WO2018175560A1

WO2018175560A1 - Nanoparticle immunogens to elicit responses against the influenza receptor binding site on the hemagglutinin head domain

Info

Publication number: WO2018175560A1
Application number: PCT/US2018/023534
Authority: WO
Inventors: William Schief; Dan KULP; Sergey MENIS
Original assignee: Scripps Research Institute; International AIDS Vaccine Initiative Inc
Current assignee: Scripps Research Institute; International AIDS Vaccine Initiative Inc
Priority date: 2017-03-22
Filing date: 2018-03-21
Publication date: 2018-09-27
Anticipated expiration: 2019-09-22

Abstract

Disclosed herein are designed polypeptides that can be used for treating or limiting influenza infection and generating an immune response.

Description

Nanoparticle immunogens to elicit responses against the Influenza receptor binding site on the Hemagglutinin head domain

Cross Reference

This application claims priority to U.S. Provisional Patent Application Serial No.

62/474949 filed March 22, 2017, incorporated by reference herein in its entirety.

Background

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.

Summary

In a first aspect, polypeptides are provided comprising a first domain, wherein the first domain comprises

(a) the amino acid sequence of SEQ ID NO: 1

wherein at least 1, 2, 3, 4, 5, or all 6 of the residues bounded by parentheses is the first listed residue; or

(b) the amino acid sequence of SEQ ID NO: 50

wherein at least 1, 2, 3, 4, 5, or all 6 of the residues bounded by parentheses is the first listed residue.

In various embodiments, the first domain comprises the amino acid sequence of SEQ

ID NO: 2 or 51. In another embodiment, the 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.

In another aspect, polypeptides are provided comprising:

(a) a multimerization domain comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 10, 14, and 15-28; and

(b) a hemagglutinin (HA) head domain antigen.

In various embodiments, the multimerization domain comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 10 and 15-28. In other embodiments, 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. In further embodiments, 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. In still further embodiments, the polypeptides comprise the amino acid sequence selected from the group consisting of SEQ ID NOS: 29-39.

In other embodiments, multimers comprising 2, 3, 4, 5, 6, 7, 8, 10, 20, 30, 40, 50, 60, or more of the polypeptides are provided.

Also provided are 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, and 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. Description of the Figures

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.

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

(HA 1 _H 1 _4HKX_g7_d41 m3_ Ct_60mer).

Figure 6 is a negative stain electron micrograph of C-terminal fusion of a

hyperglycosylated eHA on stabilized 60mer particle (eHAl_Hl_60mer

(HAl_Hl_4HKX_g7_d41m3_ Ct_60mer)).

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.

Figure 8 is a graph showing a vaccine test of eHA (V) and eHA-60 mer

(HAl_Hl_4HKX_g7_d41m3_Ct_60mer) based on influenza strain A/Solomon

Islands/3/2006/HlNl. Detailed Description

All references cited are herein incorporated by reference in their entirety. As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. "And" as used herein is interchangeably used with "or" unless expressly stated otherwise. All embodiments of any aspect of the disclosure can be used in combination, unless the context clearly dictates otherwise.

Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of

"including, but not limited to". Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words "herein," "above," and "below" and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application.

As used throughout the present application, the term "protein" 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 description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While the specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize.

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

95, 134,136,153, 155, 183, 190, 194,195, 226,and 228 of the HA protein. In a first aspect, the disclosure provides polypeptides comprising a first domain, wherein the first domain comprises

(a) the amino acid sequence of SEQ ID NO: 1

(b) the amino acid sequence of SEQ ID NO: 50

These 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.

In order to focus antibody responses to the RBS epitope as opposed to other antigenic regions of the head, the inventors have employed structure-guided, computational design methods to engineer H 1 domains (eHAl) that present the RBS epitope but limit the number of epitopes outside the RBS. 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.

As used herein, the 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).

Parentheses represent variable positions in the polypeptide, with the recited amino acid residues as alternatives in these positions.

In one embodiment, 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. In one embodiment, 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. In one exemplary embodiment, the first domain comprises the amino acid sequence of one or more of SEQ ID NOS: 2 and 51.

In a further embodiment, the polypeptides of the disclosure may comprise two or more (i.e.: 2, 3, 4, 5, or more) copies of the first domain.

In another aspect polypeptides are provided 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). Tn order to focus antibody responses to the RBS epitope as opposed to other antigenic regions of the head, 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. 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. In one embodiment, the engineered HA head domain is modified to add 2, 3, 4, or more N-glycosylation sites. In another embodiment, 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. In certain embodiments, the nanoparticles comprise the multimerization domains described herein.

In one non-limiting embodiment, 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. In various non-limiting embodiments, the multimerization domain comprises the amino acid sequence selected from the group consisting of SEQ ID NOS: 9-12.

synthase (LS), including a series of LS mutants described herein);

which is a genus of LS mutants described herein;

RMKQIEDKIEEILSKIYHIENEIARIKKLIGER (SEQ ID NO: 11), which is a coiled coil trimerization motif; and /or

MKVKQLEDVVEELLSVNYHLENVVARLKKLVGER (SEQ ID NO: 12), which is a tetramerization motif having 4 helices curling around each other in helical manner. For example, 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.

In one embodiment, the multimerization domain comprises the amino acid sequence selected from the group consisting of SEQ ID NOS: 13-28:

In another aspect, the disclosure provides polypeptides comprising

(b) a hemagglutinin (HA) head domain antigen.

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.

In one embodiment, the multimerization domain comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 10 and 15-28. In another embodiment, 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:

optionally wherein at least 1, 2, 3, 4, 5, or all 6 of the residues bounded by parentheses is the first listed residue;

The polypeptides of the disclosure may further comprise a linker between different domains within the polypeptide. For example, 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.

Γη various non-limiting embodiments, the polypeptides of the disclosure may comprise the amino acid sequence selected from the group consisting of SEQ ID NOS: 29- 39.

In a further embodiment, 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.

In one specific embodiment, the multimer comprises eight or more copies of the polypeptide; in another specific embodiment, the multimer comprises 60 or more copies of the polypeptide.

In another aspect, the present disclosure provides isolated nucleic acids encoding a polypeptide of the present disclosure. The isolated nucleic acid sequence may comprise RNA or DNA. As used herein, "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

embodiments, 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:

In a further aspect, 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. Thus, for example, 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.

In another aspect, 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. Such 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. (See, for example, Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells: A Manual of Basic Technique, 2^nd Ed. (R.I. Freshney. 1987. Liss, Inc. New York, NY). 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.

In another aspect, 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. In a further aspect, 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. In another embodiment, 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.

For treating an influenza virus infection, 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. As used herein, "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. In one embodiment method, 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. In a further aspect, 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. By "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. The detection of the influenza binding antibodies allows the influenza-induced disease in the subject to be monitored. In addition, 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. The polypeptides of the disclosure for use in this aspect may be conjugated to a detectable tag, to facilitate detection technique.

In a still further aspect, 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

(b) detecting influenza antibody complexes with the polypeptide, multimer, recombinant host cell, or pharmaceutical composition of the disclosure.

In this aspect, 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. In a further embodiment, the HA head antigen- binding antibodies are isolated using standard procedures.

In another aspect, the present disclosure provides methods for producing influenza antibodies, comprising

(a) administering to a subject an amount effective to generate an antibody response of the polypeptide, multimer, nucleic acid, expression vector, host cell, or pharmaceutical composition of the disclosure; and

(b) isolating antibodies produced by the subject.

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.

As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. "And" as used herein is interchangeably used with "or" unless expressly stated otherwise.

All embodiments of any aspect of the disclosure can be used in combination, unless the context clearly dictates otherwise.

Examples:

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.

Focusing antibody responses to the RBS epitope In order to focus antibody responses to the RBS epitope as opposed to other antigenic regions of the head we started with the wild-type head domain (HA1), which can be expressed as a monomeric protein without loss of structure or antigenic profile (PDB ID: 4HKX). We 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. We modeled glycans using ROSETTA™ at various positions distal to the RBS with the goal to maximally cover HA head surface area and at the same time not interfere with antibody binding to the RBS. These 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

We started from two HA-head sequences (PDBid: 4HKX and 4M5Z) and we added 3 additional glycosylation sites to each of those sequences in order to mask surfaces on the protein outside our target epitope. The starting sequences for the HA-head domains in 4HKX and 4M5Z were:

Additional glycans were then added at strategic locations:

# This strain has 4 natural N-glycosylation sites, adding 3 extra shown in black, bold, underline

# This strain has 1 natural N-glycosylation sites, adding 3 extra

We then constructed fusions of our two hyperglycosylated HA-head domains to our d41m3-stabilized version of lumazine synthase. We used a specific codon optimization of the lumazine synthase and the HA-head domains, based on past experience with another protein- fusion that this codon-optimization of the stabilized lumazine synthase was successful while another codon-optimized form was not. Below are the HA-head 60mer sequences that we have tested.

# Connect C-term d41m3-60mer to N-term immunogen

Biophysical characterization of eHAl immunogen

To assess the purity and oligomeric state of the eHAl monomer, we ran an analytical size-exclusion chromatography-multi-angle light scattering experiment and analyzed the data using the protein conjugate methods from Wyatt Technology. The results (Figure 2) confirm that the protein can be purified to homogeneity and that the observed molecular weight of 23.9 kDa is close to the expected molecular weight based on the gene sequence of 24.8 kDa. To assess the stability of the eHAl monomer, we conducted differential scanning calorimetry experiments which show that the melting temperature is 60°C. To examine the antigenic profile of the eHAl monomer, we conducted surface plasmon resonance(SPR) experiments. 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. Together these results demonstrate that the new HA molecule is stable, monomelic and has high affinity for the class of broadly neutralizing RBS-directed antibodies.

Amplifying immune response to the RBS epitope

To further amplify immune responses to the RBS and help ensure that one or two immunizations of eHAl vaccines will suffice, we engineered self-assembling nanoparticles presenting the eHAl. The linker and connectivity between eHAl and the nanoparticle was structurally modeled to identify genetic fusions which could display 60 copies of eHAl and present a fully exposed RBS. Self-assembling nanoparticles have additional advantages: ease of production (no conjugation required) and compatibility with DNA, RNA or viral vector delivery. The C-terminus of lumazine synthase was fused to the N-terminus of eHAl with a

15-amino acid GlySer linker to create eHAl-Hl-60mers (Figure 3). We created two versions: one containing only native glycans: HAl_Hl_4HKX_d41m3_Ct_60mer and one hyperglycoslated to focus immune responses: HAl_Hl_4HKX_g7_d41m3_Ct_60mer. The particles were purified using lectin affinity beads. A reducing 4-12% BIS TRIS SDS PAGE gel was run to determine the expression and purity of eHAl_Hl_60mer demonstrating that the eluted fraction contained a single major band. 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). 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 10⁶ Da, which is close to the expected molecular weight for the 60mer of 2.5 x 10⁶ Da (Figure 5). To confirm the intended particulate assembly, we stained eHAl-Hl-60mers with uranyl formate at a concentration of O.Olmg/ml and took images by negative stain electron microscopy. The results are consistent with fully formed particles of 20nm in diameter (Figure 6). To demonstrate that the RBS is exposed and capable of binding to RBS-directed bnAbs on eHAl-Hl-60mers, we conducted an SPR experiment. The specific binding to RBS-directed bnAbs was determined using 60mer as analyte and 5J8, CH65 and C05 as ligands (as described above for monomeric eHAl). The 60mer bound to the same influenza antibodies as the monomer, but not to an HIV antibody, VRCOl . Together these results demonstrate a novel RBS-focused HI nanoparticle that fully assembles into a 60mer and exposes the RBS to antibody binding (Figure 7).

Vaccine test of eHA (HAl_Hl_4HKX_g7) and eHA-60mer

(HAl_Hl_4HKX_g7_d41m3_Ct_60mer SEQ ID NO:29) based on A/Solomon

Islands/3/2006/HlNl. Two groups of female CB6F1/J mice (8 animals/group) were immunized at weeks 0, 3, and 10 with either eHA monomer or eHA-60mer (10 ug dose) and Sigma Adjuvant System by sub-cutaneous injection at the base of the tail. Animals were bled at weeks 0, 2, 5, and 12. HAI titers against A/Solomon Islands/3/2006/HlNl were assessed at weeks 0 and 12. We combined vaccinated mouse sera with receptor-destroying enzyme

(RDE, Denka Seiken Co) in a 1:3 ratio of sera to RDE and incubated 12 hours at 37 degrees C. 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.

The results are shown in Figure 8, and demonstrate that vaccination induced robust

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.

Claims

We claim:

1. A polypeptide comprising a first domain, wherein the first domain comprises

(a) the amino acid sequence of SEQ ID NO: 1

(b) the amino acid sequence of SEQ ID NO: 50

2. The polypeptide of claim 1, wherein the first domain comprises the amino acid sequence of SEQ ID NO: 2.

3. The polypeptide of claim 1, wherein the first domain comprises the amino acid sequence of SEQ ID NO: 51.

4. A polypeptide comprising an engineered hemagglutinin (FLA) head domain comprising the receptor binding site (RBS) and limiting the number of epitopes outside the RBS, wherein the engineered (FLA) head domain is modified to add one or more N- glycosylation sites.

5. The polypeptide of claim 4, wherein the engineered HA head domain is modified to add two or more N-glycosylation sites.

6. The polypeptide of claim 4 or 5, wherein the engineered polypeptide is presented on the surface of a nanoparticle, such as a self-assembling polypeptide nanoparticle.

7. The polypeptide of any one of claims 1 -6, comprising a multimerization domain.

8. The polypeptide of claim 7, wherein the multimerization domain comprises the amino acid sequence selected from the group consisting of SEQ ID NOS:9-12.

9. The polypeptide of claim 7 or 8 wherein the multimerization domain comprises one or more copies of the amino acid sequence selected from the group consisting of SEQ ID

NOS: 13-28.

10. A polypeptide comprising:

(b) a hemagglutinin (HA) head domain antigen.

11. The polypeptide of claim 10, wherein the multimerization domain comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 10 and 15-28.

12. The polypeptide of claim 10 or 1 1, wherein 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.

13. The polypeptide of any one of claims 7-12, further comprising an amino acid linker between the first domain and the multimerization domain, or between the multimerization domain and the HA antigen.

14. The polypeptide of any one of claims 1-13, comprising the amino acid sequence selected from the group consisting of SEQ ID NOS : 29-39.

15. A multimer, comprising two or more copies of the polypeptide of any one of claims 7- 14.

16. The multimer of claim 15, comprising eight or more copies of the polypeptide of any one of claims 4-12

17. A nucleic acid encoding the polypeptide of any one of claims 1-16.

18. The nucleic acid of claim 17, wherein the nucleic acid comprises the sequence selected from the group consisting of SEQ ID NOS:42-49.

19. A recombinant expression vector comprising the nucleic acid of claim 17 or 18 operatively linked to a promoter.

20. A recombinant host cell comprising the recombinant expression vector of claim 19.

21. A pharmaceutical composition comprising

(a) the polypeptide of any one of claims 1-14, the multimer of claims 15- 16, the nucleic acid of claims 17-18, the recombinant expression vector of claim 19, or the recombinant host cell of claim 20; and

(b) a pharmaceutically acceptable carrier.

22. A method for treating an influenza infection, comprising administering to a subject infected with influenza an amount effective to treat the infection of the polypeptide of any one of claims 1-14, the multimer of claims 15-16, the nucleic acid of claims 17-18, the recombinant expression vector of claim 19, the recombinant host cell of claim 20;, or the pharmaceutical composition of claim 21.

23. A method for limiting development of an influenza infection, comprising

administering to a subject at risk of an influenza infection an amount effective to limit development of an influenza infection of the polypeptide of any one of claims 1-14, the multimer of claims 15-16, the nucleic acid of claims 17-18, the recombinant expression vector of claim 19, the recombinant host cell of claim 20;, or the pharmaceutical composition of claim 21.

24. A method for generating an immune response in a subject, comprising administering to the subject an amount effective to generate an immune response of the polypeptide of any one of claims 1-14, the multimer of claims 15-16, the nucleic acid of claims 17-18, the recombinant expression vector of claim 19, the recombinant host cell of claim 20;, or the pharmaceutical composition of claim 21.

25. A method for monitoring an influenza-induced disease in a subject and/or monitoring response of the subject to immunization by an influenza vaccine, comprising contacting the polypeptide of any one of claims 1-14, the multimer of claims 15-16, the nucleic acid of claims 17-18, the recombinant expression vector of claim 19, the recombinant host cell of claim 20;, or the pharmaceutical composition of claim 21with a bodily fluid from the subject and detecting influenza-binding antibodies in the bodily fluid of the subject.

26. The method of claim 25, wherein the bodily fluid comprises serum or whole blood.

27. A method for detecting influenza binding antibodies, comprising

(a) contacting the polypeptide of any one of claims 1-14, the multimer of claims 15- 16, the nucleic acid of claims 17-18, the recombinant expression vector of claim 19, the recombinant host cell of claim 20;, or the pharmaceutical composition of claim 21with a composition comprising a candidate influenza binding antibody under conditions suitable for binding of anti-HA antibodies to the polypeptide, multimer, or composition; and

(b) detecting anti-HA antibody complexes with the polypeptide, multimer, or composition.

28. The method of claim 27, further comprising isolating the anti-HA antibodies.

29. A method for producing anti-HA antibodies, comprising

(a) administering to a subject an amount effective to generate an antibody response of the polypeptide of any one of claims 1-14, the multimer of claims 15-16, the nucleic acid of claims 17-18, the recombinant expression vector of claim 19, the recombinant host cell of claim 20;, or the pharmaceutical composition of claim 21 ; and

(b) isolating anti-HA antibodies produced by the subject.