2013
  • H10 strain e.g., A/Jiangxi/IPB13b/2013
  • Neutralization can be measured using known techniques, including the neutralization assays described herein.
  • the bnAbs bind the stem region and neutralize two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all nine of the following group 2 strains: A/Singapore/INFIMH-16-0019/2017 (H3N2), A/Hong Kong/4801/2014 (H3N2), A/Victoria/361/2011 (H3N2), A/Wisconsin/67/2005 (H3N2), A/Shangdong/9/ 1993 (H3N2), A/Port Chalmers/1/1973 (H3N2), A/Ai chi/02/1968, A/Jiangxi/IPB13b/2013 (H10N8), A/Jiangxi-Donghu/346-2/2013 (H10N8) and A/Anhui/1/2013 (H7N9).
  • the bnAbs bind the stem region and neutralize two or more, three or more, four or more, five or more, or all six of the following H3 strains: A/Singapore/INFIMH- 16-0019/2017, A/Hong Kong/4801/2014,
  • A/Victoria/361/2011 A/Wisconsin/67/2005, A/Shangdong/9/1993, A/Port Chalmers/1/1973, and A/Aichi/02/1968.
  • the bnAbs bind the stem region and neutralize influenza viruses having an HA2 polypeptide that shares at least 60% identity with the wild type HA2 polypeptide from A/Finland/486/2004 (SEQ ID NO: 4), including, for example, any of A/Singapore/INFIMH- 16-0019/2017, A/Hong Kong/4801/2014, A/Victoria/361/2011, A/Wisconsin/67/2005, A/Shangdong/9/1993, A/Port Chalmers/1/1973, A/Aichi/02/1968, A/Jiangxi/IPB 13b/2013, and, A/Anhui/1/2013, which share 96.83%, 98.19%, 98.64%, 100%, 97.28%, 95.92%, 94%, 63.34%, and 63.80%, respectively, with the wild type HA2 polypeptide from A/Finland/486/2004 (SEQ ID NO: 4).
  • the bnAbs bind the stem region and neutralize influenza viruses having an HA2 polypeptide that shares at least 63%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identity with the wild type HA2 polypeptide from A/Finland/486/2004 (SEQ ID NO: 4). In certain embodiments, the bnAbs bind the stem region and neutralize influenza viruses having an HA2 polypeptide that shares at least 94% identity with the wild type HA2 polypeptide from A/Finland/486/2004 (SEQ ID NO: 4).
  • the bnAbs elicit neutralization of divergent H3N2 strains from at least 1968 to 2016, as measured by an in vitro functional microneutralization reporter assay as described herein.
  • the divergent H3N2 strains comprise A/Singapore/INFIMH- 16-0019/2017, A/Hong Kong/4801/2014, A/Victoria/361/2011, A/Wisconsin/67/2005, A/Shangdong/9/1993, A/Port Chalmers/1/1973, and A/Aichi/02/1968.
  • the vaccine composition elicits an average antibody titer of at least 4 (loglO) to the HA protein from both the H3 strain A/Perth/16/2009 and the HA protein from the H10 strain A/Jiangxi/IPB13b/2013 when 50 pg of the vaccine composition is administered to cynomolgus macaques at weeks 0, 4, and 10, and wherein the antibody titer is measured by ELISA in sera samples obtained from the cynomolgus macaques at week 12.
  • Vaccine compositions comprising HA-ss-np, as described herein, and a squalene-based oil-in-water adjuvant emulsion, such as AF03, as described herein may be administered prior to or after development of one or more symptoms of an influenza infection. That is, in some embodiments, the vaccine compositions described herein may be administered prophylactically to prevent influenza infection or ameliorate the symptoms of a potential influenza infection.
  • a subject is at risk of influenza virus infection if the subject will be in contact with other individuals or livestock (e.g., swine) known or suspected to have been infected with pandemic influenza virus and/or if the subject will be present in a location in which influenza infection is known or thought to be prevalent or endemic.
  • the vaccine compositions are administered to a subject considered to be suffering from an influenza infection, or the subject is displaying one or more symptoms commonly associated with influenza infection.
  • the subject is known or believed to have been exposed to an influenza virus.
  • a subject is considered to be at risk or susceptible to an influenza infection if the subject is known or believed to have been exposed to the influenza virus.
  • a subject is known or believed to have been exposed to the influenza virus if the subject has been in contact with other individuals or livestock (e.g., swine) known or suspected to have been infected with pandemic influenza virus and/or if the subject is or has been present in a location in which influenza infection is known or thought to be prevalent or endemic.
  • Vaccine compositions in accordance with the disclosure may be administered in any dose appropriate to achieve a desired outcome.
  • the desired outcome is induction of a lasting adaptive immune response against a broad spectrum of influenza strains, including both seasonal and pandemic strains.
  • the desired outcome is reduction in intensity, severity, and/or frequency, and/or delay of onset of one or more symptoms of influenza infection.
  • the dose required may vary from subject to subject depending on the species, age, weight and general condition of the subject, the severity of the infection being treated, the particular composition being used and its mode of administration.
  • the vaccine compositions described herein are administered to a human subject.
  • a human subject is 6 months of age or older, is 6 months through 35 months of age, is 36 months through 8 years of age, or 9 years of age or older.
  • the human subject is an infant (less than 36 months).
  • the human subject is a child or adolescent (less than 18 years of age).
  • the human subject is elderly (at least 60 years of age).
  • the human subject is a non-elderly adult (at least 18 years of age and less than 60 years of age).
  • the methods and uses of the vaccine compositions described herein include prime-boost vaccination strategies.
  • Prime-boost vaccination comprises administering a priming vaccine and then, after a period of time has passed, administering to the subject a boosting vaccine.
  • the immune response is “primed” upon administration of the priming vaccine, and is “boosted” upon administration of the boosting vaccine.
  • the priming vaccine can include a vaccine composition comprising HA-ss-np, as described herein, and a squalene- based oil-in- water adjuvant emulsion, such as AF03, as described herein.
  • the boosting vaccine can include a vaccine composition comprising HA-ss-np, as described herein, and a squalene-based oil-in-water adjuvant emulsion, such as AF03, as described herein.
  • the priming vaccine composition can be, but need not be, the same as the boosting vaccine.
  • Administration of the boosting vaccine is generally weeks or months after administration of the priming composition, preferably about 2-3 weeks or 4 weeks, or 8 weeks, or 16 weeks, or 20 weeks, or 24 weeks, or 28 weeks, or 32 weeks.
  • the vaccine composition can be administered using any suitable route of administration, including, for example, parenteral delivery, as discussed above.
  • the HA-ss-np and adjuvant are administered together as components of the same vaccine composition.
  • the HA-ss-np and adjuvant it is not necessary for the HA-ss-np and adjuvant to be administered as part of the same vaccine composition. That is, if desired, the HA-ss-np and the squalene-based oil-in-water adjuvant emulsion, such as AF03, can be administered to the subject sequentially.
  • NHP non-human primates
  • bnAbs broadly reactive neutralizing antibodies
  • H3-SS-np was designed from A/Finland/486/2004 (H3N2) HA template sequences and was prepared and purified as described in Corbett et ak, Therapeutics and Prevention, 2019, 10(1), 1-18.
  • the H3-ss-F fusion protein comprises the amino acid sequence of SEQ ID NO: 61. When expressed in cells, the signal sequence is cleaved resulting in the amino acid sequence of SEQ ID NO: 62, which forms uniform nanoparticles.
  • the ectodomain of the HAs from A/Perth/16/2009 (H3N2) and A/Jiangxi/IPB13b/2013 (H10N8) were fused to C-terminal Thrombin cleavage site, followed by the trimeric foldon domain of T4 fibritin and a hexahistidine tag sequence (SEQ ID NO: 83). Plasmids were purified with the Powerprep kit (Origene #NP100009) and used to transfect Expi293 cells (Thermo Fisher Scientific #A14635).
  • FectoPRO DNA transfection reagent (Polyplus #116-100) was used (0.5 pg of DNA per mL, 0.75 pL of FectoPRO reagent per mL, and 0.45 pL of enhancer per mL).
  • supernatant was harvested by centrifugation at 3,488 g for 15 min at 4 °C and filtered through a 0.45 pm vacuum-driven filter unit (Thermo Fisher Scientific #167-0045).
  • Foldon-trimerized antigens used for in vitro assays were purified with Nickel Sepharose Excel resin (GE Healthcare, catalog# 17371201) in 50 mM Tris buffer pH 7.5 with 300 mM NaCl and eluted with 150 mM Imidazole.
  • HA trimers were purified by size-exclusion chromatography using a Superdex 200 10/300 column (GE catalog# 28990944) in phosphate- buffered saline.
  • B/Brisbane/60/2008 by ELISA.
  • age of the monkeys ranged from 6 to 15 years old, and body weight ranged between 4 and 7 kg.
  • Subjects received three doses (50 pg each) of vaccine with H3-SS-np at weeks 0, 4 and 10 with AF03 adjuvant. Serum samples were collected at weeks 0, 2, 4, 6, 8, 10 and 12. PBMC were collected and isolated at weeks 0, 2, 5, 6 and 12. Serum and PBMCs were isolated and cryopreserved following standard operating procedures.
  • Antibody responses were measured by Enzyme-Linked Immunosorbent Assay (ELISA).
  • ELISA assays serum was heat-inactivated at 65 °C for 30 min.
  • Nunc MaxiSorp 96-well plates (Thermo Fisher Scientific # 44-2404-21) were coated with 100 ng of antigen per well overnight at 4 °C.
  • Antigens included HA-foldon proteins from A/P erth/16/2009 (H3N2) wildtype or ASteml(I45N, Q47T) or AStem2 (D19N, G33E) and A/Jiangxi/IPB13/2013 (H10N8).
  • NHP sera or recombinant monkey antibodies were serially-diluted in 5% milk-PBST and allowed to bind for 1 hour at room temperature. Binding to the antigens were detected with anti-monkey-HRP (Southern Biotech catalog# 4700-05, at 1:5,000) or anti-human-HRP (Jackson H+L anti-human IgG secondary catalog#709-035-149, at 1:5,000). HRP was developed with SureBlueTMB substrate (Seracare # 52-00-02). Absorbance was measured at 450 nm in a Spectramax instrument. Endpoint titers were calculated with Graphpad Prism with a threshold value of 0.2 and typical background level of 0.05 or 0.1.
  • H3-SS-foldon, wildtype (A/Perth/16/2009 (H3N2)) or Astern (D19N, G33E) mutant proteins (1 pg) were pre-incubated with varying amount of serum for 1 hour before lentivirus was added.
  • MN microneutralization
  • Binding antibodies were detected to both the homologous H3 (A/Perth/16/2009) strain (Fig. 1A, left) and a heterosubtypic H10 (A/Jiangxi-Donghu/346-2/2013) strain (Fig. 1A, right).
  • the group 2 stabilized HA stem vaccine composition stimulated neutralizing Abs to the group 2 HA stem epitope as the activity was absorbed by wild type H3 HA but not by Astern mutant (Fig. IB; stem vs. Astern competitor).
  • this vaccine induced broad H3 reactivity, neutralizing divergent H3N2 strains from 1968 to 2016 (Fig. 1C).
  • PBMCs from immunized monkeys were isolated following standard methodology, using Leucosep 50 mL tubes (VWR#89048-938) with a 95% Ficoll-Paque Plus density gradient medium (GE Healthcare #17-1440). Isolated PBMCs were cryopreserved using commercial freezing media (Gibco catalog# 12648-010). On the day of single cell sorting, PBMC aliquots were thawed, washed with RPMI media (Gibco/Invitrogen, 11875-093), and treated with 50 units per mL of Benzonase (EMD Millipore #70664-3) before staining.
  • Antibody cocktails were prepared in PBS-1% BSA (Bovine Albumin fraction V (BSA) from Fisher Bioreagents, catalog# BP 1600- 100).
  • BSA Bovine Albumin fraction V
  • the following antibody markers were used: CD20- FITC (BD Biosciences, catalog #347673), CD3-PerCP-Cy5.5 (BD Biosciences, catalog #552852), IgD-PE (Southern Biotech, catalog #2030-09), CD8-PE Texas Red (Invitrogen, catalog #MHCD0817), IgM-BV786 (BD Biosciences catalog #740998), CD 16- PE-Cy7 (BD Biosciences, catalog #557744), CD14-BV650 (BioLegend, catalog #301836), IgG-BV605 (BD Biosciences catalog #563246), and CD27- APC-Cy7 (BioLegend catalog #302816).
  • H3 (2009) and H10, or H3 (1968) and H3 (2011) fluorescent probes were used at 0.5 pg of HA per 5-20 million cells. Viability was assessed with Aqua stain (Invitrogen, catalog #L34957).
  • HA+ IgG+ B cells were isolated by single-cell sorting on a BD Influx cell sorter gating for either H3+, H10+, or H3+/H3+ as indicated.
  • PBMCs were gated sequentially for forward and side scatter, viability, singlet cell dispersion, CD 16 negative, CD 14 negative, CD3 negative, CD20 positive, IgD negative, IgM negative, IgG positive, HA positive gates.
  • the bnAbs elicited by the H3-SS-np + AF03 vaccine were isolated using two screening protocols and sequenced. In one screening protocol, bnAbs were sorted using a single H3 HA probe (H3+) derived from A/Perth/16/2009 (Fig. 2A). In a second screening protocol, bnAbs were counter sorted using two H3 HA probes (H3+/H3+) derived from divergent strains, A/Hong Kong/1/1968 and A/Victoria/361/2011 (Fig. 2B).
  • bnAbs antibodies were derived from diverse VH-DH-JH germline gene combinations, with 75% of the heavy chain sequences derived from the IGHV4 family, and >60% using the IGHJ4 gene (Figs. 2A-B).
  • human anti-H3 stem antibodies are derived from diverse families, including IGHV4-30 and IGHV4-34, but without preferential IGHV gene usage (M. G. Joycen et al., Cell 166, 609-623 (2016)).
  • H3+ or H3+/H3+ light chains were also derived from a diverse combination of VJ germline genes, with >40% of lambda chains derived from IGLV1-15 and >25% of Kappa chains derived from IGKV1S26 (Figs. 3A-B).
  • H3+ immune B cells from NHP immunized with the vaccine composition were sorted and 20 mAbs characterized (Table 1). Sixteen mAbs bound to H3 HA, and 8 reacted with both H3 and H10 HA (Table 1). Ten mAbs neutralized at least two H3 influenza strains, and 7 of these had Pan-H3 activity, since they neutralized all strains tested, spanning viral isolates derived from 1973 to 2014 (Fig. 4A). Furthermore, 6 mAbs cross-neutralized a heterosubtypic H10 influenza strain and two mAbs neutralized an H7 subtype strain (Fig. 4A). Binding of these mAbs to H3 HA was affected by AStem (D19N/G33E) mutations on HA stem (Fig. 4B and Fig. 6), indicating that they were also HA stem directed.
  • the CDR-H1 and CDR-H2 made contacts with the N-terminus of the fusion peptide through van der Waals and hydrogen-bonding interactions, while CDR-H3 largely contacted the C-terminus of the fusion peptide through additional cation-p interactions (R25 HA2 -W101 3B10 - cdr'H3 ) (Fig. 7A- C).
  • CDR-H3 largely contacted the C-terminus of the fusion peptide through additional cation-p interactions (R25 HA2 -W101 3B10 - cdr'H3 ) (Fig. 7A- C).
  • Fig. 7D The epitope of 3B 10 overlay well with that of another human monoclonal antibody, CR8020 (Fig. 5B).
  • a broadly protective vaccine is desired to effectively control seasonal influenza infection and to avert pandemic outbreaks.
  • pan-H3 broadly neutralizing responses can be achieved by immunization of cynomolgus macaques using engineered stabilized stem nanoparticle vaccines with a squalene-based, oil-in-water adjuvant emulsion adjuvant, like AF03.
  • the mAbs derived from immune NHP resembled human bnAbs prototypes induced by infection or vaccination, with similar potency and binding specificity.
  • the CDR-H3 of 3B10 also plays an important role in recognizing the HA stem epitope.
  • a hallmark of influenza outbreaks is the ability of the virus to give rise to new strains quickly and unpredictably (H. V. Fineberg, /VE/M, 2014, 370:1335-1342).
  • Stem-based universal vaccines have the potential to limit the global spread of new strains because they can be deployed before pandemics begin, and the immune response is directed to a conserved structure, reducing the chances of immune escape (K. E. Neu et ah, Current opinion in immunology , 2016, 42:48-55).
  • immunogens that present the conserved stem epitope have been designed, as previously discussed herein.
  • the structural superimposition of the vaccine-induced macaque H3-3B10 to human CR8020 demonstrates with atomic resolution that broadly neutralizing anti-stem antibodies can be elicited through diverse antibody germline genes in antigen-naive non-human primates by vaccination with a composition comprising a rationally-designed HA stem immunogen and a squalene-based, oil-in-water adjuvant emulsion adjuvant, like AF03, which composition can be developed and deployed in humans.
  • Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.
  • the disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process.
  • the disclosure also includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.

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Abstract

Provided are vaccine compositions comprising a squalene-based adjuvant emulsion, like AF03, and a stem-stabilized, group 2 influenza hemagglutinin immunogen without the immunodominant head region and presented on nanoparticles derived from a protein with self-assembling multimerization properties, the vaccine compositions eliciting broadly neutralizing antibody responses to diverse influenza viruses. Also provided are methods of using the vaccine compositions to vaccinate a subject against influenza virus, wherein administration of the vaccine composition elicits broadly neutralizing influenza antibodies in the subject.

Description

ADJUVANTED STABILIZED STEM HEMAGGLUTININ NANOPARTICLES AND METHODS OF USING THE SAME TO INDUCE BROADLY NEUTRALIZING ANTIBODIES AGAINST INFLUENZA
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of, and relies on the filing date of, U.S. provisional patent application number 63/023,978, filed 13 May 2020, and U.S. provisional patent application number 63/071,244, filed 27 August 2020, the entire disclosures of which are herein incorporated by reference
GOVERNMENT INTEREST
[0002] This invention was created in the performance of a Cooperative Research and Development Agreement with the National Institutes of Health, an Agency of the Department of Health and Human Services. The Government of the United States has certain rights in this invention.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 13, 2021, is named 0171_0019-PCT_SL.txt and is 208,218 bytes in size.
BACKGROUND
[0004] Influenza is caused by a virus that attacks mainly the upper respiratory tract - the nose, throat and bronchi and rarely also the lungs. The infection usually lasts for about a week. It is characterized by sudden onset of high fever, myalgia, headache and severe malaise, nonproductive cough, sore throat, and rhinitis. Most people recover within one to two weeks without requiring any medical treatment. However, in the very young, the elderly and people suffering from medical conditions, such as lung diseases, diabetes, cancer, kidney or heart problems, influenza poses a serious risk. In these people, the infection may lead to severe complications of underlying diseases, pneumonia and death. Annual influenza epidemics are thought to result in between three and five million cases of severe illness and between 250,000 and 500,000 deaths every year around the world.
[0005] Influenza virus is a member of Orthomyxoviridae family. There are three main subtypes of influenza viruses, designated influenza A, influenza B, and influenza C. The influenza virion contains a segmented negative-sense RNA genome, which encodes the following proteins: hemagglutinin (HA), neuraminidase (NA), matrix (Ml), proton ion-channel protein (M2), nucleoprotein (NP), polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), polymerase acidic protein (PA), and nonstructural protein 2 (NS2). The HA, NA, Ml, and M2 are membrane associated, whereas NP, PB1, PB2, PA, and NS2 are nucleocapsid associated proteins. The Ml protein is the most abundant protein in influenza particles. The HA and NA proteins are envelope glycoproteins, responsible for virus attachment and penetration of the viral particles into the cell. Specifically, HA binds the influenza virus to host cells with sialic acid-containing on surface structures on their membranes.
[0006] Both HA and NA proteins are the sources of the major immunodominant epitopes for virus neutralization and protective immunity, making them important components for prophylactic influenza vaccines. The genetic makeup of influenza viruses allows frequent minor genetic changes, known as antigenic drift. Thus, the amino acid sequence of the major antigens of influenza, particularly HA, is highly variable across groups, subtypes and strains. For this reason, current seasonal influenza vaccines must be administered every year and require yearly updates to account for mutations in HA and NA proteins (antigenic drift) and to match rapidly-evolving viral strains. HI and H3 subtypes account for about 75% of the confirmed influenza infections (M. Tafalla et al., Human Vaccines & Immunotherapeutics , 2016, 12:993-1002). H3 subtypes are most frequently revised in seasonal vaccines as they harbor the highest sequence variability (J. D. Allen et al., Human Vaccines & Immunotherapeutics, 2018, 14:1840-1847). Seasonal influenza vaccines confer protection against specific viral strains but have restricted breadth that limit their protective efficacy. Furthermore, the influenza strains used in the vaccine are selected by the WHO/CDC based on the agencies’ best guess as to the prevalent influenza strains for the upcoming flu season. Often times, the guess is not accurate and the vaccine strains do not match the seasonal influenza strains, limiting the effectiveness of the seasonal vaccines. Seasonal vaccines are also not designed to provide protection against pandemic strains that can result from antigen shift.
[0007] Pandemic outbreaks of influenza are caused by the emergence of a pathogenic and transmissible virus to which the human population is immunologically naive. Because the virus is new, the human population has little to no immunity against it. The virus spreads quickly from person-to-person worldwide. Three times in the last century, the influenza A viruses have undergone major genetic changes mainly in their HA-component, resulting in global pandemics and large tolls in terms of both disease and deaths. HI and H3 are the major seasonal subtypes that have caused pandemics (E. D. Kilboume et al., Emerging infectious diseases, 2006, 12:9-14). The most infamous pandemic was the “Spanish Flu” which affected large parts of the world population and is thought to have killed at least 40 million people in 1918-1919. More recently, two other influenza A pandemics occurred in 1957 (“Asian influenza”) and 1968 (“Hong Kong influenza”) and caused significant morbidity and mortality globally. In contrast to current influenza epidemics, these pandemics were associated with severe outcomes also among healthy younger persons, albeit not on such a dramatic scale as the “Spanish flu” where the death rate was highest among healthy young adults. In 2009, a descendant from the 1918 influenza virus, named (HlNl)pdm09 virus was transmitted from swine to humans (CJ Wei et al., Science Translational Medicine, 2010, 2(24):24ra21) and spread from person-to-person worldwide, causing the 2009 H1N1 pandemic which infected over 60 million people in one year (www.cdc.gov/flu/pandemic-resources/2009-hlnl- pandemic.html). Swine influenza has also been associated with influenza A subtypes H1N2, H2N3, H3N1, and H3N2. Due to antigenic drift, and even more dramatic alterations known as antigenic shift, pandemic influenza antigens (e.g., the HA amino acid sequence of the pandemic strain) are highly unpredictable. Thus, vaccines have traditionally been unavailable until the later stages of a pandemic.
[0008] A substantial factor underlying the low efficacy of influenza vaccines, observed in many seasons, lies in the tendency of the virus to mutate through error-prone replication, which allows the virus to evade the binding of neutralizing antibodies in vaccinated individuals. The existence of conserved epitopes on the influenza virus suggests a possible path towards an influenza vaccine that can resist viral drift. However, in order to elicit antibodies against these conserved epitopes, recombinant protein engineering is required to present these epitopes in a favorable manner to the immune system. For example, when full-length HA is used as an immunogen, the antibodies elicited are predominantly those that bind to the highly-variable, immunodominant head domain. A strategy has been devised to induce antibodies to the subdominant highly-conserved stem region and involves removing the head region of HA, stabilizing the remaining HA stem regions through protein engineering, and fusing the stabilized stem region (HA-ss) to a multimerizing protein, such as ferritin. This approach was first used to generate group 1 HA stem immunogens. See e.g., PCT International Publication No. WO2015/183969; and Yassine et al., 2015, Nat. Med. 21:1065-70. It was also used to generate group 2 HA stem immunogens. See e.g., PCT International Publication No. WO2018/045308; and Corbeh et al., Therapeutics and Prevention, 2019, 10(1). However, stabilizing group 2 HA stem was unexpectedly more difficult than stabilizing group 1 HA stem and required the use of different strategies other than the simpler hydrophobic repacking used to stabilize group 1 HA stem nanoparticles.
[0009] While, these group 2 stem-stabilized ferritin protein constructs were immunogenic in mice and elicited antibody responses that protected mice against lethal challenges, the murine species tested did not have the appropriate B-cell repertoire to evaluate whether the group 2 protein constructs were capable of eliciting broadly neutralizing antibodies (bnAbs), similar to the bnAbs that have been observed in humans with cross-subtype binding. Thus, until now, the question of whether these group 2 influenza immunogens could elicit bnAbs akin to the ones discovered in humans remained unanswered.
[0010] There is an unmet need for influenza vaccines that can better address the current problems of antigenic drift, antigenic shift, and virus mismatch by providing broader protection against multiple influenza strains, including the HI and/or H3 subtypes that account for the majority of seasonal infections and pandemic outbreaks. A vaccine that could provide broad protection against these viruses would provide a significant public health benefit, extending protection to drifted seasonal strains and/or pandemic strains and reducing or eliminating the need for annual vaccine reformulations. A broadly protective vaccine is desired to effectively control seasonal influenza infection and to avert pandemic outbreaks. The elicitation of broadly neutralizing antibodies that prevent infection would be a transformative solution to prevent influenza.
SUMMARY
[0011] When administered in combination with a squalene-based adjuvant emulsion, like AF03, stem-stabilized group 2 influenza hemagglutinin (HA) immunogens without the immunodominant head region and presented on nanoparticles derived from a protein with selfassembling multimerization properties (e.g., ferritin) elicit broadly neutralizing antibody (bnAb) responses to diverse influenza viruses in non-human primates (NHPs), including diverse group 2 strains. The potency and breadth of these NHP antibodies were comparable to human bnAbs and extended to mismatched subtypes. These bnAbs neutralized diverse influenza viruses and shared a mode of recognition similar to human bnAbs indicating that this vaccine composition can be used to protect against diverse influenza viruses, including influenza viruses generated through antigenic drift and antigen shift that give rise to seasonal infections and pandemic outbreaks. [0012] A first aspect is directed to a vaccine composition comprising stem-stabilized influenza hemagglutinin-multimerizing protein (e.g., ferritin) nanoparticles and a squalene-based oil-inwater adjuvant emulsion, wherein the stem-stabilized influenza hemagglutinin-multimerizing protein nanoparticles comprise a protein with self-assembling multimerization properties (e.g., ferritin) joined to a modified, group 2 influenza hemagglutinin (HA) protein (e.g., modified HA protein from H3 influenza virus) to form a protein construct and wherein the protein construct forms stem-stabilized influenza hemagglutinin nanoparticles when expressed in cells, wherein the modified influenza virus HA protein lacks an antigenic head region and comprises a modified stem region from a group 2 influenza HA protein wherein the modified stem region comprises at least one immunogenic epitope, and wherein the vaccine composition elicits broadly neutralizing influenza antibodies when administered to a subject. Various features and embodiments of this aspect of the present disclosure are described throughout this application.
[0013] A second aspect is directed to a method of vaccinating against influenza virus, the method comprising administering to a subject in need thereof an immunologically effective dose of the vaccine composition, as described herein, wherein administration of the vaccine composition elicits broadly neutralizing influenza antibodies in the human subject. Various features and embodiments of this aspect of the present disclosure are described throughout this application.
[0014] Various features and embodiments of the first and second aspects of the present disclosure, as well as other aspects of the compositions and methods of using the same, are described throughout this application. The foregoing general summary and the following detailed description are exemplary and explanatory and are not restrictive of the claims.
BRIEF DESCRIPTION OF THE DRAWING
[0015] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments, and together with the written description, serve to explain certain principles of the compositions and methods disclosed herein.
[0016] Figures 1A-C show antibody responses after group 2 stem nanoparticle vaccination (H3-SS-np + AF03) in cynomolgus macaques. (A) Anti-H3 and anti-HIO IgG responses determined by ELISA after three immunizations administered at weeks 0, 4 and 10 (see vertical arrows) with H3-SS-np with AF03 adjuvant. (B) Left: Neutralization measured by a reporter- microneutralization (MN) assay for 2005 H3 (A/Wisconsin/67/05, H3N2) in NHP serum, in the presence of competitors, either 2005 HA (WT) or Astern (I45N, Q47T). Right: HA lentiviral vector reporter assay for 2013 H10 (A/Jiangxi/IPB13b/2013, H10N8) in NHP serum. Specificity was determined with protein competitors, either wildtype 2009 HA (WT) or Astern (D19N, G33E). The IC50 is the dilution factor of serum that achieves 50% neutralization. (C) Microneutralization activity of macaque antisera was assayed 2 weeks after the third immunization using the following diverse H3N2, H10N8 or H7N9 strains:
A/Singapore/INFIMH- 16-0019/2016 (H3N2), A/Hong Kong/4801/2014 (H3N2), A/Victoria/361/2011 (H3N2), A/Wisconsin/67/2005 (H3N2), A/Shangdong/9/1993 (H3N2), A/Port Chalmers/1/1973 (H3N2), A/Aichi/02/1968 (H3N2), A/Jiangxi-Donghu/346-2/2013 (H10N8) and A/Anhui/1/2013 (H7N9).
[0017] Figures 2A-B show isolation and repertoire analysis of monoclonal antibodies from monkeys immunized with a vaccine composition comprising H3-SS-np and AF03 using antigen-specific B cell sorting. The bnAbs were sorted using a single H3 HA probe (H3+) derived from A/Perth/16/2009 (A) or counter sorted using two H3 HA probes (H3+/H3+) derived from divergent strains, A/Hong Kong/1/1968 and A/Victoria/361/2011 (B). Frequencies of HA+ IgG B cells from after immunizations (week 12) are shown and pie charts depict the frequency of usage of specific heavy chain variable (IGHV), diversity (IGHD) and joining (IGHJ) germline genes for H3+ IgG+ B cell populations.
[0018] Figures 3A-B show repertoire analysis of lambda and kappa light chains from H3-SS- np-immunized monkeys. Pie charts depict the frequency of usage of specific variable and joining germline genes, in lambda (A) and kappa (B) light chains sequenced from single-sorted B cells that were either H3+ (Perth 2009 probe) or H3+/H3+ (Hong Kong 1968 and Victoria 2011 probes). As indicated, some germline genes could not be assigned unambiguously despite >90% sequence match.
[0019] Figures 4A-B show the breadth and specificity of mAbs isolated from immune macaques, including (A) microneutralization (MN) activity of mAbs isolated from group 2 immune macaques in a reporter assay with H3N2, H7N9 and H10N8 strains (two previously reported human bnAbs, CR6261 and CR8020, were included as controls in the assay); and (B) binding of H3-3B10 mAb to wildtype 2009 H3 HA (WT) and a Astern mutant (D19N, G33E).
[0020] Figures 5A-C show that vaccine-induced NHP Fabs bind to the conserved epitope on the influenza hemagglutinin stem. (A) Trimeric 2011 H3 HA-3B10 complex, with different shading to show the HA1, HA2, Fab heavy chain and Fab light chain of one HA/Fab protomer; the other two HA monomers and Fabs in the trimer are not shaded. Glycans are depicted as their component atoms. (B) Epitope footprint of bnAbs, from left to right: NHP 3B10 on 2011 H3, CR9114 on H3N2 (PDB: 4FQY), CR8020 on 1968 H3 (3SDY), and FI6v3 Fab on H3N2 (PDB: 3ZTJ), with epitopes shown in dark gray. (C) Superimposition of 3B 10-HA structure with CR8020 (PDB: 3SDY) shows extensive epitope overlap and conservation of the structural mechanism of binding. Contacts with the epitope are observed for the CDR-H1, H2 and H3 of the heavy chain. Epitope of 3B10 on Vicl 1 HA is shown in dark gray.
[0021] Figure 6 shows the binding of antibodies isolated from H3-SS-np-immunized cynomolgus macaques to wildtype and AStem (D19N, G33E) H3 HA trimers. ELISA plates were coated with either wildtype or mutant HA (A/Perth/16/2009) trimers and probed for binding with indicated monkey and human mAbs.
Figure 7A-D shows where the 3B10 antibody makes contacts with the HA1 and HA2 portions of the stabilized stem region of the fusion protein. (A) The CDR-H1 make contacts with the N- terminus of the fusion peptide through van der Waals and hydrogen-bonding interactions. Residues from CDRHl and the fusion peptide involved in interactions are shown as sticks. (B) The CDR-H2 make contacts with the N- and C-terminus of the fusion peptide through hydrogen-bonding interactions. (C) The CDR-H3 make contacts with the C-terminus of the fusion peptide through van der Waals and hydrogen-bonding interactions. (D) Continuous Cryo-EM density was observed in the HA1-HA2 connection region. The model is shown as cartoon with relevant residue numbers labeled.
DETAILED DESCRIPTION
[0022] This application discloses vaccine compositions comprising a squalene-based oil-inwater adjuvant emulsion, such as AF03, and nanoparticles formed from a protein construct comprising a stem-stabilized group 2 influenza hemagglutinin (HA) joined to a protein with self-assembling multimerization properties (e.g., ferritin). The stem-stabilized HA substantially lacks the head region. When expressed in cells, the protein construct forms nanoparticles that are antigenic with respect to the stem region of the HA polypeptide. The vaccine compositions induce broadly neutralizing antibodies (bnAbs) to the conserved stem region of the influenza HA. The bnAbs induced by the vaccine compositions neutralize a broad range of group 2 influenza viruses comparable to human bnAbs and extending to mismatched subtypes, and share a mode of recognition similar to other known human bnAbs, indicating that vaccine compositions, as described herein, can be used to protect against diverse influenza viruses, including those responsible for influenza pandemics in humans. [0023] For convenience, certain abbreviations can be used to refer to protein constructs, and portions thereof, described herein. For example, HA can refer to influenza hemagglutinin protein, or a portion thereof. HA-ss refers to a stabilized stem region, or a portion of the stem region, from an influenza HA protein. The HA portion of such a designation can also refer to the subtype of the hemagglutinin protein. For example, a stabilized stem region from a subtype 3 hemagglutinin can be referred to as H3-ss. A protein construct comprising HA-ss joined to a ferritin protein can be referred to as HA-ss-F, or H3-ss-F if the stabilized stem region is from a subtype 3 HA. When expressed in cells the HA-ss-F fusion protein forms antigenic nanoparticles, or HA-ss-np for short, H3-ss-np if the stabilized stem region is from a subtype 3 HA.
[0024] All nomenclature used to classify influenza virus is that commonly used by those skilled in the art. Thus, a Ty pe, or Group, of influenza virus refers to influenza Type A, influenza Type B or influenza type C. It is understood by those skilled in the art that the designation of a virus as a specific Type relates to sequence difference in the respective Ml (matrix) protein or P (nucleoprotein). Type A influenza viruses are further divided into group 1 and group 2. These groups are further divided into subtypes, which refers to classification of a virus based on the sequence of its HA protein. Examples of current commonly recognized subtypes are H1, H2, H3, H4, H5, H6, H7, H8, H8, H10, H1l, H12, H13, H14, H15, H16, H17 or H18. Group 1 influenza subtypes are HI, H2, H5. H6. H8, H9, H11, H12. H13. H16, H17 and H18. Group 2 influenza subtypes are H3, H4, H7, H 10, H14, and H15. Finally, the term strain refers to viruses within a subtype that differ from one another in that they have small, genetic variations in their genome.
A. Definitions
[0025] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms may be set forth through the specification. If a definition of a term set forth below is inconsistent with a definition in an application or patent that is incorporated by reference, the definition set forth in this application should be used to understand the meaning of the term.
[0026] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.
[0027] Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.
[0028] Antigen: As used herein, the term “antigen”, refers to an agent that elicits an immune response; and/or (ii) an agent that is bound by a T cell receptor (e.g., when presented by an MHC molecule) or to an antibody (e.g., produced by a B cell) when exposed or administered to an organism. In some embodiments, an antigen elicits a humoral response (e.g., including production of antigen-specific antibodies) in an organism; alternatively or additionally, in some embodiments, an antigen elicits a cellular response (e.g., involving T-cells whose receptors specifically interact with the antigen) in an organism. It will be appreciated by those skilled in the art that a particular antigen may elicit an immune response in one or several members of a target organism (e.g., mice, rabbits, primates, humans), but not in all members of the target organism species. In some embodiments, an antigen elicits an immune response in at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the members of a target organism species. In some embodiments, an antigen binds to an antibody and/or T cell receptor, and may or may not induce a particular physiological response in an organism. In some embodiments, for example, an antigen may bind to an antibody and/or to a T cell receptor in vitro, whether or not such an interaction occurs in vivo. In some embodiments, an antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. Antigens include the stem-stabilized influenza hemagglutinin-ferritin protein constructs (HA-ss-F) or nanoparticles obtained therefrom (HA-ss-np) as described herein.
[0029] Approximately: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). [0030] Broadly neutralizing antibodies: As used herein, “broadly neutralizing antibodies” are antibodies that neutralize more than one subtype and/or strain of influenza virus. For example, broadly neutralizing antibodies elicited against a vaccine composition comprising a modified influenza HA protein construct from one sub-type of virus, may neutralize another sub-type of virus. For example, broadly neutralizing antibodies elicited against a vaccine composition comprising a modified influenza HA protein construct from an H3 influenza virus may neutralize viruses from two or more of the following sub-types: HI, H2, H4, H5, H6, H7, H8, H8, H10, Hll, H12, H13, H14, H15, H16, H17 or H18, or from two or more of the following group 2 sub-types: H3, H4, H7, H10, H14, or H15. As an additional example, broadly neutralizing antibodies elicited against a vaccine composition comprising a modified influenza HA protein construct from one sub-type may neutralize other strains of the same sub-type of virus. For example, broadly neutralizing antibodies elicited against a vaccine composition comprising a modified influenza HA protein construct from an H3 influenza virus may neutralize viruses from other divergent group 2 strains, including, for example, one or more H3 and/or H10 strains. Exemplary H3 strains include, but are not limited to, A/Singapore/INFIMH- 16-0019/2016, A/Hong Kong/4801/2014, A/Victoria/361/2011, A/Wisconsin/67/2005, A/Shangdong/9/1993, A/Port Chalmers/ 1/1973, and A/Aichi/02/1968. Exemplary H10 strains include, but are not limited to, A/Jiangxi/IPB13b/2013. Divergent group 2 strains may also have an HA2 polypeptide sharing at least 60% identity with the HA2 polypeptide from with the wild type HA2 polypeptide from A/Finland/486/2004 (SEQ ID NO: 4).
[0031] Carrier: As used herein, the term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which a composition is administered. In some exemplary embodiments, carriers can include sterile liquids, such as, for example, water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as, for example, peanut oil, soybean oil, mineral oil, sesame oil and the like. In some embodiments, carriers are or include one or more solid components.
[0032] Epitope: As used herein, the term “epitope” includes any moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component in whole or in part. In some embodiments, an epitope is comprised of a plurality of chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface- exposed when the antigen adopts a relevant three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically near to each other in space when the antigen adopts such a conformation. In some embodiments, at least some such chemical atoms are groups are physically separated from one another when the antigen adopts an alternative conformation (e.g., is linearized).
[0033] Excipient : As used herein, the term “excipient” refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example to provide or contribute to a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
[0034] Fusion protein: As used herein, the term “fusion protein” refers to a protein encoded by a nucleic acid sequence engineered from nucleic acid sequences encoding at least a portion of two different (e.g., heterologous) proteins. To create a fusion protein nucleic acid sequences are joined such that the resulting reading does not contain an internal stop codon.
[0035] “Glycosylation,” as used herein, refers to the addition of a saccharide unit to a protein. “N-glycan,” as used herein, refers to a saccharide chain attached to a protein at the amide nitrogen of an N (asparagine) residue of the protein. As such, an N-glycan is formed by the process ofN-glycosylation. This glycan may be a polysaccharide.
[0036] Immune response : As used herein, the term “immune response” refers to a response of a cell of the immune system, such as a B cell, T cell, dendritic cell, macrophage or polymorphonucleocyte, to a stimulus such as an antigen, immunogen, or vaccine. An immune response can include any cell of the body involved in a host defense response, including for example, an epithelial cell that secretes an interferon or a cytokine. An immune response includes, but is not limited to, an innate and/or adaptive immune response. As used herein, a protective immune response refers to an immune response that protects a subject from infection (prevents infection or prevents the development of disease associated with infection). Methods of measuring immune responses are well known in the art and include, for example, measuring proliferation and/or activity of lymphocytes (such as B or T cells), secretion of cytokines or chemokines, inflammation, antibody production and the like. An antibody response or humoral response is an immune response in which antibodies are produced. A “cellular immune response” is one mediated by T cells and/or other white blood cells.
[0037] Immunogen : As used herein, the term “immunogen” or “immunogenic” refers to a compound, composition, or substance which is capable, under appropriate conditions, of stimulating an immune response, such as the production of antibodies or a T cell response in an animal, including compositions that are injected or absorbed into an animal. As used herein, “immunize” means to render a subject protected from an infectious disease, such as by vaccination.
[0038] In some embodiments: As used herein, the term “in some embodiments” refers to embodiments of all aspects of the disclosure, unless the context clearly indicates otherwise.
[0039] Neutralizing antibodies : As used herein, “neutralizing antibodies” are antibodies that prevent influenza virus from completing one round of replication. As defined herein, one round of replication refers the life cycle of the virus, starting with attachment of the virus to a host cell and ending with budding of newly formed virus from the host cell. This life cycle includes, but is not limited to, the steps of attaching to a cell, entering a cell, cleavage and rearrangement of the HA protein, fusion of the viral membrane with the endosomal membrane, release of viral ribonucleoproteins into the cytoplasm, formation of new viral particles and budding of viral particles from the host cell membrane. Accordingly, a neutralizing antibody is one that inhibits one or more such steps.
[0040] Pandemic strain: A “pandemic” influenza strain is one that has caused or has capacity to cause pandemic infection of human populations. In some embodiments, a pandemic strain has caused pandemic infection. In some embodiments, such pandemic infection involves epidemic infection across multiple territories; in some embodiments, pandemic infection involves infection across territories that are separated from one another (e.g., by mountains, bodies of water, as part of distinct continents, etc.) such that infections ordinarily do not pass between them.
[0041] Prevention: The term “prevention”, as used herein, refers to prophylaxis, avoidance of disease manifestation, a delay of onset, and/or reduction in frequency and/or severity of one or more symptoms of a particular disease, disorder or condition (e.g., infection for example with influenza virus). In some embodiments, prevention is assessed on a population basis such that an agent is considered to “prevent” a particular disease, disorder or condition if a statistically significant decrease in the development, frequency, and/or intensity of one or more symptoms of the disease, disorder or condition is observed in a population susceptible to the disease, disorder, or condition. [0042] Seasonal strain: A “seasonal” influenza strain is one that has caused or has capacity to cause a seasonal infection (e.g., annual epidemic) of human populations. In some embodiments, a seasonal strain has caused seasonal infection.
[0043] Sequence identity: The similarity between amino acid or nucleic acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. “Sequence identity” between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences. “Sequence identity” between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. Homologs or variants of a given gene or protein will possess a relatively high degree of sequence identity when aligned using standard methods.
[0044] The terms “% identical”, “% identity” or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing said sequences, after optimal alignment, with respect to a segment or “window of comparison”, in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm by Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).
[0045] Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100.
[0046] In some embodiments, the degree of identity is given for a region which is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments in continuous nucleotides. In some embodiments, the degree of identity is given for the entire length of the reference sequence.
[0047] Nucleic acid sequences or amino acid sequences having a particular degree of identity to a given nucleic acid sequence or amino acid sequence, respectively, may have at least one functional property of said given sequence, e.g., and in some instances, are functionally equivalent to said given sequence. One important property includes the ability to act as a cytokine, in particular when administered to a subject. In some embodiments, a nucleic acid sequence or amino acid sequence having a particular degree of identity to a given nucleic acid sequence or amino acid sequence is functionally equivalent to said given sequence.
[0048] Stem-stabilized influenza hemagglutinin-ferritin protein construct: As used herein, “stem-stabilized influenza hemagglutinin-ferritin protein construct” or the like is used herein to refer to a non-naturally occurring polypeptide comprising a ferritin polypeptide joined to a modified influenza hemagglutinin (HA) polypeptide, where the immunodominant head region of the HA is substantially removed, and the polypeptide forms nanoparticles that are antigenic with respect to the stem region of the HA polypeptide when expressed in cells. Antigenicity may be a feature of the modified influenza HA polypeptide as part of the larger construct. That is, it is sufficient that the construct can serve as an antigen that generates antibodies against the modified influenza HA polypeptide, regardless of whether the modified influenza HA polypeptide without the ferritin could do so.
[0049] Subject : As used herein, the term “subject” means any member of the animal kingdom. In some embodiments, “subject” refers to humans. In some embodiments, “subject” refers to non-human animals. In some embodiments, subjects include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In certain embodiments, the nonhuman subject is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a ferret, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, a subject may be a transgenic animal, genetically-engineered animal, and/or a clone. In certain embodiments, the subject is an adult, an adolescent or an infant. In some embodiments, terms “individual” or “patient” are used and are intended to be interchangeable with “subject.”
[0050] Vaccination: As used herein, the term “vaccination” or “vaccinate” refers to the administration of a composition intended to generate an immune response, for example to a disease-causing agent such as influenza. Vaccination can be administered before, during, and/or after exposure to a disease-causing agent, and/or to the development of one or more symptoms, and in some embodiments, before, during, and/or shortly after exposure to the agent. Vaccines may elicit both prophylactic (preventative) and therapeutic responses. Methods of administration vary according to the vaccine, but may include inoculation, ingestion, inhalation or other forms of administration. Inoculations can be delivered by any of a number of routes, including parenteral, such as intravenous, subcutaneous, intraperitoneal, intradermal, or intramuscular. Vaccines may be administered with an adjuvant to boost the immune response. In some embodiments, vaccination includes multiple administrations, appropriately spaced in time, of a vaccinating composition.
B. Stem-Stabilized Influenza Hemagglutinin Nanoparticles (HA-ss-np)
[0051] The vaccine compositions for immunization against influenza virus described in this application include a squalene-based adjuvant emulsion, like AF03, and a protein construct comprising a stem-stabilized group 2 influenza HA and a protein with self-assembling multimerization properties (e.g., ferritin), where the protein construct self assembles into immunogenic nanoparticles when expressed in cells. By joining an immunogenic portion of the influenza HA stem region to a protein with self-assembling multimerization properties, like ferritin, protein constructs as described herein (also called HA-ss-F) are capable of self- assembling into 24-mer nanoparticles (also called HA-ss-np) expressing eight trimers of HA stem protein on each vertex formed by three subunits coming together and displaying octahedral symmetry. When administered to a subject, the vaccine composition induces broadly neutralizing antibodies to the conserved stem region of the influenza HA. HA-ss-F is formed by joining two proteins that normally do not exist in nature: a modified stem region of an influenza HA protein and a ferritin protein. HA-ss-F protein constructs and the nanoparticles (HA-ss-np) formed by those protein constructs have been previously described. For example, PCT International Publication No. W02018/045308, which is hereby incorporated by reference in its entirety, describes in detail the production of group 2 HA-ss- F. See also, Corbett et ak, Therapeutics and Prevention, 2019, 10(1), 1-18, which is hereby incorporated by reference in its entirety.
1. Modified Stem Region of Influenza Hemagglutinin (HA-ss)
[0052] Hemagglutinin (HA) is the immunodominant surface influenza protein and is composed of two structurally distinct domains or regions: the globular head and the stem (also referred to as the stalk). The HA protein is formed from a precursor protein called HAO, which is transported through the golgi complex and transported to the plasma membrane where it is cleaved by cellular proteases into HA1 and HA2, which are linked by a disulfide bond. The head region of the influenza HA is composed of the majority of the HA1 polypeptide, including the receptor-binding domain (about 145 amino acids) and the vestigial esterase domain (about 75 amino acids). The stem region, which anchors the HA protein into the viral lipid envelope, includes the HA2 polypeptide and a portion of the HA1 polypeptide. A first portion of the stem region is located upstream of the N-terminus of the head region, while a second portion of the stem region is located downstream of the C-terminus of the head region. As used herein, “a first stem region” refers to the portion of the HA stem region that is located upstream of the N- terminus of the HA head region. As used herein “a second stem region” refers to the portion of the HA stem region that is located downstream of the C-terminus of the HA head region.
[0053] Mutations occur frequently in the head region, giving rise to the constant antigenic drift of influenza viruses and seasonal influenza endemics. In contrast, the HA stem is highly conserved and experiences little antigenic drift. Five topologically distinct antigenic sites have been identified in the head domain of influenza HA, A to E in H3 strains and Sa, Sb, Cal , Ca2, and Cb in HI strains. Broeker et ak, Journal of Virology, 2018, 92(20): 1-13 and Liu et ak, Journal of Clinical Investigation, 2018, 128(11), 4992-96. Each antigenic site contains many epitopes.
[0054] By way of reference, the head region corresponds to amino acids 60-329 of the full length HA1 polypeptide of the H3N2 influenza virus A/Finland/486/2004 (SEQ ID NO: 2). Similarly, by way of reference, the first stem region (located upstream of the N-terminus of the head region) corresponds to amino acids 1-59 (or amino acids 17-59 when the signal sequence is removed) of the full length HA1 polypeptide of the H3N2 influenza virus A/Finland/486/2004 (SEQ ID NO: 2), and the second stem region (located downstream of the C-terminus of the head region) corresponds to 1) amino acids 330-345 of the full length HA1 polypeptide of the H3N2 influenza virus A/Finland/486/2004 (SEQ ID NO: 2) and 2) amino acids 1-221 of the HA2 polypeptide of the H3N2 influenza virus A/Finland/486/2004 (SEQ ID NO: 4).
[0055] The amino acid numbering of the A/Finland/486/2004 H3N2 influenza is used merely by way of reference. With regard to HA proteins, it is understood by those skilled in the art that HA proteins from different influenza viruses may have different lengths due to sequence differences (insertions, deletions) in the protein. Thus, reference to a corresponding region refers to a region of another protein that is identical, or nearly so (e.g., at least 90% identical, at least 95%, identical, at least 98% identical or at least 99% identical), in sequence, structure and/or function to the region being compared. For example, with regard to the stem region of an HA protein, the corresponding region in another HA protein may not have the same residue numbers but will have a nearly identical sequence and will perform the same function. To better clarify sequence comparisons between viruses, numbering systems are used by those in the field, which relate amino acid positions to a reference sequence. Thus, corresponding amino acid residues in HA proteins from different strains of influenza may not have the same residue number with respect to their distance from the N-terminal amino acid of the protein. For example, using the H3 numbering system, reference to residue 100 in A/New Caledoma';2Q/1999 (1999 NC, HI) does not mean it is the 100* residue from the N-terminal amino acid. Instead, residue 100 of A/New Caledonia/20/1999 (1999 NC, HI) aligns with residue 100 of influenza H3N2 strain. The use of such numbering systems is understood by those skilled in the art. While the H3 numbering system can be used to identify the location of ammo acids, unless otherwise noted, the location of amino acid residues in HA proteins will be identified by general reference to the position of a corresponding amino acid from a sequence disclosed herein. Thus, when embodiments are described by reference to an A/Finland/486/2004 sequence, it should be understood that such embodiments are not limited to the A/Finland/486/2004 sequence and that, in accordance with amino acid numbering systems used in the field, other influenza A sequences can be used in place of the reference sequence A/Finland/486/2004. a. Removal of the HA Head Region
[0056] The HA-ss portion of the HA-ss-F protein construct comprises at least one immunogenic epitope from the stem region (Embodiment HAla). The HA-ss portion is constructed by removing the immunodominant head region of the influenza HA protein (Embodiment HAlb). This can be achieved by removing all five antigenic sites of the head region (e.g., antigenic sites A-E in the head region of group 2 influenza strains) (Embodiment HA1 c). The entire antigenic site may be removed or a sufficient number of amino acid residues of each antigenic site such that no immunogenic epitopes remain in the head region (Embodiment HAld). Accordingly, it is not necessary to remove all amino acids of the head region. For example, in certain embodiments, the head region is “substantially removed” or “substantially replaced” meaning that all but up to 5 contiguous amino acids of the head region are removed (e.g., 5, 4, 3, 2, 1 or 0 contiguous amino acids of the head region are removed) (Embodiment HAle). In other embodiments, at least 95% of the head region is removed (Embodiment HA11). In certain embodiments, all amino acid residues of the head region are removed (Embodiment HAlg).
[0057] Upon deletion of the head region, the first stem region and the second stem region can be joined together directly, or they can be joined with a linker sequence (Embodiment HA2a). Typically, the head region is replaced with a linker sequence that connects the first and second stem regions (Embodiment HA2b). Any linker sequence may be used so long as the first and second stem region sequences are able to form the desired structure (Embodiment HA2c). While any amino acids may be used to make the linker sequence, it is preferred to use amino acids lacking large or charged side chains (Embodiment HA2d). Preferred amino acids include, but are not limited to, serine, glycine, valine and alanine (Embodiment HA2e). In one embodiment, the linker is made from serine and glycine residues (Embodiment HA21). The length of the linker sequence may vary, but preferred embodiments use the shortest possible sequence in order to allow the stem sequences to form the desired structure (Embodiment HA2g). In one embodiment, the linker sequence is less than 10 amino acids in length (Embodiment HA2h). In one embodiment, the linker sequence is less than 5 amino acids in length (Embodiment HA2i). In one embodiment, the linker sequence is 6-7 amino acids in length (Embodiment HA2j). In one embodiment, the linker sequence consists of the amino acid sequence GSG (Embodiment HA2k). In one embodiment, the linker sequence forms a loop (Embodiment HA21). In certain embodiments, the linker sequence is comprised of the following amino acid residues: valine, isoleucine, phenylalanine, cysteine, proline, glycine, and asparagine (Embodiment HA2m). In one embodiment, the linker sequence consists of one of the following amino acid sequences: VFPGCGV (SEQ ID NO: 5), VFPCGV (SEQ ID NO: 6), VFPGCV (SEQ ID NO: 7), CFNGIC (SEQ ID NO: 8), and VFPNCGV (SEQ ID NO: 9), wherein optionally a first cysteine residue in the linker sequence forms a disulfide bond with a second cysteine residue in the second stem region downstream of the C-terminus of the head region (Embodiment HA2n). In certain embodiments, the linker sequence comprises less than 5 contiguous amino acids from the head region of an HA protein (Embodiment HA2o). b. Modifications to the Stem Region
[0058] In addition to replacing the head region, it is also possible to modify the remaining stem region sequences to improve the stability of the protein construct (Embodiment HA3). The second stem region includes a small portion of the HA1 polypeptide and the entire the HA2 polypeptide. The HA2 polypeptide comprises two central helices (helix A and helix C) that form the majority of the stem backbone that extends from the transmembrane portion of the HA2 polypeptide to the head region. As used herein, “a helix A sequence” refers to a sequence from an influenza A (e.g., group 2 influenza virus, such as H3) HA protein that forms helix A in an HA2 polypeptide and “a helix C sequence” refers to a sequence from an influenza A (e.g., group 2 influenza virus, such as H3) HA protein that forms helix C in an HA2 polypeptide. The HA2 polypeptide also includes an interhelical region of about 32 amino acids that connects helix A and helix C (“HA2 interhelical region”). By way of reference, the HA2 polypeptide and HA2 interhelical region of the H3N2 strain A/Finland/486/2004, correspond to SEQ ID NO: 4 and amino acids 60-92 of SEQ ID NO: 4, respectively. As used herein, the “HA2 interhelical region” refers to a portion of the HA2 influenza polypeptide that connects helix A to helix C in a wild type HA protein. i. HA2 Interhelical Region
[0059] Additional modifications of the stem region can improve stability. For example, shortening or deleting the HA2 interhelical region can improve the stability of the protein construct (Embodiment HA4a). In certain embodiments, the HA2 interhelical region that is shortened or removed comprises amino acids 60-92 of SEQ ID NO: 4 or an analogous amino acid sequence from an influenza A virus other than A/Finland/486/2004 (Embodiment HA4b).
[0060] Upon shortening or deleting this region, helix A (with or without a helix A extension sequence) and helix C amino acid sequences flanking this region can be joined together directly, or they can be joined with a linker sequence (Embodiment HA4c). It is not necessary to delete the entire HA2 interhelical region, so long as helix A and helix C are able to be joined and form the desired structure (Embodiment HA4d). For example, in certain embodiments, the HA2 interhelical region is “substantially removed” or “substantially replaced,” meaning that up to 5 amino acids of the HA2 interhelical region remain, such as 5, 4, 3, 2, 1, or 0 amino acid(s) of the HA2 interhelical region (Embodiment HA4e). In other embodiments, at least 90% of the HA2 interhelical region is removed (Embodiment HA4I). In certain embodiments, all amino acid residues of the HA2 interhelical region are removed (Embodiment HA4g). Any linker sequence may be used so long as the helix A and helix C sequences are able to form the desired structure (Embodiment HA4h). While any amino acids may be used to make the linker sequence, it is preferred to use amino acids lacking large or charged side chains (Embodiment HA4i). Preferred amino acids include, but are not limited to, serine, glycine, valine and alanine (Embodiment HA4j). In certain embodiments, the linker sequences that replaces the HA2 interhelical region is a glycine-rich loop (Embodiment HA4k). In one embodiment, the linker is made from serine and glycine residues (Embodiment HA41). The length of the linker sequence may vary, but preferred embodiments use the shortest possible sequence in order to allow the stem sequences to form the desired structure (Embodiment HA4m). In one embodiment, the linker sequence is less than 10 amino acids in length (Embodiment HA4n). In one embodiment, the linker sequence is less than 5 amino acids in length (Embodiment HA4o). In one embodiment, the linker sequence is 3-6 amino acids in length (Embodiment HA4p). In one embodiment, the linker sequence is 3-6 amino acids in length and includes at least two glycine residues (Embodiment HA4q). In one embodiment, the linker sequence comprises or consists of the amino acid sequence GSG (Embodiment HA4r). In one embodiment, the linker sequence consists of the amino acid sequence GGP or GGPD (SEQ ID NO: 10) (Embodiment HA4s). ii. Helix A Extension
[0061] The outer helix A in certain H3 strains is approximately 5 amino acids shorter at its C- terminus than in HI HA. In certain embodiments, the stability of the protein construct can be improved by extending the length of helix A at its C-terminus, particularly for group 2 HA-ss constructs or H3-ss constructs (Embodiment HA5a). Typically, the helix A extension sequence comprises amino acid residues with helix-forming propensities (Embodiment HA5b). In one embodiment, the helix A extension sequence is less than 10 amino acids in length (Embodiment HA5c). In one embodiment, the helix A extension sequence is 5 or fewer amino acids in length (Embodiment HA5d). In certain embodiments, the helix A extension sequence comprises, or consists of, X1LMX2Q, or helix-forming variants thereof, wherein the amino acids at positions Xiand X2 are acidic amino acids, such as glutamic acid or aspartic acid (SEQ ID NO: 11; Embodiment HA5e). It should be noted that Xi and X2 can, but need not, be the same amino acid residue. In one embodiment, the helix A extension sequence comprises, or consists of, X1LMX2Q, or helix-forming variants thereof, wherein the amino acids at positions Xiand X2 are independently selected from the group consisting of glutamine, glutamic acid, asparagine, aspartic acid, glycine, and proline (SEQ ID NO: 12; Embodiment HA5f). In one embodiment, the helix A extension sequence comprises or consists of ALMAQ (SEQ ID NO: 13) or ELMEQ (SEQ ID NO: 14), or helix-forming variants thereof (Embodiment HA5g). In one embodiment, the helix A extension sequence comprises or consists of the amino acid sequence of ALMAQ (SEQ ID NO: 13) or ELMEQ (SEQ ID NO: 14) (Embodiment HA5h). iii. Mutations to HA2 Polypeptide
[0062] The stability of the protein construct can be further stabilized by one or more mutations (Embodiment HA6a). In certain embodiments, the second stem region comprises one or more mutations (Embodiment HA6b). For example, the second stem region may contain one or more mutations that improve side chain repacking and/or resurfacing (Embodiment HA6c). Repacking mutations generally refer to mutations at residues on the core of the protein (inside) to reduce or eliminate atomic voids that would cause structural instability. See e.g., Liang et ak, Biophysical Journal, 2001, 81:751-66. Resurfacing mutations generally refers to mutations on the protein surface (outside) to reduce or eliminate issues such as hydrophobic patches that can make the protein sticky or prone to aggregation and, therefore, unstable. See e.g., Chapman et ak, Cell Chem Biol, 2016, 23(5):543-53. For example, after removal of the HA head a new surface would be generated (analogous to a wound) and this new surface could include hydrophobic amino acids that would normally be shielded from water in the wild type HA. Such residues can be replaced by others with more hydrophilic properties while conserving the protein structure.
[0063] In certain embodiments, the one or more mutations that improve side chain repacking and/or resurfacing are at residues K51 and/or El 03, where the amino acid numbering, by way of reference, is based on the HA2 polypeptide of the A/Finland/486/2004 H3N2 influenza virus (SEQ ID NO: 4) (Embodiment HA6d). In certain embodiments, the one or more mutations that improve side chain repacking and/or resurfacing further comprise mutations at one or more of amino acid residues L52, L55, N95, T107, and/or N116, where the amino acid numbering, by way of reference, is based on the HA2 polypeptide of the A/Finland/486/2004 H3N2 influenza virus (SEQ ID NO: 4) (Embodiment HA6e). In certain embodiments, the one or more mutations that improve side chain repacking and/or resurfacing are at amino acid residues K51, L52, L55, N95, E103, T107, and N116, where the amino acid numbering, by way of reference, is based on the HA2 polypeptide of A/Finland/486/2004 (SEQ ID NO: 4) (Embodiment HA61). In certain embodiments, the one or more mutations that improve side chain repacking and/or resurfacing are K51M and/or E103L (Embodiment HA6g). In certain embodiments, the one or more mutations that improve side chain repacking and/or resurfacing further comprises one or more of L52V, L55V, N95L, T107V, and/or N116R (Embodiment HA6h). In certain embodiments, the one or more mutations that improve side chain repacking and/or resurfacing are K51M, L52V, L55V, N95L, E103L, E103L, T107V, and N116R (Embodiment HA6i). [0064] The second stem region may also contain one or more mutations that stabilize the N- terminus of helix C (Embodiment HA7a). In certain embodiments, the one or more mutations that stabilize the N-terminus of helix C increase the helix-forming propensities of helix C (Embodiment HA7b). In certain embodiments, the one or more mutations that stabilize the N- terminus of helix C are at residues W92 and/or S93, where the amino acid numbering, by way of reference, is based on the HA2 polypeptide of A/Finland/486/2004 (SEQ ID NO: 4) (Embodiment HA7c). In certain embodiments, the one or more mutations that stabilize the N- terminus of helix C are W92D, S93A, and/or S93C (Embodiment HA7d). In certain embodiments, the one or more mutations that stabilize the N-terminus of helix C comprise is at residue W92, including, for example, W92D (Embodiment HA7e). In certain embodiments, the one or more mutations that stabilize the N-terminus of helix C comprise W92D and S93C (Embodiment HA7f). In certain embodiments, when the second region contains the S93C mutation, the cysteine residue corresponding to amino acid residue 93 forms a disulfide bond with the cysteine residue in the linker sequence that joins the first and second stem regions, optionally where the linker sequence joining the first and second stem regions consists of one of the following amino acid sequences: VFPGCGV (SEQ ID NO: 5), VFPCGV (SEQ ID NO: 6), VFPGCV (SEQ ID NO: 7), and VFPNCGV (SEQ ID NO: 9) (Embodiment HA7g). c. Group 2 Influenza
[0065] As disclosed herein, the modified HA stem region is from a group 2 influenza HA (Embodiment HA8a). In certain embodiments, the group 2 influenza HA is from one of the currently recognized group 2 subtypes: H3, H4, H7, H10, H14, and HI 5 (Embodiment HA8b). In certain embodiments, the group 2 influenza HA is an H3 HA (Embodiment HA8c). In certain embodiments, the modified stem region is from the HA of one of the following H3 influenza strains (where 3 sequences are provided, they correspond to the full length hemagglutinin, HA1, and HA2 in that order): A/Finland/486/2004 (SEQ ID NOs 1, 2, and 4), A/Singapore/INFIMH- 16-0019/2016 (SEQ ID NOs 15-17), A/Hong Kong/4801/2014 (SEQ ID NOs 18-20), A/Victoria/361/2011 (SEQ ID NOs 21-23), A/Wisconsin/67/2005 (SEQ ID NOs 24-26), A/Shangdong/9/1993 (SEQ ID NOs 27-29), A/Port Chalmers/ 1/1973 (SEQ ID NOs 30-32), or A/Aichi/02/1968 (SEQ ID NOs 80-82) (Embodiment HA8d). In certain embodiments, the modified stem region is from the HA of one of the following H3 influenza strains: A/Denmark/35/2005 (SEQ ID NO: 33), A/Bangladesh/558/2012 (SEQ ID NO: 34), A/Sao Paulo/89403/2010 (SEQ ID NO: 35), A/Bangladesh/541/2012 (SEQ ID NO: 36), A/Bangladesh/542/2012 (SEQ ID NO: 37), A/Tocantins/979/2010 (SEQ ID NO: 38), A/Tunisia/17332/2011 (SEQ ID NO: 39), A/Norway/88/2003 (SEQ ID NO: 40), A/Japan/ AF2844/2012 (SEQ ID NO: 41), A/Texas/2977/2012 (SEQ ID NO: 42), A/North Carolina/AF2716/2011 (SEQ ID NO: 43), or A/Norway /70/2005 (SEQ ID NO: 44) (Embodiment HA8e). In certain embodiments, the H3 influenza strain is an H3N2 strain (Embodiment HA81).
[0066] In certain embodiments, the group 2 influenza HA is an H7 HA (Embodiment HA8g). In certain embodiments, the modified stem region is from the HA of one of the following H7 influenza strains: A/duck/Chiba/24-203-44/2012 (SEQ ID NO: 45), A/chicken/ Germany/2003 (SEQ ID NO: 46), A/chicken/Italy /444/1999 (SEQ ID NO: 47), A/mallard/Italy/4810-7/2004 (SEQ ID NO: 48), A/Anhui/1/2013 (SEQ ID NOs 49-51), A/Anhui/DEWH72-03/2013 (SEQ ID NO: 52), A/Shanghai/JS01/2013 (SEQ ID NO: 53), A/Guangdong/02/2013 (SEQ ID NO: 54), A/Shenzhen/ S P44/ 2014 (SEQ ID NO: 55), or A/Beijing/3/2013 (SEQ ID NO: 56), A/Hong Kong/470129/2013 (SEQ ID NO: 57) (Embodiment HA8h). In certain embodiments, the H7 influenza strain is an H7N1 and/or H7N9 strain (Embodiment HA8i).
[0067] In certain embodiment, the group 2 influenza HA is an H10 HA (Embodiment HA8j). In certain embodiments, the modified stem region is from an H10 influenza strain, including, but not limited to A/Jiangxi/IPB13b/2013 (SEQ ID NOs 58-60) (Embodiment HA8k). In certain embodiments, the H10 strain is an H10N8 strain (Embodiment HA81).
[0068] Exemplary group 2 HA-ss protein constructs that can be used also include those disclosed in PCT International Publication No. WO2018/045308 and Corbett et ak, Therapeutics and Prevention, 2019, 10(1), 1-18, each of which is hereby incorporated by reference in its entirety (Embodiment HA8m).
[0069] In certain embodiments, the group 2 influenza HA is an H3 HA and the H3-ss comprises a first linker sequence that substantially replaces the head region of the H3 HA and a modified stem region from the H3 HA, wherein the modified stem region comprises at least one immunogenic epitope (Embodiment HA8n).
[0070] In certain embodiments, the H3-ss comprises a first HA stem region covalently joined to a second HA stem region by a first linker sequence, wherein the head region of the H3 HA has been substantially replaced by the first linker sequence, wherein the second stem region comprises a first portion comprising a helix A sequence, a helix A extension sequence at the C-terminus of the helix A sequence, a second linker sequence, and a second portion comprising a helix C sequence, wherein the second linker sequence covalently joins the helix A extension sequence and the helix C sequence and substantially replaces the HA2 interhelical region, wherein the helix A extension sequence extends the length of the helix A sequence by up to 5 amino acid residues, and wherein the second stem region comprises at least one first mutation that improves side chain repacking and/or resurfacing and at least one second mutation at the N-terminus of the helix C sequence that increases the helix-forming propensity of the helix C sequence (Embodiment HA9a).
[0071] In certain embodiments, the at least one first mutation comprises a mutation at K51, L52, L55, N95, E103, T107, and N116, wherein the amino acid numbering, by way of reference, is based on the HA2 polypeptide of A/Finland/486/2004 H3N2 influenza virus (SEQ ID NO: 4) (Embodiment HA9b). In certain embodiments, the at least one second mutation comprises a mutation at W92 and S93, wherein the amino acid numbering, by way of reference, is based on the HA2 polypeptide of A/Finland/486/2004 H3N2 influenza virus (SEQ ID NO: 4) (Embodiment HA9c). In certain embodiments, the at least one first mutation comprises a mutation at K51, L52, L55, N95, E103, T107, and N116 and the at least one second mutation comprises a mutation at W92 and S93, wherein the amino acid numbering, by way of reference, is based on the HA2 polypeptide of the A/Finland/486/2004 H3N2 influenza virus (SEQ ID NO: 4) (Embodiment HA9d). In certain embodiments, the at least one first mutation comprises K51M, L52V, L55V, N95L, E103L, T107V, and N116R (Embodiment HA9e). In certain embodiments, the at least one second mutation comprises W92D and S93C (Embodiment HA9f). In certain embodiments, the at least one first mutation comprises K51M, L52V, L55V, N95L, E103L, T107V, and N116R and the at least one second mutation comprises W92D and S93C (Embodiment HA9g).
[0072] In certain embodiments, the first linker forms a loop and contains a first cysteine residue that forms a disulfide bond with a second cysteine residue in the second stem region (Embodiment HAlOa). In certain embodiments, the first linker consists of 6-7 amino acids (Embodiment HAlOb). In certain embodiments, the first linker sequence consists one of the following amino acid sequences: VFPGCGV (SEQ ID NO: 5), VFPCGV (SEQ ID NO: 6), VFPGCV (SEQ ID NO: 7), CFNGIC (SEQ ID NO: 8), and VFPNCGV (SEQ ID NO: 9), wherein optionally a first cysteine residue in the linker sequence forms a disulfide bond with a second cysteine residue in the N-terminus of the helix C sequence in the second stem region (Embodiment HAlOc).
[0073] In certain embodiments, the second linker consists of 3-6 amino acids including at least two glycine residues (Embodiment HAlla). In certain embodiments, the second linker consists of the amino acid sequence GGP (Embodiment HA1 lb).
[0074] In certain embodiments, the helix A extension sequence consists of ALMAQ (SEQ ID NO: 13) or ELMEQ (SEQ ID NO: 14) (Embodiment HA12).
[0075] In certain embodiments, the H3 HA comprises a first HA stem region covalently joined to a second HA stem region by a first linker sequence, wherein the head region of the H3 HA has been substantially replaced by the first linker sequence, wherein the second stem region comprises a first portion comprising a helix A sequence, a helix A extension sequence at the C-terminus of the helix A sequence, a second linker sequence, and a second portion comprising a helix C sequence, wherein the first linker sequence consists of the amino acid sequence VFPGCGV (SEQ ID NO: 5), wherein a first cysteine residue in the linker sequence forms a disulfide bond with a second cysteine residue in the N-terminus of the helix C sequence in the second stem region, wherein the second linker sequence consists of the amino acid sequence GGP and covalently joins the helix A extension sequence and the helix C sequence, and substantially replaces the HA2 interhelical region, wherein the helix A extension sequence consists of the amino acid sequence ELMEQ (SEQ ID NO: 14), wherein the second stem region comprises at least one first mutation that improves side chain repacking and/or resurfacing and at least one second mutation at the N-terminus of the helix C sequence that increases the helix-forming propensity of the helix C sequence, and wherein the at least one first mutation comprises K51M, L52V, L55V, N95L, E103L, T107V, and N116R and the at least one second mutation comprises W92D and S93C, wherein the amino acid numbering is based on the HA2 polypeptide of the A/Finland/486/2004 H3N2 influenza virus (SEQ ID NO: 4) (Embodiment HA13). [0076] In certain embodiments, the H3-ss is from the A/Finland/486/2004 H3N2 strain (SEQ ID NO: 1) (Embodiment HA14a). In certain embodiments, the HA-ss comprises the amino acid sequence (signal sequence in bold italics):
1 MKTIIALSYI LCLVFAQKLP GNDNSTATLC LGHHAVPNGT IVKTITNDQI EVTNATELVF
61 PGCGVLKLAT GMRNVPEKQT RGIFGAIAGF IENGWEGMVD GWYGFRHQNS EGIGQAADLK
121 STQAAINQIN GMVNRVIELM EQGGPDCYLA ELLVALLNQH VIDLTDSEMR KLFERTKKQL
181 RENAEDMGNG CFKIYHKCDN ACIGSIRNGT YDHDVYRDEA LNNRFQIK (SEQ ID NO: 63)
(Embodiment HA14b)
[0077] In certain embodiments, the H3-ss comprises the amino acid sequence (no signal sequence):
1 QKLPGNDNST ATLCLGHHAV PNGTIVKTIT NDQIEVTNAT ELVFPGCGVL KLATGMRNVP 61 EKQTRGIFGA IAGFIENGWE GMVDGWYGFR HQNSEGIGQA ADLKSTQAAI NQINGMVNRV 121 IELMEQGGPD CYLAELLVAL LNQHVIDLTD SEMRKLFERT KKQLRENAED MGNGCFKIYH 181 KCDNACIGSI RNGTYDHDVY RDEALNNRFQ IK (SEQ ID NO: 64)
(Embodiment HA14c).
2. Multimerizing Protein
[0078] As discussed herein, the modified HA sequence is linked to a multimerizing protein or portion thereof. As used herein, a multimerizing protein refers to a protein monomer that is capable of binding to other monomeric subunit proteins such that the monomeric subunit proteins self-assemble into a nanoparticle. Any monomeric subunit protein can be used to produce the protein construct, so long as the protein construct is capable of forming a multimeric structure displaying HA protein on its surface. Typically, the multimerizing protein is ferritin. a. Ferritin
[0079] Ferritin is an iron storage protein found in almost all living organisms. Ferritin has been extensively studied and engineered for a number of potential bioeliemical/biomedical purposes [Iwahori, K. U.S. Patent 2009/0233377 (2009); Mel drum, F.C. et al. Science 257, 522-523 (1992); Naitou, M. et al. U.S. Patent 2011/0038025 (2011); Yamashita, I. Biochim BiophysActa 1800, 846-857 (2010)], including its use as a vaccine platform for displaying exogenous epitope peptides [Carter, D.C. et al. U.S. Patent 2006/0251679 (2006); Li, C.Q. et al. Industrial Biotechnol 2, 143-147 (2006)], Ferritin’s use as a vaccine platform is particularly interesting because of its self-assembly and multivalent presentation of antigen which induces stronger B cell responses than monovalent form as well as induce T-cell independent antibody responses [Bachmann, M.F. et al. Annu Rev Immunol 15, 235-270 (1997); Dintzis, H.M. et al. Proc Natl Acad Sci USA 73, 3671-3675 (1976)]. Further, the molecular architecture of ferritin, which consists of 24 subunits assembling into an octahedral cage with 432 symmetry can display multimeric antigens on its surface.
[0080] Ferritin genes are found in many species and generally show a conserved highly alpha- helical structure despite sequence variation. As such, any ferritin can be used in the fusion proteins described herein, including bacterial, insect, and human ferritin, despite its sequence identity to any particularly described ferritin.
[0081] In some embodiments, the ferritin is bacterial, insect, fungal, bird, or mammalian (Embodiment Fla). In some embodiments, the ferritin is human, optionally with one or more mutations described herein (Embodiment Fib). In some embodiments, the ferritin is bacterial (Embodiment Flc), optionally with one or more mutations described herein. In some embodiments, the ferritin is H. pylori ferritin ( see SEQ ID NO: 65 for exemplary H. pylori ferritin sequences), optionally with one or more mutations described herein (Embodiment Fid). In some embodiments, the ferritin is Pyrococcus furiosus ferritin (NCBI seq WP_011011871.1; SEQ ID NO: 70), optionally with one or more mutations described herein (Embodiment Fie). The lower sequence homology between H. pylori ferritin (or other bacterial ferritins) and human ferritin may decrease the potential for autoimmunity when used as a vaccine platform (see Kanekiyo et al., Cell 162, 1090-1100 (2015)).
[0082] In some embodiments, the ferritin is a light chain and/or heavy chain ferritin (Embodiment F2a). In some embodiments, the ferritin is human heavy chain ferritin (FTH1, GENE ID No: 2495; SEQ ID NO: 71) or human light chain ferritin (FTL, GENE ID No: 2512; SEQ ID NO: 72), optionally with one or more modifications described herein (Embodiment F2b). In some embodiments, the ferritin is Trichoplusia ni heavy chain ferritin (GenBank: AY970291.1; SEQ ID NO: 73) or Trichoplusia ni light chain ferritin (GenBank: AY970292.1; SEQ ID NO: 74), optionally with one or more mutations described herein (Embodiment F2c).
[0083] HA-ss-F protein constructs need not comprise the full-length sequence of a ferritin protein. Portions, or regions, of the ferritin protein can be used as long as the portion comprises an amino acid sequence that directs self-assembly of monomeric ferritin subunits into the globular form of the protein (Embodiment 3a). In certain embodiments, a region comprising N-terminal amino acids of the ferritin protein are removed (Embodiment 3b). For example, amino acids 1-4 of the wild typ Q Helicobacter pylori ferritin protein may be removed (see e.g., SEQ ID NOs: 66-69) (Embodiment 3c). More specific regions are described in Zhang, Y. 2011, Int. J. Mol. Sci., 12, 5406-5421, which is incorporated herein by reference in its entirety (Embodiment 3d).
[0084] In some embodiments, the ferritin comprises a sequence having greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 97%, greater than 98%, or greater than 99% identity to a wild-type ferritin, including, but not limited to H. pylori ferritin, P. furiosus ferritin or human ferritin (Embodiment F4).
[0085] A ferritin monomer is a single ferritin molecule (or, where applicable, a single ferritin heavy or light chain) that has not assembled with other ferritin molecules. Ferritin protein self- assembles into a globular protein complex comprising multiple individual monomers. The self- assembled ferritin complex may be referred to as a ferritin particle or nanoparticle.
[0086] In some embodiments, the ferritin comprises one or more mutations (Embodiment F5a). In some embodiments, the one or more mutations comprise changes to the amino acid sequence of a wild-type ferritin and/or an insertion, e.g., at the N- or C-terminus (Embodiment F5b). In some embodiments, one, two, three, four, five, or more different amino acids are mutated in the ferritin as compared to wild-type ferritin (in some embodiments, in addition to any N- terminal insertion) (Embodiment F5c). In general, a mutation simply refers to a difference in the sequence (such as a substituted, added, or deleted amino acid residue or residues) relative to the corresponding wild-type ferritin. In some embodiments, the ferritin is a H. pylori ferritin (see e.g., SEQ ID NO: 65 or 66) with one or more mutations described herein (Embodiment F5d).
[0087] Human-compatible glycosylation can contribute to safety and efficacy in recombinant drug products. Regulatory approval may be contingent on demonstrating appropriate glycosylation as a critical quality attribute (see Zhang et al., Drug Discovery Today 21(5): 740- 765 (2016)). N-glycans can result from glycosylation of asparagine side chains and can differ in structure between humans and other organisms such as bacteria and yeast. Thus, it may be desirable to reduce or eliminate non-human glycosylation and/or N-glycan formation in ferritin according to the disclosure. In some embodiments, controlling glycosylation of ferritin improves the efficacy and/or safety of the composition, especially when used for human vaccination. [0088] In some embodiments, ferritin is mutated to inhibit formation of an N-glycan (Embodiment F6a). In some embodiments, a mutated ferritin has reduced glycosylation as compared to its corresponding wild type ferritin (Embodiment F6b).
[0089] In some embodiments, the ferritin comprises a mutation at an asparagine residue that is glycosylated in the wild type ferritin (Embodiment F6c). In some embodiments, the asparagine is N19 of H. pylori ferritin or a position that corresponds to N19 of H. pylori ferritin as determined by pair-wise or structural alignment (Embodiment F6d). In some embodiments, mutating such an asparagine, e.g., N 19 of H. pylori ferritin, decreases glycosylation of ferritin (Embodiment F6e). In some embodiments, the mutation replaces the asparagine with a glutamine (Embodiment F6f). In some embodiments, the ferritin is an H. pylori ferritin (e.g., SEQ ID NO: 65 or 66) comprising an N19Q mutation (Embodiment F6g). SEQ ID NOs 68 and 69 are exemplary ferritin sequences comprising aN19Q mutation (Embodiment F6h).
[0090] A mammal exposed to a glycosylated protein produced in bacteria or yeast may generate an immune response to the glycosylated protein, because the pattern of glycosylation of a given protein in bacterial or yeast could be different from the pattern of glycosylation of the same protein in a mammal. Thus, some glycosylated therapeutic proteins may not be appropriate for production in bacteria or yeast.
[0091] In some embodiments, decreased glycosylation of ferritin by amino acid mutation facilitates protein production in bacteria or yeast (Embodiment F7a). In some embodiments, decreased glycosylation of ferritin reduces the potential for adverse effects in mammals upon administration of mutated ferritin that is expressed in bacteria or yeast (Embodiment F7b). In some embodiments, the reactogenicity in a human subject of a mutated ferritin produced in bacteria or yeast is lower because glycosylation is decreased (Embodiment F7c). In some embodiments, the incidence of hypersensitivity responses in human subjects is lower following treatment with a mutated ferritin with reduced glycosylation compared to wildtype ferritin (Embodiment F7d).
[0092] In some embodiments, degradation in a subject of a composition comprising a mutated ferritin with reduced glycosylation is slower compared with a composition comprising a wild- type ferritin, or a composition comprising a corresponding ferritin with wild-type glycosylation (Embodiment F7e). In some embodiments, a composition comprising a mutated ferritin with reduced glycosylation has reduced clearance in a subject compared with a composition comprising a wild-type ferritin, or a composition comprising a corresponding ferritin with wild- type glycosylation (Embodiment F7f). In some embodiments, a composition comprising a mutated ferritin with reduced glycosylation has a longer-serum half-life compared to wild-type ferritin, or a composition comprising a corresponding ferritin with wild-type glycosylation (Embodiment F7g).
[0093] In one embodiment, the ferritin comprises a H. pylori ferritin comprising the amino acid sequence:
1 DIIKLLNEQV NKEMQSSNLY MSMSSWCYTH SLDGAGLFLF DHAAEEYEHA KKLIIFLNEN 61 NVPVQLTSIS APEHKFEGLT QIFQKAYEHE QHISESINNI VDHAIKSKDH ATFNFLQWYV 121 AEQHEEEVLF KDILDKIELI GNENHGLYLA DQYVKGIAKS RKSGS (SEQ ID NO: 69)(Embodiment F8) b. Other Multimerizing Proteins
[0094] In place of ferritin, other multimerizing proteins or portions thereof may be used (Embodiment F9a). For example, in certain embodiments, the multimerizing protein is lumazine synthase or a portion thereof (Embodiment F9b). In certain embodiments, the influenza HA-ss is joined to at least 50, at least 100 or least 150 amino acids from lumazine synthase, wherein the protein construct is capable of forming a nanoparticle (Embodiment F9c). In certain embodiments, the influenza HA-ss is joined to a protein at least 85%, at least 90%, at least 95%, or at least 98% identical to lumazine synthase, wherein the protein construct is capable of forming a nanoparticle (Embodiment F9d).
3. Structural Alignment
[0095] As discussed herein, positions of mutations corresponding to those described with respect to a given polypeptide (e.g., H. pylori ferritin or influenza HA) can be identified by pairwise or structural alignment. Structural alignment is relevant to large protein families such as ferritin where the proteins share similar structures despite considerable sequence variation and many members of the family have been structurally characterized.
[0096] Structural alignment involves identifying corresponding residues across two (or more) polypeptide sequences by (i) modeling the structure of a first sequence using the known structure of the second sequence or (h) comparing the structures of the first and second sequences where both are known and identifying the residue in the first sequence most similarly positioned to a residue of interest in the second sequence. Corresponding residues are identified in some algorithms based on distance minimization in the overlaid structures (e.g., what set of paired alpha carbons provides a minimized root-mean-square deviation for the alignment). Structural modeling and alignment is well known in the art, as discussed, for example, in U.S. Patent Nos 6,859,736 and 8,738,343; Aslam et al., Journal of Biotechnology, 2016, 20:9-13 and Bordoli et al., Nature Protocols, 2009, (4): 1-13.
4. Stem-Stabilized Influenza Hemagglutinin-Ferritin Protein Constructs ( HA - ss-F)
[0097] As discussed above, HA-ss-F protein constructs have been previously described. For example, PCT International Publication No. WO2018/045308, which is hereby incorporated by reference in its entirety, describes in detail the production of group 2 HA-ss-F protein constructs and H3-ss-np formed when the protein constructs are expressed in cells. See also, Corbett et al., Therapeutics and Prevention, 2019, 10(1), 1-18, which is hereby incorporated by reference in its entirety. Any of these previously described group 2 HA-ss-F constructs can be used to produce the HA-ss-np used in the vaccine compositions described herein. The HA-ss- np component of the vaccine composition can also be formed using any of the protein constructs described in this application, including any of the HA-ss-F protein constructs.
[0098] Typically, the modified stem region of the influenza HA and ferritin are genetically fused as a fusion protein. In certain embodiments, a linker sequence connects the C-terminus of the second stem region to the N-terminus of the ferritin protein. Any linker sequence may be used so long as the HA-ss and ferritin are able to form the desired immunogenic nanoparticles. While any amino acids may be used to make the linker sequence, it is preferred to use amino acids lacking large or charged side chains. Preferred amino acids include, but are not limited to, serine, glycine, valine and alanine. In one embodiment, the linker is made from serine and glycine residues. In some embodiments, the glycine-serine linker is GS, SGG, GGGS (SEQ ID NO: 75), 2XGGGS (SEQ ID NO: 76), 3XGGGS (SEQ ID NO: 77), 4XGGGS (SEQ ID NO: 78), or 5XGGGS (SEQ ID NO: 79). The length of the linker sequence may vary, but preferred embodiments use the shortest possible sequence in order to allow the stem sequences to form the desired structure. In one embodiment, the linker sequence is less than 10 amino acids in length. In one embodiment, the linker sequence is less than 5 amino acids in length. In one embodiment, the linker sequence is 3 amino acids in length. In one embodiment, the linker sequence consists of the amino acid sequence SGG. The modified stem region of the influenza HA and ferritin may also be joined by other mechanisms, including non- genetically linked, for example, by chemical conjugation.
[0099] Various embodiments of the modified stem region of the influenza HA are described in this application, and each embodiment described herein or otherwise know in the art (“HA-ss Embodiments”) can be combined with any of the ferritin embodiments described herein or otherwise known in the art (“F Embodiments”) to generate a desired HA-ss-F protein construct that assemble into nanoparticles (HA-ss-np) expressing trimers of HA stem protein on their surface when expressed in cells. For example, the HA-ss component of the HA-ss-F construct may be represented by any one of the HA-ss Embodiments described herein (e.g., HAla-g, HA2a-o, HA3, HA4a-s, HA5a-h, HA6a-i, HA7a-g, HA8a-n, HA9a-g, HAlOa-c, HAlla-b, HA12, HA13, and HA14a-c) or any combination of two or more of those HA Embodiments. Similarly, the ferritin component of the HA-ss-F construct may be represented by any one of the described F Embodiments described herein (e.g., Fla-c, F2a-c, F3a-d, F4, F5a-d, F6a-h, F7a-g, F8, F9a-d) or any combination of two or more of those F Embodiments. To generate the HA-ss-F construct, any of the HA Embodiments or combinations thereof may be combined with any of the F Embodiments or combinations thereof.
[00100] By way of example, in certain embodiments, Embodiment HA8n is combined with any of the F Embodiments described herein. For example, Embodiment HA8n can be combined with Embodiment Fid, F5d, or F8.
[00101] In certain embodiments, Embodiment HA9a is combined with any of the F Embodiments described herein. For example, Embodiment HA9a can be combined with Embodiment Fid, F5d, or F8.
[00102] In certain embodiments, any of Embodiments HA9b to HA9g, HAlOa to HAlOc, HAlla to HAllb, and/or HA12 or Embodiment HA9a further comprising any of Embodiments HA9b to HA9g , HAlOato HAlOc, HAlla to HAllb, and/or HA12 is combined with any of the F Embodiments described herein. For example, any of Embodiments HA9b to HA9g or Embodiment HA9a further comprising any of Embodiments HA9b to HA9g, HA12a to HA12c, HAlla to HAllb, and/or HA12 can be combined with Embodiment Fid, F5d, or F8.
[00103] In certain embodiments, Embodiment HA13 is combined with any of the F Embodiments described herein. For example, Embodiment HA13 can be combined with Embodiment Fid, F5d, or F8.
[00104] In certain embodiments, the Embodiment HA14a is combined with any of the
F Embodiments described herein. For example, Embodiment HA14a can be combined with Embodiment Fid, F5d, or F8. [00105] In certain embodiments, the Embodiment HA14b or HA14c is combined with any of the F Embodiments described herein. For example, Embodiment HA14b or HA14c can be combined with Embodiment Fid, F5d, or F8.
[00106] In certain embodiments, the FG-ss-F comprises the amino acid sequence (signal sequence in bold italics):
1 MKTIIALSYI LCLVFAQKLP GNDNSTATLC LGHHAVPNGT IVKTITNDQI EVTNATELVF
61 PGCGVLKLAT GMRNVPEKQT RGIFGAIAGF IENGWEGMVD GWYGFRHQNS EGIGQAADLK
121 STQAAINQIN GMVNRVIELM EQGGPDCYLA ELLVALLNQH VIDLTDSEMR KLFERTKKQL
181 RENAEDMGNG CFKIYHKCDN ACIGSIRNGT YDHDVYRDEA LNNRFQIKSG GDIIKLLNEQ
241 VNKEMQSSNL YMSMSSWCYT HSLDGAGLFL FDHAAEEYEH AKKLIIFLNE NNVPVQLTSI
301 SAPEHKFEGL TQIFQKAYEH EQHISESINN IVDHAIKSKD HATFNFLQWY VAEQHEEEVL
361 FKDILDKIEL IGNENHGLYL ADQYVKGIAK SRKSGS (SEQ ID NO: 61)
[00107] In certain embodiments, the FG-ss-F comprises the amino acid sequence (no signal sequence):
1 QKLPGNDNST ATLCLGHHAV PNGTIVKTIT NDQIEVTNAT ELVFPGCGVL KLATGMRNVP
61 EKQTRGIFGA IAGFIENGWE GMVDGWYGFR HQNSEGIGQA ADLKSTQAAI NQINGMVNRV
121 IELMEQGGPD CYLAELLVAL LNQHVIDLTD SEMRKLFERT KKQLRENAED MGNGCFKIYH
181 KCDNACIGSI RNGTYDHDVY RDEALNNRFQ IKSGGDIIKL LNEQVNKEMQ SSNLYMSMSS
241 WCYTHSLDGA GLFLFDHAAE EYEHAKKLII FLNENNVPVQ LTSISAPEHK FEGLTQIFQK
301 AYEHEQHISE SINNIVDHAI KSKDHATFNF LQWYVAEQHE EEVLFKDILD KIELIGNENH
361 GLYLADQYVK GIAKSRKSGS (SEQ ID NO: 62)
C. Adjuvant
[00108] The HA-ss-F nanoparticles described herein are administered with a squalene- based adjuvant emulsion, as described in PCT International Publication No. W02007/006939 and U.S. Patent No. 8,703,095, which are hereby incorporated by reference in their entireties. As described in U.S. Patent No. 8,703,095, such an emulsion can be obtained by means of a phase inversion temperature process, which permits the production of a monodisperse emulsion, the droplet size of which is very small, thus permitting the emulsion to be filtered with sterilizing filters having a cutoff threshold of 200 nm.
[00109] The squalene-based adjuvant comprises an oil-in-water adjuvant emulsion comprising at least: squalene, an aqueous solvent, a polyoxyethylene alkyl ether hydrophilic nonionic surfactant, and a hydrophobic nonionic surfactant. In certain embodiments, the emulsion is thermoreversible, optionally wherein 90% of the population by volume of the oil drops has a size less than 200 nm.
[00110] In certain embodiments, the polyoxyethylene alkyl ether is of formula CFE- (CH2)x-(0-CH2-CH2)n-0H, in which n is an integer from 10 to 60, and x is an integer from 11 to 17. In certain embodiments, the polyoxyethylene alkyl ether surfactant is polyoxyethylene(12) cetostearyl ether.
[00111] In certain embodiments, 90% of the population by volume of the oil drops has a size less than 160 nm. In certain embodiments, 90% of the population by volume of the oil drops has a size less than 150 nm. In certain embodiments, 50% of the population by volume of the oil drops has a size less than 100 nm. In certain embodiments, 50% of the population by volume of the oil drops has a size less than 90 nm.
[00112] In certain embodiments, the adjuvant further comprises at least one alditol, including, but not limited to, glycerol, erythritol, xylitol, sorbitol and mannitol.
[00113] In certain embodiments the hydrophilic/lipophilic balance (HLB) of the hydrophilic nonionic surfactant is greater than or equal to 10. In certain embodiments, the HLB of the hydrophobic nonionic surfactant is less than 9. In certain embodiments, the HLB of the hydrophilic nonionic surfactant is greater than or equal to 10 and the HLB of the hydrophobic nonionic surfactant is less than 9.
[00114] In certain embodiments, the hydrophobic nonionic surfactant is a sorbitan ester, such as sorbitan monooleate, or a mannide ester surfactant. In certain embodiments, the amount of squalene is between 5 and 45%. In certain embodiments, the amount of polyoxyethylene alkyl ether surfactant is between 0.9 and 9%. In certain embodiments, the amount of hydrophobic nonionic surfactant is between 0.7 and 7%. In certain embodiments, the adjuvant comprises: i) 32.5% of squalene, ii) 6.18% of polyoxyethylene(12) cetostearyl ether, iii) 4.82% of sorbitan monooleate, and iv) 6% of mannitol.
[00115] In certain embodiments, the adjuvant further comprises an alkylpolyglycoside and/or a cryoprotective agent, such as a sugar, in particular dodecylmaltoside and/or sucrose.
[00116] In certain embodiments, the adjuvant comprises AF03, as described in Klucker et ak, J. Pharm. Sci. 2012, 101(12):4490-500, which is hereby incorporated by reference in its entirety.
D. Compositions and Methods 1. Compositions
[00117] The present disclosure provides vaccine compositions comprising HA-ss-np, as described herein, in combination with a squalene-based oil-in-water adjuvant emulsion, such as AF03, as described herein. As disclosed in the examples, administration of the vaccine composition induces broadly neutralizing antibodies in non-human primates.
[00118] In addition to the HA-ss-np and squalene-based oil-in-water adjuvant emulsion, the vaccine composition may also include other pharmaceutically acceptable excipients. In general, the nature of the excipient will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, vaccine compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pharmaceutically acceptable salts to adjust the osmotic pressure, preservatives, stabilizers, buffers, sugars, amino acids, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
[00119] Typically, the vaccine composition is a sterile, liquid solution formulated for parenteral administration, such as intravenous, subcutaneous, intraperitoneal, intradermal, or intramuscular. The vaccine composition may also be formulated for intranasal or inhalation administration. The vaccine composition can also be formulated for any other intended route of administration.
[00120] In some embodiments, a vaccine composition is formulated for intradermal injection, intranasal administration or intramuscular injection. In some embodiments, injectables are prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. In some embodiments, injection solutions and suspensions are prepared from sterile powders or granules. General considerations in the formulation and manufacture of pharmaceutical agents for administration by these routes may be found, for example, in Remington ’s Pharmaceutical Sciences, 19th ed., Mack Publishing Co., Easton, PA, 1995; incorporated herein by reference. At present the oral or nasal spray or aerosol route ( e.g . , by inhalation) are most commonly used to deliver therapeutic agents directly to the lungs and respiratory system. In some embodiments, the vaccine composition is administered using a device that delivers a metered dosage of the vaccine composition. Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices such as those described in U.S. Patent No. 4,886,499, U.S. Patent No. 5,190,521, U.S. Patent No. 5,328,483, U.S. Patent No. 5,527,288, U.S. Patent No. 4,270,537, U.S. Patent No. 5,015,235, U.S. Patent No. 5,141,496, U.S. Patent No. 5,417,662 (all of which are incorporated herein by reference). Intradermal compositions may also be administered by devices which limit the effective penetration length of a needle into the skin, such as those described in WO1999/34850, incorporated herein by reference, and functional equivalents thereof. Also suitable are jet injection devices which deliver liquid vaccines to the dermis via a liquid jet injector or via a needle which pierces the stratum comeum and produces a jet which reaches the dermis. Jet injection devices are described for example in U.S. Patent No. 5,480,381, U.S. Patent No. 5,599,302, U.S. Patent No. 5,334,144, U.S. Patent No. 5,993,412, U.S. Patent No. 5,649,912, U.S. Patent No. 5,569,189, U.S. Patent No. 5,704,911, U.S. Patent No. 5,383,851, U.S. Patent No. 5,893,397, U.S. Patent No. 5,466,220, U.S. Patent No. 5,339,163, U.S. Pat. No. 5,312,335, U.S. Pat. No. 5,503,627, U.S. Pat. No. 5,064,413, U.S. Patent No. 5,520,639, U.S. Patent No. 4,596,556, U.S. Patent No. 4,790,824, U.S. Patent No. 4,941,880, U.S. Patent No. 4,940,460, WO1997/37705, and W01997/13537 (all of which are incorporated herein by reference). Also suitable are ballistic powder/particle delivery devices which use compressed gas to accelerate vaccine in powder form through the outer layers of the skin to the dermis. Additionally, conventional syringes may be used in the classical mantoux method of intradermal administration.
[00121] Preparations for parenteral administration typically include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti- oxidants, chelating agents, and inert gases and the like.
2. Methods
[00122] The present disclosure provides methods of administering the vaccine compositions described herein to a subject. The methods may be used to vaccinate a subject against an influenza virus. In certain embodiments, the vaccination method comprises administering to a subject in need thereof an immunologically effective dose of a vaccine composition comprising HA-ss-np, as described herein, and a squalene-based oil-in-water adjuvant emulsion, such as AF03, as described herein, wherein administration of the vaccine composition elicits broadly neutralizing influenza virus antibodies in the subject. Likewise, the present disclosure provides a vaccine composition comprising HA-ss-np, as described herein, and a squalene-based oil-in-water adjuvant emulsion, such as AF03, as described herein, for use in vaccinating a subject against an influenza virus.
[00123] The broadly neutralizing antibodies (bnAbs) elicited by the vaccine compositions described herein neutralize or protect against influenza viruses of more than one subtype and/or strain. In certain embodiments, the bnAbs bind the stem region and neutralize influenza viruses from two or more of the following sub-types: HI, H2, H4, H5, H6, H7, H8, H8, H10, Hll, H12, H13, H14, H15, H16, H17 or H18. In certain embodiments, the bnAbs bind the stem region and neutralize influenza viruses from two or more of the following group 2 sub-types: H3, H4, H7, H10, H14, or H15. In certain embodiment, the bnAbs bind the stem region and neutralize influenza viruses from one or more of the following group 1 sub-types: HI, H2, H5, H6, H8, H9, Hll, H12, H13, H16, H17 or H18 and one or more ofthe following group 2 sub-types: H3, H4, H7, H10, H14, or H15. In certain embodiments, the bnAbs bind the stem region and neutralize H3 influenza strains and one or more group 2 influenza strains selected from one or more of H4, H7, H10, H14, and HI 5 influenza strains. In certain embodiments, the bnAbs bind the stem region and neutralize at least one H3 influenza strain and at least one H7 strain (e.g., AJ Anhui/1/2013) or H10 strain (e.g., A/Jiangxi/IPB13b/2013). In certain embodiments, the bnAbs bind the stem region and neutralize at least one H3, H7 (e.g., A/Anhui/1/2013), and H10 (e.g., A/Jiangxi/IPB13b/2013) influenza strain.
Neutralization can be measured using known techniques, including the neutralization assays described herein.
[00124] In certain embodiments, the bnAbs bind the stem region and neutralize two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all nine of the following group 2 strains: A/Singapore/INFIMH-16-0019/2016 (H3N2), A/Hong Kong/4801/2014 (H3N2), A/Victoria/361/2011 (H3N2), A/Wisconsin/67/2005 (H3N2), A/Shangdong/9/ 1993 (H3N2), A/Port Chalmers/1/1973 (H3N2), A/Ai chi/02/1968, A/Jiangxi/IPB13b/2013 (H10N8), A/Jiangxi-Donghu/346-2/2013 (H10N8) and A/Anhui/1/2013 (H7N9). In certain embodiments, the bnAbs bind the stem region and neutralize two or more, three or more, four or more, five or more, or all six of the following H3 strains: A/Singapore/INFIMH- 16-0019/2016, A/Hong Kong/4801/2014,
A/Victoria/361/2011, A/Wisconsin/67/2005, A/Shangdong/9/1993, A/Port Chalmers/1/1973, and A/Aichi/02/1968.
[00125] In certain embodiments, the bnAbs bind the stem region and neutralize influenza viruses having an HA2 polypeptide that shares at least 60% identity with the wild type HA2 polypeptide from A/Finland/486/2004 (SEQ ID NO: 4), including, for example, any of A/Singapore/INFIMH- 16-0019/2016, A/Hong Kong/4801/2014, A/Victoria/361/2011, A/Wisconsin/67/2005, A/Shangdong/9/1993, A/Port Chalmers/1/1973, A/Aichi/02/1968, A/Jiangxi/IPB 13b/2013, and, A/Anhui/1/2013, which share 96.83%, 98.19%, 98.64%, 100%, 97.28%, 95.92%, 94%, 63.34%, and 63.80%, respectively, with the wild type HA2 polypeptide from A/Finland/486/2004 (SEQ ID NO: 4). In certain embodiments, the bnAbs bind the stem region and neutralize influenza viruses having an HA2 polypeptide that shares at least 63%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identity with the wild type HA2 polypeptide from A/Finland/486/2004 (SEQ ID NO: 4). In certain embodiments, the bnAbs bind the stem region and neutralize influenza viruses having an HA2 polypeptide that shares at least 94% identity with the wild type HA2 polypeptide from A/Finland/486/2004 (SEQ ID NO: 4).
[00126] In certain embodiments, the bnAbs elicit neutralization of divergent H3N2 strains from at least 1968 to 2016, as measured by an in vitro functional microneutralization reporter assay as described herein. In one embodiment, the divergent H3N2 strains comprise A/Singapore/INFIMH- 16-0019/2016, A/Hong Kong/4801/2014, A/Victoria/361/2011, A/Wisconsin/67/2005, A/Shangdong/9/1993, A/Port Chalmers/1/1973, and A/Aichi/02/1968.
[00127] In certain embodiments, the vaccine composition elicits an average antibody titer of at least 4 (loglO) to the HA protein from both the H3 strain A/Perth/16/2009 and the HA protein from the H10 strain A/Jiangxi/IPB13b/2013 when 50 pg of the vaccine composition is administered to cynomolgus macaques at weeks 0, 4, and 10, and wherein the antibody titer is measured by ELISA in sera samples obtained from the cynomolgus macaques at week 12.
[00128] Vaccine compositions comprising HA-ss-np, as described herein, and a squalene-based oil-in-water adjuvant emulsion, such as AF03, as described herein may be administered prior to or after development of one or more symptoms of an influenza infection. That is, in some embodiments, the vaccine compositions described herein may be administered prophylactically to prevent influenza infection or ameliorate the symptoms of a potential influenza infection. In some embodiments, a subject is at risk of influenza virus infection if the subject will be in contact with other individuals or livestock (e.g., swine) known or suspected to have been infected with pandemic influenza virus and/or if the subject will be present in a location in which influenza infection is known or thought to be prevalent or endemic. In some embodiments, the vaccine compositions are administered to a subject considered to be suffering from an influenza infection, or the subject is displaying one or more symptoms commonly associated with influenza infection. In some embodiments, the subject is known or believed to have been exposed to an influenza virus. In some embodiments, a subject is considered to be at risk or susceptible to an influenza infection if the subject is known or believed to have been exposed to the influenza virus. In some embodiments, a subject is known or believed to have been exposed to the influenza virus if the subject has been in contact with other individuals or livestock (e.g., swine) known or suspected to have been infected with pandemic influenza virus and/or if the subject is or has been present in a location in which influenza infection is known or thought to be prevalent or endemic. Vaccine compositions in accordance with the disclosure may be administered in any dose appropriate to achieve a desired outcome. In some embodiments, the desired outcome is induction of a lasting adaptive immune response against a broad spectrum of influenza strains, including both seasonal and pandemic strains. In some embodiments, the desired outcome is reduction in intensity, severity, and/or frequency, and/or delay of onset of one or more symptoms of influenza infection. The dose required may vary from subject to subject depending on the species, age, weight and general condition of the subject, the severity of the infection being treated, the particular composition being used and its mode of administration.
[00129] In some embodiments, the vaccine compositions described herein are administered to a human subject. In particular embodiments, a human subject is 6 months of age or older, is 6 months through 35 months of age, is 36 months through 8 years of age, or 9 years of age or older. In certain embodiments, the human subject is an infant (less than 36 months). In certain embodiments, the human subject is a child or adolescent (less than 18 years of age). In certain embodiments, the human subject is elderly (at least 60 years of age). In certain embodiments, the human subject is a non-elderly adult (at least 18 years of age and less than 60 years of age).
[00130] The methods and uses of the vaccine compositions described herein include prime-boost vaccination strategies. Prime-boost vaccination comprises administering a priming vaccine and then, after a period of time has passed, administering to the subject a boosting vaccine. The immune response is “primed” upon administration of the priming vaccine, and is “boosted” upon administration of the boosting vaccine. The priming vaccine can include a vaccine composition comprising HA-ss-np, as described herein, and a squalene- based oil-in- water adjuvant emulsion, such as AF03, as described herein. Likewise, the boosting vaccine can include a vaccine composition comprising HA-ss-np, as described herein, and a squalene-based oil-in-water adjuvant emulsion, such as AF03, as described herein. The priming vaccine composition can be, but need not be, the same as the boosting vaccine. Administration of the boosting vaccine is generally weeks or months after administration of the priming composition, preferably about 2-3 weeks or 4 weeks, or 8 weeks, or 16 weeks, or 20 weeks, or 24 weeks, or 28 weeks, or 32 weeks.
[00131] The vaccine composition can be administered using any suitable route of administration, including, for example, parenteral delivery, as discussed above.
[00132] Typically, the HA-ss-np and adjuvant are administered together as components of the same vaccine composition. However, it is not necessary for the HA-ss-np and adjuvant to be administered as part of the same vaccine composition. That is, if desired, the HA-ss-np and the squalene-based oil-in-water adjuvant emulsion, such as AF03, can be administered to the subject sequentially.
[00133] The present disclosure will be more fully understood by reference to the following Examples.
EXAMPLES
[00134] To determine if non-human primates (NHP) can generate broadly reactive neutralizing antibodies (bnAbs) to diverse group 2 strains of influenza, NHPs were immunized with H3-SS-np vaccines and antibody responses were analyzed genetically and structurally.
[00135] Example 1 - Immunization of NHP
[00136] All nucleotide sequences were human codon-optimized and cloned into the SIB002 vector for mammalian expression. H3-SS-np was designed from A/Finland/486/2004 (H3N2) HA template sequences and was prepared and purified as described in Corbett et ak, Therapeutics and Prevention, 2019, 10(1), 1-18. The H3-ss-F fusion protein comprises the amino acid sequence of SEQ ID NO: 61. When expressed in cells, the signal sequence is cleaved resulting in the amino acid sequence of SEQ ID NO: 62, which forms uniform nanoparticles. [00137] To produce antigen for in vitro assays, the ectodomain of the HAs from A/Perth/16/2009 (H3N2) and A/Jiangxi/IPB13b/2013 (H10N8) were fused to C-terminal Thrombin cleavage site, followed by the trimeric foldon domain of T4 fibritin and a hexahistidine tag sequence (SEQ ID NO: 83). Plasmids were purified with the Powerprep kit (Origene #NP100009) and used to transfect Expi293 cells (Thermo Fisher Scientific #A14635). FectoPRO DNA transfection reagent (Polyplus #116-100) was used (0.5 pg of DNA per mL, 0.75 pL of FectoPRO reagent per mL, and 0.45 pL of enhancer per mL). Four days after transfection, supernatant was harvested by centrifugation at 3,488 g for 15 min at 4 °C and filtered through a 0.45 pm vacuum-driven filter unit (Thermo Fisher Scientific #167-0045). Foldon-trimerized antigens used for in vitro assays were purified with Nickel Sepharose Excel resin (GE Healthcare, catalog# 17371201) in 50 mM Tris buffer pH 7.5 with 300 mM NaCl and eluted with 150 mM Imidazole. HA trimers were purified by size-exclusion chromatography using a Superdex 200 10/300 column (GE catalog# 28990944) in phosphate- buffered saline.
[00138] Four cynomolgus macaques (Macaca fascicularis ) were immunized with a H3- SS-np vaccine with a squalene-based oil-in-water emulsion adjuvant, AF03. Cynomolgus macaques were housed and cared for by Bioqual Inc. in compliance with all federal regulations, including USD A regulations and the Animal Welfare Act. Animals were pre-screened and selected for lack of reactivity to 2016-2017 Fluzone Quadrivalent antigens (A/Califomi a/07/2009; A/HongKong/4801/2014 X-263B; B/Phuket/3073/2013;
B/Brisbane/60/2008) by ELISA. At the time of the study the age of the monkeys ranged from 6 to 15 years old, and body weight ranged between 4 and 7 kg. Subjects received three doses (50 pg each) of vaccine with H3-SS-np at weeks 0, 4 and 10 with AF03 adjuvant. Serum samples were collected at weeks 0, 2, 4, 6, 8, 10 and 12. PBMC were collected and isolated at weeks 0, 2, 5, 6 and 12. Serum and PBMCs were isolated and cryopreserved following standard operating procedures.
[00139] Antibody responses were measured by Enzyme-Linked Immunosorbent Assay (ELISA). For ELISA assays, serum was heat-inactivated at 65 °C for 30 min. Nunc MaxiSorp 96-well plates (Thermo Fisher Scientific # 44-2404-21) were coated with 100 ng of antigen per well overnight at 4 °C. Antigens included HA-foldon proteins from A/P erth/16/2009 (H3N2) wildtype or ASteml(I45N, Q47T) or AStem2 (D19N, G33E) and A/Jiangxi/IPB13/2013 (H10N8). NHP sera or recombinant monkey antibodies were serially-diluted in 5% milk-PBST and allowed to bind for 1 hour at room temperature. Binding to the antigens were detected with anti-monkey-HRP (Southern Biotech catalog# 4700-05, at 1:5,000) or anti-human-HRP (Jackson H+L anti-human IgG secondary catalog#709-035-149, at 1:5,000). HRP was developed with SureBlueTMB substrate (Seracare # 52-00-02). Absorbance was measured at 450 nm in a Spectramax instrument. Endpoint titers were calculated with Graphpad Prism with a threshold value of 0.2 and typical background level of 0.05 or 0.1.
[00140] Neutralization was measured using a lentiviral reporter assay performed as previously described (N. Darricarrere et ak, Journal of Virology, 2018, published online Epub: Sep 5 (10.1128/JVI.01349-18)). Serum or antibodies were serially diluted and pre-incubated with a fixed amount of lentivirus and used to infect target 293 A cells. Infection was quantified 72 hours later with the Promega Luciferase Assay System (catalog# E1500). EC50 were calculated with Graphpad Prism from neutralization curves. For the HA competition assays, H3-SS-foldon, wildtype (A/Perth/16/2009 (H3N2)) or Astern (D19N, G33E) mutant proteins (1 pg) were pre-incubated with varying amount of serum for 1 hour before lentivirus was added. The microneutralization (MN) reporter assay was described in (A. Creanga et ak, bioRxiv, 2020 doi.org/10.1101/2020.02.24.963611) and used reporter knock-in influenza viruses from the following strains: A/Singapore/INFIMH-16-0019/2016 (H3N2), A/Hong Kong/4801/2014 (H3N2), A/Victoria/361/2011 (H3N2), A/Wisconsin/67/2005 (H3N2), A/Shangdong/9/1993 (H3N2), A/Port Chalmers/1/1973 (H3N2), A/Aichi/02/1968 (H3N2), A/Jiangxi-Donghu/346-2/2013 (H10N8) and A/Anhui/1/2013 (H7N9). For the competition reporter-MN assay, either wildtype (WT) or Astern (I45N, Q47T) mutant HA protein from A/Wisconsin/67/2005 strain were used.
[00141] Binding antibodies were detected to both the homologous H3 (A/Perth/16/2009) strain (Fig. 1A, left) and a heterosubtypic H10 (A/Jiangxi-Donghu/346-2/2013) strain (Fig. 1A, right). The group 2 stabilized HA stem vaccine composition stimulated neutralizing Abs to the group 2 HA stem epitope as the activity was absorbed by wild type H3 HA but not by Astern mutant (Fig. IB; stem vs. Astern competitor). Moreover, this vaccine induced broad H3 reactivity, neutralizing divergent H3N2 strains from 1968 to 2016 (Fig. 1C). Three out of four animals neutralized the oldest strain tested from the 1968 H3N2 pandemic, and all animals developed neutralization activity towards selected heterosubtypic H10N8 and H7N9 strains. Therefore, a vaccine composition comprising group 2 HA-ss-F nanoparticles and AF03 elicited bnAbs to a conserved domain of the HA stem. In general, cross-group neutralization was sub- optimal (data not shown). [00142] EXAMPLE 2 - Characterization of broadly neutralizing antibodies from immunized NHP
[00143] PBMCs from immunized monkeys were isolated following standard methodology, using Leucosep 50 mL tubes (VWR#89048-938) with a 95% Ficoll-Paque Plus density gradient medium (GE Healthcare #17-1440). Isolated PBMCs were cryopreserved using commercial freezing media (Gibco catalog# 12648-010). On the day of single cell sorting, PBMC aliquots were thawed, washed with RPMI media (Gibco/Invitrogen, 11875-093), and treated with 50 units per mL of Benzonase (EMD Millipore #70664-3) before staining. Antibody cocktails were prepared in PBS-1% BSA (Bovine Albumin fraction V (BSA) from Fisher Bioreagents, catalog# BP 1600- 100). The following antibody markers were used: CD20- FITC (BD Biosciences, catalog #347673), CD3-PerCP-Cy5.5 (BD Biosciences, catalog #552852), IgD-PE (Southern Biotech, catalog #2030-09), CD8-PE Texas Red (Invitrogen, catalog #MHCD0817), IgM-BV786 (BD Biosciences catalog #740998), CD 16- PE-Cy7 (BD Biosciences, catalog #557744), CD14-BV650 (BioLegend, catalog #301836), IgG-BV605 (BD Biosciences catalog #563246), and CD27- APC-Cy7 (BioLegend catalog #302816). H3 (2009) and H10, or H3 (1968) and H3 (2011) fluorescent probes were used at 0.5 pg of HA per 5-20 million cells. Viability was assessed with Aqua stain (Invitrogen, catalog #L34957). HA+ IgG+ B cells were isolated by single-cell sorting on a BD Influx cell sorter gating for either H3+, H10+, or H3+/H3+ as indicated. PBMCs were gated sequentially for forward and side scatter, viability, singlet cell dispersion, CD 16 negative, CD 14 negative, CD3 negative, CD20 positive, IgD negative, IgM negative, IgG positive, HA positive gates. Single cells were sorted directly onto reverse transcriptase lysis buffer (1.25x Superscript Buffer (Invitrogen, catalog #18080- 085), 6.25 mM DTT (Invitrogen, catalog #18080-085), 0.4% Igepal (Sigma catalog # 18896) and 20 units/well of RNase OUT inhibitor (Invitrogen catalog #10777-019) in 20 pL/well) and frozen on dry ice before further processing.
[00144] The bnAbs elicited by the H3-SS-np + AF03 vaccine were isolated using two screening protocols and sequenced. In one screening protocol, bnAbs were sorted using a single H3 HA probe (H3+) derived from A/Perth/16/2009 (Fig. 2A). In a second screening protocol, bnAbs were counter sorted using two H3 HA probes (H3+/H3+) derived from divergent strains, A/Hong Kong/1/1968 and A/Victoria/361/2011 (Fig. 2B). These bnAbs antibodies were derived from diverse VH-DH-JH germline gene combinations, with 75% of the heavy chain sequences derived from the IGHV4 family, and >60% using the IGHJ4 gene (Figs. 2A-B). For comparison, human anti-H3 stem antibodies are derived from diverse families, including IGHV4-30 and IGHV4-34, but without preferential IGHV gene usage (M. G. Joycen et al., Cell 166, 609-623 (2016)). The H3+ or H3+/H3+ light chains were also derived from a diverse combination of VJ germline genes, with >40% of lambda chains derived from IGLV1-15 and >25% of Kappa chains derived from IGKV1S26 (Figs. 3A-B).
[00145] H3+ immune B cells from NHP immunized with the vaccine composition were sorted and 20 mAbs characterized (Table 1). Sixteen mAbs bound to H3 HA, and 8 reacted with both H3 and H10 HA (Table 1). Ten mAbs neutralized at least two H3 influenza strains, and 7 of these had Pan-H3 activity, since they neutralized all strains tested, spanning viral isolates derived from 1973 to 2014 (Fig. 4A). Furthermore, 6 mAbs cross-neutralized a heterosubtypic H10 influenza strain and two mAbs neutralized an H7 subtype strain (Fig. 4A). Binding of these mAbs to H3 HA was affected by AStem (D19N/G33E) mutations on HA stem (Fig. 4B and Fig. 6), indicating that they were also HA stem directed.
[00146] Among the six non-neutralizing mAbs, one had lower binding affinity to HA (H3-2E9, Table 1) and another recognized a different epitope on the stem since the Astern did not affect its binding affinity (H3-2E2, Fig. 6). The other four had similar affinity to wild type stem as other neutralizing mAbs but binding to Astern was either reduced (H3-1C7, H3-1 A3), or completely abolished (H3-1B5, H3-2A6), suggesting that these 4 non-neutralizing antibodies target the same region but have a different binding footprint (H3-1C7, H3-1 A3, H3- 1B5, H3-2A6, Fig. 6).
[00147] Table 1: Repertoire analysis of IgG heavy chain and light chain variable region from 20 antibodies cloned from H3+ B cells. The variable, diversity and joining germline genes are listed next to the % match of the antibody nucleotide sequence to the germline gene and EC50 values (pg/mL) are reported to H3/Perth/16/2009 and A/Jiangxi/IPB 13/2013. NB = No binding. LB = Low binding.
Figure imgf000046_0001
[00148] EXAMPLE 3 - Cryo-electron microscopy analysis of H3-3B10 mAb bound
[00149] To elucidate how H3-3B10 mAb (3B10) neutralized group 2 influenza viruses, the cryoEM structure of 3B10 Fab in complex with an H3 HA (A/Victoria/361/2011) was determined. The structure showed that 3B10 bound to the lower part of the HA stem, in close proximity to the viral membrane (Fig. 5A-B). The epitope residues were mostly located on the HA2 polypeptide and the N- and C-terminal end of the fusion peptide region (Fig.7A-D). The complete buried HA- 3B10 interface area is 970 A2, of which 90% was contributed from heavy chain binding and 10% from the light chain. The fusion peptide is extensively targeted by all three heavy chain CDRs. The CDR-H1 and CDR-H2 made contacts with the N-terminus of the fusion peptide through van der Waals and hydrogen-bonding interactions, while CDR-H3 largely contacted the C-terminus of the fusion peptide through additional cation-p interactions (R25HA2-W1013B10-cdr'H3) (Fig. 7A- C). In the HA-3B10 EM map, continuous density was observed in the HA1-HA2 connection region, which may be due to the stabilizing effect of extensive 3B10 interactions (Fig. 7D). The epitope of 3B 10 overlay well with that of another human monoclonal antibody, CR8020 (Fig. 5B). The structure superimposition between CR8020-HA and 3B 10-HA showed substantial epitope overlap and identical orientation with conservation of some paratope residues between the two mAbs (Fig. 5C). These observations are consistent with the equivalent potency and neutralizing breadth of 3B10 and CR8020 (Fig. 4A) and support a similar mode of binding of the human and macaque bnAbs.
[00150] A broadly protective vaccine is desired to effectively control seasonal influenza infection and to avert pandemic outbreaks. In this study, it was found that pan-H3 broadly neutralizing responses can be achieved by immunization of cynomolgus macaques using engineered stabilized stem nanoparticle vaccines with a squalene-based, oil-in-water adjuvant emulsion adjuvant, like AF03. Interestingly, the mAbs derived from immune NHP resembled human bnAbs prototypes induced by infection or vaccination, with similar potency and binding specificity. Likewise, the CDR-H3 of 3B10 also plays an important role in recognizing the HA stem epitope. The elicitation of bnAbs in non-human primates through vaccination with a rationally-designed protein nanoparticle vaccine suggests that rational vaccine design, together with the judicious use of adjuvant, facilitates the development of a broadly protective influenza vaccine for humans.
[00151] A hallmark of influenza outbreaks is the ability of the virus to give rise to new strains quickly and unpredictably (H. V. Fineberg, /VE/M, 2014, 370:1335-1342). Stem-based universal vaccines have the potential to limit the global spread of new strains because they can be deployed before pandemics begin, and the immune response is directed to a conserved structure, reducing the chances of immune escape (K. E. Neu et ah, Current opinion in immunology , 2016, 42:48-55). With the aim of developing a truly universal vaccine that will address the threat of influenza pandemics, immunogens that present the conserved stem epitope have been designed, as previously discussed herein. However, a question that remained unaddressed is whether these immunogens could elicit bnAbs akin to the ones discovered in humans. The structural superimposition of the vaccine-induced macaque H3-3B10 to human CR8020 demonstrates with atomic resolution that broadly neutralizing anti-stem antibodies can be elicited through diverse antibody germline genes in antigen-naive non-human primates by vaccination with a composition comprising a rationally-designed HA stem immunogen and a squalene-based, oil-in-water adjuvant emulsion adjuvant, like AF03, which composition can be developed and deployed in humans.
[00152] While the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be clear to one of ordinary skill in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the disclosure and may be practiced within the scope of the appended claims. For example, all the protein constructs, methods, and/or component features, steps, elements, or other aspects thereof can be used in various combinations.
[00153] Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure also includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists, (e.g., in Markush group or similar format) it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where embodiments or aspects of the disclosure, is/are referred to as comprising particular elements, features, etc., certain embodiments or aspects consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the disclosure can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification.
[00154] All patents, patent applications, websites, other publications or documents, accession numbers and the like cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number, if applicable. Likewise, if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant, unless otherwise indicated.

Claims

We claim:
1. A vaccine composition, comprising stem-stabilized influenza hemagglutinin-ferritin nanoparticles and an adjuvant, wherein the adjuvant comprises an oil-in-water adjuvant emulsion comprising:
- squalene,
- an aqueous solvent,
- a polyoxyethylene alkyl ether hydrophilic nonionic surfactant, and
- a hydrophobic nonionic surfactant; wherein the stem-stabilized influenza hemagglutinin-ferritin nanoparticles comprise a ferritin protein joined to a modified H3 influenza virus hemagglutinin (HA) protein to form a protein construct and wherein the protein construct forms stem- stabilized influenza hemagglutinin-ferritin nanoparticles when expressed in cells; wherein the modified H3 influenza virus HA protein lacks an antigenic head region and comprises a modified stem region from an H3 influenza virus HA protein wherein the modified stem region comprises at least one immunogenic epitope; and wherein the vaccine composition elicits broadly neutralizing influenza antibodies when administered to a subject.
2. The vaccine composition of claim 1, wherein the modified H3 influenza virus HA protein comprises a first stem region covalently joined to a second stem region by a first linker sequence, wherein the HA head region has been substantially replaced by the first linker sequence, wherein the second stem region comprises a first portion comprising a helix A sequence, a helix A extension sequence at the C-terminus of the helix A sequence, a second linker sequence, and a second portion comprising a helix C sequence, wherein the second linker sequence covalently joins the helix A extension sequence and the helix C sequence and substantially replaces the HA2 interhelical region, wherein the helix A extension sequence extends the length of the helix A sequence by up to 5 amino acid residues, and wherein the second stem region comprises at least one first mutation that improves side chain repacking and/or resurfacing and at least one second mutation at the N-terminus of the helix C sequence that increases the helix-forming propensity of the helix C.
3. The vaccine composition according to claim 1 or 2, wherein the at least one first mutation is selected from the group consisting of one or more mutations at amino acid residues K51, L52, L55, N95, E103, T107, andN116, wherein the amino acid numbering is based on the HA2 polypeptide of the A/Finland/486/2004 H3N2 influenza virus (SEQ ID NO: 4)·
4. The vaccine composition according to any one of the preceding claims, wherein the at least one second mutation is selected from the group consisting of one or more mutations at amino acid residues W92 and S93, wherein the amino acid numbering is based on the HA2 polypeptide of the A/Finland/486/2004 H3N2 influenza virus (SEQ ID NO: 4).
5. The vaccine composition according to claim 3, wherein the at least one first mutation is selected from the group consisting of one or more of K51M, L52V, L55V, N95L, E103L, T107V, and Nil 6R.
6. The vaccine composition according to claim 4, wherein the at least one second mutation is selected from the group consisting of one or more of W92D and S93C.
7. The vaccine composition according to any one of the preceding claims, wherein the at least one first mutation and at least one second mutation are located at amino acid residues K51, L52, L55, W92, S93, N95, E103, T107, andN116, wherein the amino acid numbering is based on the HA2 polypeptide of A/Finland/486/2004 H3N2 influenza virus (SEQ ID NO: 4).
8. The vaccine composition according to claim 7, wherein the at least one first mutation comprises K51M, L52V, L55V, N95L, E103L, T107V, and N116R and the at least one second mutation comprises W92D and S93C, wherein the amino acid numbering is based on the HA2 polypeptide of A/Finland/486/2004 H3N2 influenza virus (SEQ ID NO: 4).
9. The vaccine composition according to any one of the preceding claims, wherein all amino acid residues of the head region are removed.
10. The vaccine composition according to any one of the preceding claims, wherein the first linker forms a loop and contains a first cysteine residue that forms a disulfide bond with a second cysteine residue in the N-terminus of the helix C sequence in the second stem region.
11. The vaccine composition of any one of the preceding claims, wherein the first linker consists of 6-7 amino acids.
12. The vaccine composition of claim 11, wherein the first linker sequence consists of the amino acid sequence of VFPGCGV (SEQ ID NO: 5).
13. The vaccine composition of any one of claims 2-12, wherein the second linker consists of 3-6 amino acids including at least two glycine residues.
14. The vaccine composition of claim 13, wherein the second linker consists of the amino acid sequence GGP.
15. The vaccine composition of any one of claims 2-14, wherein the helix A extension sequence consists of the amino acid sequence ELMEQ (SEQ ID NO: 14).
16. The vaccine composition of any one of the preceding claims, wherein the ferritin protein is joined to the modified H3 influenza virus hemagglutinin (HA) protein by a third linker sequence.
17. The vaccine composition of claim 16, wherein the third linker sequence comprises glycine and serine residues.
18. The vaccine composition of claim 17, wherein the third linker sequence consists of the amino acid sequence SGG.
19. A vaccine composition, comprising stem-stabilized influenza hemagglutinin-ferritin nanoparticles and an adjuvant, wherein the adjuvant comprises an oil-in-water adjuvant emulsion comprising:
- squalene,
- an aqueous solvent,
- a polyoxyethylene alkyl ether hydrophilic nonionic surfactant, and
- a hydrophobic nonionic surfactant; wherein the stem-stabilized influenza hemagglutinin-ferritin nanoparticles comprise a ferritin protein joined to a modified H3 influenza virus hemagglutinin (HA) protein to form a protein construct and wherein the protein construct forms stem- stabilized influenza hemagglutinin-ferritin nanoparticles when expressed in cells; wherein the modified H3 influenza virus HA protein comprises a first stem region covalently joined to a second stem region by a first linker sequence, wherein the HA head region has been substantially replaced by the first linker sequence, wherein the second stem region comprises a first portion comprising a helix A sequence, a helix A extension sequence at the C-terminus of the helix A sequence, a second linker sequence, and a second portion comprising a helix C sequence, wherein the second linker sequence covalently joins the helix A extension sequence and the helix C sequence, and substantially replaces the HA2 interhelical region, wherein the second stem region comprises at least one first mutation that improves side chain repacking and/or resurfacing and at least one second mutation at the N-terminus of the helix C sequence that increases the helix-forming propensity of the helix C sequence, and wherein the vaccine composition elicits broadly neutralizing influenza antibodies when administered to a human subject.
20. The vaccine composition according to claim 19, wherein the first linker sequence consists of the amino acid sequence VFPGCGV (SEQ ID NO: 5), wherein a first cysteine residue in the linker sequence forms a disulfide bond with a second cysteine residue in the N- terminus of the helix C sequence in the second stem region, wherein the second linker sequence consists of the amino acid sequence GGP, wherein the helix A extension sequence consists of the amino acid sequence ELMEQ (SEQ ID NO: 14), wherein the at least one first mutation is selected from the group consisting of one or more mutations at amino acid residues K51, L52, L55, N95, E103, T107, and N116, wherein the amino acid numbering is based on the HA2 polypeptide of A/Finland/486/2004 H3N2 influenza virus (SEQ ID NO: 4), and wherein the at least one second mutation is selected from the group consisting of one or more mutations at amino acid residues W92 and S93, wherein the amino acid numbering is based on the HA2 polypeptide of A/Finland/486/2004 H3N2 influenza virus (SEQ ID NO:
4)·
21. The vaccine composition of claim 20, wherein the at least one first mutation comprises K51M, L52V, L55V, N95L, E103L, T107V, and N116R and the at least one second mutation comprises W92D and S93C, wherein the amino acid numbering is based on the HA2 polypeptide of A/Finland/486/2004 H3N2 influenza virus (SEQ ID NO: 4)
22. The vaccine composition according to any one of the preceding claims, wherein the modified H3 influenza virus HA protein comprises the amino acid sequence:
1 QKLPGNDNST ATLCLGHHAV PNGTIVKTIT NDQIEVTNAT ELVFPGCGVL KLATGMRNVP 61 EKQTRGIFGA IAGFIENGWE GMVDGWYGFR HQNSEGIGQA ADLKSTQAAI NQINGMVNRV 121 IELMEQGGPD CYLAELLVAL LNQHVIDLTD SEMRKLFERT KKQLRENAED MGNGCFKIYH 181 KCDNACIGSI RNGTYDHDVY RDEALNNRFQ IK (SEQ ID NO:64).
23. The vaccine composition according to any one of the preceding claims, wherein the ferritin protein is from H. pylori.
24. The vaccine composition according to claim 23, wherein the ferritin protein comprises the amino acid sequence:
1 DIIKLLNEQV NKEMQSSNLY MSMSSWCYTH SLDGAGLFLF DHAAEEYEHA KKLIIFLNEN 61 NVPVQLTSIS APEHKFEGLT QIFQKAYEHE QHISESINNI VDHAIKSKDH ATFNFLQWYV 121 AEQHEEEVLF KDILDKIELI GNENHGLYLA DQYVKGIAKS RKSGS (SEQ ID NO: 69).
25. The vaccine composition according to any one of the preceding claims, wherein the protein construct comprises the amino acid sequence:
1 QKLPGNDNST ATLCLGHHAV PNGTIVKTIT NDQIEVTNAT ELVFPGCGVL KLATGMRNVP 61 EKQTRGIFGA IAGFIENGWE GMVDGWYGFR HQNSEGIGQA ADLKSTQAAI NQINGMVNRV 121 IELMEQGGPD CYLAELLVAL LNQHVIDLTD SEMRKLFERT KKQLRENAED MGNGCFKIYH 181 KCDNACIGSI RNGTYDHDVY RDEALNNRFQ IKSGGDIIKL LNEQVNKEMQ SSNLYMSMSS 241 WCYTHSLDGA GLFLFDHAAE EYEHAKKLII FLNENNVPVQ LTSISAPEHK FEGLTQIFQK 301 AYEHEQHISE SINNIVDHAI KSKDHATFNF LQWYVAEQHE EEVLFKDILD KIELIGNENH 361 GLYLADQYVK GIAKSRKSGS (SEQ ID NO: 62).
26. The vaccine composition according to any one of the preceding claims, wherein the protein construct is expressed in a eukaryotic cell and comprises native glycans from the eukaryotic cell.
27. The vaccine composition according to any one of the preceding claims, wherein the emulsion is thermoreversible and optionally, wherein 90% of the population by volume of the oil drops has a size less than 200 nm.
28. The vaccine composition according to any one of the preceding claims, wherein the polyoxyethylene alkyl ether is of formula CH3-(CH2)x-(0-CH2-CH2)n-0H, in which n is an integer from 10 to 60, and x is an integer from 11 to 17.
29. The vaccine composition of claim 28, wherein the polyoxyethylene alkyl ether surfactant is polyoxyethylene(12) cetostearyl ether.
30. The vaccine composition according to any one of the preceding claims, wherein the adjuvant further comprises an alditol.
31. The vaccine composition according to any one of the preceding claims, wherein a hydrophilic/lipophilic balance (HLB) of the hydrophilic nonionic surfactant is greater than or equal to 10 and wherein the HLB of the hydrophobic nonionic surfactant is less than 9.
32. The vaccine composition according to any one of the preceding claims, wherein the adjuvant comprises: i) 32.5% of squalene, ii) 6.18% of polyoxyethylene(12) cetostearyl ether, iii) 4.82% of sorbitan monooleate, and iv) 6% of mannitol.
33. The vaccine composition according to any one of the preceding claims, wherein the adjuvant comprises AF03.
34. The vaccine composition according to any one of the preceding claims, wherein the vaccine composition elicits neutralization of one or more divergent group 2 strains having an HA2 polypeptide that shares at least 60% identity with the wild type HA2 polypeptide from A/Finland/486/2004 (SEQ ID NO: 4), as measured by an in vitro functional microneutralization reporter assay
35. The vaccine composition according to any one of the preceding claims, wherein the vaccine composition elicits neutralization of one or more divergent H3 strains having an HA2 polypeptide that shares at least 95% identity with the wild type HA2 polypeptide from A/Finland/486/2004 (SEQ ID NO: 4), as measured by an in vitro functional microneutralization reporter assay.
36. The vaccine composition according to any one of the preceding claims, wherein the vaccine composition elicits neutralization of divergent H3N2 strains from 1968 to 2016, as measured by an in vitro functional microneutralization reporter assay.
37. The vaccine composition according to any one of the preceding claims, wherein the divergent H3N2 strains comprise A/Singapore/INFIMH- 16-0019/2016, A/Hong Kong/4801/2014, A/Victoria/361/2011, A/Wisconsin/67/2005, A/Shangdong/9/1993, A/Port Chalmers/1/1973, and A/Aichi/02/1968.
38. The vaccine composition according to any one of the preceding claims, wherein the vaccine composition elicits an average antibody titer of at least 4 (logio) to the HA protein from both the H3 strain A/Perth/16/2009 and the HA protein from the H10 strain A/Jiangxi/IPB13b/2013 when 50 pg of the vaccine composition is administered to cynomolgus macaques at weeks 0, 4, and 10, and wherein the antibody titer is measured by ELISA in sera samples obtained from the cynomolgus macaques at week 12.
39. The vaccine composition according to any one of the preceding claims, wherein the broadly neutralizing influenza antibodies neutralize H3 influenza strains and one or more group 2 influenza strains selected from one or more of H4, H7, H10, H14, and H15 influenza strains, as measured by an in vitro functional microneutralization reporter assay.
40. The vaccine composition according to any one of the preceding claims, wherein the broadly neutralizing influenza antibodies neutralize at least one H3 influenza strain and at least one H7 or H10 influenza strain, as measured by an in vitro functional microneutralization reporter assay.
41. The vaccine composition according to any one of the preceding claims, wherein the broadly neutralizing influenza antibodies neutralize at least one H3 influenza strain, at least one H7 influenza strain, and at least one H10 influenza strain as measured by an in vitro functional microneutralization reporter assay.
42. A method of vaccinating against influenza virus, the method comprising administering to a subject in need thereof an immunologically effective dose of the vaccine composition of any one of the preceding claims, wherein administration of the vaccine composition elicits broadly neutralizing influenza antibodies in the human subject.
43. The method according to claim 42, wherein the broadly neutralizing influenza antibodies neutralize one or more divergent group 2 strains having an HA2 polypeptide that shares at least 60% identity with the wild type HA2 polypeptide from A/Finland/486/2004 (SEQ ID NO: 4), as measured by an in vitro functional microneutralization reporter assay
44. The method according to claim 42, wherein the broadly neutralizing influenza antibodies neutralize divergent H3 strains having an HA2 polypeptide that shares at least 95% identity with the wild type HA2 polypeptide from A/Finland/486/2004 (SEQ ID NO: 4), as measured by an in vitro functional microneutralization reporter assay.
45. The method according to claim 42, wherein the broadly neutralizing influenza antibodies neutralize divergent H3N2 strains from at least 1968 to 2016, as measured by an in vitro functional microneutralization reporter assay.
46. The method according to claim 45, wherein the divergent H3N2 strains comprise A/Singapore/INFIMH- 16-0019/2016, A/Hong Kong/4801/2014, A/Victoria/361/2011, A/Wisconsin/67/2005, A/Shangdong/9/1993, A/Port Chalmers/1/1973, and A/Aichi/02/1968.
47. The method according to any one of claims 42-46, wherein the vaccine composition elicits an average antibody titer of at least 4 (logio) to the HA protein from both the H3 strain A/Perth/16/2009 and the HA protein from the H10 strain A/Jiangxi/IPB13b/2013 when 50 pg of the vaccine composition is administered to cynomolgus macaques at weeks 0, 4, and 10, and wherein the antibody titer is measured by ELISA in sera samples obtained from the cynomolgus macaques at week 12.
48. The method according to any one of claims 42-47, wherein the broadly neutralizing influenza antibodies neutralize H3 influenza strains and one or more group 2 influenza strains selected from one or more of H4, H7, H10, H14, and H15 influenza strains, as measured by an in vitro functional microneutralization reporter assay.
49. The method according to any one of claims 42-48, wherein the broadly neutralizing influenza antibodies neutralize at least one H3 influenza strain and at least one H7 or H10 influenza strain, as measured by an in vitro functional microneutralization reporter assay.
50. The method according to claim 49, wherein the broadly neutralizing influenza antibodies neutralize at least one H3 influenza strain, at least one H7 influenza strain, and at least one H10 influenza strain as measured by an in vitro functional microneutralization reporter assay.
51. The method according to any one of claims 42-50, wherein the subject is a human.
PCT/US2021/032252 2020-05-13 2021-05-13 Adjuvanted stabilized stem hemagglutinin nanoparticles and methods of using the same to induce broadly neutralizing antibodies against influenza WO2021231729A1 (en)

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CN110167585A (en) * 2016-09-02 2019-08-23 美国卫生与公众服务部 Stabilized 2nd group of influenza hemagglutinin stem area tripolymer and application thereof
CN114522226A (en) * 2022-02-14 2022-05-24 江南大学 Chiral tumor nano vaccine and application thereof

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CN110167585A (en) * 2016-09-02 2019-08-23 美国卫生与公众服务部 Stabilized 2nd group of influenza hemagglutinin stem area tripolymer and application thereof
US11793871B2 (en) 2016-09-02 2023-10-24 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Stabilized group 2 influenza hemagglutinin stem region trimers and uses thereof
CN110167585B (en) * 2016-09-02 2024-03-08 美国卫生与公众服务部 Stabilized group 2 influenza hemagglutinin stem trimer and uses thereof
CN114522226A (en) * 2022-02-14 2022-05-24 江南大学 Chiral tumor nano vaccine and application thereof
CN114522226B (en) * 2022-02-14 2024-01-26 江南大学 Chiral tumor nano vaccine and application thereof

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