EA044592B1 - MODIFICATION OF CONSTRUCTED POLYPEPTIDES OF INFLUENZA VIRUS HEMAGGLUTIININ - Google Patents

Description

Cross reference to related applications

The invention claims benefit from U.S. Provisional Patent Application No. 62/345502, filed June 3, 2016, the entire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Influenza has a long history of pandemics, epidemics, surges and outbreaks. Vaccines provided the most effective protection against influenza. However, efforts to develop and produce vaccines that induce strain-specific immunity that lasts for a year have been difficult as influenza continues to cause significant health problems worldwide. Indeed, currently marketed influenza virus vaccines need to be updated annually based on the predicted strains that will be present in human populations in the upcoming season.

Modern influenza virus vaccines rely on inducing immunity to the hemagglutinin antigen present on the surface of influenza viruses. Hemagglutinin (HA) is a glycoprotein responsible for binding the influenza virus to cells by interacting with sialic acid-containing structures on their membranes. It differs greatly among different strains of the influenza virus due to the continuous mutation of the virus and the pressure of the host immune system. The variability (also known as antigenic drift) of the HA molecule means that the HA polypeptide vaccine typically counteracts only a small subset of related circulating viruses. Over time, the number of strains to which cross-reactive immunity is formed decreases as the virus continues to mutate. Consequently, HA formulations of influenza virus vaccines are constantly modified if the change in the HA molecule is such that the existing vaccine is no longer effective. In addition to existing influenza virus vaccination strategies, the development of a universal vaccine remains promising to increase the breadth of existing strain-specific vaccines and their immunity to antigenic drift. Universal influenza virus vaccines have the ability to protect people and animals against a wide range of influenza virus types, subtypes, and strains, including pandemic and/or seasonal strains. Several approaches to the development of universal influenza antigens are known from the prior art. However, existing approaches often result in the production of an HA polypeptide that still has an effect against seasonal or pandemic strains.

Brief description of the invention

In some embodiments, the present invention provides methods for altering the immunogenicity profile of an engineered HA polypeptide, comprising the steps of detecting the presence or absence of one or more putative N-glycosylation sites in a head region of the engineered HA polypeptide compared to a corresponding head region of an HA polypeptide with a distinct immunogenicity profile; introduction into the head region of the engineered HA polypeptide of one or more amino acid substitutions, deletions or insertions to destroy one or more putative N-glycosylation sites or insertion of additional N-glycosylation sites based on the corresponding sequence of the HA polypeptide with a distinguishable immunogenicity profile, thereby providing a redesigned HA polypeptide with an altered immunogenicity profile. In some embodiments, one or more putative or additional N-glycosylation sites are defined by the consensus sequence NxS/Ty, where x and y are not P. In some embodiments, the engineered HA polypeptide has a predominantly seasonal immune profile, and the HA polypeptide with a distinct immunogenicity profile characterized by a predominantly pandemic immune profile, wherein the engineered HA polypeptide has one or more amino acid substitutions, deletions or insertions introduced to disrupt one or more putative N-glycosylation sites, and wherein the redesigned HA polypeptide is modified to be more consistent with the pandemic . In some embodiments, an engineered HA polypeptide has a predominantly pandemic immune profile, and an HA polypeptide with a distinct immunogenicity profile has a predominantly seasonal immune profile, wherein the engineered HA polypeptide has one or more amino acid substitutions, deletions, or insertions introduced to insert one or more putative additional sites N-glycosylation, and the redesigned HA polypeptide is modified to be more seasonal.

In some embodiments, the present invention provides methods for altering the immunogenicity profile of an engineered HA polypeptide, including introducing one or more amino acid substitutions within 15 (e.g., within 10, 9, 8, 7, 6, 5, etc.) angstroms from the site receptor binding binding system (RBS), with an RBS defined as all amino acid residues within 15 (e.g., within 10, 9, 8, 7, 6, or 5) angstroms of the position corresponding to residue W167 (as designated by CA09 numbering) in three-dimensional ( 3D) structure; wherein each of the one or more amino acid substitutions involves replacing an amino acid residue at a particular position with an amino acid residue observed at a corresponding position in an HA polypeptide with a distinct immunogenicity profile, thereby providing a redesigned HA polypeptide with an altered immunogenicity profile. Alternatively, an RBS may be defined as an epitope bound by a paratope of the monoclonal antibody CH65, and wherein one or more amino acid substitutions are present adjacent to (e.g., within 100 amino acid residues, within 75 amino acid residues, within 50 amino acid residues, within 40 amino acid residues, within 30 amino acid residues, within 25 amino acid residues, within 20 amino acid residues, within 15 amino acid residues, within 10 amino acid residues, within 5 amino acid residues, etc.) with the CH65 epitope. In some embodiments, the HA polypeptide with a distinct immunogenicity profile is derived from a circulating seasonal or pandemic strain of influenza virus. In some embodiments, an engineered HA polypeptide has a predominantly seasonal immune profile, and an HA polypeptide with a distinct immunogenicity profile has a predominantly pandemic immune profile, and wherein the redesigned HA polypeptide is altered to be more consistent with a pandemic immune profile. In some embodiments, the engineered HA polypeptide has a predominantly pandemic immune profile, and the HA polypeptide with a distinct immunogenicity profile has a predominantly seasonal immune profile, and wherein the redesigned HA polypeptide is altered to be more consistent with the seasonal one.

In some embodiments, the present invention provides methods for changing the immunogenicity profile of an engineered HA polypeptide, including introducing one or more modifications selected from those shown in Table. 4, 5, 6, 7, 8 or 9, into one or more corresponding positions of the engineered HA polypeptide, thereby providing a redesigned HA polypeptide with an altered immunogenicity profile. In some embodiments, one or more modifications are present at positions corresponding to 137, 144, 145, 154, 155, 156, 157, 158, 159, 177, 210, 211, 212, 213, 214, 244, 245 and/or 262 constructed polypeptide HA (according to CA09 numbering). In some embodiments, one or more modifications are present at positions corresponding to positions 137, 144, 145, 154, 155, 156, 157, 158, 159, 177, 210, 211, 212, 213, and/or 214 of the engineered HA polypeptide (as numbered CA09). In some embodiments, one or more modifications include two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more modifications selected from those , which are shown in table. 4, 5, 6, 7, 8 or 9. In some embodiments, one or more modifications include at least 2, 3, 4, 5 or 10 consecutive replacements selected from table. 4, 5, 6, 7, 8 or 9.

In some embodiments, the present invention provides methods for modifying the immunogenicity profile of an engineered HA polypeptide, comprising selecting two or more modifications selected from the group consisting of the following:

(i) selecting a head region of the designed HA polypeptide and replacing the selected head region of the designed HA polypeptide with a corresponding head region of the HA polypeptide with a distinct immunogenicity profile;

(ii) identifying the presence or absence of one or more putative N-glycosylation sites in the head region of the engineered HA polypeptide as compared to a corresponding head region of the HA polypeptide with a distinct immunogenicity profile and introducing one or more amino acid substitutions, deletions or insertions into the head region of the engineered HA polypeptide for disrupting one or more putative N-glycosylation sites or inserting additional N-glycosylation sites into a designed HA polypeptide based on a corresponding HA polypeptide sequence with a distinct immunogenicity profile;

(iii) introducing one or more amino acid substitutions within 15 (for example, within 10, 9, 8, 7, 6 or 5) angstroms of the receptor binding site (RBS), wherein RBS is defined as all amino acid residues within 15 angstroms from the position corresponding to W167 (according to CA09 numbering) in the three-dimensional (3D) structure; wherein each of the one or more amino acid substitutions involves replacing an amino acid residue at a particular position with an amino acid residue observed at a corresponding position in the HA polypeptide with a distinct immunogenicity profile; alternatively, an RBS can be defined as an epitope bound by a paratope of the monoclonal antibody CH65, and wherein one or more amino acid substitutions are present adjacent to (e.g., within 100 amino acid residues, within 75 amino acid residues, within 50 amino acid residues, within 40 amino acid residues, within 30 amino acid residues, within 25 amino acid residues, within 20 amino acid residues, within 15 amino acid residues, within 10 amino acid residues, within 5 amino acid residues, etc.) epitope CH65; and (iv) introducing one or more modifications selected from those shown in table. 4, 5, 6, 7, 8 or 9, at one or more corresponding positions of the designed HA polypeptide,

- 2 044592 due to which the production of a redesigned HA polypeptide with an altered immunogenicity profile is ensured.

In various embodiments, the method of the present invention further includes assessing the expression and conformation of the redesigned HA polypeptide.

In various embodiments, the method of the present invention further includes the step of determining whether the redesigned HA polypeptide produces neutralizing antibodies against seasonal and/or pandemic strains of influenza virus.

In some embodiments, producing a redesigned HA polypeptide with an altered immunogenicity profile includes increasing the binding index for one or more head region monoclonal antibodies to seasonal and/or pandemic strains of influenza virus. In some embodiments, the production of a redesigned HA polypeptide with an altered immunogenicity profile involves increasing the binding spectrum for monoclonal antibodies to the head region against pandemic strains of influenza virus. In some embodiments, producing a redesigned HA polypeptide with an altered immunogenicity profile includes increasing the binding spectrum for the anti-stem region monoclonal antibodies. In such embodiments, modifications in the head region induce increased binding to monoclonal antibodies to the stem region, indicating that substitutions at one site can have long-range allosteric effects at a distant location. In some embodiments, increased binding rates are determined by flow cytometric detection of monoclonal antibodies bound to redesigned HA polypeptides expressed on the surface of mammalian cells. In some embodiments, the level of monoclonal antibodies that bind to redesigned HA polypeptides expressed on the surface of mammalian cells is quantified.

In the invention, the use of or means and/or, unless otherwise indicated. As used herein, the term contain and variations of the term, such as containing and containing, are not exclusive of other additives, components, integers or steps. As used herein, the terms approximately and approximately are used interchangeably. Any numerals used in the invention with or without the term approximately/approximately are intended to cover any normal variations as would be understood by one of ordinary skill in the art.

Other features, objects and advantages of the present invention will be apparent from the following detailed description, drawings and claims. However, it should be understood that the detailed description, drawings and claims, although they indicate embodiments of the present invention, are provided for purposes of illustration and not limitation. Various changes and modifications within the scope of the present invention will become apparent to one skilled in the art.

Brief description of graphic materials

Graphics are provided for illustrative purposes only and not as a limitation.

In fig. Figure 1 shows three different regions, distinguished by the specific residue sites at which the globular head was truncated.

In fig. 2 shows a flow cytometry-based assay used to demonstrate the proper folding, expression, and antibody binding abilities of influenza virus HA polypeptides.

In fig. 3 shows the results of flow cytometry analysis demonstrating proper folding and expression of recombinant HA polypeptides obtained by grafting influenza RBS globular head regions onto recipient HA stem regions.

In fig. 4 shows the results of a flow cytometry analysis demonstrating an improved seasonal immune profile (as demonstrated by increased mAb binding) of recombinant HA polypeptides produced by grafting influenza RBS globular head regions onto recipient HA stem regions.

In fig. 5 shows a digital representation of the enlarged spectrum of immunological activity of recombinant HA polypeptides obtained by grafting regions of the globular head of influenza RBS onto recipient stem regions of HA.

In fig. Figure 6 shows a graphical representation of putative N-glycosylation sites, lysine loop insertion sites, and modification of residues around the RBS site.

In fig. 7 shows the results of flow cytometry analysis demonstrating the proper folding and expression of recombinant HA polypeptides produced by modifications of putative N-glycosylation sites, lysine loop insertions, or amino acid residues in the globular head region.

In fig. Figure 8 shows the results of a flow cytometry analysis demonstrating an increased spectrum of immunological activity of recombinant HA polypeptides obtained through modifications of putative N-glycosylation sites, lysine loop insertions, or amino acid residues in the globular head region.

In fig. 9 shows a graphical representation of amino acid residues in the globular head region that can be modified to change the spectrum of immunological activity of the recombinant HA polypeptide.

In fig. 10 shows the results of flow cytometry analysis demonstrating the increased spectrum of immunological activity of the recombinant HA polypeptides, as demonstrated by the increase in 4K8 binding to deglycosylated constructs.

In fig. 11 shows an exemplary schedule of immunizations and subsequent in vivo assessments.

In fig. Figure 12 shows a typical Kaplan-Meier survival curve for animals immunized with the next generation modifications of DO2a compared to the original SMARtDO2a.

In fig. Figure 13 shows typical weight loss curves for animals immunized with the next generation of DO2a modifications compared to the original SMARtDO2a.

In fig. 14 shows typical virus titers in the lungs of animals immunized with SMARtDO2a constructs compared to PBS on day 4 post-virus challenge.

In fig. 15 shows typical hemagglutination inhibition (HAI) assay results for sera obtained from animals immunized with SMARtDO2a constructs.

Definitions

To facilitate understanding of the present invention, certain terms are first defined below. Additional definitions of the following terms and other terms are set forth throughout the specification.

Adjuvant. As used herein, the term adjuvant refers to a substance or carrier medium that nonspecifically enhances the immune response to an antigen. Adjuvants may include a suspension of mineral substances (aluminum alum, aluminum hydroxide or phosphate) on which the antigen is adsorbed; or a water-in-oil emulsion in which the antigen solution is emulsified in mineral oil (eg, MF59®), sometimes with the inclusion of deactivated mycobacteria (Freund's complete adjuvant) to further enhance antigenicity. Immunostimulatory oligonucleotides (such as those comprising a CpG motif) can also be used as adjuvants (for example, see US Pat. Nos. 6,194,388, 6,207,646, 6,214,806, 6,218,371, 6,239,116, 6,339,068, 6,406,705, and 6,429,199). Adjuvants also include biological molecules such as co-stimulatory molecules for TLR ligands. Exemplary biological adjuvants include IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41 BBL.

Animal. As used herein, the term animal refers to any member of the animal kingdom. In some embodiments, the animal refers to humans at any stage of development. In some embodiments, animal refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (eg, rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cattle, primate, and/or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In some embodiments, the animal may be a transgenic animal, a genetically engineered animal, and/or a clone.

Antibody. As used herein, the term antibody refers to an immunoglobulin molecule produced by B lymphocytes with a specific amino acid sequence. In some embodiments, antibody production in humans or other animals is induced by a specific antigen (immunogen). Antibodies are characterized by a specific reaction with an antigen in some descriptive way, with each antibody and antigen defined relative to the other. The terms mediating an antibody response, mediating a neutralizing antibody, mediating an immunogenic response, or grammatical equivalents, refer to the ability of an antigen or other molecule to induce the production of antibodies. In some embodiments, the term antibodies refers to any recombinant antibodies used in in vitro assays, such as HA screening assays, including one or more polypeptides primarily encoded by immunoglobulin genes or fragments of immunoglobulin genes. Accordingly, such antibodies can exist in the form of intact immunoglobulins or in the form of fragments of immunoglobulins of the classes IgG, IgM, IgA, IgD and IgE. Exemplary antibody fragments include, but are not limited to, F(ab)'2, Fab', and single chain Fv (scFv).

Antigen. As used herein, the term antigen refers to an agent that mediates an immune response; and/or to (ii) an agent that is bound by a T cell receptor (eg, when presented by an MHC molecule) or antibody (eg, produced by a B cell) when exposed to or introduced into the body. In some embodiments, the antigen provides a humoral response (eg, including the production of antigen-specific antibodies) in the body; alternatively or additionally, in some embodiments, the antigen mediates a cellular response (eg, involving T cells whose receptors specifically interact with the antigen) in the body. Those skilled in the art will appreciate that a particular antigen may elicit an immune response in one or more members of the target organism (eg, mice, rabbits, primates, humans), but not all members of the target organism's species. In some embodiments, the antigen provides 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% representatives of the target organism species. In some embodiments, the antigen binds to an antibody and/or a T cell receptor and may or may not induce a specific physiological response in the body. In some embodiments, for example, an antigen can bind to an antibody and/or a T cell receptor in vitro, regardless of whether such interaction occurs in vivo or not. In some embodiments, the antigen reacts with specific humoral or cellular immune products, including inducible heterologous immunogens. In some embodiments of the disclosed compositions and methods, the antigen is an influenza virus HA polypeptide or an immunogenic fragment thereof.

Approximately. As used herein, the term approximately or approximately, when applied to one or more values of interest, refers to a value similar to a specified reference value. In certain embodiments, the term about or approximately refers to a range of values 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 any direction (more or less) from the specified reference value, unless otherwise stated or otherwise obvious from the context (unless such number would exceed 100% of the possible value).

Biological activity. As used herein, the phrase biological activity refers to the observable biological effect or result achieved by the agent or molecule of interest. For example, in some embodiments, the biological activity is an interaction through specific binding. In some embodiments, the biological activity is a modulation (eg, induction, enhancement, or inhibition) of a biological pathway or event. In some embodiments, the presence or extent of biological activity is assessed by detecting a direct or indirect product produced through a biological pathway or biological event of interest. In some embodiments, the biological activity of the HA polypeptide refers to the ability of the HA polypeptide to produce a neutralizing antibody. In these cases, the term biological activity is used interchangeably with immunogenic activity.

California 09 Numbering As used herein, the phrase California 09 numbering or CA09 numbering refers to a normalized biological sequence alignment that provides a comparison of a query sequence (e.g., an engineered HA polypeptide sequence that has been or will be subject to one or more modifications described herein) with the sequence in question (e.g., the polypeptide sequence from A/California/07/2009 (H1N1)), thereby identifying amino acid residues in the target sequence that correspond to the same position of A/California/07/2009 (H1N1 ), and therefore all other biological sequence similarities normalized to A/California/07/2009 (H1N1). In most cases, the target sequence and the query sequence share characteristic parts or features, but differ slightly in length and/or sequence identity. For example, the numbering of residues in a particular target sequence or for targeted modification can be identified and described based on the sequence of the A/California/07/2009 (H1N1) protein. The sequences are aligned with the full-length protein sequence (including signal peptide, transmembrane and cytoplasmic tail domains) A/California/07/2009 (NCBI accession number: ACP44189, NCBI gi number 227977172, 566 amino acids). Residue 1 is the N-terminal methionine of the signal peptide.

Carrier. As used herein, the term carrier refers to the diluent, adjuvant, excipient or carrier medium with which the composition is administered. In some illustrative embodiments, carriers may include sterile liquids, such as, for example, water and oils, including petroleum, animal, vegetable, or synthetic oils, such as, for example, peanut oil, soybean oil, mineral oil, sesame oil, and etc. In some embodiments, the carriers are or include one or more solid components.

A characteristic part or sign. As used herein, the term characteristic part or feature is used, in its broadest sense, to refer to a part of a substance whose presence (or absence) correlates with the presence (or absence) of a particular feature, attribute or activity of the substance. In some embodiments, a characteristic part or feature of a substance is a part or feature that is found in the substance and in related substances that share a particular characteristic, attribute, or activity, but not in those that

- 5 044592 which do not have such a common specific feature, attribute or activity. For example, the term characteristic pandemic trait is a trait that is found in at least one reference pandemic strain and not found in at least one non-pandemic strain. In some embodiments, a characteristic pandemic trait is a trait that is widely found in pandemic strains and rarely found in non-pandemic strains. Similarly, the term characteristic seasonal trait is a trait that is found in at least one reference seasonal strain and not found in at least one non-seasonal strain. In some embodiments, the characteristic seasonal trait is a trait that is widely found in seasonal strains and rarely found in non-seasonal strains. In some embodiments, the characteristic portion or feature is a characteristic sequence element that designates a sequence element found in a polymer (eg, a polypeptide or nucleic acid) that is a characteristic portion of such polymer. In some embodiments, the presence of a characteristic sequence element correlates with the presence or level of a particular activity or property of the polymer. In some embodiments, the presence (or absence) of a characteristic sequence element identifies a particular polymer as a member (or non-member) of a particular family or group of such polymers (eg, pandemic or seasonal). A representative sequence element typically includes at least two monomers (eg, amino acids or nucleotides). In some embodiments, the characteristic sequence element includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45 , 50 or more monomers (eg, contiguously linked monomers). In some embodiments, the characteristic sequence element includes at least first and second fragments of contiguous monomers separated by one or more spacer regions, the length of which may or may not vary among polymers that possess such a sequence element. In some embodiments, the characteristic sequence element includes the presence or absence of one or more putative glycosylation sites.

Computational Optimized Broad Spectrum Reactivity Antigen (COBRA) technology. As used herein, the terms computationally optimized broad spectrum antigens or COBRA technology refer to the methodology and methods for developing, and resulting compounds, engineered recombinant influenza virus HA antigens described, for example, in those published 18 months after the priority date patent publications WO 2012/036993, US No. 2015/0030628, WO 2013/119683, WO 2013/148164, WO 2014/085616 and WO 2013/122827. These applications are incorporated herein by reference in their entirety.

Corresponding. As used herein, the term corresponding is often used to refer to the position/identity of an amino acid residue in a polypeptide of interest (eg, an HA polypeptide). One of ordinary skill in the art will recognize that, for simplicity, residues in a polypeptide are often designated using a canonical numbering system based on the reference related polypeptide so that the amino acid corresponding to the residue at position 190, for example, need not be the 190th amino acid in a particular amino acid chain, but rather corresponds to the residue found at position 190 in the reference polypeptide; one of ordinary skill in the art will readily understand how to identify the corresponding amino acids using, for example, various sequence alignment tools.

Constructed. As used herein, the term engineered describes a polypeptide the amino acid sequence of which was designed by a person skilled in the art and/or whose existence and production requires the action of a person skilled in the art (ie, at the hands of a person). For example, the engineered HA polypeptide has an amino acid sequence that differs from the amino acid sequences of HA polypeptides found in naturally occurring influenza isolates. In some embodiments, the engineered HA polypeptide has an amino acid sequence that differs from the amino acid sequence of HA polypeptides included in the NCBI database.

Epitope. As used herein, the term epitope includes any fragment that is specifically recognized in whole or in part by an immunoglobulin binding component (eg, antibody or receptor). In some embodiments, an epitope consists of a plurality of chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are exposed on the surface when the antigen adopts the appropriate three-dimensional conformation. In some embodiments, such chemical atoms or groups are in physical proximity to each other in space when the antigen adopts such a conformation. In some embodiments, at least some of such chemical atoms are groups that are physically separated from each other when the antigen adopts an alternative conformation (eg, a linearized appearance).

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Excipient. As used herein, the term excipient refers to a non-therapeutic agent that can be included in a pharmaceutical composition, for example, to provide or help achieve 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, skim milk powder, glycerin, propylene, glycol, water , ethanol, etc.

Head area. As used herein, the term head region refers to the segment encompassed by amino acid residues 59-292 (as numbered CA09) of an engineered HA polypeptide or a wild-type HA polypeptide. From a morphological point of view, the head region can be defined as a globular HA domain.

Hemagglutinin (HA) polypeptide. As used herein, the term hemagglutinin polypeptide (or HA polypeptide) refers to a polypeptide whose amino acid sequence includes at least one characteristic HA sequence. A wide variety of HA sequences from influenza isolates are known in the art; indeed, the National Center for Biotechnology Information (NCBI) maintains a database (http://www.ncbi.nlm.nih.gov/genomes/FLU/) that, at the time of filing of this application, included at least 15,491 complete HA polypeptides from influenza A (subtype H1N1) in the database (of which 6974 are unique). One skilled in the art, by reference to this database, can readily identify sequences that are characteristic of HA polypeptides in general and/or specific HA polypeptides (e.g., H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16; or NAs that mediate infection of specific hosts, e.g. bird, camel, dog, cat, civet, environmental, horse, human, leopard, mink, mouse, seal, stone marten, pig, tiger, whale, etc.). For example, in some embodiments, the HA polypeptide includes one or more characteristic sequence elements found between residues about 97 and about 185, about 324 and about 340, about 96 and about 100, and/or about 130 and about 230 of the HA protein found in naturally occurring influenza virus isolate.

Polypeptide HA H1N1. An H1N1 HA polypeptide, as that term is used herein, is an HA polypeptide whose amino acid sequence includes at least one sequence element that is characteristic of H1N1 and distinguishes H1N1 from other HA subtypes. Such typical sequence elements can be determined by alignment, as will be appreciated by one skilled in the art.

Master. The term host is used herein to refer to the system (eg, cell, organism, etc.) in which the polypeptide of interest is present. In some embodiments, the host is a system that is susceptible to infection by a particular infectious agent. In some embodiments, the host is a system that expresses the particular polypeptide of interest.

Host cell. As used herein, the phrase host cell refers to a cell into which exogenous DNA (recombinant or other type) has been incorporated. For example, host cells can be used to produce the engineered influenza virus hemagglutinin polypeptides described herein using standard recombinant techniques. After reading this disclosure, one skilled in the art will understand that such terms refer not only to the particular cell in question, but also to the progeny of such cell. Since certain modifications may be present in subsequent generations due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term host cell as used herein. In some embodiments, host cells include any prokaryotic and eukaryotic cells that are suitable for expression of exogenous DNA (eg, a recombinant nucleic acid sequence). Exemplary cells include prokaryotic and eukaryotic cells (unicellular or multicellular), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., etc.), mycobacterial cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g. SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or fusion cells such as, for example, hybridomas or quadromas. In some embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, the cell is eukaryotic and is selected from the following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), CoS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (eg BHK21), Jurkat, Daudi, A431 (epidermal), CV- 1, U937, 3T3, L cells, C127 cells, SP2/0, NS-0, MMT 060562, Sertoli cells, BRL 3A cells, HT1080 cells, myeloma cell, tumor cell and a cell line derived from the above cell.

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In some embodiments, the cell contains one or more viral genes, such as a retinal cell that expresses a viral gene (eg, a PER.C6™ cell).

Immune response. As used herein, the term immune response refers to the response of an immune system cell, such as a B cell, T cell, dendritic cell, macrophage, or polymorphonuclear leukocyte, to a stimulus, such as an antigen or vaccine. The immune response may include any cell of the body involved in the host defense response, including, for example, an epithelial cell that secretes interferon or a cytokine. The immune response includes, but is not limited to, the 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 for measuring immune responses are well known in the art and include, for example, measuring the proliferation and/or activity of lymphocytes (such as B or T cells), cytokine or chemokine secretion, inflammation, antibody production, and the like.

Immunogen. As used herein, the term immunogen refers to a compound, composition or substance that, under appropriate conditions, is capable of stimulating an immune response, such as antibody production or T cell response, in an animal, including compositions that are injected into the animal or absorbed in an animal. As used herein, an immunogenic composition is a composition containing an immunogen (such as an HA polypeptide). As used herein, to immunize means to render a subject protected against an infectious disease, for example, by vaccination.

In vitro. As used herein, the term in vitro refers to events that occur in an artificial environment, such as a test tube or reaction vessel, cell culture, etc., rather than in a multicellular organism.

In vivo. As used herein, the term in vivo refers to events that occur in a multicellular organism, such as humans and non-human animals. In the context of cellular systems, the term can be used to refer to events that occur in a living cell (as opposed to, for example, in vitro systems).

Influenza virus. As used herein, the term influenza virus refers to a negative-strand RNA virus with a segmented genome that belongs to the family Orthomyxoviridae.

Vaccine against influenza virus. As used herein, the term influenza virus vaccine refers to an immunogenic composition capable of stimulating an immune response that is administered to prevent, alleviate, or treat an infection caused by an influenza virus. An influenza virus vaccine may include, for example, attenuated or inactivated (eg, cleaved) influenza virus, virus-like particles (VLPs), and/or antigenic polypeptides (eg, engineered hemagglutinins described herein), or DNA derived therefrom, or any recombinant version of such immunogenic materials.

Dedicated. As used herein, the term isolated refers to an agent or molecule that has either (i) been separated from at least some of the components with which it was initially associated (whether under natural or experimental conditions); or (ii) obtained through human manipulation. The isolated agents or molecules can be separated from at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least from about 60%, at least about 70%, at least about 80%, at least about 90% or more of the other components with which they were originally associated. In some embodiments, the isolated agents have a purity greater than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.

Flash. As used herein, an influenza virus outbreak refers to a collection of virus isolates from a single country in a given year.

Pandemic or seasonal strain. A pandemic strain of influenza virus is a strain that has caused or has the potential to cause pandemic infection in populations. In some cases, a pandemic is a global outbreak of disease that occurs when a new virus appears or emerges in a population, causes severe illness, and then spreads easily to people around the world. In most cases, pandemic strains span the period from 2009 to the present and form a single cluster of genetic sequences antigenically similar to A/California/07/2009. More generally, pandemic influenza strains include those that arise from reassortment (an antigenic shift that occurs approximately every 20 to 30 years) between human and avian or swine influenza viruses, resulting in a virus with a new NA of avian or swine origin against which people have no immunity to. In other words, the population is considered to be unexposed, unresistant, or marginally resistant, either as a result of previous vaccination or previous exposure. Pandemic and seasonal strains are antigenically distinct and very different in sequence. In most cases, seasonal influenza strains can be defined as circulating strains from 1986 to 2009 (including sequences from the 2009 strains that are not pandemic) and other strains that have substantially similar genetic sequences encoding antigenic regions (i.e., i.e. spatially similar antigenic sequences). Exemplary pandemic strains include A/California/07/2009, A/California/04/2009, A/Belgium/145/2009, A/South Carolina/01/1918, and A/New Jersey/1976. Pandemic subtypes include, but are not limited to, the H5N1, H2N2, H9N2, H7N7, H7N3, H7N9, and H10N7 subtypes. Illustrative seasonal strains include A/Texas/36/1991, A/Singapore/1986, A/New Caledonia/20/1999, A/Solomon Islands/03/2006 and A/Brisbane/59/2007, as well as A/Wisconsin/ 67/2005.

Warning. As used herein, the term prevention refers to preventing, preventing the onset of a disease, delaying the onset and/or reducing the frequency and/or severity of one or more symptoms of a particular disease, disorder or condition (eg, infection with, for example, an influenza virus). In some embodiments, the extent of prevention is assessed on a population basis such that an agent is considered to prevent a particular disease, disorder, or condition if, in a population susceptible to the disease, disorder, or condition, there is a statistically significant reduction in the development, frequency, and/or intensity of one or more symptoms of a disease, disorder or condition.

Receptor binding site (RBS). As used herein, the term receptor binding site or RBS refers to contiguous or non-contiguous amino acid residues of the head region of the influenza virus HA polypeptide that include amino acids involved in the direct binding of sialic acids to target cell receptor proteins. The region of HA responsible for receptor binding is located at the membrane-distal end of each monomer of the HA trimer and has several basic structural features. For example, the binding site is flanked by loops 220 and 130, which contain amino acids that interact with sialic acid or internal sugars of the glycan chain. The membrane-distal region of the site is formed by helix 190, which also includes residues that have the ability to bind to the receptor either at sialic acid (residue 194) or to internal glycans on the receptor (approximately residues 190 and 193). The base of the site contains several highly conserved residues that form an extensive network of hydrogen bonds. The amino acid residues that constitute the receptor or RBS binding site of the influenza virus HA polypeptide can be described from the three-dimensional crystal structures of the HA polypeptides complexed with sialic acid analogues and the identification of amino acid residues in specific proximity to the analogue or can be described in relation to the HA polypeptide sequence from specific viral strain (for example, A/New Caledonia/20/99 or A/California/07/2009). Thus, in some embodiments, the receptor binding site or RBS of an engineered HA polypeptide described herein can be determined using a reference HA polypeptide sequence. In some embodiments, the receptor binding site or RBS of an engineered HA polypeptide described herein can be determined using crystal structures of the HA polypeptide sequence in complex with human and avian receptor analogues (eg, LSTa, LSTc). An exemplary reference crystal structure of the HA polypeptide complex with LSTc includes A/Puerto Rico/8/1934 (H1N1) pdb|1RVZ. In some embodiments, an RBS may be defined as an epitope bound by the broadly binding neutralizing monoclonal antibody CH65 (see, e.g., Whittle J.R. et al. Broadly neutralizing human antibody that recognizes the receptorbinding pocket of influenza virus hemagglutinin. Proc Natl Acad Sci USA. 2011;108:14216-21). Alternatively or additionally, the RBS can be defined as the region comprising all amino acid residues within 15 angstroms of the universally conserved tryptophan corresponding to position 167 in (as numbered CA09 09) (for example, see Xu, R. et al. Nat Struct Mol Biol 2013 Mar;20(3):363-70).

Recombinant. As used herein, the term recombinant is intended to refer to polypeptides (e.g., the HA polypeptides described herein) that are designed, engineered, produced, expressed, generated, or isolated by recombinant methods, such as polypeptides expressed using a recombinant expression vector transfected into a host cell, polypeptides isolated from a library of recombinant combinatorial polypeptides, or polypeptides produced, expressed, generated or isolated by any other method that involves splicing selected sequence elements with each other. In some embodiments, one or more such selected sequence elements are naturally occurring. In some embodiments, one or more such selected sequence elements are designed in silico. In some embodiments, one or more such selected sequence elements are obtained by mutagenesis (eg, in vivo or in vitro) of a known sequence element, such as from a natural or synthetic source. In some embodiments

- 9 044592 one or more such selected sequence elements are obtained by combining several (eg, two or more) known sequence elements that are not naturally present in the same polypeptide (eg, two epitopes from two separate HA polypeptides).

Recombinant vaccine against influenza virus. As used herein, the term recombinant influenza virus vaccine refers to an immunogenic composition specific for influenza containing engineered influenza hemagglutinins described herein, including, without limitation, influenza virus, preparations based on its subunits, virus-like particles, recombinant protein (i.e. preparations consisting of recombinant HA purified to varying degrees) and vaccines based on DNA and viral vectors. The recombinant influenza virus vaccines described herein may optionally contain one or more adjuvants.

Recombinant hemagglutinin polypeptide. As used herein, the term recombinant hemagglutinin (HA) polypeptide refers to any modified hemagglutinin polypeptide. In particular, the term refers to further modified or engineered hemagglutinin polypeptides.

Specificity. As is known in the art, specificity is a measure of the ability of a particular ligand (e.g., antibody, HA polypeptide, etc.) to discriminate its binding partner (e.g., antigen, human HA receptor and, in particular, human upper respiratory tract HA receptor ) from other potential binding partners (e.g. avian HA receptor).

Stem region. As used herein, the term stem region or stem region can refer to a heterogeneous region of an engineered HA polypeptide or a wild-type HA polypeptide, the region comprising approximately amino acid residues 18-58 and 293-519 (as numbered CA09). In terms of morphology, the stalk region can be defined as an elongated domain that extends from the globular head.

Subject. As used herein, the term subject means any mammal, including humans. In certain embodiments of the present invention, the subject is an adult, adolescent, or infant. In some embodiments, the terms individual or patient are used and are intended to be interchangeable with the term subject. The present invention also covers the administration of pharmaceutical compositions and/or treatment methods in utero.

Practically. As used herein, the term practically refers to a qualitative condition in which a characteristic or property of interest is demonstrated to its full or nearly full extent or extent. The average biologist will appreciate that biological and chemical phenomena rarely, if ever, occur to completion and/or continue until completion or the achievement or avoidance of an absolute outcome. The term is thus practically used in this document to cover the potential lack of completeness inherent in many biological and chemical phenomena.

Almost similar. As used herein, the term substantially similar refers to the comparison of two molecules. In most cases, molecules are considered substantially similar to each other when they are characterized by sufficient structural similarity (eg, a characteristic structural feature) that they have a comparable probability of having one or more additional attributes or features. As just one example, a characteristic, such as a pattern of glycosylation sites, that is either the same or sufficiently similar in two strains of influenza virus means that the risk of a human pandemic is the same for each strain.

Almost homologous sequence. The phrase substantially homologous is used herein to refer to comparisons between amino acid sequences or nucleic acid sequences. As will be appreciated by one of ordinary skill in the art, two sequences are generally considered substantially homologous if they contain homologous residues at corresponding positions. Homologous residues may be identical residues. Alternatively, homologous residues may be non-identical residues having correspondingly similar structural and/or functional characteristics. For example, as is well known to one of ordinary skill in the art, certain amino acids are generally classified as hydrophobic or hydrophilic amino acids and/or as having polar or nonpolar side chains. Replacing one amino acid with another of the same type can often be considered a homologous substitution. Typical categories of amino acids are summarized in Table. 1 and 2.

- 10 044592

Table 1

Alanin Ala A non-polar neutral 1.8 Arginine Arg R polar positive -4.5 Asparagine Asn N polar neutral -3.5 Aspartic acid Asp D polar negative -3.5 Cysteine Cys C non-polar neutral 2.5 Glutamic acid Glu E polar negative -3.5 Glutamine Gin Q polar neutral -3.5 Glycine Gly G non-polar neutral -0.4 Histidine His H polar positive -3.2 Isoleucine He I non-polar neutral 4.5 Leucine Leu L non-polar neutral 3.8 Lysine Lys TO polar positive -3.9 Methionine Met M non-polar neutral 1.9 Phenylalanine Phe F non-polar neutral 2.8 Proline Pro P non-polar neutral -1.6 Serin Ser S polar neutral -0.8 Threonine Thr T polar neutral -0.7 Tryptophan Trp w non-polar neutral -0.9 Tyrosine Tyr Y polar neutral -1.3 Valin Vai V non-polar neutral 4.2

table 2

Ambiguous amino acids 3-letter designation 1-letter designation Asparagine or aspartic acid Asx IN Glutamine or glutamic acid Glx Z Leucine or isoleucine Xie J Unspecified or unknown amino acid Haa X

As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a number of algorithms, including those available in commercial computer programs, such as BLASTN for nucleotide sequences and BLASTP, gap BLAST and PSI-BLAST for amino acid sequences . Such exemplary programs are described in Altschul et al., Basic local alignment search tool, J. Mol. Biol., 215(3):403-410, 1990; Altschul et al., Methods in Enzymology; Altschul et al., Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener et al. (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, vol. 132), Humana Press, 1999; all of the foregoing are incorporated herein by reference. In addition to identifying homologous sequences, the programs mentioned above usually provide an indication of the degree of homology. In some embodiments, two sequences are considered substantially homologous if at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% at least 97%, at least 98%, at least 99% or more of their corresponding residues are homologous over a significant portion of the residues. In some embodiments, the significant fragment is the entire sequence. In some embodiments, the significant fragment is at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, at least 500 or more residues.

Almost identical. The phrase substantially identical or substantially identical is used herein to refer to comparisons between amino acid sequences or nucleic acid sequences. As will be appreciated by one of ordinary skill in the art, two sequences are generally considered substantially identical if they contain identical residues at corresponding positions. As is well known in the art, amino acid sequences or nucleic acid sequences can be compared using any of a number of algorithms, including those available in commercial computer programs,

- 11 044592 such as BLASTN for nucleotide sequences and BLASTP, BLAST with gaps and PSI-BLAST for amino acid sequences. Such exemplary programs are described in Altschul et al., Basic local alignment search tool, J. Mol. Biol., 215(3):403-410, 1990; Altschul et al., Methods in Enzymology; Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener et al. (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, vol. 132), Humana Press, 1999. In addition to identifying identical sequences, the programs mentioned above usually provide an indication of the degree of identity. In some embodiments, two sequences are considered substantially identical if at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more of their respective residues are identical over a significant portion of the residues. In some embodiments, the significant fragment is the entire sequence. In some embodiments, the significant portion is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more balances. In the context of an HA polypeptide, reference to substantially identical generally refers to an HA polypeptide (or HA epitope) having an amino acid sequence that is at least 90%, preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identical to the reference HA polypeptide (or HA epitope).

Vaccination. As used herein, the term vaccination or vaccination refers to the administration of a composition intended to produce an immune response, for example, to a pathogen. Vaccination can be administered before, during, and/or after exposure to a pathogen and/or development of one or more symptoms, and in some embodiments, before, during, and/or shortly after exposure to such an agent. In some embodiments, vaccination involves multiple administrations of the vaccination composition with appropriate time intervals between them.

Virus-like particle (VLP). As used herein, the phrase virus-like particle or VLP refers to particles that resemble a virus but lack any viral genetic material and, as a result, are not infectious. The virus-like particle or VLP can be produced through heterologous expression in a number of cell culture systems, including mammalian cell lines, insect cell lines, yeast cells and plant cells. In addition, VLPs can be purified by methods known in the art. In some embodiments, an influenza virus-based VLP described herein comprises hemagglutinin (HA) polypeptides and neuraminidase (NA) polypeptides. In some embodiments, an influenza virus-based VLP described herein comprises HA polypeptides, NA polypeptides, and/or viral structural polypeptides (eg, influenza structural protein, such as influenza M1). In some certain embodiments, an influenza virus-based VLP described herein comprises HA polypeptides, NA polypeptides, and/or M1 polypeptides. In some embodiments, an influenza virus-based VLP described herein comprises HA polypeptides, NA polypeptides, and/or HIV gag polypeptides. As will be known to one skilled in the art, other viral structural proteins may be used as alternatives to those illustrated herein. Influenza virus-based VLPs can be produced by transfecting host cells (eg, mammalian cells) with plasmids encoding the HA and NA proteins and optionally the HIV gag proteins. After incubating the transfected cells for an appropriate time to allow protein expression (such as about 72 hours), VLPs can be isolated from cell culture supernatants. In some embodiments, the influenza virus-based VLPs described herein are produced by transient transfection in mammalian cells (eg, human cells). In some embodiments, the influenza virus-based VLP is analyzed using one or more assays. As just a few examples, influenza virus-based VLPs can be analyzed for hemagglutinin activity, dynamic light scattering, and to quantify hemagglutinin content through protein staining. Other assays will become readily apparent to one skilled in the art upon familiarization with the present invention.

Wild type. As understood in the art, the phrase wild type generally refers to the normal form of a protein or nucleic acid found in nature. For example, wild-type HA polypeptides are present in natural isolates of influenza virus. A number of different wild-type HA sequences can be found in the NCBI influenza virus sequence database, available online at ncbi.nlm.nih.gov/genomes/FLU/FLU.

Detailed Description of Certain Embodiments

The present invention provides, among other things, methods for modifying engineered HA polypeptides to alter immune profiles and increase cross-reactivity to different influenza strains. Embodiments of the present invention include

- 12 044592 personal strategies for designing HA polypeptides to expand the seasonal response profile to cover pandemic strains. In some embodiments, the strategy is designed to introduce modifications near the receptor binding site (RBS) of a host HA polypeptide based on sequences derived from an HA polypeptide with a distinct immunogenicity profile. Similar strategies can be used to expand the pandemic response profile to include seasonal strains.

The present invention provides improved modified recombinant HA polypeptides with an expanded immunogenicity profile, the coverage of which extends (ie, the ability to provide an immune response to the HA polypeptide) to antigenically distinct influenza strains, for example, new or additional pandemic and/or seasonal influenza virus strains. The present invention is based, in part, on modifications derived from in silico analysis of sequence variation among circulating influenza virus strains, mapping of the HA antigenic region and/or epitope profiles, and structural analysis of the HA peptide. Targeted modifications can subsequently be introduced into various amino acid residue locations and/or specific regions of an HA polypeptide with a known immune profile to produce new HA polypeptides with improved and more balanced immune profiles. As described in detail below, including in the Examples section, the present inventors have developed, among other things, excellent specific strategies for designing HA polypeptides to expand the seasonal response profile to cover pandemic strains or, conversely, these strategies expand the immune profile across sequence clusters (or clades) antigenically different strains; they can be applied to an engineered recombinant HA molecule over time so that it continues to provide an immune response to circulating seasonal strains subject to antigenic drift. In all cases, this strategy is designed generally to retain specific receptor binding site (RBS) residues of the host HA polypeptide with modifications designed in the region close to the RBS. Similar strategies can be used to expand the pandemic response profile to include seasonal strains. The compositions and methods of the present invention are applicable to a wide variety of recombinant HA polypeptides, including those constructed by a variety of methods and which primarily contain wild-type sequences.

Thus, in one aspect, the present invention provides a recombinant hemagglutinin (HA) polypeptide comprising an engineered head region or segment thereof derived from an engineered HA polypeptide with a predominantly seasonal immune profile and a stalk region derived from a pandemic strain. In some embodiments, the engineered head region or segment thereof comprises a sequence that is at least 95%, 96%, 97%, 98%, 99% or more identical to the amino acids corresponding to positions 135-269, 125-277, or 63-278 of SEQ ID NO: 1. In some embodiments, the engineered head region or segment thereof contains a sequence that is identical to amino acids corresponding to positions 135-269, 125-277, or 63-278 of SEQ ID NO: 1 (sequence SMARt_DO2a).

In some embodiments, the stem region is derived from a naturally occurring pandemic strain or a wild-type pandemic strain. In some embodiments, a suitable naturally occurring pandemic strain is selected from A/California/07/2009, A/New Jersey/10/1976, or A/South Carolina/1/1918. In some embodiments, the stem region is derived from an engineered HA polypeptide that has a pandemic immune profile.

In some embodiments, an engineered HA polypeptide that is characterized by a pandemic immune profile is constructed using computationally optimized broad spectrum reactivity antigen (COBRA) technology, mosaic technology, strain combinations based on influenza consensus sequences, deletion and/or rearrangement of structural domains , domain substitutions, or combinations of neutralizing or cross-reactive epitopes shared by multiple influenza virus strains.

In another aspect, the present invention provides a recombinant influenza virus hemagglutinin (HA) polypeptide comprising an engineered head region derived from an HA polypeptide with a predominantly seasonal immune profile and comprising one or more amino acid substitutions, deletions or insertions within one or more putative N-glycosylation sites , which are defined by the consensus sequence NxS/Ty, where x and y are not proline (P), thereby ensuring the destruction of one or more putative N-glycosylation sites. In specific embodiments, each of the one or more amino acid substitutions, deletions, or insertions is derived from a corresponding sequence of the pandemic strain.

In yet another aspect, the present invention provides a recombinant influenza virus hemagglutinin (HA) polypeptide comprising an engineered head region derived from poly

- 13044592 HA peptide with a predominantly pandemic immune profile into which one or more engineered putative N-glycosylation sites have been inserted, which are defined by the consensus sequence NxS/Ty, where x and y are not P. In specific embodiments, each of one or more engineered putative N-glycosylation sites are constructed using amino acid substitutions, deletions, or insertions based on the corresponding sequence of the seasonal strain.

In some embodiments, the hemagglutinin is influenza A. In some embodiments, influenza A is of the H1N1 subtype.

In some embodiments, one or more putative N-glycosylation sites correspond to positions 142-145 and/or 177-179 (normalized sequence alignment results to A/California/07/2009 HA (SEQ ID NO: 2); as numbered CA09). In some embodiments, one or more putative N-glycosylation sites are within 15 angstroms of a receptor binding site (RBS), wherein an RBS is defined as all amino acid residues within 15 angstroms of a position corresponding to W167 (as numbered CA09) in three dimensions (3D) structure. Alternatively, an RBS may be defined as an epitope bound by a paratope of the monoclonal antibody CH65, and wherein one or more amino acid substitutions are present adjacent to (e.g., within 100 amino acid residues, within 75 amino acid residues, within 50 amino acid residues, within 40 amino acid residues, within 30 amino acid residues, within 25 amino acid residues, within 20 amino acid residues, within 15 amino acid residues, within 10 amino acid residues, within 5 amino acid residues, etc.) with the CH65 epitope. In some embodiments, one or more amino acid substitutions, deletions, or insertions are selected from lists or tables provided herein, such as Table. 4 or 5. In some embodiments, the one or more amino acid substitutions, deletions or insertions involve modifying the NxS/Ty consensus sequence to z1z ² z ³ z ⁴ where z ¹ is N, D, K or S; z ² represents Y or remains unchanged; z ³ represents E, D or N; and z ⁴ is I, L, P, S or T or remains unchanged.

In some embodiments, the pandemic or seasonal strain from which one or more amino acid substitutions, deletions, or insertions originate is a circulating strain of influenza virus. In some embodiments, the circulating influenza virus strain is selected from the group consisting of A/California/07/2009 and A/South Carolina/1/1918. In some embodiments, the amino acid substitutions, deletions, or insertions involve the introduction of a lysine (K) or arginine (R) residue within 1-5 amino acids (e.g., within 1-4, 1-3, 1-2 amino acids) of the NxS consensus sequence /Ty. In some embodiments, a lysine (K) or arginine (R) residue is within 1-5 amino acids (eg, within 1-4, 1-3, 1-2 amino acids) upstream of the NxS/Ty consensus sequence. In some embodiments, the one or more amino acid substitutions, deletions, or insertions involve insertion at a position corresponding to residue 147 (as numbered CA09). In some embodiments, the insertion at the position corresponding to residue 147 involves the insertion of a lysine (K) or an arginine (R).

In a further aspect, the present invention provides a recombinant influenza virus HA polypeptide comprising an engineered head region derived from an HA polypeptide with a predominantly seasonal immune profile and comprising one or more amino acid substitutions between positions corresponding to positions 60 and 291 (as numbered CA09), wherein each of one or more amino acid substitutions are derived from the corresponding sequence of the pandemic strain. In some embodiments, one or more amino acid substitutions are selected from lists or tables provided herein, such as Table 6. In some embodiments, the one or more amino acid substitutions are between positions corresponding to 137 and 262 (according to CA09 numbering), and the one or more amino acid substitutions are selected from table. 7. In some embodiments, the one or more amino acid substitutions include two, three, four, five, six, seven, eight, nine, ten, or more amino acid substitutions selected from table. 3 or 4. In some embodiments, the one or more amino acid substitutions include at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive substitutions selected from table. 6 or 7. In some embodiments, one or more amino acid substitutions are present at positions corresponding to 137, 144, 145, 154, 155, 156, 157, 158, 159, 177, 210, 211, 212, 213, 214, 244, 245 and/or 262 (according to CA09 numbering).

In a related aspect, the present invention provides a recombinant influenza virus HA polypeptide comprising an engineered head region derived from an HA polypeptide with a predominantly seasonal immune profile and comprising one or more amino acid substitutions within 15 angstroms of a receptor binding site (RBS), wherein the RBS is defined as all amino acid residues within 15 angstroms of the position corresponding to W167 (as numbered CA09) in a three-dimensional (3D) structure, where each of the one or more amino acid substitutions originates from the corresponding pandemic strain sequence. As an alternative,

- 14 044592

An RBS can be defined as an epitope bound by a paratope of the monoclonal antibody CH65, and wherein one or more amino acid substitutions are present adjacent to (e.g., within 100 amino acid residues, within 75 amino acid residues, within 50 amino acid residues, within 40 amino acid residues, within 30 amino acid residues, within 25 amino acid residues, within 20 amino acid residues, within 15 amino acid residues, within 10 amino acid residues, within 5 amino acid residues, etc.) with the CH65 epitope.

In some embodiments, the pandemic strain is a circulating influenza virus. In some embodiments, the one or more amino acid substitutions are within 10 (eg, within 9, 8, 7, 6, 5, etc.) angstroms of the receptor binding site (RBS). In some embodiments, the one or more amino acid substitutions include two, three, four, five, six, seven, eight, nine, ten, or more amino acid substitutions selected from Table. 8. In some embodiments, the one or more amino acid substitutions include at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive substitutions selected from table. 8. In some embodiments, one or more amino acid substitutions are present at positions corresponding to positions 137, 144, 145, 154, 155, 156, 157, 158, 159, 177, 210, 211, 212, 213, and/or 214 (as numbered CA09).

In yet another aspect, the present invention provides a recombinant influenza virus HA polypeptide comprising an engineered head region derived from an HA polypeptide with a predominantly seasonal immune profile and containing one or more amino acid modifications selected from Table 1. 9 (for example, two or more, three or more, four or more, five or more modifications selected from Table 9).

In some embodiments, an HA polypeptide with a predominantly seasonal immune profile or an HA polypeptide with a predominantly pandemic immune profile suitable for the present invention is designed using computationally optimized broad spectrum reactivity antigen (COBRA) technology, mosaic technology, influenza virus strain combinations based on influenza virus consensus sequences, deletion and/or rearrangement of structural domains, domain replacement, or combinations of neutralizing or cross-reactive epitopes shared by multiple influenza virus strains.

In some embodiments, the recombinant HA polypeptide produces neutralizing antibodies against both seasonal and pandemic strains of influenza virus. In specific embodiments, the recombinant HA polypeptide produces neutralizing antibodies against one or more seasonal strains and another pandemic strain of influenza virus. In some embodiments, the recombinant HA polypeptide has a more balanced immunogenicity profile against both seasonal and pandemic strains of influenza virus.

Among other things, the present invention provides an isolated nucleic acid encoding a recombinant HA polypeptide described herein, and a vector containing such nucleic acid. In some embodiments, the present invention provides a cell containing such a vector or nucleic acid. In some embodiments, a suitable cell is a human cell. In specific embodiments, the human cell is a HEK-293 cell. In some embodiments, a suitable cell is a monkey cell. In specific embodiments, the monkey cell is a Vero cell.

In various additional aspects, the present invention provides virus-like particles (VLPs) comprising a recombinant HA polypeptide described herein. In some embodiments, the VLP of the present invention further comprises an influenza virus neuraminidase (NA) protein, a human immunodeficiency virus gag protein ( HIV) or both. The present invention also provides pharmaceutical compositions containing the recombinant HA polypeptide or VLP described herein. In addition, the present invention provides methods for generating an immune response (eg, immunization or vaccination) to seasonal and pandemic influenza viruses in a subject by administering a pharmaceutical composition described herein.

In another aspect, the present invention provides methods for changing the immunogenicity profile of an engineered HA polypeptide. In some embodiments, a method for altering the immunogenicity profile of an engineered HA polypeptide involves selecting a head region of the engineered HA polypeptide and replacing the selected head region of the engineered HA polypeptide with a corresponding head region of the HA polypeptide with a distinct immunogenicity profile, thereby producing a redesigned HA polypeptide with an altered immunogenicity profile. In some embodiments, the engineered HA polypeptide has a predominantly seasonal immune profile, and the HA polypeptide with a distinct immunogenicity profile has a predominantly pandemic immune profile.

In some embodiments, a selected region of the head of the engineered polypipe

- 15 044592 HA type with a predominantly seasonal immune profile corresponds to residues 63-278, 125-277 or 135-269 (according to CA09 numbering). In some embodiments, a corresponding head region of a HA polypeptide with a distinct immunogenicity profile that is predominantly pandemic comprises an amino acid sequence selected from residues 63-277 of SEQ ID NO: 2 [full-length wild-type CA09 sequence (HA sequence A/Califomia/07/ 2009)], residues 63-277 from SEQ ID NO: 3 [wild-type SC1918 full-length sequence], residues 63-277 from SEQ ID NO: 4 [wild-type NJ1976 full-length sequence], residues 125-277 from SEQ ID NO: 2 [full-length wild-type CA09 sequence], residues 125-277 from SEQ ID NO: 3 [full-length wild-type SC1918 sequence], residues 125-277 from SEQ ID NO: 4 [full-length wild-type NJ1976 sequence], residues 135-269 from SEQ ID NO: 2 [full-length wild-type CA09 sequence], residues 135-269 of SEQ ID NO: 3 [full-length wild-type SC1918 sequence], or residues 135-269 of SEQ ID NO: 4 [full-length wild-type NJ1976 sequence].

In some embodiments, the HA polypeptide with a distinct immunogenicity profile that is predominantly pandemic is the HA polypeptide from a wild-type influenza virus. In some embodiments, an HA polypeptide with a distinct immunogenicity profile that is predominantly pandemic is an engineered HA polypeptide. In some embodiments, an HA polypeptide with a distinct immunogenicity profile that is predominantly pandemic comprises SEQ ID NO: 5 (full length sequence for DO1A).

In some embodiments, the engineered HA polypeptide has a predominantly pandemic immune profile, and the HA polypeptide with a distinct immunogenicity profile has a predominantly seasonal immune profile. In some embodiments, the selected head region of the engineered HA polypeptide with a predominantly pandemic immune profile is selected from the group consisting of residues 63-277 of SEQ ID NO: 2 [full-length wild-type CA09 sequence (HA sequence A/California/07/2009)]; residues 63-277 of SEQ ID NO: 3 [full-length wild-type SC1918 sequence]; residues 63-277 of SEQ ID NO: 4 [full-length wild-type NJ1976 sequence]; residues 125-277 of SEQ ID NO: 2 [full-length wild-type CA09 sequence]; residues 125-277 of SEQ ID NO: 3 [full-length wild-type SC1918 sequence]; residues 125-277 of SEQ ID NO: 4 [full-length wild-type NJ1976 sequence]; residues 135-269 of SEQ ID NO: 2 [full-length wild-type CA09 sequence]; residues 135-269 of SEQ ID NO: 3 [full-length wild-type SC1918 sequence]; and residues 135-269 of SEQ ID NO: 4 [full-length wild-type NJ1976 sequence]. In some embodiments, a corresponding head region of an HA polypeptide with a distinct immunogenicity profile that is predominantly seasonal comprises residues 63-278, 125-277, or 135-269 of SEQ ID NO: 2 (the full-length sequence for SMARt_DO2A).

HA polypeptides can be engineered to provide a specific immunogenic response profile. In other words, one may choose or stumble upon various design strategies used to create engineered HA polypeptides to produce HA polypeptides that provide a significant immune response (eg, a neutralizing antibody response) against predominantly circulating seasonal (i.e., endemic) strains influenza virus and/or historical or pandemic strains of influenza virus. Thus, the term seasonal response profile can be used to describe a recombinant HA polypeptide that induces the production of cross-neutralizing antibodies against seasonal influenza virus strains rather than pandemic influenza virus strains. In most cases, seasonal influenza strains can be defined as circulating strains from 1986 to 2009 (including sequences from the 2009 strains that are not pandemic) and other strains that have substantially similar genetic sequences encoding antigenic regions (i.e., i.e. spatially similar antigenic sequences). Specific examples include A/New Caledonia/20/1999 and A/Wisconsin/67/2005. Thus, the seasonal response profile can be used to describe a recombinant HA polypeptide that induces the production of cross-neutralizing antibodies against one or more seasonal influenza virus strains, but not the standard pandemic strain A/California/07/2009. Likewise, the term pandemic response profile can be used to describe a recombinant HA polypeptide that induces the production of cross-neutralizing antibodies to a greater extent against pandemic strains of influenza virus than against seasonal strains of influenza virus; The pandemic response profile can also be used to describe a recombinant HA polypeptide that induces the production of cross-neutralizing antibodies against one or more pandemic strains of influenza virus, but not the standard seasonal strain A/New Caledonia/20/1999. In most cases, pandemic strains span the period from 2009 to the present and form a single cluster of sequences antigenically similar to A/California/07/2009. More generally, pandemic influenza strains include those that arise in

- 16 044592 as a result of reassortment (an antigenic shift that occurs approximately every 20-30 years) between human and avian or swine influenza viruses, which leads to the emergence of a virus with a new NA of avian or swine origin, against which humans have no immunity. In other words, the population is considered to be unexposed, unresistant, or marginally resistant, either as a result of previous vaccination or previous exposure. Thus, pandemic strains include A/South Carolina/01/1918 and A/New Jersey/1976, which differ in sequence and antigenic distance from the California 2009 sequence cluster. Pandemic subtypes include, but are not limited to, subtypes H5N1, H2N2, H9N2, H7N7, H7N3, H7N9 and H10N7.

The modifications described herein can be used to further tailor or optimize the immunogenicity profile so as to redesign the engineered HA polypeptide to produce antibodies against more or fewer seasonal strains (or demonstrate an improved response or a response with more antibodies to seasonal strains) or more or fewer pandemic strains (or demonstrating an improved response or a response with greater antibody production to pandemic strains). Thus, these modifications expand the immune profile across sequence clusters (or clades) of antigenically distinct strains. They can be applied to an engineered recombinant HA molecule to provide an immune response to new pandemic strains arising due to antigenic shift (i.e. to cover antigenically distinct strains that are distantly separated in genetic sequence space in an extended time frame). They can also be used to direct genetic changes that occur over relatively short periods of time so that the engineered HA polypeptide continues to be effective in providing an immune response to antigenically drifted circulating seasonal strains (eg, improved response to seasonal strains). In specific embodiments, the modifications described herein can be used to (1) expand the coverage (i.e., the ability to provide a neutralizing immune response) of the engineered pandemic-like HA polypeptide (i.e., the pandemic response profile) to one or more seasonal strains (i.e. more seasonal immune profile); (2) to expand the coverage of the engineered seasonal-like HA polypeptide to any pandemic strain (to address the issue of antigenic drift); and (3) to expand the coverage of the seasonal-like HA polypeptide to any other antigenically distinct seasonal strains (ie, an improved seasonal immune profile that overcomes the problem of antigenic shift).

Various aspects of the present invention are described in more detail in the following subsections. The use of subclauses is not intended to limit the present invention. Each subsection can be applied to any aspect of the present invention. In the invention, the use of or means and/or, unless otherwise indicated.

The present invention is not limited to the specific methods and experimental conditions described, since such methods and conditions may vary. It should also be understood that the terminology used herein is intended to describe specific embodiments only and is not intended to be limiting unless otherwise stated, as the scope of the present invention will be limited only by the appended claims.

Unless otherwise specified, all technical and scientific terms and phrases used herein have the same meaning as would be commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein may be used in practicing or testing the present invention, preferred methods and materials will now be described. All publications mentioned in this document are incorporated herein by reference.

Standard techniques (eg, electroporation, lipofection) can be used to synthesize recombinant DNA, oligonucleotides, and tissue culture and transformation. Enzymatic reactions and purification procedures can be performed in accordance with the manufacturer's technical documents or as they are routinely performed in the art or as described herein. The above techniques and procedures can generally be performed in accordance with conventional methods well known in the art and described in various general and more specific literature sources, which are mentioned and discussed in the present description. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989)), which is incorporated herein by reference for all purposes.

Designed HA polypeptides.

The present invention can be used to modify any engineered hemagglutinin (HA) polypeptides, including any HA polypeptides produced using various recombinant techniques. Embodiments of the present invention can be applied to products of any method used by one skilled in the art to produce polypep

- 17 044592 types of HA with improved properties for the purpose of vaccination. Applicable methods for producing engineered HA polypeptides for use in embodiments of the present invention include producing engineered HA polypeptides using computationally optimized broad spectrum reactivity antigen (COBRA) technology, mosaic technology, reverse genetics, protein engineering, influenza virus strain combinations based on influenza virus consensus sequences, deletion and/or rearrangement of structural domains, domain replacements, or combinations of neutralizing or cross-reactive epitopes shared by multiple influenza virus strains. Such prior attempts include those described in 62/005670, WO 2012/177760, WO 2013/148164, US 20140147459, WO 2013/043729, US 20140286981, US 2014-0050759, US 8685410, WO 20 15/028478, US 2010-0074915, each of which are incorporated herein by reference.

However, these technologies often result in the production, intentionally or otherwise, of HA polypeptides that have an immunogenicity profile that is effective against either seasonal or pandemic strains. The immunogenicity profile of an HA polypeptide can be defined as the spectrum of neutralizing antibodies induced by immunization with the HA polypeptide. Typically, the HA polypeptide may exhibit a seasonal or predominantly seasonal immune profile, a pandemic or predominantly pandemic immune profile, or a balanced immune profile. Among other things, the present invention can be used to improve the immunogenicity profile of an engineered HA polypeptide so that it is capable of providing neutralizing antibodies against both seasonal and pandemic strains of influenza virus, or improving the quality or quantity of neutralizing antibodies against seasonal and/or pandemic strains. In some embodiments, the present invention can be used to improve an engineered HA polypeptide so that it has a balanced immunogenicity profile. Likewise, embodiments of the present invention can be used to alter the immune profile of an HA polypeptide with a balanced immune profile so that it becomes more or less seasonal or more or less pandemic.

As used herein, the term neutralizing antibodies refers to immunoglobulin molecules that are produced by B lymphocytes in humans or other animals in response to stimulation by a specific antigen (immunogen). For example, neutralizing antibodies can be induced by the HA polypeptide of the influenza virus. Neutralizing antibodies raised by a specific HA polypeptide are generally capable of neutralizing (eg, blocking infectivity) influenza viruses containing that specific influenza virus HA polypeptide, or influenza viruses containing related HA polypeptides that share certain immunogenic features.

As used herein, an HA polypeptide with a seasonal or predominantly seasonal immune profile is an HA polypeptide that provides an immune response (eg, neutralizing antibodies) against one or more seasonal influenza virus strains. In some embodiments, an HA polypeptide with a seasonal or predominantly seasonal immune profile produces antibodies that are capable of neutralizing at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seasonal strains of influenza virus. In some embodiments, an HA polypeptide with a seasonal or predominantly seasonal immune profile produces antibodies that are capable of neutralizing 2 or more seasonally circulating influenza virus strains. In some embodiments, the HA polypeptide with a predominantly seasonal immune profile is an HA polypeptide that produces antibodies that do not neutralize pandemic influenza strains. In some embodiments, an HA polypeptide with a seasonal or predominantly seasonal immune profile produces antibodies that do not neutralize A/California/07/2009. In some embodiments, an HA polypeptide with a seasonal or predominantly seasonal immune profile produces antibodies that are capable of neutralizing more or substantially more seasonal influenza virus strains compared to pandemic influenza strains. In some embodiments, an HA polypeptide with a seasonal or predominantly seasonal immune profile produces antibodies that are capable of neutralizing at least 2, 3, 4, 5, etc., 6, 7, 8, 9, or 10 more seasonal strains influenza virus than pandemic influenza virus strains. In some embodiments, the HA polypeptide with a seasonal or predominantly seasonal immune profile is an HA polypeptide that produces antibodies that are capable of neutralizing two or more seasonal circulating strains than pandemic strains.

As used herein, an HA polypeptide with a pandemic or predominantly pandemic immune profile is an HA polypeptide that provides an immune response (eg, neutralizing antibodies) against one or more pandemic strains of influenza virus. In particular, an HA polypeptide with a pandemic or predominantly pandemic immune profile is an HA polypeptide that produces antibodies that are capable of neutralizing A/California/07/2009. In some variants

- 18 044592 embodiments, an HA polypeptide with a pandemic or predominantly pandemic immune profile is an HA polypeptide that produces antibodies that are capable of neutralizing one or more of A/California/07/2009, A/South Carolina/01/1918, A/New Jersey/1976 or any other pandemic influenza virus strains identified herein. In some embodiments, an HA polypeptide with a pandemic or predominantly pandemic immune profile produces antibodies that are capable of neutralizing at least 1, 2, 3, 4, 5, etc. pandemic strains of influenza virus. In some embodiments, an HA polypeptide with a pandemic or predominantly pandemic immune profile produces antibodies that are capable of neutralizing two or more pandemic influenza virus strains. In some embodiments, an HA polypeptide with a pandemic or predominantly pandemic immune profile is an HA polypeptide that produces antibodies that do not neutralize seasonal influenza strains. In some embodiments, an HA polypeptide with a pandemic or predominantly pandemic immune profile produces antibodies that do not neutralize A/New Caledonia/20/1999. In some embodiments, an HA polypeptide with a pandemic or predominantly pandemic immune profile produces antibodies that are capable of neutralizing a greater number of pandemic influenza virus strains compared to seasonal influenza strains. In some embodiments, an HA polypeptide with a pandemic or predominantly pandemic immune profile produces antibodies that are capable of neutralizing at least 2, 3, 4, 5, etc. There are more pandemic influenza virus strains than seasonal influenza virus strains. In some embodiments, the HA polypeptide with a pandemic or predominantly pandemic immune profile is an HA polypeptide that produces antibodies that are capable of neutralizing two or more pandemic strains than seasonal strains.

As used herein, a balanced immune profile HA polypeptide is an HA polypeptide that produces antibodies that are capable of neutralizing both seasonal and pandemic strains. In some embodiments, the immune-balanced HA polypeptide is an HA polypeptide that produces antibodies that are capable of neutralizing at least 1, 2, 3, 4, or 5 seasonal strains (e.g., A/New Caledonia/20/1999), and one or more of A/California/07/2009, A/South Carolina/01/1918, A/New Jersey/1976, or any other pandemic strains described herein. In some embodiments, the immune-balanced HA polypeptide is an HA polypeptide that produces antibodies that are capable of neutralizing at least 1, 2, 3, 4, or 5 seasonal strains as well as A/California/07/2009. In some embodiments, the HA polypeptide with a balanced immune profile is an HA polypeptide that produces antibodies that are capable of neutralizing A/New Caledonia/20/1999 and A/California/07/2009. In some embodiments, the HA polypeptide with a balanced immune profile is an HA polypeptide that produces antibodies that are capable of neutralizing substantially the same number of seasonal and pandemic strains. For example, the difference in the numbers of seasonal and pandemic strains neutralized by antibodies, the production of which is ensured by the HA polypeptide with a balanced immune profile, does not exceed 1, 2, 3, 4 or 5.

As used herein, the phrases improve the immunogenicity profile, increase the breadth of the immune profile, more balanced immune profile, less impactful immune profile, more seasonal, less seasonal, more pandemic, less pandemic or grammatical equivalents indicate the spectrum of neutralizing antibodies that are induced by the modified HA polypeptide, relative to the spectrum of a reference HA polypeptide, such as the parent HA polypeptide before the modifications described herein are made.

Modification of engineered NA to change the immunogenicity profile.

Embodiments of the present invention can be used to modify or alter the immunogenicity profile of engineered HA polypeptides, particularly to increase the diversity of influenza virus strains against which the engineered HA polypeptide is capable of producing an immune response (eg, a neutralizing antibody response). In some embodiments, the method of the present invention is based on modifications derived from in silico analysis of sequence variation among circulating influenza virus strains, mapping of antigenic region(s) and/or epitope patterns, and structural analyzes of the HA polypeptide relative to HA polypeptides with different or different immune profiles. Targeted modifications can be introduced at various amino acid residue locations and/or specific regions of a HA polypeptide with a known immune profile based on corresponding sequences derived from the HA polypeptide with a distinguishable immune profile, yielding new HA polypeptides with an improved and more balanced immune profile. The location, type and number of modifications can be selected and combined to produce a redesigned HA polypeptide with an immunogenicity profile that has been tailored to provide a specific immune response (e.g., a balanced immune profile, an improved more pandemic response to pandemic strains, etc. ). In some embodiments, the modification strategy is designed generally to preserve specific receptor binding site (RBS) residues of the host HA polypeptide with modifications designed in the region proximal to the RBS. Exemplary modification strategies are described below.

Unless otherwise indicated, specific positions for modifications (eg, amino acid substitutions, deletions or insertions) in the target HA polypeptide are determined by reference to the sequence of the HA polypeptide A/California/07/2009 (H1N1) presented below (as numbered CA09).

MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHN

GKLCKLRGVAPLHLGKCNIAGWILGNPECESLST AS SWS YIVETPS SDNGTC YPGDF ID YEE

LREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKL SKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRX XEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPK GAINTSLPF QNIHPITIGKCPKYVKSTKLRLATGLRNIPSIQSRGLFGAIAGFIEGGWTGMVD GWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIEN LNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFE FYHKCDNTCMESVKNGTYDY PKYSEEAKLNREEIDGVKLESTRIYQILAIYSTVASSLVLV VSLGAISFWMCSNGSLQCRICI (SEQ ID NO:2)

Grafting the designed area of the head.

In some embodiments, a method of altering the immunogenicity profile of an engineered HA polypeptide is based on grafting structured globular head regions of an HA polypeptide with a known immune profile onto stem regions of HA polypeptides with distinct immune profiles. For example, the method of the present invention may involve selecting a head region of an engineered HA polypeptide with a known immune profile and replacing the selected head region of the engineered HA polypeptide with a corresponding head region of an HA polypeptide with a distinguishable immunogenicity profile. In some embodiments, a selected head region of an engineered HA polypeptide with a predominantly seasonal immune profile can be grafted onto stem regions of an HA polypeptide with a predominantly pandemic immune profile. Conversely, in some embodiments, a selected head region of an engineered HA polypeptide with a predominantly pandemic immune profile can be grafted onto stem regions of an HA polypeptide with a predominantly seasonal immune profile.

In some embodiments, structured globular head regions of an HA polypeptide with a known immune profile are grafted onto a region of a HA polypeptide with a distinguishable immune profile, wherein the region comprises a stem region plus a portion of a head region of an HA polypeptide with a distinguishable immune profile. In other embodiments, an entire HA globular domain with a known immune profile is grafted onto a stem region of an HA molecule with a distinct immunological profile. In most cases, the head region suitable for grafting is selected to ensure that the structural integrity of the resulting full-length hybrid molecule is maintained. Typically, a suitable head region is selected to preserve the receptor binding site (RBS) of the HA polypeptide. The RBS of an HA polypeptide can generally be defined as the epitope recognized by the CH65 antibody (see, for example, Whittle JR et al. Proc Natl Acad Sci USA. 2011; 108:14216-21). Alternatively, the RBS can be defined as the region comprising all amino acid residues within 15 angstroms of the universally conserved tryptophan corresponding to position 167 (as numbered CA09) (for example, see Xu, R et al. Nat Struct Mol Biol. 2013 Mar; 20(3):363-70.) A suitable region of the head containing or consisting of RBS can be selected for grafting onto stem recipients based on the preservation of secondary structure, the compact globular configuration of the detached RBS, and the preservation of border contact areas when integrating the donor RBS into the recipient stem molecule. Non-limiting examples of head regions selected from an engineered HA polypeptide with a predominantly seasonal immune profile and suitable for grafting are described in Example 1. In some embodiments, the HA polypeptide with a predominantly seasonal immune profile has an amino acid sequence that is substantially identical to the sequence shown in SEQ ID NO: 1.

- 20 044592

MKAKLLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNILEDSHNG

KLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGYFADYEEL REQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKS YANNKEKEVLVLWGVHHPPNIGDQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQE GRINYYWTLLEPG DTHFEANGNLIAPWYAFALSRGFGSGIITSNAPMDKCDAKCQTPQGAI NSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRRNIPFIQSRGLFGAIAGFIEGGWTGMVDG WYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKLERRMEN LNKKVDDGFLDIWTYNAELLVLLENERTL DFHDSNVKNLYEKVKSQLKNNAKEIGNGCFE FYHI<CNDECMESVI<NGTYDYPI<YSEESI<LNREI<IDGVI<LESMGVYQILAIYSTVASSLVLL VSLGAISFWMCSNGSLQCRICI (SEQ ID NO:1)

In some embodiments, a suitable head region is selected to contain an amino acid sequence corresponding to residues 63-278, 125-277, or 135-269 of SEQ ID NO:1. As used herein, the term corresponding is used to refer to the position/identity of an amino acid residue in the HA polypeptide of interest. One of ordinary skill in the art will appreciate that, for simplicity, residues in an HA polypeptide are designated using a canonical numbering system based on the reference cognate polypeptide so that the amino acid corresponding to the residue at position 63, for example, need not be the 63rd amino acid in specific amino acid chain, but rather corresponds to the residue located at position 63 in the reference polypeptide; one of ordinary skill in the art will readily understand how to identify the corresponding amino acids using, for example, various sequence alignment tools. In some embodiments, a suitable head region may comprise an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to amino acid residues 63-278, 125-277, or 135 -269 from SEQ ID NO: l.

The selected head region can then be used to replace or replace a corresponding head region of the HA polypeptide with a distinguishable immune profile, i.e. pandemic or predominantly pandemic. Such a suitable HA polypeptide with a distinct immune profile (i.e., pandemic or predominantly pandemic) may be naturally occurring or engineered, including, but not limited to, polypeptides engineered using computationally optimized broad spectrum reactivity antigen (COBRA) technology. mosaics, reverse genetics, protein engineering, combinations of influenza virus strains based on influenza virus consensus sequences, deletion and/or rearrangement of structural domains, domain replacement, or combinations of neutralizing or cross-reactive epitopes shared by multiple influenza virus strains.

For example, the selected head region can be used to replace or replace the corresponding head region of a naturally occurring pandemic strain selected from residues 63-277 of SEQ ID NO: 2 [full-length wild-type CA09 sequence (HA sequence A/Califomia/07/2009)], residues 63-277 from SEQ ID NO: 3 [wild-type SC1918 full-length sequence], residues 63-277 from SEQ ID NO: 4 [wild-type NJ1976 full-length sequence], residues 125-277 from SEQ ID NO: 2 [CA09 full-length sequence wild type], residues 125-277 from SEQ ID NO: 3 [full-length wild-type SC1918 sequence], residues 125-277 from SEQ ID NO: 4 [full-length wild-type NJ1976 sequence], residues 135-269 from SEQ ID NO: 2 [full-length wild-type CA09 sequence], residues 135-269 of SEQ ID NO: 3 [full-length wild-type SC1918 sequence], or residues 135-269 of SEQ ID NO: 4 [full-length wild-type NJ1976 sequence].

In some embodiments, the selected head region can be used to replace or replace the corresponding head region of an engineered HA polypeptide with a distinct immunogenicity profile that is predominantly pandemic. As a non-limiting example, the engineered HA polypeptide with a predominantly pandemic immune profile has an amino acid sequence that is substantially identical to SEQ ID NO: 6.

- 21 044592

MKAKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCK LKGIAPLQLGKCSVAGWILGNPECESLSTASSWSYIVETSSPDNGTCYPGYFADYEELREQL SSVSSFERFEIFPKTSSWPNHDSNGVTASCPHAGAKSFYRNLLWLVKKGNSYPKLSKSYIND KGKEVLVLWGVH HPSTSADQQSLYQNANAYVSVVTSRYSRRFTPEIAIRPKVRDQEGRMN YYWTLVEPGDTIIFEATGNLIAPWYAFALSRGFGSGIITSDTPVHDCNTTCQTPQGAINSSLP FQNVHPVTIGECPKYVRSAKLRMATGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYGY HHQNEQGSGYAADLI<STQNAIDG ITNI<VNSVIEI<MNTQFTAVGI<EFNI<LERRMENLNI<I< VDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHK CNNTCMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLG AISFWMCSNGSLQCRICI.

A suitable head region is selected to contain an amino acid sequence corresponding to residues 63-278, 125-277 or 135-269 of SEQ ID NO: 6.

Modifications to remove or engineer putative N-glycosylation sites.

In some embodiments, a method for altering the immunogenicity profile of an engineered HA polypeptide is based on modifications to residues associated with predicted or putative N-glycosylation sites in the globular head region of the HA polypeptide. Typically, putative or predicted N-glycosylation sites are defined by the consensus sequence NxS/Ty, where x and y are not P. Seasonal HA polypeptides typically contain additional N-glycosylation sites in the receptor binding site (RBS) region relative to pandemic HA polypeptides or HA polypeptides , similar to pandemic ones. In a target seasonal HA polypeptide or a seasonal-like engineered HA polypeptide, specific amino acid residues can be mutated to remove glycosylation sites and produce an engineered HA polypeptide with a more pandemic glycosylation profile. In specific embodiments, the target seasonal HA polypeptide or engineered seasonal-like HA polypeptide may be mutated or specific amino acid residues may be replaced with those observed at corresponding positions in the pandemic HA polypeptides or pandemic-like HA polypeptides (e.g., California/07/2009 ) in order to change the glycosylation profile and immunogenicity profile of the target HA polypeptide so that it is more pandemic.

Thus, in some embodiments, the method of the present invention involves detecting the presence or absence of one or more putative N-glycosylation sites in the head region of an engineered HA polypeptide with a known immune profile compared to a corresponding head region of an HA polypeptide with a distinguishable immunogenicity profile; introducing into the head region of the engineered HA polypeptide one or more amino acid substitutions, deletions or insertions to disrupt one or more putative N-glycosylation sites or introduce additional N-glycosylation sites based on the corresponding sequence of the HA polypeptide with a distinct immunogenicity profile.

In some embodiments, one or more amino acid substitutions, deletions, or insertions are introduced into an engineered HA polypeptide with a predominantly seasonal immune profile to disrupt one or more putative N-glycosylation sites so as to alter the engineered HA polypeptide to be more pandemic. Conversely, in some embodiments, one or more amino acid substitutions, deletions, or insertions are introduced into a redesigned HA polypeptide with a predominantly pandemic immune profile to insert one or more putative N-glycosylation sites so as to alter the redesigned HA polypeptide so that it is to more seasonal.

In some embodiments, putative N-glycosylation sites are removed or added at or near the receptor binding site (RBS) region. In some embodiments, putative N-glycosylation sites can be found within 15 (e.g., within 10, 9, 8, 7, 6, or 5) angstroms of the receptor binding site (RBS), wherein an RBS is defined as all amino acid residues in within 15 (eg, within 10, 9, 8, 7, 6 or 5) angstroms from the position corresponding to W167 (as numbered CA09) in a three-dimensional (3D) structure. In specific embodiments, predicted N-glycosylation sites may correspond to positions 142-145 and/or 177-179 (as numbered CA09).

Thus, a recombinant HA polypeptide that provides a balanced immune profile can be produced by amino acid substitution, disruption or deletion to disrupt or remove the N-glycosylation site in a HA polypeptide with a predominantly seasonal immune profile. Alternatively, a recombinant HA polypeptide that provides a balanced immune profile can be produced by amino acid substitution, disruption, or deletion to introduce an N-glycosylation site into the HA polypeptide with predominantly

- 22 044592 pandemic immune profile. Examples of amino acid substitutions, disruptions or deletions that can be performed to produce a recombinant HA polypeptide that provides a balanced immune profile can be found in Table. 4 or 5. Amino acid substitutions, disruptions or deletions can be obtained from the corresponding regions of the circulating influenza virus strain.

Targeted substitutions or deletions at N-glycosylation sites can be combined with one or more additional modifications. For example, positively charged amino acid residues can be inserted near the RBS to produce a redesigned HA polypeptide with a more pandemic immune profile. In specific embodiments, an engineered HA polypeptide with a seasonal or predominantly seasonal immune profile can be made to have a more pandemic (e.g., more balanced) immune profile by inserting one or more positively charged amino acids at or adjacent to one or more putative N- sites. glycosylation or in conformational loop structures that bound the RBS (eg, loops 220 and 130; see, eg, Bradley, K.C. et al., J. Virol., 2011, 85(23), 12387-12398). In some embodiments, a lysine or arginine residue is inserted into the loop or loops delimiting the RBS (lysine loop insertion). In some embodiments, the loop insertions may include the insertion of a lysine (K) or arginine (R) at positions corresponding to residue 147 (as numbered CA09) of the target engineered HA polypeptide. For example, loop insertions may include the insertion of a lysine (K) or arginine (R) residue within 1-5 (eg, within 1-4, 1-3, 1-2 amino acids) amino acids from the NxS/Ty consensus sequence. In some embodiments, a lysine (K) or arginine (R) residue is within 1-5 amino acids (e.g., within 1-4, 1-3, 1-2 amino acids) 5' or 3' from the NxS consensus sequence /Ty.

Targeted modifications of residues in the RBS region.

In some embodiments, changing the immunogenicity profile of the engineered HA polypeptide can be accomplished by introducing one or more amino acid substitutions at or near the RBS region. For example, one or more amino acid substitutions can be introduced at amino acid positions within the region spanning residues 60 and 291 (CA09 numbering) of the target engineered HA polypeptide. One or more amino acid substitutions may also be introduced within 15 (e.g., within 10, 9, 8, 7, 6, 5, etc.) angstroms of the receptor binding site (RBS), with RBS defined as all amino acids residues within 15 (eg, within 10, 9, 8, 7, 6, 5, etc.) angstroms of the position corresponding to the conserved residue W167 (as numbered CA09) in the three-dimensional (3D) structure. For example, in embodiments where modifications are present within 10 angstroms of the RBS, they are present in the range of 15-25 angstroms from the conservative W167. In some embodiments, the RBS may be defined by an epitope bound by the broadly binding neutralizing monoclonal antibody CH65 (see, e.g., Whittle J.R. et al. Broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin. Proc Natl Acad Sci USA 2011;108:14216-21). In such embodiments, one or more amino acid substitutions are present adjacent to (e.g., within 100 amino acid residues, within 75 amino acid residues, within 50 amino acid residues, within 40 amino acid residues, within 30 amino acid residues, within 25 amino acid residues , within 20 amino acid residues, within 15 amino acid residues, within 10 amino acid residues, within 5 amino acid residues, etc.) with the CH65 epitope or within 15 angstroms of the CH65 epitope. In some embodiments, each amino acid substitution involves replacing an amino acid residue at a particular position with an amino acid residue observed at a corresponding position in an HA polypeptide with a distinct immunogenicity profile (eg, a circulating seasonal or pandemic strain of influenza virus). For example, an engineered HA polypeptide with a predominantly seasonal immune profile can be modified to be more pandemic-specific by substituting amino acids at specific positions based on amino acid residues that are present at corresponding positions of the HA polypeptide with a predominantly pandemic immune profile. Conversely, an engineered HA polypeptide with a predominantly pandemic immune profile can be modified to be more seasonal by substituting amino acids at specific positions based on amino acid residues that are present at corresponding positions of the HA polypeptide with a predominantly seasonal immune profile.

Exemplary amino acid substitutions are given in table. 4, 5, 6, 7, 8 or 9. As non-limiting examples, one or more amino acid substitutions may be present at positions of the target HA polypeptide corresponding to 137, 144, 145, 154, 155, 156, 157, 158, 159, 177 , 210, 211, 212, 213, 214, 244, 245 and/or 262 (according to CA09 numbering). In specific embodiments, one or more amino acid substitutions may be present at positions corresponding to 137, 144, 145, 154, 155, 156, 157, 158, 159, 177, 210, 211, 212, 213 and/or 214 (as designated by CA09 numbering ). In some embodiments, one or more modifications include two or more, three or more,

- 23 044592 four or more, five or more, six or more, seven or more, eight or more, nine or more or ten or more modifications selected from those shown in table. 4, 5, 6, 7, 8, or 9. In some embodiments, one or more modifications may include at least 2, 3,

4, 5, 6, 7, 8, 9 or 10 consecutive substitutions selected from the table. 4, 5, 6, 7, 8 or 9.

A combination of various methods described herein can be used to alter the immunogenicity profile of an engineered HA polypeptide. For example, targeted modifications to residues in the RBS region can be used in combination with modifications at putative N-glycosylation sites and loop insertion(s). Grafting of the head region can also be used in combination with targeted modifications to residues around the RBS and/or modifications at putative N-glycosylation sites and loop insertion(s).

Evaluation of redesigned HA polypeptides.

In some embodiments, modified recombinant HA polypeptides produced in accordance with various methods described herein can be assessed for desired expression and conformation. Screening methods are well known in the art and include cell-free assays, cell-based assays and animal-based assays. In vitro detection assays can be performed against either a soluble target molecule or a solid state target molecule by a variety of methods known in the art, including the use of a label or detectable moiety capable of detecting an engineered HA polypeptide that is associated with the target molecule (e.g. immunoglobulin). Detectable tags can be used in combination with assays using the engineered HA polypeptides of the present invention. For example, the recombinant HA polypeptide described herein can be selected based on expression characteristics and conformational characteristics, which are determined through the assays described in international patent application PCT/US2015/033205 entitled Expression Analysis and Conformational Analysis of Engineered Influenza Virus Hemagglutinin, filed May 29, 2015 G.

The present invention provides methods for testing recombinant HA polypeptides in accordance with the present invention in an animal host. As used herein, an animal host includes any animal model suitable for influenza research. For example, animal hosts suitable for the present invention can be any mammalian host, including primates, ferrets, cats, dogs, cows, horses, rodents such as mice, hamsters, rabbits and rats. In some embodiments, the host animal used for the purposes of the present invention is a ferret. Particularly, in some embodiments, the host animal is naïve to a virus or infection prior to administration of a binding agent in accordance with the present invention (optionally in a composition in accordance with the present invention). In some embodiments, the host animal is inoculated, infected, or otherwise exposed to a virus prior to or concurrently with administration of the recombinant HA polypeptide of the present invention. An animal host used in the practice of the present invention may be inoculated, infected, or otherwise exposed to the virus by any method known in the art. In some embodiments, the host animal can be inoculated, infected, or exposed to the virus intranasally.

The modified recombinant HA polypeptides of the present invention can also be evaluated in screening assays to identify and/or select those that can produce antibodies that are protective (i.e., neutralizing) immune responses against both seasonal and pandemic strains of influenza virus in an animal (e.g. , mouse, ferret or human). In certain embodiments, the provision of a protective immune response can be determined, for example, using a well-known hemagglutination inhibition (HAI) assay as a surrogate measure of influenza virus vaccine efficacy. HAI assays may use chicken, turkey, or horse red blood cells to detect H1N1-specific antibodies. In specific embodiments, protective immune responses are demonstrated by the occurrence of an average HAI titer greater than 1:40, which correlates with the prevention and reduction of influenza virus disease. HAI antibody titers of approximately 1:32 to 1:40 will generally protect against infection in approximately 50% of subjects following immunization with an inactivated human influenza virus vaccine. See Treanor, J. & Wright, P.F. Immune correlates of protection against influenza in the human challenge model. Dev. Biol. (Basel), 2003, 115:97-104; incorporated herein by reference). In some embodiments, the provision of a protective immune response can be detected by serum conversion rates. A protective serum conversion level can be defined as at least a 4-fold increase in the HAI titer, for example, a pre-challenge or vaccination HAI titer of less than 1:10 and a post-vaccination titer of greater than or equal to 1:40. That is, successful serum conversion rates can be defined as the percentage of subjects with either a pre-vaccination HAI titer less than approximately 1:10 and a post-vaccination HAI titer greater than approximately 1:40, or a pre-vaccination HAI titer greater than approximately 1:40. approximately 1:10, and a minimum fourfold increase in HAI antibody titer after vaccination.

Naive and/or inoculated animals can be used for any of a number of studies. For example, such animal models can be used for viral transmission studies, as is known in the art. It is suggested that the use of ferrets in viral transmission studies may serve as a reliable predictor of viral transmission in humans. For example, airborne transmission of influenza virus from inoculated animals (eg, ferrets) to unexposed animals is known in the art (Tumpey et al., 2007, Science 315; 655-59; incorporated herein by reference). Viral transmission studies can be used to test recombinant HA polypeptides in accordance with the present invention. For example, the recombinant HA polypeptides of the present invention can be administered to a suitable animal host to determine the effectiveness of the engineered HA polypeptide in providing a broad immune response in the host animal. Using information gathered from studies in animal hosts, one skilled in the art can predict the effectiveness of a recombinant HA polypeptide in providing broad protection in a human host.

Design and expression of nucleic acids.

The recombinant influenza virus HA polypeptides described herein can be obtained from nucleic acid molecules using molecular biological methods known in the art. The nucleic acid molecules are inserted into a vector that is capable of expressing HA polypeptides when introduced into an appropriate host cell. Suitable host cells include, but are not limited to, bacterial, yeast, insect and mammalian cells. To construct expression vectors encoding the fusion proteins of the present invention, under the control of transcription/translation control signals, any of the methods known to one skilled in the art can be used to insert DNA fragments into the vector. These methods may include in vitro techniques using recombinant DNA and synthesis techniques, as well as in vivo recombination (see Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory; Current Protocols in Molecular Biology, Eds. Ausubel et al ., Greene Publ. Assoc, Wiley-Interscience, New York).

In some embodiments, the present invention provides nucleic acids that encode an HA polypeptide or a characteristic or biologically active portion of an HA polypeptide. In some embodiments, the present invention provides nucleic acids that are complementary to nucleic acids that encode an HA polypeptide or a characteristic or biologically active portion of an HA polypeptide.

In some embodiments, the present invention provides nucleic acid molecules that hybridize to nucleic acids encoding an HA polypeptide or a characteristic or biologically active portion of an HA polypeptide. Such nucleic acids can be used, for example, as primers or as probes. As just a few examples, such nucleic acids can be used as primers in polymerase chain reaction (PCR), as probes for hybridization (including in situ hybridization), and/or as primers for reverse transcription-PCR (RT-PCR). ).

In some embodiments, the nucleic acids may be DNA or RNA and may be single-stranded or double-stranded. In some embodiments, the nucleic acids of the present invention may include one or more non-natural nucleotides; in some embodiments, the nucleic acids of the present invention include only naturally occurring nucleotides.

The expression of the nucleic acid molecules of the present invention can be controlled by the second nucleic acid sequence such that the molecule is expressed in a host transformed with the recombinant DNA molecule. For example, the expression of the nucleic acid molecules of the present invention can be controlled by promoter and/or enhancer elements known in the art.

The nucleic acid constructs of the present invention are inserted into an expression vector or viral vector by methods known in the art, and the nucleic acid molecules are operably linked to an expression control sequence.

An expression vector containing a nucleic acid molecule is transformed into a suitable host cell to allow production of the protein encoded by the nucleic acid constructs. Exemplary host cells include prokaryotes (eg, E. coli) and eukaryotes (eg, COS, 293, or CHO cell). Host cells transformed with the expression vector are grown under conditions to produce the engineered HA polypeptide of the present invention, followed by recovery of the engineered HA polypeptide.

The recombinant HA polypeptides of the present invention can be purified using any technique known in the art. For example, without being bound by any theory, engineered HA polypeptides can be recovered from cells either as soluble polypeptides or as inclusion bodies, from which they can be quantitatively extracted using 8 M gua-nidinium hydrochloride and dialysis. To further purify the recombinant HA polypeptides of the present invention, conventional ion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography, or gel filtration can be used. The recombinant HA polypeptides of the present invention can also be recovered from conditioned media after secretion from eukaryotic or prokaryotic cells.

Virus-like particles (VLPs) based on the influenza virus.

In some embodiments, the present invention provides influenza virus-based virus-like particles (VLPs) comprising a modified recombinant HA polypeptide described herein. In some embodiments, influenza virus VLPs are generally composed of HA, NA, and viral structural (eg, HIV gag) proteins. The preparation of VLPs from influenza virus is known in the art and will be readily apparent to one skilled in the art upon reading the present disclosure. For example, influenza virus-based VLPs can be produced by transfecting host cells with plasmids encoding the HIV HA, NA, and gag proteins. As one example, a suitable host cell includes a human cell (eg, HEK293T). After incubating the transfected cells for an appropriate time to allow protein expression (such as about 72 hours), VLPs can be isolated from cell culture supernatants. In some embodiments, the influenza virus VLPs described herein can be used as influenza virus vaccines to provide a broadly neutralizing immune response to H1N1 influenza viruses.

Pharmaceutical compositions.

In some embodiments, the present invention provides pharmaceutical compositions comprising a modified recombinant HA polypeptide and/or related molecules as described herein. For example, in some embodiments, the pharmaceutical compositions of the present invention include modified recombinant HA polypeptides, nucleic acids encoding such polypeptides, characteristic or biologically active fragments of such polypeptides or nucleic acids, antibodies that bind and/or compete with such polypeptides, or fragments, small molecules that interact or compete with such polypeptides or glycans that bind to them, etc.

In some embodiments, the present invention provides methods for preventing or treating influenza infections by administering such pharmaceutical compositions in accordance with the present invention. In some embodiments, the pharmaceutical compositions of the present invention are administered to a subject suffering from or susceptible to influenza infection. In some embodiments, the subject is an animal, including, without limitation, birds (eg, chickens, ducks, turkeys, etc.), dogs, horses, and pigs. In some embodiments, a subject is considered to be suffering from an influenza infection if the subject exhibits one or more symptoms typically associated with an influenza infection. In some embodiments, the subject is known or suspected to have been exposed to an influenza virus. In some embodiments, a subject is considered susceptible to influenza infection if the subject is known or suspected to have been exposed to an influenza virus. In some embodiments, the subject is known or suspected to have been exposed to an influenza virus if the subject has been in contact with other individuals who are known or suspected to be infected with an influenza virus, and/or if the subject is or has been present in a location where infection is known or suspected to be widespread flu

In some embodiments, subjects suffering from or susceptible to influenza infection are tested for antibodies to the modified recombinant HA polypeptides of the present invention before, during, or after administration of the pharmaceutical compositions of the present invention. In some embodiments, subjects having such antibodies are not administered pharmaceutical compositions containing modified recombinant HA polypeptides in accordance with the present invention. In some embodiments, the appropriate dosage of the pharmaceutical composition and/or modified recombinant HA polypeptide is selected based on the detection of such antibodies (or lack thereof).

In some embodiments, the selection of a particular subject to be treated, a particular modified recombinant HA polypeptide or composition to be administered, and/or a particular dosage or regimen of administration is documented, for example, in written, printed, or electronic storage form.

Compositions containing the described modified recombinant HA polypeptide can be administered before or after the development of one or more symptoms of influenza infection. In some embodiments, influenza virus-based VLPs containing a modified recombinant HA polypeptide described herein (or a modified recombinant HA polypeptide itself) can be administered before or after the development of one or more symptoms of an influenza infection.

In some embodiments, the present invention provides for the treatment of influenza infections by administering modified recombinant HA polypeptides described herein. In some embodiments, treatment of influenza infections in accordance with the present invention is carried out by administering an influenza virus-based VLP containing a modified recombinant HA polypeptide described herein. In some embodiments, treatment of influenza infections in accordance with the present invention is carried out by administering a vaccine. To date, although significant advances have been made in the development of influenza virus vaccines, there is room for further improvement. The present invention provides vaccines containing modified recombinant HA polypeptides in accordance with the present invention and, in particular, containing engineered HA polypeptides that provide broad-spectrum protective immune responses to multiple neutralizing antigenic determinants (eg, epitope) of the modified recombinant HA polypeptides.

In some embodiments, the present invention provides an influenza virus VLP, influenza virus vaccine, fusion protein, and/or modified recombinant HA polypeptide as described herein for the prevention of influenza.

In some embodiments, the present invention provides immunogenic compositions (eg, vaccines) and the administration of these immunogenic compositions to a human subject. In specific embodiments, the human subject's age is 6 months or older, 6 months to 35 months, 36 months to 8 years, or 9 years or older. In some embodiments, the immunogenic compositions are pharmaceutical compositions comprising one or more of the following: (1) an inactivated virus, (2) a live attenuated influenza virus, such as a replication-deficient virus, (3) virus-like particles (VLPs), (4) a modified recombinant HA polypeptide, (5) a nucleic acid encoding a modified recombinant HA polypeptide or a characteristic or biologically active portion thereof, (6) a DNA vector that encodes a modified recombinant HA polypeptide according to the present invention or a characteristic or biologically active portion thereof, and/or (7) an expression system, for example cells expressing one or more influenza proteins for use as antigens.

Whole influenza viruses containing the engineered and redesigned HA polypeptides described herein can be produced through plasmid-based reverse genetics (see, e.g., Neumann, G. et al., Reverse Genetics of Influenza Viruses, Methods Mol Biol., 2012, 865:193-206; incorporated herein by reference) and germ-based technologies; for example, a recombinant virus containing the computationally optimized H1 HA polypeptide described herein, a wild-type NA polypeptide from an H1N1 influenza virus strain, and an internal protein gene backbone from a donor virus (e.g., influenza A/Puerto Rico/8/34 (PR8) ), which provides a high yield in embryos. For example, six plasmids encoding the internal proteins of the rapidly growing influenza A/Puerto Rico/8/34 (PR8) donor virus can be cotransfected with two plasmids encoding the computationally optimized H1N1 HA polypeptide described herein and the wild neuraminidase (NA) glycoprotein. type, into suitable mammalian cells (eg Vero cells) followed by isolation of the recombinant virus. One skilled in the art will appreciate that reverse genetics systems with 12 plasmids can also be used (see, for example, Pekosz, A. et al. Reverse genetics of negative-strand RNA viruses: Closing the circle. Proc. Natl. Acad. Sci., 1999, 96, 884-8806). Recombinant viruses containing internal protein genes from the PR8 virus can be used to produce inactivated influenza virus vaccines (see, for example, Fodor, E. et al. Rescue of influenza A virus from Recombinant DNA. J. Virol., 1999, 73 , 9679-9682; incorporated herein by reference). Whole influenza viruses can be administered as components of a live attenuated or digestion-inactivated vaccine.

Thus, in some embodiments, the present invention provides inactivated influenza virus vaccines. In some embodiments, inactivated influenza virus vaccines contain one of three types of antigenic preparation: inactivated whole virus, subvirions in which purified virus particles are destroyed using detergents or other reagents to solubilize the lipid envelope (cleaved vaccine), or purified HA polypeptide (subunit vaccine). In some embodiments, the virus can be inactivated by treatment with formaldehyde, beta-propiolactone, ether, detergent ester (such as TWEEN-80®), cetyltrimethylammonium bromide (CTAB) and Triton N101, sodium deoxycholate and tri(n-butyl)phosphate . Inactivation can be carried out after or before purification of allantoic fluid (from the virus produced in the embryos); virions are isolated and purified by centrifugation (Nicholson et al., eds., 1998, Textbook of Influenza, Blackwell Science, Malden, MA; incorporated herein by reference). The one-dimensional radial immunodiffusion (SRD) test can be used to evaluate the specific activity of a vaccine (Schild et al., 1975, Bull. World Health Organ., 52:43-50 &223-31; Mostow et al., 1975, J. Clin Microbiol., 2:531, both of which are incorporated herein by reference).

In some embodiments, the engineered or redesigned HA polypeptides of the present invention are used as a component of vaccines against seasonal and/or

- 27 044592 pandemic influenza or as part of an influenza virus vaccination regimen designed to confer long-term (multi-seasonal) protection.

In some embodiments, the influenza virus for use in vaccines is grown in embryos, for example, in embryonated chicken eggs, in which case the collected material is allantoic fluid. Alternatively or additionally, influenza virus or engineered/reengineered hemagglutinin polypeptides can be produced by any tissue culture method for growing the virus. Suitable cellular substrates for growing virus or otherwise recombinantly producing engineered or re-engineered hemagglutinin polypeptides include, for example, canine kidney cells such as MDCK or MDCK clone cells, MDCK-like cells, monkey kidney cells such as AGMK cells, including Vero cells , cultured epithelial cells as continuous cell lines, 293T cells, BK-21 cells, CV-1 cells, or any other mammalian cell types suitable for the production of influenza virus (including upper respiratory tract epithelial cells) to produce vaccines that are readily available from commercial sources (eg, ATCC, Rockville, Md.). Suitable cell substrates also include human cells, such as MRC-5 cells. Suitable cell substrates are not limited to cell lines; for example, germ cells such as chick embryo fibroblasts are also included.

Designed or redesigned hemagglutinin polypeptides can also be expressed/produced in various eukaryotic cell-based expression systems, including microalgae cells (e.g., Schizochytrium sp.; see, e.g., Bayne, A-C.V. et al., PLOS ONE, 8(4) ):e61790, April 2013), plant cell based systems (e.g. tobacco plants; see, e.g., Jul-Larsen, A. et al., Hum Vaccin Immunother., 8(5):653-61, 2012) , yeasts (see, for example, Athmaram, T.N. et al., Virol J., 8:524, 2011) and fungi (see, for example, Allgaier, S. et al., Biologicals, 37:128-32, 2009 ). Bacterial cell-based expression systems are also covered by the present invention (see, for example, Davis, A.R. et al., Gene, 21:273-284, 1983).

In some embodiments, the vaccines of the present invention further contain one or more adjuvants. Examples of adjuvants in human vaccines include aluminum salts (Baylor et al., 2002, Vaccine, 20:S18; incorporated herein by reference) and monophosphoryl lipid A (MPL; Ribi et al., 1986, Immunology and Immunopharmacology of Bacterial Endotoxins, Plenum Publ. Corp., New York, p. 407; incorporated herein by reference). Alternatively or additionally, new compounds such as MF59 (Chiron Corp., http://www.chiron.com/investors/pressreleases/2005/051028.html) are currently being tested as adjuvants in human vaccines. CPG 7909 (Cooper et al., 2004, Vaccine, 22:3136; incorporated herein by reference) and saponins such as QS21 (Ghochikyan et al., 2006, Vaccine, 24:2275; incorporated herein by reference) .

In addition, certain adjuvants known in the art, such as poly[di(carboxylatophenoxy)phosphazene] (PCCP; Payne et al., 1998, Vaccine, 16:92; incorporated herein by reference), interferon-γ (Cao et al., 1992, Vaccine, 10:238; incorporated herein by reference), P1205 block copolymer (CRL1005; Katz et al., 2000, Vaccine, 18:2177; incorporated herein by reference), interleukin -2 (IL-2; Mbwuike et al., 1990, Vaccine, 8:347; incorporated herein by reference) and polymethyl methacrylate (PMMA; Kreuter et al., 1981, J. Pharm. Sci., 70:367; incorporated herein by reference) enhance the immunogenicity of influenza virus vaccines.

In addition to immunogenic compositions (eg, vaccines containing VLPs with engineered or re-engineered influenza hemagglutin polypeptides described herein), the present invention provides other therapeutic compositions useful in the treatment of viral infections. Therapeutic compositions include, for example, influenza virus-based VLPs, fusion proteins, and the engineered or redesigned HA polypeptide itself as described herein. In some embodiments, treatment is carried out by administering an agent that interferes with the expression or activity of the HA polypeptide.

In some embodiments, immunogenic compositions described herein (eg, influenza virus-based VLPs or engineered/reengineered HA polypeptides themselves) are administered alone or in combination with one or more therapeutic agents to enhance the immune response. For example, in some embodiments, the influenza virus-based VLPs described herein can be administered with an adjuvant, such as Freund's incomplete adjuvant or Freund's complete adjuvant. In some embodiments, one or more cytokines, such as IL-2, IL-6, IL-12, RANTES, GM-CSF, TNF-α or IFN-γ, one or more growth factors such as as GM-CSF or G-CSF; one or more molecules, such as OX-40L or 41 BBL, or combinations of these molecules (eg, Salgaller et al., 1998, J. Surg. Oncol. 68(2):122-38; Lotze et al., 2000, Cancer J. Sci. Am. 6(Suppl 1):S61-6; Cao et al., 1998, Stem Cells 16(Suppl 1):251-60; Kuiper et al., 2000, Adv. Exp. Med. Biol 465:381-90).

- 28 044592

In some embodiments, the present invention provides pharmaceutical compositions containing antibodies or other agents related to the provided HA polypeptides. For example, the present invention provides compositions containing antibodies that recognize viral particles containing a particular engineered or redesigned HA polypeptide, nucleic acids (such as nucleic acid sequences complementary to HA sequences that can be used for RNAi), glycans that compete for binding to HA receptors, small molecules or glycomimetics that compete for the interaction of the glycan and the HA polypeptide, or any combination thereof. In some embodiments, combinations of different means having different structures are used. In some embodiments, the therapeutic compositions contain one or more multivalent agents. In some embodiments, the treatment involves immediate administration soon after exposure or suspected exposure.

In some embodiments, any of the immunogenic compositions (eg, vaccines) described herein provide broad cross-protection against various forms of influenza viruses. For example, in some embodiments, the immunogenic compositions described herein provide cross-protection against avian, porcine, and/or human influenza A viruses. In some embodiments, any of the immunogenic compositions described herein provide cross-protection against one or more subtypes of influenza A. In some embodiments, the immunogenic compositions described herein provide cross-protection against multiple strains of influenza A subtype H1 viruses (see, e.g., Fig. 4 and 5).

In most cases, the immunogenic and/or pharmaceutical composition will include a therapeutic agent in addition to one or more inactive agents, such as a sterile biocompatible carrier, including, without limitation, sterile water, saline, buffered saline, or dextrose solution. Alternatively or additionally, the composition may contain any of a variety of additives, such as stabilizers, buffers, excipients (eg, sugars, amino acids, etc.) or preservatives.

In some embodiments, the pharmaceutical compositions described herein include a therapeutically effective amount of an influenza virus-based VLP (containing an engineered or redesigned HA polypeptide described herein) alone or in combination with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. In some embodiments, the carrier and composition are sterile and the composition is suitable for the route of administration. In some embodiments, the pharmaceutical composition contains minor amounts of wetting or emulsifying agents or pH buffering agents. In some embodiments, the pharmaceutical composition is a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. In some embodiments, the pharmaceutical composition is formulated for intradermal injection, intranasal administration, or intramuscular injection. Any of the generally known pharmaceutical carriers, such as sterile saline or sesame oil, may be used. In some embodiments, the medium may also contain conventional pharmaceutical excipients, such as, for example, pharmaceutically acceptable osmotic adjustment salts, buffers, preservatives, and the like. In some embodiments, other media that can be used with the compositions and methods presented herein are saline and sesame oil.

In some embodiments, the therapeutic agent present in the pharmaceutical composition in accordance with the present invention will consist of one or more engineered or redesigned HA polypeptides described herein.

In some embodiments, the pharmaceutical composition will include a therapeutic agent that is encapsulated, entrapped or embedded in a lipid vesicle, bioavailable and/or biocompatible and/or biodegradable matrix or other microparticle. In some embodiments, the immunogenic or pharmaceutical composition comprises nanoparticles having on their surface engineered or re-engineered hemagglutinin polypeptides described herein. In some embodiments, the nanoparticles are ferritin nanoparticles (see, for example, US Provisional Patent Application Publication 2014/0072958).

The pharmaceutical compositions of the present invention can be administered either alone or in combination with one or more other therapeutic agents, including, without limitation, vaccines and/or antibodies. By in combination with, it is not meant that the agents must be administered simultaneously or in a co-delivery formulation, although these delivery methods are within the scope of the present invention. In most cases, each agent will be administered at the dose and schedule established for that agent. In addition, the present invention covers

- 29 044592 supply of pharmaceutical compositions in accordance with the present invention in combination with agents that can improve their bioavailability, slow down or modify their metabolism, inhibit their excretion or modify their distribution in the body. Although the pharmaceutical compositions of the present invention can be used for treatment (eg, vaccination) of any subject in need thereof (eg, any animal), they are most preferably used in the treatment of humans. In some embodiments, the pharmaceutical compositions of the present invention and/or the engineered or redesigned HA polypeptides described herein are administered in combination with one or more antiviral agents (e.g., oseltamivir [TAMIFLU®], zanamavir [RELEZA®], etc. .) and/or sialidase.

The pharmaceutical compositions of the present invention can be administered by various routes, including oral, intravenous, intramuscular, intraarterial, subcutaneous, intraventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal, topical (as through powders, ointments, creams or drops), mucosal, nasal, transbuccal, enteral, sublingual; by intratracheal instillation, bronchial instillation and/or inhalation; and/or in the form of an oral spray, nasal spray and/or aerosol. In most cases, the most appropriate route of administration will depend on a number of factors, including the nature of the agent (eg, its stability in the gastrointestinal tract environment), the condition of the patient (eg, whether the patient is able to tolerate oral administration), etc.

In some embodiments, parenteral administration, such as subcutaneous, intravenous, or intramuscular administration, is accomplished by injection. In some embodiments, injectables are provided in conventional forms, either as liquid solutions or suspensions, solid forms suitable for dissolution or suspension in liquid prior to injection, or as emulsions. In some embodiments, injectable solutions and suspensions are prepared from sterile powders, granules, etc. In some embodiments, administration of an influenza virus VLP described herein is systemic or topical.

In some embodiments, influenza virus VLPs or compositions thereof are administered by any suitable method, such as through pharmaceutically acceptable carriers. One skilled in the art will recognize that pharmaceutically acceptable carriers are determined in part by the particular composition administered as well as the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical compositions described herein.

In some embodiments, preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Exemplary non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Exemplary aqueous vehicles include water, alcohol/aqueous solutions, emulsions or suspensions, including saline and buffer media. In some embodiments, carrier media for parenteral administration include sodium chloride solution, dextrose Ringer's, dextrose and sodium chloride, lactated Ringer's, or fixed oils. In some embodiments, intravenous carrier media include fluid and nutrient supplements, electrolyte supplements (such as Ringer's dextrose), and the like. In some embodiments, preservatives and/or other additives may also be present. Exemplary preservatives and/or other additives include antimicrobial agents, antioxidants, chelating agents and inert gases, and the like.

In some embodiments, the compositions (comprising the HA polypeptide described herein as an influenza virus-based VLP or otherwise) are administered as a pharmaceutically acceptable acid or base addition salt formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl- and arylamines and substituted ethanolamines.

Currently, oral or nasal spray or aerosol routes (eg, inhalation) are most often used to deliver therapeutic agents directly to the lungs and respiratory system. However, the present invention covers the delivery of a pharmaceutical composition in accordance with the present invention by any suitable route, taking into account likely advances in the field of drug delivery.

In some embodiments, formulations for inhalation or aerosol delivery contain multiple particles. In some embodiments, such formulations have an average particle size of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, approx.

- 30 044592 specifically 9, approximately 10, approximately 11, approximately 12 or approximately 13 microns. In some embodiments, the formulations for inhalation or aerosol delivery are formulated as a dry powder. In some embodiments, formulations for inhalation or aerosol delivery are formulated as a wet powder, for example, by including a wetting agent. In some embodiments, the wetting agent is selected from the group consisting of water, saline, or other liquid having a physiological pH value.

In some embodiments, the compositions of the present invention are administered as drops into the nasal or oral cavity. In some embodiments, the dose may comprise multiple drops (eg, 1-100, 1-50, 1-20, 1-10, 1-5, etc.).

In some embodiments, the compositions of the present invention are administered using a device that delivers a metered dose of the composition (eg, an engineered or redesigned HA polypeptide).

Suitable devices for use in the delivery of pharmaceutical compositions described herein for intradermal administration include short needle devices such as those described in US Patent No. 4886499, US Patent No. 5190521, US Patent No. 5328483, US Patent No. 5527288, US Patent No. 4270537, US Patent No. 5,015,235, US Patent No. 5,141,496, US Patent No. 5,417,662 (all of which are incorporated herein by reference). Compositions for intradermal administration can also be administered using devices that limit the effective length of penetration of the needle into the skin, such as those described in WO 1999/34850, incorporated herein by reference, and their functional equivalents. Also suitable are needleless injection devices that deliver liquid vaccines into the dermis through a liquid jet injector or through a needle that pierces the stratum corneum and creates a jet that reaches the dermis. Needleless injection devices are described, for example, in US Patent No. 5480381, US Patent No. 5599302, US Patent No. 5334144, US Patent No. 5993412, US Patent No. 5649912, US Patent No. 5569189, US Patent No. 5704911, US Patent No. 5383851, patent US Patent No. 5893397, US Patent No. 5466220, US Patent No. 5339163, US Patent No. 5312335, US Patent No. 5503627, US Patent No. 5064413, US Patent No. 5520639, US Patent No. 4596556, US Patent No. 4790824, US Patent No. 49 41880, patent US No. 4940460, WO 1997/37705 and WO 1997/13537 (all of which are incorporated herein by reference). Ballistic powder/particle delivery devices are also suitable, which use compressed gas to accelerate the delivery of vaccine in powder form through the outer layers of the skin into the dermis. In addition, in the classic method of intradermal administration according to Mantoux, traditional syringes can be used.

General considerations for the formulation and manufacture of pharmaceuticals can be found, for example, in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Co., Easton, PA, 1995; incorporated herein by reference.

Pharmaceutical compositions in accordance with the present invention can be administered in any dose suitable to achieve the desired result. In some embodiments, the desired result is the induction of a durable adaptive immune response to multiple strains of influenza virus. In some embodiments, the desired result is a reduction in the intensity, severity, and/or frequency, and/or delay in the onset of one or more symptoms of influenza infection.

In some embodiments, the pharmaceutical compositions of the present invention are administered in single or multiple doses. In some embodiments, the pharmaceutical compositions of the present invention are administered in multiple doses administered on different days (eg, prime-boost vaccination strategies). In some embodiments, the pharmaceutical compositions of the present invention are administered according to a continuous dosing regimen such that the subject does not have periods that are less than the therapeutic dosing between therapeutic dosing periods. In some embodiments, the pharmaceutical compositions of the present invention are administered according to an intermittent dosing schedule such that between two therapeutic dosing periods the subject has at least one period that is less than the therapeutic dosing.

In some embodiments, the dosage administered to the subject must be sufficient to induce a beneficial therapeutic response in the subject over time, or to suppress or prevent H1N1 influenza virus infection. The required dosage will vary between subjects depending on the species, age, weight and general condition of the subject, the severity of the infection being treated, the specific composition used and the route of administration.

A more complete understanding of the present invention will be achieved by reference to the following examples. All references cited are incorporated by reference.

Examples

Example 1: Grafting receptor binding sites improves the seasonal immune profile (binding strength) of the engineered HA polypeptide.

This example describes the structure and testing of engineered HA polypeptides, which

- 31 044592 These are characterized by increased breadth with respect to the immunological profile, by grafting the globular head region of the influenza virus HA protein, including the RBS, onto the stem regions of the recipient HA. Structured regions of the globular head of the HA polypeptide, characterized by a seasonal immune profile, were grafted onto the stem regions of HA molecules from pandemic-like strains (New Jersey/1976, South Carolina/1918, California/07/2009 and a new engineered pandemic HA). For grafting, three different regions of the HA globular head were tested (defined as RBS 00, RBS 01 and RBS 02, Fig. 1).

For the purposes of this example, the three RBS-containing grafting regions were identified as G63-G277 (CA09 numbering), V125-G277 (CA09 numbering) and P135-P269 (CA09 numbering). The regions of RBS selected for grafting were selected based on the criteria that they would cause minimal disruption of the entire protein fold upon detachment of the RBS from the rest of the HA molecule. More specifically, the RBS regions were chosen such that (i) the start and end positions were located in the loop regions delimiting the RBS, which would help preserve local secondary structure, (ii) the compact globular structure of the resulting detached RBS was preserved, and (iii) the boundary contact sites during integration of the donor RBS into the recipient molecule.

Twelve separate combinations of RBS donor regions from an engineered HA with a predominantly seasonal immune profile combined with a recipient stem region from a pandemic strain were synthesized (Table 3) and tested in vitro for cell surface expression and proper antigenic conformation using a flow cytometry-based assay , as described in International Application No. PCT/US2015/033205, which is incorporated herein by reference, and depicted in FIG. 2. This assay provides a reliable and rapid screening assay to identify structures that produce functional influenza virus hemagglutinin (HA) antigens for universal vaccines. It involves the use of a panel of neutralizing antibodies to analyze the expression and conformation of engineered HA antigens exposed on the surface. It not only allows the identification and verification of engineered HA antigens that are properly expressed and structurally correct, but also allows prediction of the breadth and/or specificity of the immunogenicity of engineered HA antigens. A panel of antibodies can use antibodies that are known to bind to conformational epitopes (eg, epitopes close to the receptor binding site) of the HA head and conserved conformational epitopes (eg, helix A) of the HA stem region. As non-limiting examples, suitable neutralizing antibodies to the head may include: CH65 (current seasonal strains prior to the 2009 pandemic strain) (Whittle, J.R.R. et al. PNAS 2011), 5J8 (current seasonal and historical strains) (Krause, J.C. et al. J. Virology 2011), 4K8 (pandemic strains only) (Krause, J.C. et al., J. Immunology 2011), AH4 and AH5. Suitable stem region neutralizing antibodies may include: C179 (Group 1 HA) (Okuno, Y. et al., J. Virology 1993), AS2 (Group 1 HA), AS3 (Group 1 and Group 2 HA) and AS4 (Group 1 and 2 HA). group 1).

Table 3

SEQ ID NO: ID structures RBS Donor (designed AN with a predominantly seasonal immune profile) Start RBS End of RBS RBS recipient (pandemic strain) SEQ ID NO: 7 DO2a_trl SMARt_DO2a G63 G277 SMARtDOla * SEQ ID NO: 8 DO2a_tr2 SMARt_DO2a G63 G277 Cal2009 SEQ ID NO: 9 DO2a_tr3 SMARt_DO2a G63 G277 SC1918 SEQ ID NO: 10 DO2a_tr4 SMARt_DO2a G63 G277 NJ1976 SEQ ID NO: 11 DO2a_tr5 SMARt_DO2a V125 G277 SMARTDOla * SEQ ID NO: 12 DO2a_tr6 SMARt_DO2a V125 G277 Cal2009 SEQ ID NO: 13 DO2a_tr7 SMARt_DO2a V125 G277 SC1918 SEQ ID NO: 14 DO2a_tr8 SMARt_DO2a V125 G277 NJ1976 SEQ ID NO: 15 DO2a_tr9* SMARt_DO2a P135 P269 SMARTDOla *

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SEQ ID NO: 16 D02a_trlO SMARt_DO2a P135 P269 Cal2009 SEQ ID NO: 17 DO2a_trll SMARt_DO2a P135 P269 SC1918 SEQ ID NO: 18 DO2a_trl2 SMARt_DO2a P135 P269 NJ1976

* Engineered HA with a predominantly pandemic immune profile.

The assay consisted of transfection of HEK293FT plasmid DNA using Lipofectamine. At 24 h post-transfection, cells were labeled with the LIVE/DEAD® Far-Red Fixed Dead Cell Stain Kit to determine cell viability before surface staining. Cells resuspended in staining buffer (0.1% BSA in PBS) were then stained with 0.4 micrograms of the indicated unlabeled neutralizing hemagglutinin monoclonal antibody (eg, CH65, 5J8, 4K8, AS3, C179, AS2, or AS4).

Stained cells were washed and resuspended in 100 μl of staining buffer containing 0.2 μg of secondary anti-human or mouse IgG antibody with Alexa Fluor® 488 (depending on the primary antibody) and stained with the secondary antibody for 20 min at 4°C WITH. Finally, the stained cells were resuspended in fixation solution (1.75% formaldehyde in PBS) and stored for <1 week at 4°C.

Flow cytometry-based analysis.

Fixed cells were washed and resuspended in 200 μl PBS and then transferred to a 96-well deep well plate for sample collection using a BD HighThroughput sampler. Sample analysis was performed using a BD FACS Calibur flow cytometer equipped with a 488 nm laser (to excite Alexa Fluor® 488) and a 635 nm laser (to excite LIVE/DEAD far-red dye). A sample of mock-transfected cells stained with secondary antibody with Alexa Fluor® 488 but without primary antibody was used to determine optimal collection parameters. Specifically, forward scatter gain (FSC), side scatter voltage (SSC), and FSC threshold were adjusted to display the HEK293FT cell population on the scale and to eliminate unwanted debris. To further exclude debris, the cell population was gated on a plot of FSC versus SSC. Fluorescence detector settings were also adjusted using mock-transfected cells stained with the secondary antibody only. Specifically, the voltage of detector FL1 (to detect Alexa Fluor® 488 fluorescence) and detector FL4 (to detect LIVE/DEAD far-red dye fluorescence) was adjusted to position the fluorescence emission of the gated cell population at the first-order logarithm level. No compensation adjustments are required for this fluorophore combination because there is no spectral overlap between Alexa Fluor® 488 and the LIVE/DEAD far-red dye. All samples were obtained using the same acquisition parameters as the mock-transfected cell control. For each sample, at least 10,000 cells were counted in the FSC gate relative to the SSC and the data were saved as FCS data files.

Data analysis was performed using FlowJo software. The FCS data file corresponding to mock-transfected cells stained with the secondary antibody alone was used to generate assay gates. Specifically, the gate comprising the intact cell population was first plotted on a plot of FSC versus SSC. This subpopulation of gated cells was then analyzed in a separate graph, which plots the fluorescence intensity of FL4 (fluorescence of the LIVE/DEAD dye in the far red spectrum) relative to FSC. A new gate was obtained covering a population of cells with low FL4 fluorescence intensity. This new cell subpopulation, corresponding to intact living cells, was further analyzed in a separate graph plotting the fluorescence intensity of FL1 (Alexa Fluor® 488 fluorescence) relative to FSC. A new gate was created covering cells with positive FL1 fluorescence, determined by fluorescence values that provided a yield of 95% of mock-transfected cells in the FL1-negative fraction. All FCS files were analyzed using the same analysis gates. The median fluorescence intensity (MFI) values of the FL1 positive cell subset for each cell sample and staining results were exported to an Excel file and used to calculate the antibody binding coefficient.

The MFI value of the FL1 positive cell subset for each cell sample and the staining result were first corrected by subtracting the background fluorescence corresponding to the same cell sample stained with the secondary antibody alone. The specificity of staining with each hemagglutinin neutralizing monoclonal antibody was confirmed by examining the background-corrected MFI of mock-transfected cells (negative control) and the background-corrected MFI of cells transfected with the plasmid.

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Wild-type HA DNA (positive control). If the MFI value for the controls fell within the expected range of values, then the antibody binding coefficient for each plasmid encoding the engineered HA and the neutralizing anti-HA monoclonal antibody was determined as follows:

Coefficient MFI (HA x, primary AB y) - MFI (HA x, only secondary AB) binding = ---------------------------- -------------------------------------------------- ----------MFI (wild-type HA, primary AB) - MFI (wild-type HA, secondary AB only)

Each of the recombinant HA polypeptides was surface expressed (i.e., it is capable of intercellular processing similar to wild-type influenza antigens produced in an infected cell) and retained the folding of the stem region (comparable to or better than control wild-type strains), which was determined by binding of a panel of antibodies to the stem region in a flow cytometry-based assay (Fig. 3). This experiment also demonstrates that in some cases modifications to the head region induced a modest increase in mAb binding to the stem region. Thus, substitutions at one location can have long-range allosteric effects at a distant location. Likewise, novel recombinant HA polypeptides produced by grafting stem region RBS regions of seasonal strains onto stem regions of pandemic strains surprisingly exhibited improved seasonal immune profiles. These redesigned recombinant HA polypeptides demonstrated increased binding of seasonal strain-neutralizing antibodies relative to the original engineered parent HA molecule (SMARt_DO2a). (Fig. 4). In these assays, increased mAb binding is a measure of the average fluorescence intensity of the antibody that binds to the redesigned HA relative to the control: unmodified parental engineered HA (SMARt_DO2a) for CH65 and 5J8 and the wild-type pandemic strain A/California/7/2009 H1N1 for 4K8. Increased mAb binding is therefore a rough measure of antibody affinity. In some cases, the seasonal immune profile (measured by mAb binding) was improved 2- to 3-fold compared to the parent seasonal engineered HA molecule (Figure 5; comparison of seasonal head antibody binding results (e.g., CH65 and 5J8) from column 2 with the redesigned constructions in columns 5 and 6).

Since the RBS portion of the grafted antigens is identical to the RBS portion of the engineered HA with a predominantly seasonal immune profile, the increase in breadth relative to the recipient pandemic strain is due to a different RBS portion of the reengineered HA. The increase in breadth is better seen in in vivo experiments than in HAI assays or binding assays with antibodies that target the RBS.

Example 2. Destruction of N-glycosylation sites and/or insertion into a loop increases the spectrum of immunological activity of the constructed HA polypeptide.

This example describes a second strategy to increase the spectrum of immunological activity of engineered HA polypeptides by modifying residues associated with predicted N-glycosylation sites and introducing a lysine into the RBS bounding loop. Seasonal influenza strains contain additional putative N-glycosylation sites compared to pandemic strains. Glycosylation has the ability to block antigenic sites in HA, altering the immune response. Such glycosylation sites were identified by the sequence motif NxS/Ty, where x and y do not represent proline (P). Asparagine in this N-glycosylation pattern can be found in HA polypeptides near or directly at residues corresponding to 142 and 177 (as numbered CA09) in the receptor binding site (Fig. 6; the left panel shows the corresponding sequences in the wild-type H1N1 pandemic strain and corresponding glycosylated sequences in an exemplary engineered HA polypeptide, DO2).

Insertion of a lysine into a loop within or near predicted N-glycosylation sites of the RBS region of HA provides a signature of pandemic influenza A viruses. Insertion into a loop (e.g., insertion of a lysine or arginine residue) near N-glycosylation sites provides a signature of a pandemic strain in the engineered HA polypeptide ( Fig. 6; central part).

Modification of residues around the RBS site near the CH65 epitope further includes features of pandemic influenza A viruses (Fig. 6; right panel).

To demonstrate the increased spectrum of immunological activity conferred on an engineered HA polypeptide through modifications of the predicted N-glycosylation site and/or lysine loop insertion, specific amino acid residues in an exemplary engineered HA polypeptide (SMARt_DO2a) were modified to reflect those observed in HA polypeptides like pandemic. In table Figure 4 demonstrates the potential amino acid substitutions that can be made at these sites, which have been identified from observed residues at each position in circulating influenza A viruses.

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Table 4

Nglycosylation site (according to CA09 numbering) Residue index of the constructed NA Illustrative Remains AA Substitutions to Break the NxS/T Pattern 142 142 N D, K, S 143 N Y 144 T E, D, N 145 V I, L, P, S, T 146 T D, L, N, S 147 (loop insert) - (gap) K, R 177 177 N K, R, T 178 L I 179 S I, K, N, R

To demonstrate the effect of destruction of N-glycosylation sites on the spectrum of immunological activity of HA polypeptides, engineered HA molecules were prepared with destruction of two motifs of N-glycosylation sites. In addition, two engineered HA polypeptides were obtained in which the N-glycosylation site motifs were disrupted and a lysine was inserted into the RBS bounding loop (Table 5).

Disruption of the glycosylation motif alone, as well as in combination with loop lysine insertion, produced recombinant HA polypeptides that are surface expressed and retain stem region folding, as determined by flow cytometry analysis (FIG. 7). As in the previous case, it was observed that modifications in the head region induced a modest increase in mAb binding to the stem region, suggesting that substitutions at one site can have long-range allosteric effects at a distant location. These modifications also facilitate the achievement of an increased spectrum of immunological activity based on recognition by a panel of antibodies (FIG. 8). More specifically, several of the redesigned antigens demonstrate both improved seasonal properties (increased mAb binding of antibodies CH65 and 5J8 to the seasonal head) and an increased spectrum of immunological activity, as demonstrated by a 50-150% increase in binding of the 4K8 antibody to the pandemic head (see designs DO2a_m1-m3 in Fig. 8).

Table 5

Design Residue indices (according to CA09 numbering) Original sequence (designed HA, e.g. DO2a) Modified sequences 1 144, 145, 146 TVT DSN 177 N TO 2 144, 145, 146 TVT DSN 177 N TO 147 - (gap) TO 3 144, 145, 146 TVT ETT 177 N TO 147 - (gap) TO

Example 3. Modifications of amino acid residues in the region of RBS and N-glycosylation increase the spectrum of immunological activity of the constructed HA polypeptide.

This example describes modifications of an engineered HA polypeptide at or near the RBS of HA by introducing amino acid substitutions. The amino acid substitutions are position specific and are derived from residues identified by analysis of the HA head region of circulating influenza A viruses. Table. 6 describes a pool of residues at specific positions, spanning residues 60-291 (based on CA/09 numbering), from which specific amino acid substitutions were selected to target modification of the HA globular head. In table 7 describes a smaller pool of residues at specific positions, spanning residues 137-262 (based on CA/09 numbering), from which specific amino acid substitutions were selected to target modification of the HA globular head. In table 8 describes a pool of residues used for targeted modification of the immunological profile

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NA for residues within 10 angstroms of the RBS. These residues are indicated by shading in Fig. 9.

Table 6

Index from the residue of the constructed NA AA replacements Residue index of the constructed NA AA replacements Residual index of constructed other NA Replaced AA Index from the residue of the constructed NA AA replacements Residue index of the constructed NA AA replacements Residue index of the constructed NA AA replacements 60 K, L, N, Q, S, R 105 G,W 145 A, I, L, P, S, T, V 182 F, Y 217 F, L, S 253 Q, P, S 61 I, L 106 A, I, M, T 146 D, L, N, S, T 183 A, E, F, I, K, L, S, T, V 218 I, V 254 E, G, R 62 G, K, M, N, R 111 E, D, G, H, N, V, Y 147 I, K, N, R, - 185 D, K, N, S 219 E, G, M, S, W, V 255 E, D, G, N 63 E, G FROM A, I, N, S, T, V 149 A, E, I, t, V 186 K, Q, R 220 S, t 256 K, R, T 64 A, E, I, k, m, t, V 114 D, N 150 S, t 187 E, D, G, K, R 221 P, S, t 257 I,v 65 A, I, P, S, T, V 116 E, G, V 151 A, I, m, S, t, V 188 E, K, N, R 222 n, k, N, R, T 258 I, m, T 68 Η, N, Q 119 K, R, T 152 A, S 189 E, D, G, K, V 224 N, S, Y 259 F, L 70 D, G 120 E, K 154 A, n, L, P, S, T 190 I, V 225 G, K, Q, R 261 A, T 71 E, K, N, S, R 121 H, L, Q, R 155 n, N, Q, S, R, Y 192 I, M, L, V 226 E, I, K, M, N, R 262 N, S, T 73 D, N, S, T 123 G, N, S 156 A, E, D, G, k, N, S, 193 I, L 227 F, L, S 266 A, I, M, L, V

- 36 044592

R, T, V 74 I, v 124 S, T 157 E, G, K, R 195 A,G 228 E, I, K, N, R, T 267 A, I, V 77 R,W 125 A, I, M, L, V 158 A, E, K, M, S, T, V 196 I,v 231 I, V 269 I, k, M, s, R, W 78 I, L, V 126 L, S, T 159 K, L, N, S, R 198 Η, N 232 A, E, G, T, V 270 F, Η, Y 83 E, D, G, K 127 L, S 160 G, N, S 199 Q, P, s 233 A, E, I, K, M, R, T, V 273 A, E, T, V 85 E, D, G 128 F, L 163 E, K, N, Q,R 200 A, P, S 234 R 274 I, M, L, V 86 F, L, P, S, T 129 E, K 164 N, S 201 A, D, N, S, T 235 L,P 275 E, D, G, K, N, S, R, V 87 F, I, L, P 130 K, R 166 I, M, L, V 202 A, D, I, M, N, P, S, T, Y 236 K, R 276 G, K, R 88 F, I, L, P, S, T, V, Y 131 F, Y 168 I, L 203 A, E, D, G, I, k, N, S, R, T, V 237 I, M, V 277 D, G, K, N, S, T 89 A, I, K, P, S, T 132 E, K 169 A, I, T, V 204 A, B, E, D, G, N, V, Y 238 K, R 278 A, D, F, L, P, S, T, V, Y 90 A, E, I, k, N, S, 133 I, M, L, V 170 E, G, K, Q, 205 Q,R 239 E, D, G, K, N 279 E, D, G, N, S, R

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R, T, V R, V 91 E, D, G, I, K, N, S, R 134 F, V 171 A, E, K, N, R, T 206 K, M, L, N, Q, S, R 240 Q, R 280 A, F, P, S, Y 92 S, T 135 H, P, s 172 E, D, G, K, N 207 A, G, I, N, S, R, T 241 A, E, G, H, K, P, S, T 281 D, G, S 94 P, S 136 E, K, M, N, Q, R 173 D, G, I, K, N, S, T 208 I, J, L 242 A, G, R 282 F, I, V 97 A, I, V 137 A, E, D, G, I, s, T, V 174 L, S, T 210 H, K, N, Q, S, R 244 I, M 283 F, I, M, V 99 A I, K, N, S, R, T 138 G, I, N, S, R, T 176 P, S 211 K, N, S, T 245 D, K, N, S 284 I, K, M, L, N, S, R, T, V 100 F, P, S, T, Y 139 A, S 177 E, K, M, N, S, R, T 212 A, E, D, G, I, N, T, V 247 H,Y 285 S, T 101 D, G, I, K, N, S, T, Y 141 L, P, S 178 F, I, L, V 213 E, D, Η, N, Y 249 A, I, T 286 E, D, G, N 102 A, P, S, T 142 D, K, N, S, Y 179 G, I, K, N, S, R, T 214 A, S, T 250 I, M, L, V 287 A, I, K, S, T, V 103 E, D, G, K, N, R, Y 143 H,Y 180 E, I, K, M, N, Q, S, R, T 215 H, Y 251 I, L, V 288 H, L, Q, P, S, T 104 D, K, N 144 E, D, N, S, T 181 F, P, S, T 216 A, I, V 252 A, E, D, G, K, Ν, V 289 A, F, I, M, L, V 290 D, G, Η, N, Q, R, Y 291 E, D, G, K, N

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Table 7

Residue index of the constructed NA AA replacements Residue index of the constructed NA AA replacements Residue index of the constructed NA AA replacements Residue index of the engineered molecule AA replacements 137 A, E, D, G, I, S, T, V 171 A, E, K, N, R, T 203 A, E, D, G, I, K, N, S, R, T, V 234 R 138 G, I, N, S, R, T 172 E, D, G, K, N 204 A, B, E, D, G, Ν, V, Υ 235 L, P 139 A, S 173 D, G, I, K, N, S, T 205 Q,R 236 K, R 141 L, P, S 174 L, S, T 206 k, m, L, N, Q, S, R 237 I, m, V 142 D, K, N, S, Y 176 P, S 207 A, G, I, N, S, R, T 238 K, R 143 n, Y 177 E, K, M, N, S, R, T 208 I, J, L 239 E, D, G, K, N 144 E, D, N, S, T 178 F, I, L, V 210 N, K, N, Q, S, R 240 Q,R 145 A, I, L, P, S, T, V 179 G, I, K, Ν, S, R, t 211 K, N, S, T 241 A, E, G, N, K, P, S, T 146 D, L, N, S, t 180 E, I, K, M, N, Q, S, R, T 212 A, E, D, G, I, N, T, V 242 A, G, R 147 I, K, N, R, 181 F, P, S, T 213 E, D, N, Ν, Y 244 1M 149 A, E, I, T, V 182 F, Υ 214 A, S, T 245 D, K, N, S 150 S, t 183 A, E, F, I, K, L, S, T, V 215 H, Y 247 H, Y 151 A, I, M, S, T, V 185 D, K, N, S 216 A, I, V 249 A, I, T 152 A, S 186 K, Q, R 217 F, L, S 250 I, M, L, V 154 A, N, L, P, S, T 187 E, D, G, K, R 218 I, V 251 I, L, V 155 Η, N, Q, S, R, Y 188 E, K, N, R 219 E, G, M, S, W, V 252 A, E, D, G, K, N, V 156 A, E, D, G, K, N, S, R, T, V 189 E, D, G, K, V 220 S, t 253 Q, P, S 157 E, G, K, R 190 I, v 221 P, S, t 254 E, G, R 158 A, E, K, m, S, t, V 192 I, M, L, V 222 n, K, N, R, T 255 E, D, G, N 159 k, L, N, S, R 193 I, L 224 N, S, Y 256 K, R, T 160 G, N, S 195 A,G 225 G, K, Q, R 257 I, V 163 E, K, N, Q, R 196 I, v 226 E, I, K, M, N, R 258 I, m, T

-39044592

164 N, S 198 Η, N 227 F, L, S 259 F, L 166 I, M, L, V 199 Q, P, S 228 E, I, K, N, R, T 261 A, T 168 I, L 200 A, P, S 231 I, V 262 N, S, T 169 A, I, T, V 201 A, D, N, S, T 232 A, E, G, T, V 170 E, G, K, Q, R, V 202 A, D, I, M, N, P, S, T, Y 233 A, E, I, K, M, R, T, V

Table 8

Residue index of the constructed NA AA replacements Residue index of the constructed NA AA replacements Residue index of the constructed NA AA replacements Residue index of the constructed NA AA replacements 90 A, E, I, K, N, S, R, T, V 138 G, I, N, S, R, T 157 E, G, K, R 195 A, G 91 E, D, G, I, K, N, S, R 139 A, S 158 A, E, K, m, S, t, V 210 N, K, N, Q, S, R 92 S, T 141 L, P, S 159 k, L, N, S, R 211 K, N, S, T 125 A, I, M, L, V 142 D, K, N, S, Y 177 E, K, M, N, S, R, T 212 A, E, D, G, I, N, T, V 126 L, S, T 143 n, Y 178 F, I, L, V 213 E, D, Η, N, Y 127 L, S 144 E, D, N, S, T 179 G, I, K, N, S, R, T 214 A, S, T 128 F, L 145 A, I, L, P, S, T, V 180 E, I, K, m, N, Q, S, R, T 215 H, Y 129 E, K 146 D, L, N, S, t 181 F, P, S, T 216 A, I, V 130 K, R 147 I, K, N, R, 182 F, Y 217 F, L, S 131 F, Y 149 A, E, I, T, V 183 A, E, F, I, K, L, S, T, V 218 I, V 132 E, K 150 S, t 185 D, K, N, S 219 E, G, M, S, W, V 133 I, M, L, V 151 A, I, M, S, T, V 186 K, Q, R 220 S, t 134 F, V 152 A, S 187 E, D, G, K, R 221 P, S, t 135 N, R, S 154 A, N, L, P, S, T 188 E, K, N, R 222 N, K, N, R, T 136 E, K, M, N, Q, R 155 Η, N, Q, S, R, Y 189 E, D, G, K, V 224 N, S, Y 137 A, E, D, G, I, S, T, V 156 A, E, D, G, K, N, S, R, T, V 190 I, V 225 G, K, Q, R 192 I, M, L, V 226 E, I, K, M, N, R 193 I, L 245 D, K, N, S 247 H, Y 249 A, I, T

In table 9 describes residue modifications in an engineered HA polypeptide with a seasonal immune profile that were tested as a proof of principle. These modifications involve combining modifications to the RBS region with the modifications to the glycosylation profile described above (eg, disruption of glycosylation sites). These modifications lead to the production of a recombinant HA polypeptide, which is characterized by proper folding and extrusion.

- 40 044592 is pressed on the surface (Fig. 7). As in the previous case, it was observed that modifications in the head region induced a modest increase in mAb binding to the stem region, suggesting that substitutions at one site can have long-range allosteric effects at a distant location. The redesigned recombinant HAs were recognized by antibodies specific for both pandemic and seasonal strains (Fig. 8). Interestingly, RBS modifications alone improved the seasonal immune profile (Fig. 8, DO2a_m4-m5; modified CH65) but had little effect on the pandemic profile. However, combining RBS region modifications with glycosylation modifications significantly improved both seasonal and pandemic immune profiles. (Fig. 8, DO2a_m7-m9; Aglycosyl+mCH65). These data suggest that the modifications improve the seasonal immune profile (increased mAb binding) and make the immune profile more pandemic (increased spectrum), resulting in an overall more balanced immune profile capable of coping with the complexities caused by antigenic drift and antigenic shift , an exemplary engineered HA polypeptide (eg, SMARt_DO2a).

Table 9

Design Modification Residue indices (according to CA09 numbering) Initial sequence of the designed HA (SMARt_DO2a) Modified serial T.H. 1 E137T (charge change) 137 E T Ca antigenic site 154-159 SHNGKS PHAGAK antigenic site Sa 210-214 HTEN QNAD N262T 262 N T 2 Ca antigenic site 154-159 SHNGKS PHAGAK 3 antigenic site Sa 210-214 ΗΤΕΝ QNAD 4 E137T (charge change) 137 E T Nglyl42 144-145 TV NT Ca antigenic site 154-159 SHNGKS PHAGAK Nglyl77 177 Ν T antigenic site Sa 210-214 ΗΤΕΝ QNAD N262T 262 Ν T 5 Nglyl42 144-145 TV NT Ca antigenic site 154-159 SHNGKS PHAGAK Nglyl77 177 Ν T 6 Nglyl42 144-145 TV NT Nglyl77 177 Ν T antigenic site Sa 210-214 ΗΤΕΝ QNAD

Example 4 In Vivo Efficacy of Engineered HA Polypeptides.

This example illustrates that engineered HA polypeptides modified in accordance with the methods described herein provided broad immune responses producing antibodies to multiple influenza strains.

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Production of influenza virus-like particles (VLPs) containing engineered mosaic hemagglutinins (HAs) Influenza virus-based VLPs were produced by transiently transfecting HEK293T cells with three plasmids in serum-free Freestyle293 medium. Plasmids encoding the sequence of the engineered HA polypeptide, as well as the NA and HIV gag sequences, were mixed in a 1:1:1 ratio and used to transiently transfect HEK293T cells. At 120 h posttransfection, culture supernatants were collected and VLPs were pelleted in the supernatant by ultracentrifugation on a 20% sucrose cushion and resuspended in PBS.

Immunization of mice with VLPs expressing engineered NAs.

To assess the immunogenicity of the engineered mosaic HA structures, groups of 6-8 week old female BALB/c mice were immunized with 5 μg of influenza virus-based VLP or vehicle media alone (PBS). All immunization preparations were formulated as oil-in-water emulsions with an adjuvant and delivered subcutaneously in a total volume of 100 μl. Each group received an identical booster dose 21 days after the initial immunization. Pre- and post-immunization serum was collected from each animal on days 0 and 35, respectively. Serum pools used for the assay were prepared by mixing equal volumes of serum from each animal in the group.

Hemagglutination inhibition (HAI) assay.

Serial dilutions of pooled serum from each group were mixed with 4 hemagglutinating units of the indicated virus and incubated at room temperature for 30 min in a round bottom plate. Each serum/virus mixture was then mixed with an equal volume of 0.5% turkey red blood cell suspension in saline. Plates were scored when control wells without serum showed complete hemagglutination (~30 min). The HAI titer was defined as the maximum serum dilution resulting in complete inhibition of hemagglutination in 50% of the test wells.

Microneutralization (MN) assay.

Serial dilutions of pooled serum from each group were mixed with 100 tissue culture infection doses (TCID50) of virus and incubated at 37°C for 1 h. Each serum/virus mixture was then added to confluent Madin-Darby cell monolayers canine kidney (MDCK) and incubated at 37°C for 24 hours. The monolayers were then fixed and infected wells were identified based on ELISA detection of influenza nucleoprotein. The MN titer was defined as the highest serum dilution resulting in complete neutralization of viral infection in 50% of test wells.

Immunogenicity assays.

HAI assays were performed using modified recombinant HA polypeptides with RBS regions grafted onto recipient stem regions, modifications to remove or engineer putative N-glycosylation sites, and/or targeted modifications to residues in the RBS region. These modified recombinant HA polypeptides provide a broader immune response.

Example 5 Exemplary Modified Recombinant HA Polypeptides.

This Example illustrates examples of modified recombinant HA polypeptides produced using the various methods described herein.

DO2aRBStrunc00_resG63_G278_graftedontDola (SEQ ID NO: 7)

MKAKLLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNG

KLCKLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGYFADYEEL REQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKS YANNKEKEVLVLWGVHHPPNIGDQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQE GRINYYWTLLE PGDTIIFEANGNLIAPWYAFALSRGFGSGIITSDTPVHDCNTTCQTPQGAIN SSLPFQNVHPVTIGECPKYVRSAKLRMATGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGW YGYHHQNEQGSGYAADLKSTQNAIDGITNKVNSVIEKMNTQFTAVGKEFNKLERRMENL NKKVDDGFLDIWTYNAELLVLLENERTL DFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEF

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YHKCNNTCMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLL VSLGAISFWMC SNGSLQCRICI

DO2aRBStrunc00_resG63_G277_graftedontoCal2009 (SEQ ID NO: 8)

MKAKLLVLLCTFTATYADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHN GKLCKLRGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGYFADYEE LREQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSK SYANNKEKEVLVLWGVHHPPN IGDQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQ EGRIN YYWTLLEPGDTIIFEANGNLIAPWYAFALSRGAGSGIIISDTPVHDCNTTCQTPKGAI NTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWY GYHHQNEQGSGYAADLKSTQNAIDEITNKV NSVIEKMNTQFTAVGKEFNHLEKRIENLNK KVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYH KCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQILAIYSTVASSLVLVVSLG AISFWMCSNGSLQCRICI

DO2aRBStrunc00_resG63_G277_graftedontoSC1918 (SEQ ID NO: 9)

MKAKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNG KLCKLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGYFADYEEL REQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKS YANNKEKEVLVLWGVHHPPNIGD QRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQE GRINYYWTLLEPGDTIIFEANGNLIAPWYAFALSRGSGSGIITSDAPVHDCNTKCQTPHGAIN SSLPFQNIHPVTIGECPKYVRSTKLRMATGLRNIPSIQSRGLFGAIAGFIEGGWTGMIDGWY GYHHQNEQGSGYAADQKSTQNAIDGITNKVNSVI EKMNTQFTAVGKEFNNLERRIENLNK KVDDGFLDIWTYNAELLVLLENERTLDFHDSNVRNLYEKVKSQLKNNAKEIGNGCFEFYH KCDD ACMES VRNGTYD YPKYSEESKLNREEroGVKLESMGVYQIL AIYST VAS SLVLLVSL GAISFWMCSNGSLQCRICI

DO2aRBStrunc00_resG63_G277_graftedontoNJ1976 (SEQ ID NO: 10)

MKAKLLVLLCTFTATYADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDRHN GKLCKLGGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGYFADYEE LREQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSK SYANNKEKEVLVLWGVHHPPN IGDQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQ EGRIN YYWTLLEPGDTIIFEANGNLIAPWYAFALSRGSGSGIIISDAPVHDCNTKCQTPKGAI NTSLPFQNIHPVTIGECPKYVKSTKLRMATGLRNIPSIQSRGLFGAIAGFIEGGWTGMIDGW YGYHHQNEQGSGYAADQRSTQNAIDGIT NKVNSVIEKMNTQFTAVGKEFNHLEKRIENLN KKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVRSQLRNNAKEIGNGCFEFY

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HKCDDTCMESVKNGTYDYPKYSEESKLNREEIDGVKLESTRIYQILAIYSTVASSLVLLVSL GAISFWMCSNGSLQCRICI

DO2aRBStrunc01_resV125_G277_graftedontoDola (SEQ ID NO: 11)

MKAKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNG KLCKLKGIAPLQLGKCSVAGWILGNPECESLSTASSWSYIVETSSPDNGTCYPGYFADYEEL REQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKS YANNKEKEVLVLWGVHHPPNIG DQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQE GRINYYWTLLEPGDTIIFEANGNLIAPWYAFALSRGFGSGIITSDTPVHDCNTTCQTPQGAIN SSLPFQNVHPVTIGECPKYVRSAKLRMATGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGW YGYHHQNEQGSGYAADLKSTQNAIDGITNKVNSVI EKMNTQFTAVGKEFNKLERRMENL NKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEF YHKCNNTCMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLL VSLGAISFWMC SNGSLQCRICI

D02aRBStrunc01_resV125_G277_graftedontoCal2009 (SEQ ID NO: 12)

MKAKLLVLLCTFTATYADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHN GKLCKLRGVAPLHLGKCNIAGWILGNPECESLST AS SWS YIVETPS SDNGTC YPGDF ID YEE LREQLS S VSSFERFEIFPKES SWPNHT VTGVS ASC SHNGKS SF YRNLLWLTGKNGLYPNLSK SYANNKEKEVLVLWG VHHPPNIGDQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQ EGRIN YYWTLLEPGDTIIFEANGNLIAPWYAFALSRGAGSGIIISDTPVHDCNTTCQTPKGAI NTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWY GYHHQNEQGSGYAADLKSTQNA IDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNK KVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYH KCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQILAIYSTVASSLVLVVSLG AISFWMCSNGSLQCRICI

DO2aRBStrunc01_resV125_G277_graftedontoSC1918 (SEQ Ш NO: 13)

MKAKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNG KLCKLKGIAPLQLGKCNIAGWLLGNPECDLLLTASSWSYIVETSNSENGTCYPGDFIDYEEL REQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKS YANNKEKEVLVLWGVHHPPNIGD QRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQE GRINYYWTLLEPGDTIIFEANGNLIAPWYAFALSRGSGSGIITSDAPVHDCNTKCQTPHGAIN SSLPFQNIHPVTIGECPKYVRSTKLRMATGLRNIPSIQSRGLFGAIAGFIEGGWTGMIDGWY GYHHQNEQGSGYAADQKSTQNAroGITNKVNSVI EKMNTQFTAVGKEFNNLERRIENLNK KVDDGFLDIWTYNAELLVLLENERTLDFHDSNVRNLYEKVKSQLKNNAKEIGNGCFEFYH

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KCDD ACMES VRNGTYD YPI<YSEESI<LNREEIDGVI<LESMGVYQIL AIYST VAS SLVLLVSL GAISFWMCSNGSLQCRICI

DO2aRBStrunc01_resV125_G277_graftedontoNJ1976 (SEQ ID NO: 14)

MKAKLLVLLCTFTATYADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDRHN GKLCKLGGIAPLHLGKCNIAGWLLGNPECELLLTVSSWSYIVETSNSDNGTCYPGDFINYEE LREQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSK SYANNKEKEVLVLWGVHHPPNIG DQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQ EGRIN YYWTLLEPGDTIIFEANGNLIAPWYAFALSRGSGSGIIISDAPVHDCNTKCQTPKGAI NTSLPFQNIHPVTIGECPKYVKSTKLRMATGLRNIPSIQSRGLFGAIAGFIEGGWTGMIDGW YGYHHQNEQGSGYAADQRSTQNAIDGITNK VNSVIEKMNTQFTAVGKEFNHLEKRIENLN KKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVRSQLRNNAKEIGNGCFEFY HKCDDTCMES VKNGTYDYPKYSEESKLNREEroGVKLESTRIYQIL AIYSTVAS SLVLLVSL GAISFWMCSNGSLQCRICI

DO2aRBStrunc02_resP135_P269_graftedontoDola (SEQ ID NO: 15)

MKAKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNG KLCKLKGIAPLQLGKCSVAGWILGNPECESLSTASSWSYIVETSSPDNGTCYPGYFADYEEL REQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKS YANNKEKEVLVLWGVHHPPNIG DQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQE GRINYYWTLLEPGDTIIFEANGNLIAPWYAFALSRGFGSGIITSDTPVHDCNTTCQTPQGAIN SSLPFQNVHPVTIGECPKYVRSAKLRMATGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGW YGYHHQNEQGSGYAADLKSTQNAIDGITNKVNSVI EKMNTQFTAVGKEFNKLERRMENL NKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEF YHKCNNTCMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLL VSLGAISFWMC SNGSLQCRICI

D02aRBStrunc02_resP135_P269_graftedontoCal2009 (SEQ ID NO: 16)

MKAKLLVLLCTFTATYADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHN GKLCKLRGVAPLHLGKCNIAGWILGNPECESLST AS SWS YIVETPS SDNGTC YPGDF ID YEE LREQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSK SYANNKEKEVLVLWGVHHPPN IGDQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQ EGRIN YYWTLLEPGDTHFEANGNLIAPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAI NTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWY GYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIE KMNTQFTAVGKEFNHLEKRIENLNK KVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYH

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KCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQILAIYSTVASSLVLVVSLG

AISFWMCSNGSLQCRICI

DO2aRBStrunc02_resP135_P269_graftedontoScl918 (SEQ ID NO: 17)

MKAKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNG KLCKLKGIAPLQLGKCNIAGWLLGNPECDLLLTASSWSYIVETSNSENGTCYPGDFIDYEEL REQLSSVSSFEKFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKS YANNKEKEVLVLWGVHHPPNIGD QRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQE GRINYYWTLLEPGDTHFEANGNLIAPWYAFALNRGSGSGIITSDAPVHDCNTKCQTPHGAI NSSLPFQNIHPVTIGECPKYVRSTKLRMATGLRNIPSIQSRGLFGAIAGFIEGGWTGMIDGW YGYHHQNEQGSGYAADQKSTQNAIDGITNKVNSVI EKMNTQFTAVGKEFNNLERRIENLN KKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVRNLYEKVKSQLKNNAKEIGNGCFEFY HKCDD ACMES VRNGTYDYPI<YSEESI<LNREEIDGVI<LESMGVYQILAIYSTVASSLVLLVS LGAISFWMC SNGSLQCRICI

DO2aRBStrunc02_resP135_P269_graftedontoNj 1976 (SEQ ID NO: 18)

MKAKLLVLLCTFTATYADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDRHN GKLCKLGGIAPLHLGKCNIAGWLLGNPECELLLTVSSWSYIVETSNSDNGTCYPGDFINYEE LREQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSK SYANNKEKEVLVLWGVHHPPNIG DQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQ EGRIN YYWTLLEPGDTIIFEANGNLIAPRYAFAMNRGSGSGIIISDAPVHDCNTKCQTPK GAI NTSLPFQNIHPVTIGECPKYVKSTKLRMATGLRNIPSIQSRGLFGAIAGFIEGGWTGMIDGW YGYHHQNEQGSGYAADQRSTQNAIDGITNKV NSVIEKMNTQFTAVGKEFNHLEKRIENLN KKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVRSQLRNNAKEIGNGCEFY HKCDDTCMESVKNGTYDYPKYSEESKLNREEIDGVKLESTRIYQILAIYSTVASSLVLLVSL GAISFWMCSNGSLQCRICI

SMARt_NC_DO2a_NGlyMod (SEQ ID NO: 19)

MKAKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNILEDSHNG KLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGYFADYEEL REQLSSVSSFERFEIFPKESSWPNHDSN-GVSASCSHNGKSSFYRNLLWLTGKNGLYPKLS

KSYANNKEKEVLVLWGVHHPPNIGDQRALYHTENAYVSVVSSHYSRRFTPEIAKRP KVRDQEGRINYYWTLLEPGDTHFEANGNLIAPWYAFALSRGFGSGIITSNAPMDKCDAKC QTPQGAINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPFIQSRGLFGAIAGFIEGGWT GMVDGWYGYHHQNEQ GSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKL ERRMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEI

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GNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTV ASSLVLLVSLGAISFWMCSNGSLQCRICI

SMARt_NC_DO2a_NGlyMod+loopInsertion(CA09) (SEQ ID NO: 20)

MKAKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNILEDSHNG KLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGYFADYEEL REQLS S VS SFERFEIFPKESSWPNHDSNKGVS ASC SHNGKS SF YRNLLWLTGKNGLYPKLSK SYANNKEKEVLVLWG VHHPPNIGDQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQ EGRIN YYWTLLEPGDTIIFEANGNLIAPWYAFALSRGFGSGIITSNAPMDKCDAKCQTPQGA INSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPFIQSRGLFGAIAGFIEGGWTGMVDG WYGYHHQNEQGSGYAADQKSTQNA INGITNKVNSVIEKMNTQFTAVGKEFNKLERRMEN LNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFE FYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLL VSLGAISFWMC SNGSLQCRICI

SMART_NC_DO2A_NGLYMOD+LOOPINSERTION(SC18) (SEQ ID NO: 21)

MKAKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNILEDSHNG KLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGYFADYEEL REQLS S VS SFERFEIFPKESSWPNHETTKGVS ASC SHNGKS SFYRNLLWLTGKNGLYPKLSK SYANNKEKEVLVLWG VHHPPNIGDQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQ EGRIN YYWTLLEPGDTIIFEANGNLIAPWYAFALSRGFGSGIITSNAPMDKCDAKCQTPQGA INSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPFIQSRGLFGAIAGFIEGGWTGMVDG WYGYHHQNEQGSGYAADQKSTQNA INGITNKVNSVIEKMNTQFTAVGKEFNKLERRMEN LNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFE FYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLL VSLGAISFWMC SNGSLQCRICI

SMARt_NC_DO2a_mods_outstide_ch65_eptiopel (SEQ ID NO: 22)

MKAKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNILEDSHNG KLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGYFADYEEL REQLSSVSSFERFEIFPKTSSWPNHTVT-GVSASCPHAGAKSFYRNLLWLTGKNGLYPNLS

KSYANNKEKEVLVLWGVHHPPNIGDQRALYQNADAYVSVVSSHYSRRFTPEIAKRP KVRDQEGRINYYWTLLEPGDTIIFEATGNLIAPWYAFALSRGFGSGIITSNAPMDKCDAKCQ TPQGAINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPFIQSRGLFGAIAGFIEGGWTG MVDGWYGYHH QNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKLER RMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIG

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NGCFEFYHI<CNDECMESVI<NGTYDYPI<YSEESI<LNREI<IDGVI<LESMGVYQILAIYSTVA SSLVLLVSLGAISFWMCSNGSLQCRICI

SMARt_NC_DO2a_mods_outstide_ch65_eptiope2 (SEQ ID NO: 23)

MKAKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNILEDSHNG KLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGYFADYEEL REQLSSVSSFERFEIFPKESSWPNHTVT-GVSASCPHAGAKSFYRNLLWLTGKNGLYPNLS

KSYANNKEKEVLVLWGVHHPPNIGDQRALYHTENAYVSVVSSHYSRRFTPEIAKRP KVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPWYAFALSRGFGSGIITSNAPMDKCDAKC QTPQGAINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPFIQSRGLFGAIAGFIEGGWT GMVDGWYGYHHQNE QGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKL ERRMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEI GNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTV ASSLVLLVSLGAISFWMCSNGSLQCRIC I

SMARt_NC_DO2a_mods_outside_ch65_eptiope3 (SEQ Ш NO: 24)

MKAKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNILEDSHNG KLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGYFADYEEL REQLS S VS SFERFEIFPKESSWPNHTVT-GVS ASC SHNGKS SF YRNLLWLTGKNGLYPNLS

KSYANNKEKEVLVLWGVHHPPNIGDQRALYQNADAYVSVVSSHYSRRFTPEIAKRP KVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPWYAFALSRGFGSGIITSNAPMDKCDAKC QTPQGAINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPFIQSRGLFGAIAGFIEGGWT GMVDGWYGYHHQNE QGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKL ERRMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEI GNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTV ASSLVLLVSLGAISFWMCSNGSLQCRIC I

SMARt_NC_DO2a_mods_outside_ch65_eptiopel-noGly (SEQ ID NO: 25)

MKAKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNILEDSHNG KLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGYFADYEEL REQLS S VS SFERFEIFPKTS SWPNHNTT-GVSASCPHAGAKSFYRNLLWLTGKNGLYPKLS

KSYANNKEKEVLVLWGVHHPPNIGDQRALYQNADAYVSVVSSHYSRRFTPEIAKRP KVRDQEGRINYYWTLLEPGDTIIFEATGNLIAPWYAFALSRGFGSGIITSNAPMDKCDAKCQ TPQGAINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPFIQSRGLFGAIAGFIEGGWTG MVDGWYGYHH QNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKLER

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RMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIG

NGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVA

SSLVLLVSLGAISFWMCSNGSLQCRICI

SMARt_NC_DO2a_mods_outstide_ch65_eptiope2-noGly (SEQ ID NO: 26)

MKAKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNILEDSHNG KLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGYFADYEEL REQLSSVSSFERFEIFPKESSWPNHNTT-GVSASCPHAGAKSFYRNLLWLTGKNGLYPKLS

KSYANNKEKEVLVLWGVHHPPNIGDQRALYHTENAYVSVVSSHYSRRFTPEIAKRP KVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPWYAFALSRGFGSGIITSNAPMDKCDAKC QTPQGAINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPFIQSRGLFGAIAGFIEGGWT GMVDGWYGYHHQNE QGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKL ERRMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEI GNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTV ASSLVLLVSLGAISFWMCSNGSLQCRIC I

SMARt_NC_DO2a_mods_outstide_ch65_eptiope3-noGly (SEQ ID NO: 27)

MKAKLLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNILEDSHNG

KLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGYFADYEEL

REQLS S VS SFERFEIFPKESSWPNHNTT-GVS ASC SHNGKS SFYRNLLWLTGKNGLYPKLS

KSYANNKEKEVLVLWGVHHPPNIGDQRALYQNADAYVSVVSSHYSRRFTPEIAKRP KVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPWYAFALSRGFGSGIITSNAPMDKCDAKC QTPQGAINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPFIQSRGLFGAIAGFIEGGWT GMVDGWYGYHHQNE QGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKL ERRMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEI GNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTV ASSLVLLVSLGAISFWMCSNGSLQCRIC I

The invention also covers modified recombinant HA polypeptides that have an amino acid sequence that is at least 70, 75, 80, 85, 90, 92, 94, 95, 96, 97, 98, or 99% identical to any of those described herein sequences.

Example 6. Modifications of recombinant HA polypeptides to increase pandemic symptoms.

Additional structures were selected to test the effect of BO2a deglycosylation/RBS modification on the spectrum of Ab production responses and protection from pandemic A/California/09 infection. Redesign of the SMARtDO2a RBS improves recognition by broadly neutralizing antibodies, as demonstrated by the increase in 4K8 binding to deglycosylated constructs in the MFI assay (Figure 10). Based on in vivo evaluations, the initial structure of SMARt DO2a has an effect on seasonal strains; the structure was modified to increase the spectrum against pandemic strains. In vitro assays using a panel of mAbs indicated binding of pandemic mAbs to several modified structures. A subset of structures were evaluated in a mouse challenge model against A/California/07/2009. The results demonstrate that the modifications do improve the immune profile against pandemic influenza A.

Immunization of mice with VLPs expressing redesigned HAs.

To assess the immunogenicity of the redesigned mosaic HA structures, groups of 6-8 week old female BALB/c mice were immunized with 3 μg of influenza virus-based VLP or vehicle media alone (PBS). All immunization preparations were formulated as oil-in-water emulsions with an adjuvant and delivered intramuscularly in a total volume of 100 μl, as shown in Table. 10. Each group received two identical booster doses 21 days and 42 days after the initial immunization. Pre-immunization serum was collected from each animal on day 0. Post-immunization serum was collected from each animal on days 42, 56 and 69. FIG. 11 demonstrates the schedule of immunizations and subsequent in vivo assessments. The serum pools used for the assay were obtained by mixing equal volumes of serum from each animal in the group.

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Table 10

Group n ¹ Antigen (day 0) Antigen (day 21) Antigen (day 42) Dose a (mg) Ad yuvan t Injection route Infection (day 70) 1 24 PBS PBS PBS N.A. AF03 I.M. A/Ca1/2009 2 24 VLPSMARtDO2 a VLPSMARTD O2a VLPSMAR1DO2a 3 AF03 I.M. A/Ca1/2009 3 24 VLPSMARtDO2 a ml VLPSMARtD O2a ml VLPSMARtDO2 a ml 3 AF03 I.M. A/Ca1/2009 4 24 VLPSMARtDO2 a m2 VLPSMARtD O2a m2 VLPSMARtDO2 a m2 3 AF03 I.M. A/Ca1/2009 5 24 VLPSMARtDO2 a m8 VLPSMARTD O2a m8 VLPSMARtDO2 a m8 3 AF03 I.M. A/Ca1/2009 6 24 CalO9 IIV (SWFT) Cal09 IIV (SWFT) Cal09 IIV (SWFT) 1.5 AF03 I.M. A/Ca1/2009

Survival and body weight of mice immunized with VLPs expressing redesigned HA

Animals were challenged with ten times the LD ₅₀ of pandemic A/California/2009 on day 70 and post-challenge mortality was monitored. Immunization with parent SMARtDO2a protects against A/California/09 challenge, with 80% of animals surviving 14 days post-challenge compared to animals immunized with vehicle alone, resulting in 100% mortality by day 6 post-challenge control infection. Next generation DO2a modifications improve survival compared to the original SMARtDO2a, with immunization with SMARtDO2a_m8 being effective in protecting 100% of test animals (Figure 12).

The percentage of body weight loss of animals after challenge with the virus was monitored. Next generation modifications of DO2a improve weight maintenance compared to the original SMARtDO2a. SMARtDO2a_m8 provided the best protection against virus challenge-induced weight loss (Fig. 13).

Viral titers in the lungs of mice immunized with VLPs expressing redesigned HA.

Additionally, virus titers were monitored in the lungs of mice on day 4 after challenge. Immunization with SMARtDO2a constructs results in lower virus titers in the lungs compared to PBS. The SMARtDO2a_m8 construct resulted in a 10-fold reduction in lung titers compared to PBS and significantly lower lung viral titers than all other DO2a constructs (Fig. 14).

Hemagglutination inhibition (HAI) assay.

Serial dilutions of pooled serum from each group were mixed with 4 hemagglutinating units of the indicated virus and incubated at room temperature for 30 min in a round bottom plate. Each serum/virus mixture was then mixed with an equal volume of 0.5% turkey red blood cell suspension in saline. Plates were scored when control wells without serum showed complete hemagglutination (~30 min). The HAI titer was defined as the maximum serum dilution resulting in complete inhibition of hemagglutination in 50% of the test wells. The A/California/09 HAI responses in SMARtDO2a_m8 mice were significantly different from the HAI responses in PBS mice (P<0.001), however, only 6/24 mice had a HAI titer equal to or greater than 1:40 (Fig. 15). The mechanism of protection of all other DO2a constructs remains unclear.

All modified next-generation DO2a constructs are able to partially protect against mortality when challenged with A/California/09. SMARtDO2a_m8 shows the best reduction in mortality, weight loss and viral titer in the lungs. SMARtDO2a_m8 is also the only DO2a construct conferring HAI responses to the A/Cal/09 strain, suggesting that the improved protection is possibly due to responses at the head of the molecule. As demonstrated herein, next-generation modifications of DO2a are successful in increasing protection against pandemic influenza A challenge.

Equivalents.

The use of ordinal terms such as first, second, third, etc., in a claim to modify a claim element does not in itself imply any priority, preference or order of one claim element relative to another or temporal order, in in which the actions of the method are performed, but they are used only as marks to distinguish one element of the claim, which has a specific name, from another

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Claims (3)

element having the same name (but in the case of using an ordinal term) to distinguish the elements of the claim. As used herein, the singular number in the specification and claims, unless expressly stated to the contrary, should be understood to include references to the plural. Claims or specifications that include or between one or more members of a group are considered to be satisfactory if one, more than one, or all members of the group are present, apply to, or otherwise relate to the specified product or process, unless otherwise stated or otherwise obvious from context. The present invention includes embodiments in which exactly one member of the group is present, used or otherwise related to the specified product or method. The present invention also includes embodiments in which more than one or all members of the group are present, used or otherwise related to the specified product or method. It is further understood that the present invention covers all variations, combinations and combinations in which one or more limitations, elements, conditions, descriptive terms, etc. of one or more of the listed claims are inserted into another claim dependent on the same independent claim (or any other claim, as applicable), unless otherwise indicated or if one of ordinary skill in the art technical field, it will not be obvious that a contradiction or inconsistency would arise. If elements are presented in list form (eg, a Markush group or similar format), it should be understood that each subgroup of elements is also disclosed, and any element(s) can be removed from the group. In most cases, it will be understood that if the present invention or aspects of the present invention are/are designated as containing/containing specific elements, features, etc., then certain embodiments of the present invention or aspects of the present invention consist or are substantially composed of such elements, features, etc. For purposes of simplicity, such embodiments have not in every case been specifically set forth herein in such detail. It should also be understood that any embodiment or aspect of the present invention can be expressly excluded from the claims, regardless of whether a specific exclusion is set forth in the specification. Publications, websites and other references incorporated herein by reference to describe the prior art of the present invention and to provide additional information regarding its practice are incorporated herein by reference. CLAIM 1. A method of changing the immunogenicity profile of an engineered hemagglutinin (HA) polypeptide, wherein said engineered HA polypeptide has an amino acid sequence that differs from the amino acid sequences of HA polypeptides found in natural influenza isolates, wherein said method comprises: a) identifying the presence or absence, relative to a corresponding head region of an HA polypeptide with a distinguishable immunogenicity profile, of one or more putative N-glycosylation sites in the head region of the engineered HA polypeptide, wherein the head region is a segment of the engineered HA polypeptide at positions corresponding to residues 63 -278, 125-277 or 135-269 from SEQ ID NO: 1; b) introducing into the head region of the engineered HA polypeptide one or more amino acid substitutions, deletions or insertions to disrupt one or more putative N-glycosylation sites or introduce additional N-glycosylation sites; And c) introducing one or more amino acid substitutions within 15 angstroms of the receptor binding site (RBS) of the engineered HA polypeptide, wherein the RBS contains all amino acid residues within 15 angstroms of the position corresponding to the W167 sequence of SEQ ID NO: 2 in three-dimensional (3D) structure, and/or insertion of a lysine (K) or arginine (R) residue into the loop or loops delimiting the RBS of the engineered HA polypeptide, thereby providing a redesigned HA polypeptide with an altered immunogenicity profile compared to the engineered HA polypeptide, the altered profile The immunogenicity of the redesigned HA polypeptide includes an increase in the binding of one or more monoclonal antibodies to the head region against seasonal and/or pandemic strains of the influenza virus and/or an increase in the binding spectrum for monoclonal antibodies to the head region against pandemic strains of the influenza virus. 2. The method of claim 1, wherein the one or more putative N-glycosylation sites or additional N-glycosylation sites are defined by the consensus sequence NxS/Ty, where x and y are not proline (P). 3. The method of claim 2, wherein the one or more amino acid substitutions, deletions or insertions to disrupt one or more putative N-glycosylation sites comprise modification of the consensual -