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WO2018175560A1 - Nanoparticules immunogènes aptes à provoquer des réponses contre le site de liaison au récepteur de la grippe sur le domaine tête de l'hémagglutinine - Google Patents

Nanoparticules immunogènes aptes à provoquer des réponses contre le site de liaison au récepteur de la grippe sur le domaine tête de l'hémagglutinine Download PDF

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Publication number
WO2018175560A1
WO2018175560A1 PCT/US2018/023534 US2018023534W WO2018175560A1 WO 2018175560 A1 WO2018175560 A1 WO 2018175560A1 US 2018023534 W US2018023534 W US 2018023534W WO 2018175560 A1 WO2018175560 A1 WO 2018175560A1
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polypeptide
seq
amino acid
influenza
multimer
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PCT/US2018/023534
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English (en)
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William Schief
Dan KULP
Sergey MENIS
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The Scripps Research Institute
International Aids Vaccine Initiative
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Publication of WO2018175560A1 publication Critical patent/WO2018175560A1/fr

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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • 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
    • C12N2760/16211—Influenzavirus B, i.e. influenza B virus
    • C12N2760/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2760/16311—Influenzavirus C, i.e. influenza C virus
    • C12N2760/16334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • Nanoparticle immunogens to elicit responses against the Influenza receptor binding site on the Hemagglutinin head domain
  • Influenza virus is a member of Orthomyxoviridae family. There are three subtypes of influenza viruses designated A, B, and C.
  • the influenza virion contains a segmented negative-sense RNA genome, encoding, among other proteins, hemagglutinin (HA).
  • Influenza virus infection is initiated by the attachment of the virion surface HA protein to a sialic acid-containing cellular receptor (glycoproteins and glycolipids). Influenza presents a serious public-health challenge and new therapies are needed to combat viruses that are resistant to existing antivirals or escape neutralization by the immune system. Most of the field is focused on development of HA stem-based vaccines.
  • polypeptides comprising a first domain, wherein the first domain comprises
  • the first domain comprises the amino acid sequence of SEQ
  • polypeptides further comprise a multimerization domain, including but not limited to multimerization domains comprises the amino acid sequence selected from the group consisting of SEQ ID NOS:9-12 or 13-28.
  • polypeptides comprising:
  • HA hemagglutinin
  • the multimerization domain comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 10 and 15-28.
  • the HA head domain antigen comprises the amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 2, 50, 51, 52, or 53.
  • the polypeptides further comprise an amino acid linker between the first domain and the multimerization domain, or between the multimerization domain and the HA antigen.
  • the polypeptides comprise the amino acid sequence selected from the group consisting of SEQ ID NOS: 29-39.
  • multimers comprising 2, 3, 4, 5, 6, 7, 8, 10, 20, 30, 40, 50, 60, or more of the polypeptides are provided.
  • nucleic acids encoding the polypeptides of the disclosure
  • recombinant expression vector comprising the nucleic acids of the disclosure operatively linked to a promoter
  • recombinant host cells comprising the recombinant expression vectors of disclosure
  • pharmaceutical compositions comprising the polypeptides, multimers, nucleic acids, recombinant expression vectors, and/or host cells of the disclosure together with a pharmaceutically acceptable carrier.
  • the disclosure further provides methods for treating an influenza infection, limiting development of an influenza infection, generating an immune response in a subject, monitoring an influenza-induced disease in a subject and/or monitoring response of the subject to immunization by an influenza vaccine, detecting influenza binding antibodies, or generating anti-HA antibodies, comprising use of the polypeptides, multimers, nucleic acids, recombinant expression vectors, host cells, and/or pharmaceutical compositions thereof.
  • Figure 1 is a schematic overview of design strategy for creating an HA head nanoparticle immunogen.
  • Figure 2 shows surface plasmon resonance (SPR) analyses of the antigenic profile of an eHA monomer, determined by eHA 1 monomer being flowed as an analyte over three broadly neutralizing RBS-directed antibodies (A) 5J8, (B) CH65 and (C) C05 which were captured on a SPR chip coated with amine coupled anti-human IgG.
  • SPR surface plasmon resonance
  • Figure 3 is a schematic drawing of an eHA 60mer (B) in comparison to the eHa monomer and linker (A).
  • Figure 4 is a photograph of a reducing 4-12% BIS TRIS SDS PAGE gel run to determine the expression and purity of eHAl_Hl_60mer (HA 1 _H 1 _4HKX_g7_d41 m3_ Ct_60mer). Lanes were assigned as follows: (1) PreStained Ladder; (2) Mammalian supernatant after concentration (15ul); (3) Lectin column flowthrough (15ul); (4) Lectin column wash (15ul); (5) Lectin column beads post elution (15ul); and (6) Eluted sample after dialysis and concentration (15ul).
  • Figure 5 is a graph of an analytical size-exclusion chromatography-multi-angle light scattering experiment to confirm the oligomeric state of eHAl_Hl_60mer
  • Figure 6 is a negative stain electron micrograph of C-terminal fusion of a
  • Figure 7 shows surface plasmon resonance (SPR) analyses of the antigenic profile of an eHAl_Hl_60mer (HAl_Hl_4HKX_g7_d41m3_ Ct_60mer) using 5J8 (A), CH65 (B) and C05 (C), and an HIV antibody, VRCO 1 (D) as ligands.
  • SPR surface plasmon resonance
  • Figure 8 is a graph showing a vaccine test of eHA (V) and eHA-60 mer
  • proteins or polypeptide are used in their broadest sense to refer to a sequence of subunit amino acids.
  • the proteins or polypeptides of the disclosure may comprise L-amino acids, D-amino acids (which are resistant to L-amino acid-specific proteases in vivo), or a combination of D- and L-amino acids.
  • the proteins or polypeptides described herein may be chemically synthesized or recombinantly expressed.
  • the present disclosure focuses on development of immunogens against the receptor binding site (RBS) of the HA head domain, which is defined as amino acid positions
  • the disclosure provides polypeptides comprising a first domain, wherein the first domain comprises
  • polypeptides can be used, for example, alone or in fusion polypeptides of the disclosure, and are more effective candidates for treating influenza infection and generating a neutralizing anti-HA immune response than currently used stem-based vaccines.
  • H 1 domains eHAl
  • This strategy will enable eHAl vaccines to boost preexisting RBS-targeted responses without boosting other head-directed responses and will also allow for use of vaccines to boost RBS-directed responses primed by eHAl vaccines.
  • amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gin; Q), glycine (Gly; G), histidine (His; H), isoleucine (He; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).
  • one or more (1, 2, 3, 4, 5, or more) N-glycosylation sites are added to mask surfaces ("glycan masking") on the polypeptides outside the RBS.
  • glycan masking involved substituting one or more residue of the polypeptide outside the RBS with amino acid residue(s) that can be glycosylated (i.e.: modifying the primary amino acid sequence to generate a sequon Asn-X-Ser or Asn-X-Thr where X is any kind of amino acid except proline, or any acceptable sequon for the addition of N-linked glycans.)
  • This helps to limit antigenic cross-reactivity to wild-type HA except via the RBS, further boosting pre-existing RBS-targeted responses without boosting other head-directed responses and further allowing for use of vaccines to boost RBS-directed responses primed by the polypeptides disclosed herein.
  • the first domain comprises the amino acid sequence of one or more of SEQ ID NOS: 2 and 51.
  • polypeptides of the disclosure may comprise two or more (i.e.: 2, 3, 4, 5, or more) copies of the first domain.
  • polypeptides comprising an engineered hemagglutinin
  • HA head domain by structure-guided, computational design methods, comprising the receptor binding site (RBS), wherein the engineered hemagglutinin (FLA) head domain is modified to add one or more N-glycosylation sites (i.e.: modifying the primary amino acid sequence to generate a sequon Asn-X-Ser or Asn-X-Thr where X is any kind of amino acid except proline, or any acceptable sequon for the addition of N-linked glycans).
  • RBS receptor binding site
  • FLA hemagglutinin
  • the inventors have employed structure-guided, computational design methods to engineer HA1 domains (eHAl) that present the RBS epitope but limit the number of epitopes outside the RBS, and that include one or more engineered N-glycosylation sites to mask surfaces on the polypeptides outside the RBS.
  • eHAl HA1 domains
  • This strategy will enable eHAl vaccines to boost pre-existing RBS-targeted responses without boosting other head-directed responses and will also allow for use of vaccines to boost RBS-directed responses primed by eHAl vaccines.
  • the engineered HA head domain is modified to add 2, 3, 4, or more N-glycosylation sites.
  • the polypeptides are presented on the surface of a nanoparticle.
  • Any suitable nanoparticle may be used, including but not limited to self -assembling polypeptide nanoparticles. This embodiment helps further amplify immune responses to the RBS and help ensure that one or two immunizations of polypeptides will suffice.
  • Self-assembling nanoparticles have additional advantages: ease of production (no conjugation required) and compatibility with DNA, RNA or viral vector delivery.
  • the nanoparticles comprise the multimerization domains described herein.
  • the polypeptides of the disclosure may further comprise a multimerization domain.
  • Any suitable multimerization domain may be used that can result in a polypeptide multimer that can present multiple copies of the polypeptides of the disclosure to, for example, the immune system of a subject to which the polypeptides are administered.
  • the multimerization domain comprises the amino acid sequence selected from the group consisting of SEQ ID NOS: 9-12.
  • LS synthase
  • RMKQIEDKIEEILSKIYHIENEIARIKKLIGER SEQ ID NO: 11
  • SEQ ID NO: 11 which is a coiled coil trimerization motif
  • MKVKQLEDVVEELLSVNYHLENVVARLKKLVGER (SEQ ID NO: 12), which is a tetramerization motif having 4 helices curling around each other in helical manner.
  • a fusion with this multimerization domain may comprise fusing one copy of a polypeptide comprising a first domain of the disclosure to the N- terminus of SEQ TD NO: 12 and a second copy of a polypeptide of the disclosure to the C- terminus of SEQ ID NO: 12.
  • the multimerization domain comprises the amino acid sequence selected from the group consisting of SEQ ID NOS: 13-28:
  • polypeptides comprising
  • HA hemagglutinin
  • the polypeptides of this aspect of the disclosure are fusion proteins that comprise a lumazine synthase mutation disclosed herein fused to an HA antigen.
  • the polypeptides of this aspect of the disclosure can be used, for example, in the methods of the disclosure.
  • the HA antigen may be any suitable HA antigen, including but not limited to a HA head domain containing the receptor binding site, or an antigenic portion thereof.
  • the multimerization domain comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 10 and 15-28.
  • the HA head domain antigen comprises the amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 2, 50, 51, 52, or 53:
  • polypeptides of the disclosure may further comprise a linker between different domains within the polypeptide.
  • the polypeptides may further comprise an amino acid linker between the first domain and the multimerization domain, or between the multimerization domain and the one or more copies of the HA antigen.
  • polypeptides of the disclosure may comprise the amino acid sequence selected from the group consisting of SEQ ID NOS: 29- 39.
  • the disclosure provides multimers, comprising two or more copies (2, 3, 4, 5, 6, 7, 8, 10, 20, 30, 40, 50, 60, or more copies) of the polypeptides of the disclosure that include a multimerization domain.
  • the multimer may be a self-assembling multimer and/or may be present on a surface, including but not limited to a particle or bead.
  • the multimer comprises eight or more copies of the polypeptide; in another specific embodiment, the multimer comprises 60 or more copies of the polypeptide.
  • the present disclosure provides isolated nucleic acids encoding a polypeptide of the present disclosure.
  • the isolated nucleic acid sequence may comprise RNA or DNA.
  • isolated nucleic acids are those that have been removed from their normal surrounding nucleic acid sequences in the genome or in cDNA sequences.
  • Such isolated nucleic acid sequences may comprise additional sequences useful for promoting expression and/or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals. It will be apparent to those of skill in the art, based on the teachings herein, what nucleic acid sequences will encode the polypeptides of the disclosure. In various non-limiting
  • the nucleic acid comprises the sequence selected from the group consisting of SEQ ID NOS: 42-49, which show improved expression compared to other encoding nucleic acid sequences:
  • the present disclosure provides recombinant expression vectors comprising the isolated nucleic acid of any aspect of the disclosure operatively linked to a suitable control sequence.
  • “Recombinant expression vector” includes vectors that operatively link a nucleic acid coding region or gene to any control sequences capable of effecting expression of the gene product.
  • "Control sequences" operably linked to the nucleic acid sequences of the disclosure are nucleic acid sequences capable of effecting the expression of the nucleic acid molecules. The control sequences need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between a promoter sequence and the nucleic acid sequences and the promoter sequence can still be considered "operably linked" to the coding sequence.
  • Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites.
  • Such expression vectors can be of any type known in the art, including but not limited plasmid and viral-based expression vectors.
  • the present disclosure provides host cells that have been transfected with the recombinant expression vectors disclosed herein, wherein the host cells can be either prokaryotic or eukaryotic.
  • the cells can be transiently or stably transfected.
  • transfection of expression vectors into prokaryotic and eukaryotic cells can be accomplished via any technique known in the art, including but not limited to standard bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection.
  • a method of producing a polypeptide according to the disclosure is an additional part of the disclosure.
  • the method comprises the steps of (a) culturing a host according to this aspect of the disclosure under conditions conducive to the expression of the polypeptide, and (b) optionally, recovering the expressed polypeptide.
  • the expressed polypeptide can be recovered from the cell free extract, but preferably they are recovered from the culture medium. Methods to recover polypeptide from cell free extracts or culture medium are well known to the man skilled in the art.
  • the present disclosure provides pharmaceutical compositions (such as a vaccine), comprising one or more polypeptides, multimers, nucleic acids, recombinant expression vectors, or host cells of the disclosure and a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions of the disclosure can be used, for example, in the methods of the disclosure described below.
  • the polypeptides may be the sole active agent in the pharmaceutical composition, or the composition may further comprise one or more other agents suitable for an intended use, including but not limited to adjuvants to stimulate the immune system generally and improve immune responses overall. Any suitable adjuvant can be used.
  • the present disclosure provides methods for treating and/ or limiting an influenza virus infection, comprising administering to a subject in need thereof a therapeutically effective amount of one or more polypeptides of the disclosure, salts thereof, conjugates thereof, multimers thereof, nucleic acids of the disclosure (such as RNA), host cells or pharmaceutical compositions thereof, to treat and/or limit the influenza infection.
  • the method comprises eliciting an immune response in an individual having or at risk of an influenza infection, comprising administering to a subject in need thereof a therapeutically effective amount of one or more polypeptides of the disclosure, salts thereof, conjugates thereof, multimers thereof, nucleic acids of the disclosure (such as RNA), host cells or pharmaceutical compositions thereof, to generate an immune response.
  • the therapeutic is administered to a subject already infected with influenza, and/or who is suffering from flu symptoms (including but not limited to lower respiratory tract infections, upper respiratory tract infections, bronchiolitis, pneumonia, fever, listlessness, diminished appetite, recurrent wheezing, and asthma) indicating that the subject is likely to have been infected with influenza.
  • flu symptoms including but not limited to lower respiratory tract infections, upper respiratory tract infections, bronchiolitis, pneumonia, fever, listlessness, diminished appetite, recurrent wheezing, and asthma
  • treat or “treating” means accomplishing one or more of the following: (a) reducing influenza titer in the subject; (b) limiting any increase of influenza titer in the subject; (c) reducing the severity of flu symptoms; (d) limiting or preventing development of flu symptoms after infection; (e) inhibiting worsening of flu symptoms; (f) limiting or preventing recurrence of flu symptoms in subjects that were previously symptomatic for influenza infection.
  • the therapeutic is used as a "therapeutic vaccines" to ameliorate the existing infection and/or provide prophylaxis against infection with additional influenza virus.
  • the therapeutic can also be administered prophylactically to a subject at risk of influenza virus infection to limit development of an influenza virus infection.
  • Groups at particularly high risk include children under age 18 (particularly infants 3 years or younger), adults over the age of 65, and individuals suffering from any type of immunodeficiency.
  • a “therapeutically effective amount” is an amount of the therapeutic effective for treating and/or limiting influenza virus infection.
  • a suitable dosage range may, for instance, be 0.1 ug/kg-100 mg/kg body weight; alternatively, it may be 0.5 ug/kg to 50 mg/kg; 1 ug/kg to 25 mg/kg, or 5 ug/kg to 10 mg/kg body weight.
  • the therapeutic can be delivered in a single bolus, or may be administered more than once (e.g., 2, 3, 4, 5, or more times) as determined by an attending physician.
  • the present disclosure provides methods for monitoring an influenza virus-induced disease in a subject and/or monitoring response of the subject to immunization by an influenza vaccine, comprising contacting a polypeptide, multimer, recombinant host cell, or pharmaceutical composition of the disclosure with a bodily fluid from the subject and detecting influenza-binding antibodies in the bodily fluid of the subject.
  • influenza virus-induced disease is intended any disease caused, directly or indirectly, by influenza virus. The method comprises contacting a
  • polypeptide, multimer, recombinant host cell, or pharmaceutical composition of the disclosure with an amount of bodily fluid (such as serum, whole blood, etc.) from the subject; and detecting influenza-binding antibodies in the bodily fluid of the subject.
  • bodily fluid such as serum, whole blood, etc.
  • the detection of the influenza binding antibodies allows the influenza-induced disease in the subject to be monitored.
  • the detection of influenza binding antibody also allows the response of the subject to immunization by a flu vaccine to be monitored.
  • Any suitable detection assay can be used, including but not limited to homogeneous and heterogeneous binding immunoassays, such as radioimmunoassays (RIA), ELISA, immunofluorescence, immunohistochemistry, FACS, BIACORE and Western blot analyses.
  • the methods may be carried out in solution, or the polypeptide(s) of the disclosure may be bound or attached to a carrier or substrate, such as microtiter plates (ex: for ELISA), membranes and beads, etc.
  • a carrier or substrate such as microtiter plates (ex: for ELISA), membranes and beads, etc.
  • the polypeptides of the disclosure for use in this aspect may be conjugated to a detectable tag, to facilitate detection technique.
  • the present disclosure provides methods for detecting influenza binding antibodies, comprising contacting a polypeptide, multimer, nucleic acid, expression vector, host cell, or pharmaceutical composition of the disclosure with a composition comprising a candidate influenza binding antibody under conditions suitable for binding of influenza antibodies to the polypeptide, multimer, recombinant host cell, or pharmaceutical composition; and
  • the methods are performed to determine if a candidate influenza binding antibody recognizes the HA head antigen present in the polypeptides of the disclosure.
  • Any suitable composition may be used, including but not limited to bodily fluid samples (such as serum, whole blood, etc.) from a suitable subject (such as one who has been infected with influenza virus), naive libraries, modified libraries, and libraries produced directly from human donors exhibiting an influenza-specific immune response.
  • the assays are performed under conditions suitable for promoting binding of antibodies against the polypeptide, multimer, recombinant host cell, or pharmaceutical composition of the disclosure; such conditions can be determined by those of skill in the art based on the teachings herein. Any suitable detection assay can be used, such as those described above.
  • the polypeptides of the disclosure for use in this aspect may comprise a conjugate as disclosed above, to provide a tag useful for any detection technique suitable for a given assay.
  • the HA head antigen- binding antibodies are isolated using standard procedures.
  • the present disclosure provides methods for producing influenza antibodies, comprising
  • the antibodies can be used, for example, in influenza research.
  • the subject is preferably an animal typically used for antibody production, including but not limited to rodents, rabbits, goats, sheep, etc.
  • the antibodies can be either polyclonal or monoclonal antibodies.
  • the objective is a polypeptide that induces broadly neutralizing antibody responses against influenza viruses via the receptor binding site (RBS) on the hemagglutinin head domain.
  • Figure 1 provides an overview of the design rationale, based on engineered HA head domains for immuno-focusing to the RBS epitope.
  • the polypeptides may further include design elements for (a) glycan masking of the engineered head domains to dampen responses outside the RBS and to ensure minimal cross-reactivity to wild-type hemagglutinin outside the RBS; and (b) nanoparticles presenting the engineered head domains in arrays for increased immunogenicity.
  • HA1 wild-type head domain
  • PDB ID: 4HKX wild-type head domain
  • eHAl HA1 domains
  • ROSETTATM ROSETTATM
  • eHAl glycans can help eliminate antigenic cross- reactivity to wild-type HA except via the RBS. This strategy will enables eHAl vaccines to boost pre-existing RBS-targeted responses without boosting other head-directed responses and will also allow for use of vaccines to boost RBS-directed responses primed by eHAl vaccines.
  • HA head glycosylated variants and HA-head 60mers using fa) glycosylated HA head domains and (b " ) specifically codon-optimized d41m3 variants of lumazine synthase
  • the kinetics were determined by eHAl monomer being flowed as an analyte over three broadly neutralizing RBS-directed antibodies (5J8, CH65 and C05) which were captured on a SPR chip coated with amine coupled anti-human IgG.
  • the binding affinities for 5J8, CH65 and C05 are 1.2 uM, 137 nM and 41 nM, respectively.
  • the expected molecular weight of the heavily glycosylated eHAl-Hl-60mer monomer is 42.2 kDa and it ran between 50-60 kDa bands of unglycosylated proteins from the a pre-stained marker ( Figure 4).
  • Figure 4 To confirm the oligomeric state of the 60mer, we conducted an analytical size-exclusion chromatography- multi-angle light scattering experiment and analyzed the data using the protein conjugate methods from Wyatt Technology. The results confirmed a single peak with observed molecular weight of 2.1 x 10 6 Da, which is close to the expected molecular weight for the 60mer of 2.5 x 10 6 Da ( Figure 5).
  • RDE Denka Seiken Co
  • RDE Denka Seiken Co
  • Enzyme was heat-inactivated at 57 degrees C for 45 minutes.
  • Chicken red blood cells (RBCs) in Alsevers (Lampire) were washed and resuspended in 0.8% NaCl saline solution.
  • Serum samples were pre-absorbed to 10% chicken red blood cells in saline followed by centrifugation at 3000 rpm to remove RBCs.
  • Each H3N2 virus was titered by mixing equal volumes of serially diluted virus in PBS with 0.5% chicken RBCs for 45 minutes in a V- bottom 96 well plate. Sera were then serially diluted in PBS and combined with 4
  • agglutinating doses (4AD) of virus for 1 hour. Following incubation, an equal volume of 0.5% chicken RBCs were added and allowed to incubate 45 minutes. Scoring was performed by one of two methods: 1) Looking for the last dilution at which a dot formed; 2) Rotating each plate 90 degrees and identifying the last dilution at which a drip formed.
  • HAI activity with geometric mean titers for eHA and eHA-60mer of 761 and 783, respectively.
  • the HAI titers induced by the eHA-60mer appeared to be more consistent in magnitude than the titers induced by the eHA monomer, as all eHA-60mer-immunized animals produced titers greater than 100, whereas three eHA-monomer animals had titers less than 100, and the geometric standard deviation of the HAI titers was 1.77 for the eHA-60mer and 3.33 for the eHA-monomer.

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  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des polypeptides obtenus par conception, qui peuvent être utilisés pour traiter ou limiter une infection grippale et pour générer une réponse immunitaire.
PCT/US2018/023534 2017-03-22 2018-03-21 Nanoparticules immunogènes aptes à provoquer des réponses contre le site de liaison au récepteur de la grippe sur le domaine tête de l'hémagglutinine WO2018175560A1 (fr)

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WO2021163438A1 (fr) * 2020-02-14 2021-08-19 University Of Washington Polypeptides, compositions et leur utilisation pour traiter ou limiter le développement d'une infection
EP4110384A4 (fr) * 2020-02-26 2024-07-31 The Wistar Institute of Anatomy and Biology Compositions comprenant des vaccins à auto-assemblage et leurs méthodes d'utilisation

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WO2013043729A1 (fr) * 2011-09-20 2013-03-28 Mount Sinai School Of Medicine Vaccins contre le virus de la grippe et leurs utilisations

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021163438A1 (fr) * 2020-02-14 2021-08-19 University Of Washington Polypeptides, compositions et leur utilisation pour traiter ou limiter le développement d'une infection
EP4110384A4 (fr) * 2020-02-26 2024-07-31 The Wistar Institute of Anatomy and Biology Compositions comprenant des vaccins à auto-assemblage et leurs méthodes d'utilisation

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