EA047998B1 - RECOMBINANT INFLUENZA VIRUS ANTIGENS - Google Patents

Description

This invention was made, at least in part, with government support under HHS award HHS0100201700018C. The Government has certain rights in this invention.

Introduction

The present invention relates to the field of medicine. This document provides recombinant hemagglutinin (HA) polypeptides of influenza A virus, nucleic acids encoding said polypeptides, pharmaceutical compositions containing them, and methods for using them.

State of the art

Influenza viruses are major human pathogens that cause respiratory disease (commonly referred to as influenza) that ranges in severity from subclinical infection to primary viral pneumonia that may be fatal. The clinical effects of infection vary depending on the virulence of the influenza virus strain and the exposure, medical history, age, and immune status of the host. Each year, approximately 1 billion people worldwide are estimated to become infected with influenza virus, resulting in severe disease in 3–5 million cases and an estimated 300,000–500,000 influenza-associated deaths. Most of these infections can be attributed to influenza A viruses carrying the H1 or H3 hemagglutinin subtypes, with a smaller contribution from influenza B viruses, and hence representatives of all three are included in the seasonal vaccine. Current immunization practices rely on early identification of circulating influenza viruses to ensure timely availability of an effective seasonal influenza vaccine. In addition to the inherent difficulties in predicting which strains will predominate during the next season, antiviral resistance and immune evasion also play a role in the failure of current vaccines to prevent morbidity and mortality. In addition, the possibility of a pandemic caused by a highly virulent strain of the virus originating from animal reservoirs and reassorted with increased human-to-human transmission poses a significant and real threat to global health.

Influenza A viruses are widespread in nature and can infect a wide variety of birds and mammals. Influenza viruses are enveloped RNA viruses that belong to the Orthomyxoviridae family. Their genomes consist of eight single-stranded RNA segments that encode 11 different proteins: one nucleoprotein (NP), three polymerase proteins (PA, PB1, and PB2), two matrix proteins (M1 and M2), three nonstructural proteins (NS1, NS2, and PB1-F2), and two outer glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Viruses are classified based on differences in the antigenic structure of the HA and NA proteins, with different combinations of the two representing unique virus subtypes that are further classified into specific influenza virus strains. Although all known subtypes can be found in birds, the currently circulating human influenza A virus subtypes are H1N1 and H3N2. Phylogenetic analysis has shown that hemagglutinins are divided into two main groups: among others, the H1, H2, H5 and H9 subtypes in phylogenetic group 1, and among others, the H3, H4 and H7 subtypes in phylogenetic group 2.

Influenza B virus strains are exclusively human. Antigenic variability of HA in influenza B virus strains is less than that observed in influenza A strains. Two genetically and antigenically distinct influenza B virus lineages circulating in humans are the B/Yamagata/16/88 (also called B/Yamagata) and B/Victoria/2/87 (B/Victoria) lineages. Although the spectrum of disease caused by influenza B viruses is generally milder than that caused by influenza A viruses, severe disease requiring hospitalization is still common with influenza B infection.

It is known that antibodies that neutralize the influenza virus are mainly directed against hemagglutinin (HA). Hemagglutinin is a trimeric glycoprotein that is anchored in the viral envelope and has a dual function: it is responsible for binding to cell surface receptors containing sialic acid, and after uptake, it mediates the fusion of the viral and endosomal membranes, which leads to the release of viral RNA into the cell cytosol. HA contains the so-called head domain and the stalk domain. Attachment to the viral membrane is mediated by a C-terminal anchor sequence (also known as the transmembrane domain) connected to the stalk domain. The protein undergoes post-translational cleavage in a specific loop to produce two polypeptides - HA1 and HA2 (the complete sequence is called HA0). The membrane-distal head domain is derived primarily from HA1, and the membrane-proximal stalk domain is derived primarily from HA2 (Fig. 1).

Since the influenza virus is ubiquitous, it is almost impossible to avoid infection with the virus. Vaccination plays a crucial role in controlling influenza epidemics and pandemics. Many influenza vaccines are produced by methods that involve reassortment, adaptation, and growth of viruses in chicken eggs. However, these existing methods have several limitations. Not all influenza virus strains grow well in eggs and must be adapted or engineered reassortants of the virus. Changes in HA during production can

- 1 047998 lead to the emergence of strains that differ from circulating strains and may provide suboptimal levels of protection. Another disadvantage is that individuals with egg allergy may be hypersensitive to residual egg proteins in egg-based vaccines. In addition, egg-based methods rely on a continuous supply of eggs, which may be subject to supply disruptions, such as in the event of poultry disease. There is a need for vaccine production using methods that do not rely on egg supply and in which vaccine protein production is more tightly controlled than in egg-based methods. Recombinant forms of HA (rHA) produced in cell culture have been used as an alternative source of antigen for influenza vaccines compared to egg-based antigen. However, problems have been encountered with these methods in maintaining the immunogenicity and regular quaternary structure of rHA and in achieving high yields of trimeric rHA. Thus, there is still a need for alternative ways of delivering antigens for influenza vaccines or for diagnostics that will overcome the existing shortcomings.

Brief description of the invention

Certain aspects of the present invention are briefly described below. Additional aspects are described in the Detailed Description of the Invention, Examples, Drawings, and Claims sections of this patent.

In a first aspect, the present invention relates to recombinant influenza A virus hemagglutinin (HA) polypeptides comprising the HA1 and HA2 domains of influenza A virus HA and comprising an amino acid sequence wherein:

(a) the amino acid at position 355 is tryptophan (W), and (b) the amino acid at position 432 is isoleucine (I), and/or the amino acid at position 380 is I, and wherein the numbering of the amino acid positions in the amino acid sequence of the HA polypeptide is represented according to the numbering of the amino acids in the amino acid sequence of HA from a reference strain of the H3N2 influenza virus, in particular the reference strain H3N2 A/Aichi/2/68 (SEQ ID NO: 1).

In a further aspect, the present invention relates to multimeric polypeptides comprising at least two HA polypeptides, in particular to trimeric polypeptides comprising three HA polypeptides as described herein.

According to the present invention, it has been demonstrated that high levels of recombinant influenza virus HA polypeptides, in particular recombinant trimeric HA polypeptides, can be obtained and they are characterized by an increased melting temperature, indicating greater stability compared to wild-type HA polypeptides without the addition of heterologous amino acid sequences, such as heterologous trimerization domains. In addition, the HA polypeptides of the present invention are correctly folded, as demonstrated by binding of anti-HA antibodies to the HA polypeptides, such as, but not limited to, the antibodies CR9114, CR8020 and/or CR6261. Thus, the polypeptides are capable of inducing an immune response to HA when administered to a subject, in particular a human subject. Trimeric polypeptides are characterized by the quaternary structure of native wild-type HA and thus present natural epitopes to the immune system, including conserved epitopes of the membrane-proximal stalk of the HA molecule.

In a further aspect, the present invention provides nucleic acid molecules encoding recombinant influenza virus HA polypeptides.

In another aspect, the present invention provides vectors, in particular recombinant adenoviral vectors, comprising nucleic acid molecules encoding influenza virus HA polypeptides.

In another aspect, the present invention provides immunogenic compositions comprising an influenza virus HA polypeptide, a nucleic acid molecule and/or a vector according to the present invention and a pharmaceutically acceptable carrier.

In a further aspect, the present invention provides influenza virus HA polypeptides, nucleic acid molecules encoding said influenza virus HA polypeptides, and/or vectors containing said nucleic acid molecules, for use as a medicinal product, in particular for use as a vaccine for the prevention and/or treatment of influenza disease, in particular a disease or condition caused by an influenza A virus strain from phylogenetic group 1 and/or 2.

The present invention also provides methods for inducing an immune response to influenza virus HA in a subject in need thereof, the method comprising administering to the subject an influenza virus HA polypeptide, nucleic acid molecule and/or vector according to the present invention. In yet another further aspect, methods are provided for preventing and/or vaccinating against influenza disease comprising administering the polypeptide or

- 2 047998 of the munogenic composition described above, to a person in need thereof, such as a person identified as being at risk of infection with an influenza disease.

In yet another further aspect, there is provided a method for producing a recombinant HA polypeptide as defined above, comprising expressing the above-described nucleic acid molecule in a prokaryotic or eukaryotic cell, such as a mammalian cell, for example a CHO cell or an insect cell, optionally further comprising purifying/isolating rHA from said cell.

In another aspect, the present invention provides the use of HA polypeptides as research or diagnostic tools or as targets for the production of influenza virus-suppressing antibody agents.

Brief description of graphic materials

Fig. 1. A. Three-dimensional representation of a monomer of a polypeptide of the present invention with the indicated mutation positions. The hemagglutinin (HA) head is shown in dark gray, the stem is shown in light gray; B. Schematic representation of a monomer of a polypeptide (black: head; light gray: stem) of the present invention with the indicated mutation positions.

Fig. 2. A. Phylogenetic tree of influenza virus HA. The different subtypes of group 1 and group 2 influenza A virus and influenza B virus are indicated. Protein expression levels determined by OCTET (anti-His2 antibody sensor). The last column shows the fold increase in expression level of the stabilized soluble HA trimers of the present invention compared to wild-type (WT) HA; C. Size exclusion chromatography (SEC) profiles: the dotted lines represent WT HA and the solid black line represents the stabilized HA polypeptides of the present invention.

Fig. 3. A. Results of SEC analysis of purified trimer (T) polypeptides of the present invention (black line) and WT polypeptides with the trimerization assembly domain (gray line) (note that UFV4239 does not contain the assembly domain). Purified WT assembly polypeptides are characterized by an increase in peak width and the formation of multimers (*) after storage at -80 °C. Due to the lack of the trimerization domain in UFV4239, only the monomer (M) was expressed and purified; B and C. Results of thermal stability analysis of purified polypeptides by differential scanning fluorimetry (DSF, Tm ₅₀ values in °C); D. Binding of monoclonal antibodies (mAbs) CR6261, CR8020, CT149, CR9114 and the multidomain antibody MD3606 (ELISA, EC50 values).

Fig. 4. A. Protein expression levels determined by OCTET (anti-His2 antibody sensor); B. Results of SEC analysis of EXPI-293 cell culture supernatant. UFV181007 contains K380I and E432I mutations (dashed black line). UFV181005 contains H355W and M478I mutations (dashed gray line). Combination of stabilizing mutations (UFV1810009, black line).

Fig. 5. A. Protein expression levels determined by OCTET (anti-His2 antibody sensor). The last column shows the fold increase in expression level of the stabilized soluble HA trimers of the present invention compared to wild-type (WT) HA; B. Size exclusion chromatography (SEC) profiles: the dotted lines represent WT HA and the solid black lines represent additional stabilized HA polypeptides according to the present invention.

Fig. 6. Size exclusion chromatography (SEC) profiles of purified stabilized HA before and after temperature stress. Shown are polypeptide profiles before the experiment (dashed lines) and after 60 days of incubation at 4°C (solid black lines) and 37°C (solid gray lines).

Definitions

Definitions of terms used in the present invention are provided below.

The amino acid according to the present invention may be any of the twenty naturally occurring (or standard) amino acids or variants thereof, such as, for example, D-proline (the D-enantiomer of proline), or any variants that are not found naturally in proteins, such as, for example, norleucine. The standard amino acids can be divided into several groups based on their properties. Important factors are charge, hydrophilicity or hydrophobicity, size and functional groups. These properties are important for the structure of proteins and protein-protein interactions. Some amino acids have specific properties, such as cysteine, which is capable of forming covalent disulfide bonds (or disulfide bridges) with other cysteine residues, proline, which forms a ring with the polypeptide backbone, and glycine, which is more flexible than other amino acids. Table 3 shows the abbreviations and properties of the standard amino acids.

It is believed that the term included or including, as used in this document, should be followed by the words without limitation.

As used herein, the term infection means the invasion, replication, and/or presence of a virus in a cell or in the body of a subject. In one embodiment, the infection is an active infection, i.e., one in which the virus is replicating in the cell or in the body.

- 3,047,998 subjects. Such an infection is characterized by the spread of the virus to other cells, tissues, and/or organs from cells, tissues, and/or organs initially infected with the virus. The infection may also be a latent infection, i.e. one in which the virus does not replicate. In certain embodiments, the infection refers to a pathological condition caused by the presence of the virus in a cell or in the body of the subject or by the invasion of the virus into a cell or in the body of the subject.

Influenza viruses are generally classified into influenza virus types: genera A, B and C. The term influenza virus subtype, as used herein, refers to variants of influenza A virus that are characterized by combinations of the hemagglutinin (H) and neuraminidase (N) viral surface proteins. According to the present invention, influenza virus subtypes may be named by their H number, such as an influenza virus containing HA subtype H3, an influenza virus of subtype H3 or an influenza virus H3, or by a combination of the H number and the N number, such as an influenza virus subtype H3N2 or H3N2. The term subtype includes, in particular, all individual strains within each subtype, which are typically formed by mutations and exhibit different pathogenic profiles, including natural isolates as well as artificial mutants or reassortants, etc. Such strains may also be referred to as different isolates of a virus subtype. Accordingly, the terms strains and isolates may be used interchangeably in this document. The current nomenclature for human influenza virus strains or isolates includes the virus type (genus), i.e. A, B or C, the geographic location of first isolation, the strain number and the year of isolation, usually with a description of the HA and NA antigens given in parentheses, e.g. A/Moscow/10/2000 (H3N2). Non-human strains also include in their nomenclature the host from which they originated.

Influenza A virus subtypes can be further classified based on their phylogenetic group. Phylogenetic analysis has shown that hemagglutinins are divided into two main groups: among others, the H1, H2, H5, and H9 subtypes in phylogenetic group 1 (influenza group 1 viruses), and among others, the H3, H4, H7, and H10 subtypes in phylogenetic group 2 (influenza group 2 viruses).

As used herein, the term influenza virus disease or influenza refers to a pathological condition caused by the presence of an influenza virus, such as influenza A or B, in a subject. As used herein, the terms disease and disorder are used interchangeably. In particular embodiments, the term refers to a respiratory illness caused by infection of a subject with an influenza virus.

As used herein, the term nucleic acid or nucleic acid molecule is intended to include polynucleotides such as DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA), as well as DNA and RNA analogs produced using nucleotide analogs. Nucleic acid may be single-stranded or double-stranded. Nucleic acid molecules may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily understood by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more naturally occurring nucleotides with an analogue, modifications of internucleotide linkages such as uncharged linkages (e.g., methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.), charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.), pendant moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelating agents, alkylating agents, and modified linkages (e.g., alpha-anomeric nucleic acids, etc.). Reference to a nucleic acid sequence includes its complementary sequence unless otherwise indicated. Thus, reference to a nucleic acid molecule with a particular sequence should be understood to include its complementary strand with its complementary sequence. The complementary strand is also applicable, for example, to antisense therapeutics, hybridization probes and PCR primers.

The amino acid numbering of HA as used herein is based on the H3 numbering described in Winter et al. (Nature 292: 72-75, 1981). Thus, the numbering of amino acid residues or amino acid positions in the polypeptides of the present invention corresponds to the amino acid numbering of H3 of HA (in particular, the amino acid position numbering of HA from A/Aichi/2/68), as described and shown in Fig. 2 in Winter et al. (1981)). The numbering corresponds in particular to the amino acid position numbering in SEQ ID NO: 1. For example, the phrase amino acid at position 355 refers to the amino acid residue that is at position 355 according to the H3 numbering of Winter et al. (1981), i.e., to the amino acid residue that is at position 355 in SEQ ID NO: 1. It will be understood by one skilled in the art that by means of sequence alignment, equivalent amino acids can be determined in other influenza virus strains and/or subtypes, such as H1, H5 or H7 HA. Thus, it should be noted and one skilled in the art will understand that different HA sequences may be characterized by different numbering systems, such as if additional amino acid residues are added or deleted compared to SEQ ID NO: 1. Thus, it will be understood that if specific amino acid residues are mentioned

- 4 047 998 under its number, the disclosure is not limited to only the amino acids located at exactly that numbered position when counted from the beginning of the given amino acid sequence, but rather the equivalent/corresponding amino acid residue in all possible HA sequences is meant, even if that residue is not at exactly the same numbered position, for example, if the HA sequence is shorter or longer than SEQ ID NO: 1, or has insertions or deletions compared to SEQ ID NO: 1. One skilled in the art will be able to easily determine what the corresponding/equivalent amino acid position is for any of the specific numbered residues mentioned herein, for example by aligning the given HA sequence with SEQ ID NO: 1. Thus, in embodiments in which specific amino acid residues of the HA protein of an influenza virus are mentioned, it should be understood that the present invention is not limited to sequences containing the specified amino acid residue (e.g., the presence of a tryptophan (W) at position 355 and/or isoleucine (I) at position 432 and/or 380) only at exactly the same numbered amino acid positions.

A polypeptide refers to a polymer of amino acids linked by amide bonds, as known to those skilled in the art. The term, as used herein, may refer to a single polypeptide chain linked by covalent amide bonds. The term may also refer to multiple polypeptide chains linked by non-covalent interactions such as ionic contacts, hydrogen bonds, van der Waals contacts, and hydrophobic contacts. Those skilled in the art will appreciate that the term includes polypeptides that have been modified, for example, by post-translational processing such as signal peptide cleavage, disulfide bond formation, glycosylation (e.g., N-linked and O-linked glycosylation), protease cleavage, and lipid modification (e.g., S-palmitoylation).

The term wild type used in this document refers to HA from influenza viruses that circulate in nature.

Detailed description of the invention

Influenza viruses have a significant impact on global public health, causing millions of cases of severe illness each year, thousands of deaths, and significant economic losses. Current trivalent influenza vaccines induce strong neutralizing antibody responses to vaccine strains and closely related isolates, but these responses rarely extend to more diverse strains within a subtype or to other subtypes. Furthermore, selecting appropriate vaccine strains is complex and often results in suboptimal protection. Furthermore, predicting the subtype of the next pandemic virus, including when and where it will emerge, is currently impossible.

Hemagglutinin (HA) is the major envelope glycoprotein of influenza A viruses and is the primary target for neutralizing antibodies. Hemagglutinin has two major functions during the entry process. First, hemagglutinin mediates attachment of the virus to the surface of target cells through interaction with sialic acid-containing receptors. Second, following viral endocytosis, hemagglutinin subsequently mediates the fusion of the viral and endosomal membranes, releasing the viral genome into the cytoplasm of the target cell.

HA is a trimeric protein containing an ectodomain of approximately 500 amino acids per monomer and contains three identical subunits (monomers), each containing two polypeptides, HA1 and HA2, linked by a disulfide bond. Each monomer is initially expressed as HA0 and is subsequently cleaved by host proteases into the HA1 and HA2 domains, which are linked by the same disulfide bond.

The majority of the N-terminal domain (HA1 domain, approximately 320-330 amino acids in length) forms a membrane-distal globular domain (head domain) that contains the receptor-binding site and most of the epitopes recognized by virus-neutralizing antibodies. The smaller C-terminal domain (HA2 domain, ~180 amino acids in length) forms a stalk-like structure (stalk domain) that anchors the globular domain to the cellular or viral membrane. One of the most highly conserved regions is the sequence near the cleavage site, specifically the 23 N-terminal amino acids of HA2 (the fusion peptide), which are conserved among all influenza A virus subtypes. Part of this region is exposed as a surface loop in the HA precursor molecule (HA0) but becomes inaccessible after HA0 is cleaved into HA1 and HA2.

As noted above, the influenza virus HA protein is the major protein found on the surface of the virus. The HA found on the virion surface is in a trimeric form. The trimer is anchored to the viral membrane by transmembrane sequences at the carboxyl terminus of each of the three monomers. The major protective efficacy of influenza vaccines is due to hemagglutinin antibodies directed against the HA protein. This highlights the importance of inducing an immune response to conformationally relevant HA proteins.

To obtain soluble polypeptides characterized by the presence of a hemagglutinin ectodomain

- 5 047998 influenza A virus (HA0) HA must be expressed without its native transmembrane and cytoplasmic domain. Expression of stable, trimeric, soluble wild-type (WT) HA is often very low in mammalian cells. To improve at least the level of trimerization, a heterologous trimerization domain (e.g., a trimerization assembly domain; Stevens et al. Science 303(5665):1866–1870, 2004) is often fused to the C-terminus of the polypeptide by genetic engineering. Unfortunately, addition of a heterologous trimerization domain introduces an undesirable neoepitope and often reduces the level of expression or may alter the quaternary structure of the polypeptide.

The present invention provides stable recombinant influenza A virus hemagglutinin (HA) polypeptides comprising the HA1 and HA2 domains of influenza A virus HA and comprising an amino acid sequence wherein:

(a) the amino acid at position 355 is W, and (b) the amino acid at position 432 is I, and/or the amino acid at position 380 is I, and wherein the numbering of the amino acid positions in the amino acid sequence of the HA polypeptide is represented according to the numbering of the amino acids in the amino acid sequence of HA from a reference strain of the H3N2 influenza virus, in particular the reference strain H3N2 A/Aichi/2/68 (SEQ ID NO: 1).

In accordance with the present invention, it has been discovered that stable recombinant HA polypeptides, in particular soluble trimeric HA polypeptides, can be obtained without the addition of an assembly domain or any other heterologous trimerization domains in the presence of specific amino acid mutations in the backbone of the HA polypeptide.

In certain aspects, the present invention provides recombinant influenza A virus hemagglutinin (HA) polypeptides comprising the HA1 and HA2 domains of influenza A virus HA and comprising an amino acid sequence wherein:

(a) the amino acid at position 355 is mutated to a W substitution type, and (b) the amino acid at position 432 is mutated to an I substitution type, and/or the amino acid at position 380 is mutated to an I substitution type, and wherein the numbering of the amino acid positions in the amino acid sequence of the HA polypeptide is represented according to the numbering of the amino acids in the amino acid sequence of HA from a reference strain of the H3N2 influenza virus, in particular the reference strain H3N2 A/Aichi/2/68 (SEQ ID NO: 1). Since the mutations are silent mutations, i.e. the side chains of these residues are not exposed on the surface of the protein, the antigenicity of the HA polypeptides will not change.

In certain embodiments, the polypeptides comprise a mutation of the amino acid substitution type at position 355, in particular histidine (H), to tryptophan (W), and a mutation of the amino acid substitution type at positions 432 and/or 380 to isoleucine (I).

The HA polypeptides of the present invention comprising an amino acid residue W at position 355, such as by introducing a mutation of the amino acid substitution at position 355, in particular H, to W; in combination with an amino acid I at position 432, such as by introducing a mutation of the amino acid substitution at position 432 to I, or comprising a combination of I at position 432 and I at position 380, such as by introducing a mutation of the substitution at positions 432 and 380 to I, are characterized by an increased expression level in mammalian cells, an increased propensity for trimerization (e.g. as measured by AlphaLISA, Octet and SEC) and/or an increased level of thermal stability (e.g. as measured by dynamic scanning fluorimetry/calorimetry (DSF/DSC)) compared to HA polypeptides without these amino acid mutations. In addition, the binding strength of all the antibodies tested to the polypeptides of the present invention is less than 5 nM (as measured by Octet and ELISA). This clearly demonstrates that the polypeptides are structurally equivalent (in terms of primary, secondary, tertiary and quaternary structure) to native wild-type HA. In addition, the novel HA polypeptides do not require the presence of any artificial (heterologous) sequences, such as linker sequences, tag sequences or trimerization domain sequences.

In certain embodiments, the polypeptides comprise a mutation of the amino acid substitution type at position 355, in particular histidine (H), to tryptophan (W), and a mutation of the amino acid substitution type at positions 432 and/or 380 to isoleucine (I).

In certain embodiments, HA polypeptides comprise an amino acid sequence wherein:

(a) the amino acid at position 388 is M, and/or (b) the amino acid at position 478 is I.

These mutations, at least in certain HA subtypes, have been shown to further increase the stability of HA polypeptides.

In certain embodiments, said HA monomers do not contain a cleavage site.

- 6 047998 protease cleavage site. As described above, cleavage of the influenza virus HA0 protein (into HA1 and HA2) is required for its activity, facilitating entry of the viral genome into target cells by causing fusion of the host endosomal membrane with the viral membrane. In certain embodiments, the polypeptides of the present invention comprise a natural protease cleavage site. Thus, the Arg(R)-Gly(G) sequence spanning HA1 and HA2 (i.e., amino acid positions 329 and 330) is known to be a recognition site for trypsin and trypsin-like proteases and is typically cleaved to activate hemagglutinin (Fig. 1A). In certain embodiments, the protease cleavage site has been removed by a mutation to substituting an amino acid residue at position 329 with any amino acid other than arginine (R) or lysine (K). In certain embodiments, the amino acid residue at position 329 is not arginine (R). In a preferred embodiment, the polypeptides comprise a mutation to substituting an amino acid at position 329 with glutamine (Q). Thus, in certain embodiments, the polypeptides of the invention comprise a R329Q knockout mutation at the cleavage site to prevent the intended cleavage of the molecule during or after in vitro production or in vivo after administration. A knockout mutation at the cleavage site, such as an R329Q mutation, thus provides insensitivity to conformational changes induced by low pH and preserves the HA conformation prior to fusion.

According to the present invention, the HA1 and/or HA2 domain may comprise the entire (i.e. full-length) HA1 and/or HA2 domain of an influenza virus HA polypeptide, or they may comprise at least a portion of the HA1 and/or HA2 domain.

To produce secreted (soluble) HA polypeptides, in certain embodiments, the HA monomers comprise a truncated HA2 domain. Thus, in certain embodiments, the HA monomers in the polypeptides of the present invention do not comprise a transmembrane and cytoplasmic domain. In particular, in certain embodiments, the polypeptide monomers comprise an HA2 domain that is truncated at the C-terminus. Thus, the truncated HA2 domain of the present invention is shorter than the full-length HA2 sequence by deleting one or more amino acid residues at the C-terminus and/or N-terminus of the HA2 domain. Thus, the present invention further provides recombinant HA polypeptides comprising or consisting of an extracellular domain of HA (ectodomain, ECD).

In certain embodiments, the C-terminal portion of the HA2 domain, beginning at the amino acid corresponding to amino acid position 515, has been removed, thereby removing substantially the entire transmembrane and cytoplasmic domain.

In certain embodiments, one or more amino acids at the C-terminus of the ectodomain have also been deleted. According to the present invention, it has been found that even with the deletion of a large portion of the HA2 domain, stable soluble and trimeric HA polypeptides can be obtained. Thus, in certain embodiments, the C-terminal portion of the HA2 domain, beginning with the amino acid sequence at position 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, or 514 (according to the H3 numbering described in Winter et al., supra), has been deleted to produce a soluble polypeptide upon expression in cells.

Similarly, the HA1 domain may be the entire HA1 domain (i.e., the full-length HA1 domain) or at least a portion thereof. In a particular embodiment, the polypeptides comprise a truncated HA1 domain. The HA1 domain may be truncated at the N- and/or C-terminus of the HA1 domain.

In certain embodiments, the HA polypeptides do not contain a signal sequence. A signal sequence (sometimes referred to as a signal peptide, targeting signal, localization signal, localization sequence, transit peptide, leader sequence, or leader peptide) is a short peptide (usually 1630 amino acids in length) that is present at the N-terminus of most newly synthesized proteins that are targeted to the secretory pathway. Signal sequences function by causing the protein to move within the cell, typically to the cell membrane. In many cases, the amino acids that comprise the signal peptide are cleaved from the protein after it reaches its final destination. In influenza virus HA, signal sequences typically comprise the first 16 amino acids of the amino acid sequence of full-length HA0 (corresponding to amino acids from position -6 to position 10 according to the H3 numbering).

The present invention also provides immunogenic fragments of HA polypeptides. In certain embodiments, at least a portion of the HA1 domain comprising the head domain may be deleted to produce immunogenic fragments of the HA polypeptides of the present invention, such as HA polypeptides lacking the head domain (i.e., stalk-only polypeptides).

The polypeptides of the present invention are (or are derived from) the hemagglutinin (HA) of influenza A viruses. As described above, influenza A virus contains several HA subtypes, which can be divided into two main groups: group 1 and group 2 (Fig. 2A). Stabilizing mutations in the polypeptides of the present invention can be applied to all types of hemagglutinins of influenza A viruses.

- 7 047998 Russian flu A.

In certain embodiments, the HA1 and HA2 domains are from a group 1 or group 2 influenza A virus. In certain embodiments, the HA1 and HA2 domains are from the same group 1 or group 2 virus. In certain other embodiments, the HA1 and HA2 domains are from different group 1 viruses or from different group 2 viruses, or the HA1 and HA2 domains are from influenza A viruses from different groups, such that the HA2 domain is from a group 1 virus and the HA2 domain is from a group 2 virus, or vice versa. In certain embodiments, the head domain (i.e., at least the portion of the HA1 domain that forms the head domain) is from a different influenza virus than the stalk domain (i.e., the portion of the HA2 domain that forms the stalk domain of an influenza virus HA polypeptide).

In certain specific embodiments, the HA1 and/or HA2 domains are from an influenza A virus selected from the group consisting of an influenza virus comprising an HA subtype H1, such as an influenza A/California/07/2009 or A/Michigan/45/2015 virus; an influenza virus comprising an HA subtype H2, such as an influenza A/Env/MPU3156/2005 virus; an influenza virus comprising an HA subtype H5, such as an influenza A/Eurasian Wigeon/MPF461/2007 virus; an influenza virus comprising an HA subtype H9, such as an influenza A/Hong Kong/1073/1999 virus; an influenza virus comprising an HA subtype H3, such as an influenza H/Hong Kong/1/1968 or A/Panama/2007/1999 virus; an influenza virus comprising an HA subtype H14, such as from the influenza A/Mallard/Astrakhan/263/1982 virus; an influenza virus comprising an HA subtype H7, such as from the influenza A/Mallard/Netherlands/12/2000 virus, and an influenza virus comprising an HA subtype H10, such as from the influenza A/Chicken/Germany/N/1949 virus. It will be clear to one skilled in the art that the polypeptides of the present invention may also be derived from the HA of other influenza A virus strains from either group 1 or group 2.

In certain preferred embodiments, depending on the HA subtype (i.e., group 1 or group 2), the HA polypeptides or immunogenic fragments thereof bind to a CR9114, CR6261, CR8020 and/or MD3606 binding molecule. Thus, novel HA polypeptides are provided that are characterized by the presence of specific epitopes for the CR6261 antibody (comprising the heavy chain variable region of SEQ ID NO: 2 and the light chain variable region of SEQ ID NO: 3), and/or the CR9114 antibody (comprising the heavy chain variable region of SEQ ID NO: 6 and the light chain variable region of SEQ ID NO: 7), and/or the CR8020 antibody (comprising the heavy chain variable region of SEQ ID NO: 4 and the light chain variable region of SEQ ID NO: 5), and/or the multidomain antibody MD3606 (SEQ ID NO: 8). The polypeptides of the present invention can be used to generate influenza virus neutralizing antibodies when administered in vivo alone or in combination with other prophylactic and/or therapeutic treatments.

In certain embodiments, the HA polypeptides of the present invention or immunogenic fragments thereof are associated with nanoparticles, such as, for example, polymers, liposomes, virosomes, virus-like particles, or self-assembling nanoparticles. The polypeptides can be combined with, encapsidated in, or conjugated to (e.g., covalently linked to, or adsorbed onto) nanoparticles.

The present invention further provides multimeric polypeptides comprising at least two of the HA polypeptides or immunogenic fragments thereof described above.

In certain preferred embodiments, the multimeric polypeptides are trimeric and comprise three HA polypeptides or immunogenic fragments thereof described above.

Thus, in certain embodiments, the present invention provides stabilized recombinant stabilized trimeric influenza A virus hemagglutinin (HA) polypeptides or immunogenic fragments thereof, wherein said polypeptides comprise three HA monomers, each of said HA monomers comprises an HA1 domain and an HA2 domain of influenza A virus HA and comprises an amino acid sequence wherein:

(a) the amino acid at position 355 is W, and (b) the amino acid at position 432 is I, or the amino acid at position 432 is I and the amino acid at position 380 is I;

and wherein the numbering of the amino acid positions in the amino acid sequence of the HA polypeptide is represented according to the numbering of the amino acids in the amino acid sequence of HA from a reference strain of the H3N2 influenza virus, in particular the reference strain H3N2 A/Aichi/2/68 (SEQ ID NO: 1).

As noted above, it has been shown in accordance with the present invention that both the expression levels and the trimerization of stable HA trimers can be increased by having an amino acid residue W at position 355, such as by introducing a mutation of the amino acid substitution at position 355 to W; in combination with an amino acid I at position 432, such as by introducing a mutation of the amino acid substitution at position 432 to I; or by having a combination of I at position 432 and I at position 380, such as by introducing a mutation of the amino acid substitution at positions 432 and 380 to I. Thus, the polypeptides of the present invention are characterized by an increased

- 8 047998 expression level in mammalian cells, increased propensity to trimerization (e.g., as measured by AlphaLISA, Octet, and SEC) and/or increased thermal stability (e.g., as measured by dynamic scanning fluorimetry/calorimetry (DSF/DSC)) compared to HA polypeptides without these amino acid mutations.

In a specific embodiment, the HA polypeptides of the present invention are stable for at least 3 days at 40°C.

The present invention further provides nucleic acid molecules encoding the influenza HA polypeptides or immunogenic fragments thereof of the present invention. It will be appreciated by one skilled in the art that several different nucleic acid molecules may encode the same polypeptide due to the degeneracy of the genetic code. It will also be appreciated by one skilled in the art that nucleotide substitutions that do not affect the polypeptide sequence encoded by the disclosed polynucleotides can be made using standard techniques to reflect the codon usage frequency of any particular host organism in which the polypeptides are to be expressed. Therefore, unless otherwise indicated, the expression "a nucleic acid molecule encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.

In certain embodiments, nucleic acid molecules encoding influenza virus HA polypeptides or immunogenic fragments thereof are codon-optimized for expression in mammalian cells, such as human cells. Methods for codon optimization are known and have been described previously (e.g., WO 96/09378).

The present invention further provides methods for producing a recombinant HA polypeptide or immunogenic fragment thereof as defined above, comprising expressing the nucleic acid molecule described above in prokaryotic (e.g., E. coli) or eukaryotic cells (e.g., mammalian cells such as CHO or PER.C6), wherein said method optionally comprises a step of purifying/isolating the recombinant HA polypeptide or immunogenic fragment thereof from said cells. Recombinant influenza HA polypeptides or immunogenic fragments thereof can be produced according to any technique deemed suitable by the skilled artisan for producing recombinant polypeptides, including the techniques described herein. Thus, the polypeptides of the present invention can be synthesized as DNA sequences by standard techniques known in the art and cloned and then expressed in vitro or in vivo using suitable restriction enzymes and techniques known in the art. Nucleotide sequences encoding the HA polypeptides of the present invention, or immunogenic fragments thereof, can be synthesized and/or cloned and expressed according to techniques well known to those skilled in the art. See, for example, Sambrook, et al, Molecular Cloning, A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989). The use of recombinant DNA technology to produce influenza vaccines offers several advantages. These include the ability to avoid the steps of adapting and passaging infectious viruses in eggs and to obtain larger amounts of highly purified protein under safe and more strictly controlled conditions. Moreover, there is no need to include a virus inactivation step. Any suitable cloning and expression system can be used to recombinantly produce the HA polypeptides of the present invention.

In preferred embodiments, the polypeptides or immunogenic fragments thereof are produced in mammalian cells. In certain embodiments, the polypeptides are glycosylated when expressed in suitable cells (e.g., mammalian cells). Thus, the polypeptides may contain one or more native and/or introduced (i.e., non-native) glycosylation motifs.

Hemagglutinin sequences can be obtained by standard recombinant techniques known in the art, such as polymerase chain reaction (PCR) or reverse transcriptase PCR, reverse engineering, or DNA can be synthesized. In the case of PCR, primers can be prepared using hemagglutinin nucleotide sequences that are available in publicly available databases. Polynucleotide constructs can be assembled from PCR cassettes and subsequently cloned into a vector containing a selectable marker for replication in a host cell. The recombinant vector can then be introduced into the host cell by injection, transfection, or electroporation, or by other methods (e.g., calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation). Commercial transfection reagents such as lipofectamine (Invitrogen, Carlsbad, CA) are also available.

HA polypeptides or immunogenic fragments thereof can be recovered and isolated/purified from recombinant cell cultures by methods known in the art, including anion exchange and/or cation exchange chromatography, affinity chromatography. For analysis of protein fractions,

- 9 047998 eluted by these separation/purification techniques, techniques such as SDS-PAGE can be used. Such methods are well known to those skilled in the art and will not be described in detail herein. The purified polypeptides can also be analyzed by spectroscopic methods known in the art (e.g., circular dichroism spectroscopy, Fourier transform infrared spectroscopy and NMR spectroscopy or X-ray crystallography) to examine the presence of desired structures such as helices and beta sheets. ELISA, AlphaLISA, biolayer interferometry (Octet) and FACS and the like can be used to examine the binding of the polypeptides of the present invention to broadly neutralizing antibodies such as CR6261 and/or CR9114. In this way, polypeptides of the present invention having the desired conformation can be selected. Trimer content can be analyzed, for example, using SDS gel electrophoresis under non-reducing conditions, size exclusion chromatography in the presence of Fab fragments of antibodies from broadly neutralizing antibodies such as CR6261 and/or CR9114, and AlphaLISA using differently labeled antibodies. The stability of the polypeptides can be assessed as described above after temperature stress, freeze-thaw cycles, increasing protein concentration, or shaking. The melting temperature of the polypeptide can be additionally assessed by differential scanning fluorimetry (DSF).

In some embodiments, the present invention provides recombinant influenza virus HA polypeptides that are derived from any of the influenza virus HA amino acid sequences selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52 or any variants or fragments thereof that are characterized by at least about 40% or 50% or 60% or 65% or 70% or 75% or 80% or 85% or 90% or 95% or 98% or 99% identity to such amino acid sequences, or contain them or consist of them, wherein the influenza virus HA polypeptides comprise tryptophan (W) at position 355 and isoleucine (I) at position 432 and/or 380, wherein the amino acid numbering is based on the sequence of SEQ ID NO: 1, or at amino acid positions that correspond to such amino acid positions, for example, as determined by aligning the HA amino acid sequence with SEQ ID NO: 1.

In certain embodiments, the present invention provides recombinant influenza virus HA polypeptides that are derived from amino acid residues 18-518 of SEQ ID NO: 10, amino acid residues 18-518 of SEQ ID NO: 12, amino acid residues 16-514 of SEQ ID NO: 14, amino acid residues 17-516 of SEQ ID NO: 16, amino acid residues 19-512 of SEQ ID NO: 18, amino acid residues 17-521 of SEQ ID NO: 20, amino acid residues 17-521 of SEQ ID NO: 22, amino acid residues 18-523 of SEQ ID NO: 24, amino acid residues 19-515 of SEQ ID NO: 26, amino acid residues 17-515 of SEQ ID NO: 28, amino acid residues 17-521 of SEQ ID NO: 33, amino acids 18-518 of SEQ ID NO: 34, amino acids 18-518 of SEQ ID NO: 35, amino acids 18517 of SEQ ID NO: 36, amino acids 18-518 of SEQ ID NO: 38, amino acids 17-521 of SEQ ID NO: 40, amino acids 17-521 of SEQ ID NO: 42, amino acids 17-521 of SEQ ID NO: 44, amino acids 17-519 of SEQ ID NO: 47, amino acids 17-521 of SEQ ID NO: 50, amino acids 19-515 of SEQ ID NO: 51, or amino acids 17-514 of SEQ ID NO: 52, or comprise or consist of them.

In certain embodiments, the HA polypeptides comprise an amino acid sequence derived from, or comprising or consisting of, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52.

The present invention further relates to vectors comprising a nucleic acid molecule encoding an HA polypeptide of the present invention or an immunogenic fragment thereof.

In certain embodiments, the vector is a recombinant human adenovirus. Thus, the present invention also provides recombinant adenoviral vectors comprising a nucleic acid molecule encoding an HA polypeptide or an immunogenic fragment thereof according to the present invention. Recombinant adenoviral vectors can encode membrane-bound HA and thus encode HA polypeptides comprising an HA2 domain comprising a transmembrane and cytoplasmic domain. An adenovector can also encode soluble polypeptides and thus encode HA polypeptides comprising a truncated HA2 domain.

The production of recombinant adenoviral vectors is well known in the art. The term recombinant as used herein in relation to an adenovirus means that it has been modified by man, e.g. it contains altered termini actively cloned into it, and/or it contains a heterologous gene, i.e. it is not naturally occurring.

- 10 047998 wild-type adenovirus. In certain embodiments, an adenoviral vector of the present invention is deficient in at least one essential functional gene of the E1 region, such as the E1a region and/or the E1b region of the adenoviral genome, that is required for viral replication. In certain embodiments, an adenoviral vector of the present invention is deficient in at least a portion of the non-essential E3 region. In certain embodiments, the vector is deficient in at least one essential functional gene of the E1 region and at least a portion of the non-essential E3 region. An adenoviral vector may be multiple-deficient, meaning that the adenoviral vector is deficient in one or more essential functional genes in each of two or more regions of the adenoviral genome. For example, the above-mentioned E1-deficient or E1 and E3-deficient adenoviral vectors may additionally be deficient in at least one essential gene of the E4 region and/or at least one essential gene of the E2 region (e.g., the E2A region and/or the E2B region). Adenoviral vectors, methods for constructing them, and methods for propagating them are well known in the art and are described, for example, in U.S. Patent Nos. 5,559,099, 5,837,511, 5,846,782, 5,851,806, 5,994,106, 5,994,128, 5,965,541, 5,981,225, 6,040,174, 6,020,191, and 6,113,913.

In certain embodiments, the adenovirus is human adenovirus serotype 26.

The present invention further provides immunogenic compositions comprising an HA polypeptide, an immunogenic fragment thereof, a nucleic acid and/or a vector according to the present invention and a pharmaceutically acceptable carrier. In particular, the present invention relates to pharmaceutical compositions comprising a therapeutically effective amount of the polypeptides, immunogenic fragments, nucleic acids and/or vectors of the present invention. The pharmaceutical compositions further comprise a pharmaceutically acceptable carrier. In the context of the present invention, the term pharmaceutically acceptable means that the carrier, at the doses and concentrations used, will not cause undesirable or harmful effects in subjects to whom it is administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., Mack Publishing Company [1990]; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press [2000]). The term carrier refers to a diluent, excipient, or vehicle with which the polypeptides, nucleic acids, and/or vectors are administered. Saline solutions and aqueous dextrose and glycerol solutions can, for example, be used as liquid carriers, particularly for injection solutions.

The present invention further relates to the HA polypeptides, immunogenic fragments, nucleic acids and/or vectors described herein for use as a medicament. The present invention relates in particular to the HA polypeptides, nucleic acids and/or vectors described herein for use in inducing an immune response, preferably comprising inducing the production of neutralizing antibodies against an influenza virus, in particular against an HA molecule of an influenza virus. In a preferred embodiment, the present invention relates to the HA polypeptides, immunogenic fragment, nucleic acids and/or vectors described herein for use as an influenza vaccine.

The present invention also relates to methods for inducing an immune response, in particular to methods for inducing the production of antibodies against an influenza A virus in a subject in need thereof, wherein the method comprises administering to said subject an HA polypeptide, an immunogenic fragment, a nucleic acid molecule and/or a vector as described above. The subject according to the present invention is preferably a mammal that may be infected with an influenza virus or may otherwise benefit from the induction of an immune response to an influenza virus, wherein such a subject is, for example, a rodent, such as a mouse, a ferret, or a domestic or farm animal, or a non-human primate, or a human. Preferably, the subject is a human subject, such as a person identified as being at risk of infection with an influenza disease.

In certain embodiments, the HA polypeptides, immunogenic fragments, nucleic acid molecules, and/or vectors of the invention are administered in combination with an adjuvant. The adjuvant can be administered prior to, concurrently with, or subsequent to administration of the polypeptides, nucleic acid molecules, and/or vectors of the invention. Examples of suitable adjuvants include aluminum salts, such as aluminum hydroxide and/or aluminum phosphate; oil emulsion (or oil-in-water) compositions, including squalene in water emulsions, such as MF59 (see, e.g., WO 90/14837); saponin-based formulations, such as, e.g., QS21 and immunostimulatory complexes (ISCOMs) (see, e.g., US 5,057,540; WO 90/03184, WO 96/11711,

- 11 047998

WO 2004/004762, WO 2005/002620); derivatives produced by bacteria or microorganisms, examples of which are monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), oligonucleotides containing a CpG motif, ADP-ribosylated bacterial toxins or mutant forms thereof, such as E. coli heat-labile enterotoxin LT, cholera toxin CT, pertussis toxin PT or tetanus toxoid TT, Matrix M or combinations thereof. In addition, known immunostimulatory technologies can be used, such as fusing the polypeptides of the present invention with proteins known in the art to enhance the immune response (e.g. tetanus toxoid, CRM197, rCTB, bacterial flagellins or others), or incorporation of the polypeptides into virosomes, or combinations thereof. Genetic adjuvants that are co-delivered or co-encoded, for example, by the same adenovector, can also be used.

Administration of the HA polypeptides, immunogenic fragments, nucleic acid molecules and/or vectors according to the present invention can be carried out using standard routes of administration. Non-limiting examples include parenteral administration, such as intravenous, intradermal, transdermal, intramuscular, subcutaneous, etc., or mucosal administration, such as intranasal, oral, etc. One skilled in the art will be able to determine various possibilities for administration of the polypeptides, nucleic acid molecules and/or vectors according to the present invention for the purpose of inducing an immune response.

Furthermore, the present invention provides methods for preventing and/or treating, preferably preventing, a disease caused by an influenza virus in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of an HA polypeptide, immunogenic fragment, nucleic acid molecule and/or vector described herein.

A therapeutically effective amount refers to an amount of a polypeptide, immunogenic fragment, nucleic acid and/or vector that is effective to prevent, alleviate and/or treat a disease or condition associated with influenza virus infection. Prevention includes suppression or reduction in the spread of influenza virus or suppression or attenuation of the onset, development or progression of one or more symptoms associated with influenza virus infection. The term alleviation, as used herein, may refer to a reduction in the manifestation of visible or perceptible symptoms of disease, viremia or any other measurable manifestation of infection caused by influenza virus.

A subject in need of treatment includes subjects who already have a condition caused by an influenza virus infection, as well as those who need to be prevented from becoming infected with an influenza virus. The polypeptides, immunogenic fragments, nucleic acids and/or vectors of the present invention can thus be administered to a subject that has not previously undergone treatment, i.e. a subject that does not have a disease caused by an influenza virus infection or that has not been infected and is not currently infected with an influenza virus infection, or to subjects that have already been infected with an influenza virus.

In one embodiment, prevention and/or treatment may be targeted at patient groups that are susceptible to influenza virus infection. Such patient groups include, but are not limited to, the elderly (e.g., aged >50 years, aged >60 years, and preferably aged >65 years), young (e.g., aged <5 years, aged <1 year), hospitalized patients, immunocompromised subjects, and patients who have been treated with an antiviral compound but have had an inadequate antiviral response.

The polypeptides, immunogenic fragments, nucleic acid molecules, and/or vectors of the present invention may be administered to a subject in combination with one or more other active agents, such as alternative influenza vaccines, monoclonal antibodies, antiviral agents, antibacterial agents, and/or immunomodulatory agents. The one or more other active agents may be useful in the treatment and/or prevention of disease caused by an influenza virus, or may alleviate a symptom or condition associated with disease caused by an influenza virus. In some embodiments, the one or more other active agents are analgesics, antipyretic drugs, or therapeutic agents that facilitate or promote breathing.

The HA polypeptides of the present invention or fragments thereof can also be used as research tools, as diagnostic tools, or as targets for the production of antibody reagents or therapeutic antibodies. For example, in some embodiments, the HA polypeptides can be useful as analytes for analyzing and/or measuring the extent of binding and/or titers of antibodies to HA, such as in ELISA assays, Biacore/SPR binding assays, and/or any other antibody binding assays known in the art. As another example, the HA polypeptides of the present invention can be used

- 12 047998 for analysis and/or comparison of the effectiveness of antibodies to HA.

The HA polypeptides of the present invention or fragments thereof may also be useful for producing therapeutic antibodies and/or antibodies that can be used as research tools or for any other desired application. For example, the HA polypeptides of the present invention may be used to immunize non-human animals to produce antibodies to the HA protein for use as research tools and/or as therapeutic agents. Such antibodies, which may be monoclonal or polyclonal, and/or cells that produce such antibodies may then be obtained from the animal.

The polypeptides of the present invention for use as a diagnostic tool may comprise a label applicable to any detection technique suitable for the assay. The label used will depend on the particular detection/assay/diagnostic techniques and/or methods used. The methods may be carried out in solution, or the polypeptide(s) of the present invention may be linked to or attached to a carrier or support, such as microtiter plates (e.g., for ELISA), membranes and beads, etc.

The present invention is further illustrated by the following examples and figures. The examples are not intended to limit the scope of the present invention in any way.

Examples

Example 1. Soluble HA polypeptides - structural and schematic elements of polypeptides according to the present invention

To produce soluble polypeptides characterized by the presence of the influenza A hemagglutinin ectodomain (HA0), HA must be expressed without its native transmembrane and cytoplasmic domain. Expression of stable, trimeric, soluble wild-type (WT) HA is often very low in mammalian cells. To improve at least the level of trimerization, the trimerization assembly domain is often fused to the C-terminus of the polypeptide by genetic engineering. Addition of the assembly domain introduces an undesirable neoepitope and often reduces the level of expression or may alter the structure of the polypeptide. According to the present invention, it has been found that the expression and trimerization levels of soluble stable HA trimers can be increased without adding an assembly domain or any other non-natural trimerization sequences by introducing specific amino acid mutations into the main part of the HA polypeptide, in particular at amino acid positions 355 and 432 or at amino acid positions 355, 380 and 432. It should be noted that the numbering of the amino acid positions in the HA monomers of the present invention uses the H3 numbering of Winter et al. 1981 (supra). Thus, the numbering of the amino acid positions in the HA polypeptide monomers of the present invention corresponds to the numbering of the amino acid positions in HA from an H3N2 reference strain of influenza virus, in particular the H3N2 reference strain A/Aichi/2/68 (characterized by the amino acid sequence of SEQ ID NO: 1).

The main structural elements and positions of key mutations according to the present invention are shown in Fig. 1A in HA from the H1 A/California/07/2009 strain of influenza A virus (Fig. 1A). As shown, the HA monomer contains a truncated HA2 domain (the HA2 domain, in particular, was truncated after amino acid position 514 (i.e., the C-terminal portion of the HA2 domain, starting at amino acid position 515, was deleted) with deletion of the transmembrane and cytoplasmic domain and yielding a soluble HA ectodomain (Fig. 1B).

The polypeptides of the present invention can be made resistant to protease cleavage by mutation such as a substitution of the amino acid arginine (R) from the natural monobasic cleavage site at position 329 (Fig. 1B) to, for example, glutamine (Q). Unlike native full-length HA, polypeptides containing the R329Q mutation cannot be cleaved by serine proteases (e.g., trypsin). Cleavage of HA allows the protein to undergo a conformational change necessary for membrane fusion and viral entry.

Example 2. Expression of soluble stabilized HA compared to wild-type HA in different subtypes

In this example, several HAs from both group 1 and group 2 influenza viruses were selected and expressed as stabilized soluble trimeric HA polypeptides and compared with their corresponding soluble wild-type HA ectodomains (i.e., lacking the transmembrane and intracytoplasmic domains). According to the present invention, tryptophan (W) at position 355 and isoleucine (I) at positions 380 and 432 were introduced into the amino acid sequences of HA from five different group 1 strains and five different group 2 strains, including the eight most circulating subtypes in humans (Fig. 2A), if these amino acids were not already present in the HA amino acid sequence. In addition, methionine (M) was introduced into the upper part of the A-helix at position 388 in some polypeptides. Position 478 was either inserted with isoleucine or left unaltered if it was already present in the WT sequences, except for the polypeptide derived from A/Mallard/Netherlands/12/200 (UFV181146) and A/Chicken/Germany/N/1949 (UFV181147).

- 13 047998

The expression and trimerization levels of the polypeptides of the present invention in the supernatant of Expi293F culture were compared with the corresponding soluble WT polypeptides without the mutations of the present invention.

Table 1 shows the polypeptides according to the present invention that were obtained.

Table 1

Polypeptides of the present invention

Polypeptide (SEQ ID NO) 355W 3801 388M 4321 4781 UFV181009 (10) + + + + + UFV181091 (12) + + + + + UFV181154 (14) + + + + + UFV181159 (16) + + + + + UFV181156 (18) + + + + + UFV180660 (20) + + + + UFV181096 (22) + + + + UFV 180661 (24) + + + + + UFV 180664 (26) + + + + UFV 180662 (28) + + + +

+ indicates the presence of the indicated amino acid at the indicated position; an empty cell indicates the absence of the indicated amino acid (i.e., the presence of a wild-type amino acid residue).

DNA fragments encoding the polypeptides shown in Fig. 2 and Table 1 were synthesized (GenScript) and cloned into the expression vector pcDNA2004 (a modified pcDNA3 plasmid with an enhanced CMV promoter). The polypeptides of the invention contained a C-terminal portion of a linker-sortα3-linker-His-tag for site-specific biotinylation, screening and purification purposes and were produced on a microscale (200 μl) in suspension in the eukaryotic cell line Expi293F. Full-length (FL) wild-type (WT) HA polypeptides contained a linker-His-tag for screening purposes.

Cells were transiently transfected with industrial grade DNA (endotoxin level <0.01 EU/µg and supercoil content >90%) in 96-well half-well plates (System Duetz) at a cell density of 2.5E+06 vc/ml using the ExpiFectamine 293 Transfection Kit (Gibco, ThermoFisher Scientific) and incubated in shake flasks containing Expi293 Expression Medium (Gibco, ThermoFisher Scientific) at 37°C, 250 rpm, 8% _CO2 , and 75% humidity. Cell culture supernatant samples containing secreted polypeptides were collected on day 3 and clarified by centrifugation (10 min at 400xg) followed by filtration (96-well filter plates, 0.22 μm PVDF membrane, Corning).

The level of expressed soluble HA polypeptide in the harvested culture supernatant was assessed by biolayer interferometry using the OCTET platform (ForteBio). Briefly, a standard curve was generated using anti-HIS (HIS2) antibody biosensors (ForteBio) by measuring the binding shift in a dilution series of a strictly defined reference lot of purified UFV180436 polypeptide. Binding shifts were then measured for pre-

- 14,047,998 thoroughly diluted (in kinetic assay buffer, ForteBio) cell culture supernatant samples containing the polypeptides of the present invention were prepared and the concentration of the polypeptides was calculated using the constructed standard curve.

The presence of expressed polypeptides and their quaternary structure (which indicates whether the polypeptide is a monomer, trimer or multimer) in harvested Expi293F cell culture samples were assessed by analytical size exclusion chromatography (SEC) using ultra-high performance liquid chromatography (UHPLC) using a Vanquish system (ThermoFisher Scientific) with a BEH 200A column (Waters, injection volume 40 μl, flow rate 0.35 ml/min). Elution was monitored using a Helios light scattering detector (Wyatt Technologies). SEC profiles were analyzed using the Astra 6 software package (Wyatt Technology).

Results and conclusion.

Introduction of tryptophan at position 355 and isoleucine at positions 380 and/or 432 in wild-type HA from different strains resulted in increased expression of all tested polypeptides of the present invention, as determined by OCTET (Fig. 2B).

SEC analysis of crude cell culture supernatant samples demonstrated that upon introduction of stabilizing mutations into the polypeptides of the invention, a distinct trimer peak (T) appeared for all soluble stabilized HA at a retention time of 6 to 7 min, which was higher than the trimer peaks observed for the corresponding wild-type HA ectodomains (Fig. 2C). It should be noted that the differences in retention times between the different influenza virus HA subtypes are likely due to differences in the level and complexity of glycosylation.

Taken together, these data confirm that introduction of the 355W, 380I and/or 432I mutations into the HA polypeptides of the present invention results in increased expression and formation of stable soluble trimeric HA.

Example 3. In vitro characterization of purified trimeric full-length HA compared to wild-type HA containing the trimerization assembly domain.

To further determine the contribution of the important stabilizing mutations 355W, 380I, and/or 432I, these mutations were introduced into HA ectodomain polypeptides (i.e., excluding the TM and IC domains) obtained from H1 strains A/California/07/2009 (UFV181009), A/Michigan/45/2015 (UFV181091), and H3 strains A/Hong Kong/1/1968 (UFV180660) and A/Indiana/11/2011 (UFV181099) and compared to wild-type (WT) HA ectodomains containing the trimerization assembly domain (except UFV4239 (SEQ ID NO: 29), which did not contain the trimerization assembly domain). The polypeptides contained the amino acids shown in Table 1. 2. All polypeptides additionally contained a His-tag for purification and screening purposes and were produced in ExpiCHO cells, after which they were purified and characterized.

DNA fragments encoding the polypeptides of the present invention were synthesized as described in Example 2. The polypeptides were produced in ExpiCHO cell suspension (350 ml scale) and the cells were cultured in ExpiCHO expression medium by transient transfection with the appropriate commercial grade DNA using ExpiFectamine transfection reagent (Gibco, ThermoFisher Scientific) according to the manufacturer's protocol. ExpiFectamine CHO stimulator and ExpiCHO feed (Gibco, ThermoFisher Scientific) were added to the cell cultures 1 day after transfection according to the manufacturer's protocol. Transfected ExpiCHO cell suspensions were incubated at 32°C, 5% _CO2 and culture supernatant samples containing secreted polypeptides were collected between days 7 and 11. Samples of the culture supernatant were clarified by centrifugation followed by filtration through a 0.2 µm filter inserted into the bottle cap (Corning).

From the pooled culture supernatant samples and from the corresponding wild-type strains, the histidine-tagged polypeptides of the invention containing the trimerization assembly domain were purified in a two-step protocol using the AKTA Avant 25 system (GE Healthcare Life Sciences). First, immobilized metal affinity chromatography was performed using a preloaded cOmplete His-tag Purification Column (Roche), washed with 1 mM imidazole and eluted with 300 mM imidazole. Second, size exclusion chromatography was performed using a HiLoad Superdex 200 pg 26/600 column (GE Healthcare Life Sciences). The trimer peak fractions were pooled, frozen and stored (1 and 6 months) at -80°C.

The trimer content of the purified polypeptides of the present invention was assessed by analytical SEC using ultra-high performance liquid chromatography (UHPLC) as described in Example 2. 20 μg of each purified polypeptide was injected and run through the column.

The thermal stability of the purified polypeptides was determined by differential scanning fluorimetry (DSF) by monitoring the fluorescence emission of the orange dye Sypro (ThermoFisher Scientific) added to a 6 μg polypeptide solution. When the temperature was gradually increased from 25°C to 95°C (60°C per hour), the polypeptides unfolded and the fluorescence

- 15 047998 dye binds to exposed hydrophobic residues, resulting in a characteristic change in emission. Melting curves were measured using a ViiA7 real-time PCR instrument (Applied BioSystems) and Tm ₅₀ values were calculated using the Spotfire software package (Tibco Software Inc.). Tm ₅₀ values represent the temperature at which 50% of the protein is unfolded and are thus a measure of the thermal stability of polypeptides.

The three-dimensional conformation of the purified polypeptides was assessed by antigenicity testing in ELISA (EC ₅₀ values of antibody binding). For this purpose, the polypeptides were coated at a concentration of 10 nM and incubated with a dilution series of monoclonal antibodies (mAbs), namely CR6261 (specific for group 1), CR8020 (specific for group 2), CR9114 (specific for groups 1 and 2) and MD3606 (multidomain antibody specific for groups 1 and 2), using 70 nM as the starting concentration. Antibody binding was determined by incubation with HRP-conjugated anti-human Fc secondary antibody (mouse anti-human IgG, Jackson ImmunoResearch) and visualized by the addition of POD substrate. Readings were performed using an EnSight™ multi-mode plate reader (PerkinElmer). EC ₅₀ values from two independent experiments were calculated using the Spotfire software package (Tibco Software Inc.), and the mean and standard deviation are shown in Fig. 3D.

Results and conclusion.

Table 2

Polypeptides of the present invention

Polypeptides (SEQ Ш NO) 355W 3801 388M 4321 4781 UFV181009 (10) + + + + UFV181091 (12) + + + + UFV180660 (20) + + + + UFV181099 (33) + + +

The results of the SEC analysis confirmed that the presence (or simultaneous introduction of stabilizing) amino acids in the polypeptides of the present invention from influenza virus strains with different HA allows for the purification of highly pure and stable soluble trimeric HA polypeptides. The stabilizing effect of amino acids was best observed in the case of the purified polypeptide derived from H1 A/California/07/2009 (UFV181009), where the corresponding wild-type construct (UFV4239, SEQ ID NO: 29) did not contain the trimerization assembly domain and showed only a peak of monomers, while the stabilized polypeptide of the present invention was characterized by a peak of highly pure trimers (Fig. 3A). Wild-type HA molecules from another H1 strain A/Michigan/45/2015 and H3 strains A/Hong Kong/1/1968 and A/Indiana/11/2011 were expressed with an additional C-terminal assembly domain and indeed formed trimeric HA. However, in contrast to the corresponding stabilized polypeptides of the present invention, the trimeric peaks of wild-type HA with the assembly domain were broader, asymmetrical and characterized by the presence of shoulders, indicating the presence of alternative high-molecular-weight and/or low-molecular-weight polypeptides in an undesired conformation (*) or with a less dense fold (Seok et al., Sci. Rep. 8;7(1)-7540, 2017).

Further characterization of the polypeptides demonstrated that the polypeptides of the present invention containing stabilizing amino acids exhibited significantly higher thermal stability compared to WT polypeptides (UFV4239, SEQ ID NO: 29) with or without the trimerization assembly domain (Figs. 3B and 3C).

The introduced stabilizing mutations are silent mutations (i.e., they are located within the HA polypeptide rather than on its surface) and therefore should not affect the surface of monomeric or trimeric HA. To confirm the integrity of the HA surface, the binding of a panel of well-known broadly neutralizing antibodies to the polypeptides was assessed by ELISA. The wild-type and stabilized polypeptides of the invention exhibited comparable binding with _EC50 values in the low nanomolar range to all antibodies, consistent with their expected binding strength. An improvement (~4-8-fold) was observed in the binding of CR9114 to the HA polypeptides of the invention derived from H3 A/Hong Kong/1/1968 and H3 A/Indiana/11/2011 (Fig. 3D).

In conclusion, it should be noted that the described polypeptides of the present invention were purified from cell culture supernatant as highly pure trimeric polypeptides and they were characterized by improved thermal stability compared to WT HA (with or without the trimerization assembly domain) and were correctly folded.

Example 4. Determination of characteristics of combinations of stabilizing mutations

To assess the beneficial effect of a combination of stabilizing mutations in polypeptides,

- 16 047998 in the present invention, the combination 355W+478I and the combination 380I+432I were stepwise introduced into the ectodomain of HA from the H1 strain A/California/07/2009 (Fig. 4A, '.' indicates the unchanged presence of the wild-type (WT) H1 residue, as indicated in the first row).

DNA fragments encoding the polypeptides of the present invention were synthesized as described in Example 2. Polypeptides containing a C-terminal portion of the linker-sortase-linker-IE-tag for the purposes of site-specific biotinylation, screening and purification were produced on a microscale in Expi293F eukaryotic cells (200 μl) as described in Example 2. The level of expressed polypeptide was determined by OCTET and trimer content was analyzed by analytical SEC as described in Example 2.

Results and conclusion

Evaluation of the expression levels of the polypeptides of the invention with different combinations of stabilizing mutations revealed that the 380I and 432I mutations present in UFV181007 (SEQ ID NO: 35) had no effect on expression, but significantly increased trimer levels compared to the WT construct (Fig. 4B). The addition of the 355W and 478I mutations (e.g., UFV181005: SEQ ID NO: 34) resulted in a marked increase in expression (Fig. 4A), but no trimer formation was observed (Fig. 4B). When using a combination of 355W, 478I, 380I and 4321 (e.g. in UFV181009: SEQ ID NO: 10), the expression level was simultaneously increased (Fig. 4A) and the trimer content in the cell culture supernatant was significantly increased (Fig. 4B).

In conclusion, it should be noted that mutations 355W and 478I increased expression levels of the polypeptides of the present invention, while mutations 380I and 432I increased trimer formation. The combination of stabilizing mutations synergistically increased expression and trimer levels of the polypeptides of the present invention.

Example 5. Expression of additional soluble stabilized HA compared to wild-type HA in different HA subtypes.

In this example, additional stabilized HAs were further expressed and compared to their corresponding soluble wild-type HA ectodomains (Fig. 5A). Tryptophan (W) at position 355 and isoleucine (I) at positions 380 and 432 were introduced into the amino acid sequences of HA from two additional group 1 strains and four additional group 2 strains. Three days after transfection, the expression and trimerization levels of the polypeptides in the Expi293F culture supernatant samples were compared to the corresponding soluble WT polypeptides without the mutations of the invention. Table 4 shows the additional polypeptides of the invention that were obtained.

DNA fragments encoding the polypeptides of the present invention were synthesized as described in Example 2. The plasmids were transfected into Expi293F eukaryotic cells on a microscale (200 μl) as described in Example 2. All polypeptides were expressed with the inclusion of a C-terminal linker-His-tag sequence for screening and purification purposes, while the stabilized polypeptides contained an additional sortase-linker sequence preceding the His-tag for site-specific biotinylation. The level of expressed polypeptide was determined by OCTET and trimer content was analyzed by analytical SEC as described in Example 2.

Results and conclusion.

Similar to the observations in Example 2, the introduction of tryptophan at position 355 and isoleucine at positions 380 and/or 432 in wild-type HA from different strains resulted in increased expression of all of these additional polypeptides as measured by OCTET. One exception was seen in the case of HA derived from H1 A/South Carolina/1/1918 (UFV181084), which showed a slight decrease as determined by OCTET (Fig. 5A) but not as measured by area under the curve in SEC (Fig. 5B).

SEC analysis of crude cell culture supernatants demonstrated that upon introduction of stabilizing mutations, all additional soluble stabilized HAs contained more trimeric polypeptide (T) and less monomeric polypeptide (M) and exhibited high molecular weight species compared to the corresponding wild-type HA ectodomains (Fig. 5B). As noted in Example 2, differences in retention times between influenza virus HA subtypes are likely due to differences in the level and complexity of glycosylation.

Taken together, these data confirm that the introduction of the 355W, 380I and/or 432I mutations into the additional HA polypeptides of the present invention results in increased expression and the formation of stable soluble trimeric HA.

Example 6. In vitro characterization of purified trimeric full-length HA (Supplementary Data).

In this example, additional stabilized HAs were expressed, purified, and subjected to prolonged temperature stress. These HAs, UFV190839 (SEQ ID NO: 50), UFV190068 (SEQ ID NO: 51), and UFV190841 (SEQ ID NO: 52), were obtained from H3 A/Hong Kong/1/1968, H7 A/Mallard/NL/12/2000, and

- 17 047998

H10 A/Chick/Germany/N/1949, respectively. Briefly, the purified trimeric polypeptide was stored for 60 days at 4°C (refrigerated) and 37°C (incubator), after which the protein integrity was assessed by analytical SEC.

According to the present invention, tryptophan (W) at position 355 and isoleucine (I) at positions 380 and 432 were introduced into the amino acid sequences of HA from three different group 2 strains.

DNA fragments encoding the polypeptides of the present invention were synthesized as described in Example 2. The polypeptides were produced in ExpiCHO eukaryotic cells at medium scale (30 ml) as described in Example 3 and harvested on day 5. All polypeptides were expressed with a C-terminal linker-sortα3a-linker-His-tag sequence for site-specific biotinylation, screening and purification. The proteins were purified by a two-step method as described in Example 3, except that in this case a HiLoad Superdex 200 16/600 column (GE Healthcare Life Sciences) was used. The level of expressed polypeptide was determined by OCTET and trimer content was analyzed by analytical SEC as described in Example 2, except that in this case a Unix-C 300 A column (Sepax Technologies) was used.

Results and conclusion

The results of SEC analysis indicated that the polypeptides of the present invention containing stabilizing amino acids obtained after purification were highly pure and trimeric. Moreover, the soluble HA polypeptides were resistant to temperature stress; 60-day incubation at 4°C and 37°C did not affect the protein amount and trimeric state compared to the observed results for the material before stress (Fig. 6), and in the case of HA derived from H10, only a small amount of non-trimeric polypeptide was observed after incubation at 37°C (retention time ~ 4.75 min).

As noted in Example 2, the differences in retention times between the different influenza HA subtypes are likely due to differences in the level and complexity of glycosylation. Additionally, the small differences in retention times observed for the starting material compared to the material stressed for 60 days are likely due to column aging (i.e., a similar shift was observed for the internal control).

In conclusion, it should be noted that the polypeptides of the present invention described in this example were purified from the culture supernatant as highly pure trimeric polypeptides and were characterized by high inertness to temperature stress for 60 days.

Table 3

Standard Amino Acids, Abbreviations and Properties

Amino acid 3 letter code 1 letter code Side chain polarity Side chain charge (pH 7.4) Alanine Ala A Non-polar Neutral Arginine Arg R Polar Positive Asparagine Asn N Polar Neutral Aspartic acid Asp D Polar Negative Cysteine Cys C Non-polar Neutral Glutamic acid Glu E Polar Negative Glutamine Gin Q Polar Neutral Glycine Gly G Non-polar Neutral G istidine His H Polar Positive (10%), neutral (90%) Isoleucine He I Non-polar Neutral Leucine Leu L Non-polar Neutral Lysine Lys TO Polar Positive Methionine Met M Non-polar Neutral Phenylalanine Phe F Non-polar Neutral Proline Pro P Non-polar Neutral Serine Ser S Polar Neutral Threonine Thr T Polar Neutral Tryptophan Trp w Non-polar Neutral Tyrosine Tyr Y Polar Neutral Valin Vai V Non-polar Neutral

- 18 047998

Sequences

SEQ ID NO: 1 - hemagglutinin CAA24269.1 (influenza A virus (A/Aichi/2/1968(H3N2) (without signal sequence))

QDLPGNDNST ATLCLGHHAV PNGTLVKTIT DDQIEVTNAT ELVQSSSTGK 50

ICNNPHRILD GIDCTLIDAL LGDPHCDVFQ NETWDLFVER SKAFSNCYPY 100

DVPDYASLRS LVASSGTLEF ITEGFTWTGV TQNGGSNACK RGPGSGFFSR 150

LNWLTKSGST YPVLNVTMPN NDNFDKLYIW GIHHPSTNQE QTSLYVQASG 200

RVTVSTRRSQ QTIIPNIGSR PWVRGLSSRI SIYWTIVKPG DVLVINSNGN 250

LIAPRGYFKM RTGKSSIMRS DAPIDTCISE CITPNGSIPN DKPFQNVNKI 300

TYGACPKYVK QNTLKLATGM RNVPEKQTRG LFGAIAGFIE NGWEGMIDGW 350

YGFRHQNSEG TGQAADLKST QAAIDQINGK LNRVIEKTNE KFHQIEKEFS 400

EVEGRIQDLE KYVEDTKIDL WSYNAELLVA LENQHTIDLT DSEMNKLFEK 450

TRRQLRENAE EMGNGCFKIY HKCDNACIES IRNGTYDHDV YRDEALNNRF 500

QIKGVELKSG YKDWILWISF AISCFLLCVV LLGFIMWACQ RGNIRCNICI 550

PROTEIN VH CR6261 (SEQ ID NO: 2)

EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIF GTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVW GKGTTVTVSS

PROTEIN VL CR6261 (SEQ ID NO: 3)

QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNDYVSWYQQLPGTAPKLLIYDNNKR PSGIPDRFSGSKSGTSATLGITGLQTGDEANYYCATWDRRPTAYVVFGGGTKLTVL

- 19 047998

PROTEIN VH CR8020 (SEQ ID NO: 4)

QVQLQQSGAEVKTPGASVKVSCKASGYTFTSFGVSWIRQAPGQGLEWIGWISAY NGDTYYAQKFQARVTMTTDTSTTTAYMEMRSLRSDDTAVYYCAREPPLFYSSWSLDN WGQGTLVTVSS

PROTEIN VL CR8020 (SEQ ID NO: 5)

EIVLTQSPGTLLSLSPGERATLSCRASQSVSMNYLAWFQQKPGQAPRLLIYGASRR ATGIPDRISGSGSGTDFTLTISRLEPADFAVYYCQQYGTSPRTFGQGAKVEIK

PROTEIN VH CR9114 (SEQ ID NO: 6)

QVQLVQSGAEVKKPGSSVKVSCKSSGGTSNNYAISWVRQAPGQGLDWMGGISPI FGSTAYAQKFQGRVTISADIFSNTAYMELNSLTSEDTAVYFCARHGNYYYYSGMDVWG QGTTVTVSS

PROTEIN VL CR9114 (SEQ ID NO: 7)

SYVLTQPPAVSGTPGQRVTISCSGSDSNIGRRSVNWYQQFPGTAPKLLIYSNDQRP SVVPDRFSGSKSGTSASLAISGLQSEDEAEYYCAAWDDSLKGAVFGGGTQLTVL

PROTEIN MD3606 (SEQ ID NO: 8)

EVQLVESGGGLVQPGGSLRLSCAVSISIFDIYAMDWYRQAPGKQRDLVATSFRD GSTNYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYLCHVSLYRDPLGVAGGMG VYWGKGALVTVSSGGGGSGGGGSEVQLVESGGGLVQAGGSLKLSCAASGRTYAMGW FRQAPGKEREFVAHINALGTRTYYSDSVKGRFTIS RDNAKNTEYLEMNNLKPEDTAVYY CTAQGQWRAAPVAVAAEYEFWGQGTQVTVSSGGGGSGGGGSEVQLVESGGGLVQPG GSLRLSCAATGFTLENKAIGWFRQTPGSEREGVLCISKSGSWTYYTDSMRGRFTISRDNA ENTVYLQMDSLKPEDTAVYYCATTTAGGGLCWDGTTFSRLASSWGQGTQVTVSSGGG GSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFSTSWMYWLRQAPGKGLEWVSVI NTDGGTYYADSVKDRFTISRDNAKDTLYLQMSSLKSEDTAVYYCAKDWGGPEPTRGQ GTQVTVSSDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K

SEQ ID NO 9: UFV181157 (signal peptide and tag are underlined)

MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKH NGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFID YEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNS YPKLSKSYINDKGKEVLVLWGIHHPSTS ADQQSLYQNADAYVFVGSSRYSKKFKPEIAIR PKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCN TTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNIPSIQSRGLFGAIAGFIEG GWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGK EFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQL KNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGSHHHHHH

SEQ ID NO 10: UFV181009 (signal peptide and tag are underlined)

- 20 047998

MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKH NGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFID YEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNS YPKLSKSYINDKGKEVLVLWGIHHPSTS ADQQSLYQNADAYVFVGSSRYSKKFKPEIAIR PKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCN TTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNIPSIQSRGLFGAIAGFIEG GWTGMVDGWYGYHWQNEQGSGYAADLKSTQNAIDEITNIVNSVIEKMNTQFTAVGKE FNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLINERTLDYHDSNVKNLYEKVRSQLKN NAKEIGNGCFEFYHKCDNTCIESVKNGTYDYPKYSEEAKLNREEIDSGSLPETGGGSHH HHHH

SEQ ID NO 11: UFV181134 (signal peptide and tag are underlined)

MKAILVVLLYTFTTANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKH NGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETSNSDNGTCYPGDFIN YEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNS YPKLNQSYINDKGKEVLVLWGIHH PSTTADQQSLYQNADAYVFVGTSRYSKKFKPEIAT RPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFTMERNAGSGIIISDTPVHDC NTTCQTPEGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIE GGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDKITNKVNSVIEKMNTQFTAVG KEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRNQ LKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREKIDGSHHHHHH

SEQ ID NO 12: UFV181091 (signal peptide and tag are underlined)

MKAILVVLLYTFTTANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKH NGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETSNSDNGTCYPGDFIN YEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNS YPKLNQSYINDKGKEVLVLWGIHH PSTTADQQSLYQNADAYVFVGTSRYSKKFKPEIAT RPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFTMERNAGSGIIISDTPVHDC NTTCQTPEGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIE GGWTGMVDGWYGYHWQNEQGSGYAADLKSTQNAIDKITNIVNSVIEKMNTQFTAVG KEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLINERTLDYHDSNVKNLYEKVRNQ LKNNAKEIGNGCFEFYHKCDNTCIESVKNGTYDYPKYSEEAKLNREKIDSGSLPETGGG SHHHHHH

SEQ ID NO 13: UFV181153 (signal peptide and tag are underlined)

MAIIYLILLFAAVRGDQICIGYHSNNSTEKVDTILERNVTVTHAQDILEKTHNGKL CKLNGIPPLELGDCSIAGWLLGNPECDRLLTVPEWSYIMEKENPRNGLCYPGSFNDYEEL KHLLSSVTHFEKVKILPRDRWTQHTTTGGSRACAVSGNPSFFRNMVWLTKKGSNYPIAK GSYNNTSGEQMLIIWGVHHPNDDAEQRT LYQNVGTYVSVGTSTLNKRSVPEIATRPKV NGQGGRMEFSWTILDMLDTINFESTGNLIAPEYGFRISKRGSSGIMKTEGTLENCETKCQ TPLGAINTTLPFHNIHPLTIGECPKYVKSERLVLATGLRNVPQIESRGLFGAIAGFIEGGW QGMVDGWYGYHHSNDQGSGYAADKESTQRAIDGITNKVNSVIEKMNTQFEAVGKEFN NLEKRLENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRMQLR

- 21 047998

DNAKELGNGCFEFYHKCDDECMNSVKNGTYDYPKYEEESKLNRNEIKGSHHHHHH

SEQ ID NO 14: UFV181154 (signal peptide and tag are underlined)

MAIIYLILLFAAVRGDOICIGYHSNNSTEKVDTILERNVTVTHAQDILEKTHNGKL CKLNGIPPLELGDCSIAGWLLGNPECDRLLTVPEWSYIMEKENPRNGLCYPGSFNDYEEL KHLLSSVTHFEKVKILPRDRWTQHTTTGGSRACAVSGNPSFFRNMVWLTKKGSNYPIAK GSYNNTSGEQMLIIWGVHHPNDDAEQRTLY QNVGTYVSVGTSTLNKRSVPEIATRPKV NGQGGRMEFSWTILDMLDTINFESTGNLIAPEYGFRISKRGSSGIMKTEGTLENCETKCQ TPLGAINTTLPFHNIHPLTIGECPKYVKSERLVLATGLRNVPQIESRGLFGAIAGFIEGGW QGMVDGWYGYHWSNDQGSGYAADKESTQRAIDGITNIVNSVIEKMNTQFEAVGK EFN NLEKRLENLNKKMEDGFLDVWTYNAELLVLMINERTLDFHDSNVKNLYDKVRMQLRD NAKELGNGCFEFYHKCDDECINSVKNGTYDYPKYEEESKLNRNEIKSGSLPETGGGSHH NNNNN

SEQ ID NO 15: UFV181158 (signal peptide and tag are underlined)

MEKIVLLFAIVSLVOSDOICIGYHANNSTEOVDTIMEKNVTVTHAQDILEKTHNG KLCSLNGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKDSPINGLCYPGDFNDY EELKHLLSSTNHFEKIQIIPRSSWSNHDASSGVSSACPYNGRSSFFRNVVWLIKKNNAYPT IKRSYNNTNQEDLLVLWGIHHPNDAAEQT KLYQNPTTYVSVGTSTLNQRSVPEIATRPK VNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSGLEYGNCNTK CQTPMGAINSSMPFHNIHPLTIGECPKYVKSDRLVLATTGLRNVPQRETRGLFGAIAGFIEG GWQGMVDGWYGYLHSNEQGSGYAADKESTQKAIDGITNKINSIIDKMNTQFEAVGKEF NNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLR DNAKELGNGCFEFYHKCDDECMESVRNGTYDYPQYSEEARLNREEISGSHHHHHH

SEQ ID NO:16: UFV181159 (signal peptide and tag are underlined)

MEKIVLLFAIVSLVOSDOICIGYHANNSTEOVDTIMEKNVTVTHAQDILEKTHNG KLCSLNGVKPLILRDCSVAGWLLGNPMCDEFLNVPEWSYIVEKDSPINGLCYPGDFNDY EELKHLLSSTNHFEKIQIIPRSSWSNHDASSGVSSACPYNGRSSFFRNVVWLIKKNNAYPT IKRSYNNTNQEDLLVLWGIHHPNDAAEQT KLYQNPTTYVSVGTSTLNQRSVPEIATRPK VNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSGLEYGNCNTK CQTPMGAINSSMPFHNIHPLTIGECPKYVKSDRLVLATTGLRNVPQRETRGLFGAIAGFIEG GWQGMVDGWYGYLWSNEQGSGYAADKESTQKAIDGITNHNSIIDKMNTQFEAVGKEF NNLERRIENLNKKMEDGFLDVWTYNAELLVLMINERTLDFHDSNVKNLYDKVRLQLR DNAKELGNGCFEFYHKCDDECIESVRNGTYDYPQYSEEARLNREEISSGSLPETGGGSH NNNNN

SEQ ID NO 17: UFV181155 (signal peptide and tag are underlined)

METISLrriLLVVTASNADKICIGHOSTNSTETVDTLTETNVPVTHAKELLHTEHN GMLCATSLGHPLILDTCTIEGLVYGNPSCDLLLGGREWSYIVERSSAVNGTCYPGNVEN LEELRTLFSSASSYQRIQIFPDTTWNVTYTGTSRACSGSFYRSMRWLTQKSGFYPVQDAQ YTNNRGKSILFVWGIHHPPTYTEQTNLYIRNDTTTSVTTED LNRTFKPVIGPRPLVNGLQ GRIDYYWSVLKPGQTLRVRSNGNLIAPWYGHVLSGGSHGRILKTDLKGGNCVVQCQTE KGGLNSTLPFHNISKYAFGTCPKYVRVNSLKLAVGLRNVPARSSRGLFGAIAGFIEGGWP

- 22 047998

GLVAGWYGFQHSNDQGVGMAADRDSTQKAIDKITSKVNNIVDKMNKQYEIIDHEFSEV ETRLNMINNKIDDQIQDVWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGSNA MEDGKGCFELYHKCDDQCMETIRNGTYNRRKYREESRLERQKIEGSHHHHHH

SEQ ID NO 18: UFV181156 (signal peptide and tag are underlined)

METISLrriLLVVTASNADKICIGHOSTNSTETVDTLTETNVPVTHAKELLHTEHN GMLCATSLGHPLILDTCTIEGLVYGNPSCDLLLGGREWSYIVERSSAVNGTCYPGNVEN LEELRTLFSSASSYQRIQIFPDTTWNVTYTGTSRACSGSFYRSMRWLTQKSGFYPVQDAQ YTNNRGKSILFVWGIHHPPTYTEQTNLYIRNDTTTSVTTED LNRTFKPVIGPRPLVNGLQ GRIDYYWSVLKPGQTLRVRSNGNLIAPWYGHVLSGGSHGRILKTDLKGGNCVVQCQTE KGGLNSTLPFHNISKYAFGTCPKYVRVNSLKLAVGLRNVPARSSRGLFGAIAGFIEGGWP HH

SEQ ID NO 19: UFV181141 (signal peptide and tag are underlined)

MKTIIALSYIFCLALGODLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNAT ELVQSSSTGKICNNPHRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPY DVPDYASLRSLVASSGTLEFITEGFTWTGVTQNGGSNACKRGPGSGFFSRLNWLTKSGS TYPVLNVTMPNNDNFDKLYIWGVHHPSTNQEQTSLY VQASGRVTVSTRRSQQTIIPNIGS RPWVRGLSSRISIYWTIVKPGDVLVINSNGNLIAPRGYFKMRTGKSSIMRSDAPIDTCISE CITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLATGMRNVPEKQTRGLFGAIAGFIE NGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQINGKLNRVIEKTNEKFHQIEKE FSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRE NAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVGSHHHHHH

SEQ ID NO 20: UFV180660 (signal peptide and tag are underlined)

MKTIIALSYIFCLALGODLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNAT ELVQSSSTGKICNNPHRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPY DVPDYASLRSLVASSGTLEFITEGFTWTGVTQNGGSNACKRGPGSGFFSRLNWLTKSGS TYPVLNVTMPNNDNFDKLYIWGVHHPSTNQEQTSLY VQASGRVTVSTRRSQQTIIPNIW SRPWVRGLSSRISIYWTIVKPGDVLVINSNGNLIAPRGYFKMRTGKSSIMRSDAPIDTCISE CITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLATGMRNVPEKQTRGLFGAIAGFIE NGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQINGILNRVIEKMNEKFHQ IEKE FSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALINQHTIDLTDSEMNKLFEKTRRQLREN AEDMGNGCFKIYHKCDNACIESIRNGNYDHDVYRDEALNNRFQIKGVSGSLPETGGGSH NNNNN

SEQ ID NO 21: UFV181137 (signal peptide and tag are underlined)

MKTIIALSYILCLVFAOKLPGNDNSTATLCLGHHAVSNGTLVKTITNDQIEVTNAT ELVQSSSTGRICDSPHQILDGENCTLIDALLGDPHCDGFQNKEWDLFVERSKAYSNCYPY DVPDYASLRSLVASSGTLEFNNESFNWTGVAQNGTSSACKRRSNKSFFSRLNWLHQLK YKYPALNVTMPNNEKFDKLYIWG VHHPSTDSDQISIYAQASGRVTVSTKRSQQTVIPNIG

- 23 047998

SSPWVRGVSSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSEC ITPNGSIPNDKPFQNVNRHYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIEN GWEGMVDGWYGFRHQNSEGTGQAADLKSTQAAINQINGKLNRLIEKTNEKFHQIEKEF SEVEGRIQDLEKY VEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFERTKKQLREN AEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVGSHHHHHH

SEQ ID NO 22: UFV181096 (signal peptide and tag are underlined)

MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVSNGTLVKTITNDQIEVTNAT ELVQSSSTGRICDSPHQILDGENCTLIDALLGDPHCDGFQNKEWDLFVERSKAYSNCYPY DVPDYASLRSLVASSGTLEFNNESFNWTGVAQNGTSSACKRRSNKSFFSRLNWLHQLK YKYPALNVTMPNNEKFDKLYIWG VHHPSTDSDQISIYAQASGRVTVSTKRSQQTVIPNIG SSPWVRGVSSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSEC ITPNGSIPNDKPFQNVNRHYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIEN NNNNN

SEQ ID NO 23: UFV181145 (signal peptide and tag are underlined)

MIALILVALALSHTAYSOITNGTTGNPIICLGHHAVENGTSVKTLTDNHVEVVSA KELVETNHTDELCPSPLKLVDGQDCDLINGALGSPGCDRLQDTTWDVFIERPTAVDTCY PFDVPDYQSLRSILASSGSLEFIAEQFTWNGVKVDGSSSACLRGGRNSFFSRLNWLTKET NGNYGPINVTKENTGSYVRLYLWGVHHPSSDNEQ TDLYKVATGRVTVSTRSDQISIVPN IGSRPRVRNQSGRISIYWTLVNPGDSIIFNSIGNLIAPRGHYKISKSTKSTVLKSDKRIGSCT SPCLTDKGSIQSDKPFQNVSRIAIGNCPKYVKQGSLMLATGMRNIPGKQAKGLFGAIAGF IENGWQGLIDGWYGFRHQNAEGTGTAADLKSTQAAIDQINGKLNRLIEKTNEKYHQIEK EFEQVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDVTDSEMNKLFERVRRQLR ENAEDQGNGCFEIFHQCDNNCIESIRNGTYDHNIYRDEAINNRIKINPVGSHHHHHH

SEQ ID NO 24: UFV180661 (signal peptide and tag are underlined)

MIALILVALALSHTAYSOITNGTTGNPIICLGHHAVENGTSVKTLTDNHVEVVSA KELVETNHTDELCPSPLKLVDGQDCDLINGALGSPGCDRLQDTTWDVFIERPTAVDTCY PFDVPDYQSLRSILASSGSLEFIAEQFTWNGVKVDGSSSACLRGGRNSFFSRLNWLTKET NGNYGPINVTKENTGSYVRLYLWGVHHPSSDNEQ TDLYKVATGRVTVSTRSDQISIVPN IGSRPRVRNQSGRISIYWTLVNPGDSIIFNSIGNLIAPRGHYKISKSTKSTVLKSDKRIGSCT SPCLTDKGSIQSDKPFQNVSRIAIGNCPKYVKQGSLMLATGMRNIPGKQAKGLFGAIAGF IENGWQGLIDGWYGFRWQNAEGTGTAADLKSTQAAIDQINGILNRLIEKMNEKYHQIEK EFEQVEGRIQDLEKYVEDTKIDLWSYNAELLVALINQHTIDVTDSEMNKLFERVRRQLR ENAEDQGNGCFEIFHQCDNNCIESIRNGTYDHNIYRDEAINNRIKINPVSGSLPETGGGSH NNNNN

SEQ ID NO 25: UFV181146 (signal peptide and tag are underlined)

MNTOILVFALMAIIPTNADKICLGHHAVSNGTKVNTLTERGVEVVNATETVERT NVPRICSKGKRTVDLGQCGLLGTITGPPQCDQFLEFSADLIIERREGSDVCYPGKFVNEEA

- 24 047998

LRQILRESGGIDKETMGFTYSGIRTNGATSACRRSGSSFYAEMKWLLSNTDNAAFPQMT KSYKNTRKDPALIIWGIHHSGSTTEQTKLYGSGNKLITVGSSNYQQSFVPSPGARPQVNG QSGRIDFHWLILNPNDTVTFSFNGAFIAPDRASFLRGKSMGIQSGVQVDANCEGDCYHS GGTIISNLPFQNINSRAVGKCPRY VKQESLLLATGMKNVPEIPKGRGLFGAIAGFIENGW EGLIDGWYGFRHQNAQGEGTAADYKSTQSAIDQITGKLNRLIEKTNQQFELIDNEFTEVE KQIGNVINWTRDSMTEVWSYNAELLVAMENQHTIDLADSEMNKLYERVKRQLRENAE EDGTGCFEIFHKCDDDCMASIRNNTYDHSKYREEAMQNRIQIDPVGSHHHHHH

SEQ ID NO 26: UFV180664 (signal peptide and tag are underlined)

MNTOILVFALMAIIPTNADKICLGHHAVSNGTKVNTLTERGVEVVNATETVERT NVPRICSKGKRTVDLGQCGLLGTITGPPQCDQFLEFSADLIIERREGSDVCYPGKFVNEEA LRQILRESGGIDKETMGFTYSGIRTNGATSACRRSGSSFYAEMKWLLSNTDNAAFPQMT KSYKNTRKDPALIIWGIHHSGSTTE QTKLYGSGNKLITVGSSNYQQSFVPSPGARPQVNG QSGRIDFHWLILNPNDTVTFSFNGAFIAPDRASFLRGKSMGIQSGVQVDANCEGDCYHS GGTIISNLPFQNINSRAVGKCPRYVKQESLLLATGMKNVPEIPKGRGLFGAIAGFIENGW EGLIDGWYGFRWQNAQGEGTAADYKSTQSAIDQITGILNRLIEKMNQQFELIDNEFTEV EKQIGNVINWTRDSMTEVWSYNAELLVAMINQHTIDLADSEMNKLYERVKRQLRENAE EDGTGCFEIFHKCDDDCMASIRNNTYDHSKYREEAMQNRIQIDPVSGSLPETGGGSHHH HHH

SEQ ID NO 27: UFV181147 (signal peptide and tag are underlined)

MYKVVVIIALLGAVKGLDRICLGHHAVANGTIVKTLTNEQEEVTNATETVESTN LNKLCMKGRSYKDLGNCHPVGMLIGTPVCDPHLTGTWDTLIERENAIAHCYPGATINEE ALRQKIMESGGISKMSTGFTYGSSINSAGTTKACMRNGGDSFYAELKWLVSKTKGQNFP QTTNTYRNTDTAEHLIIWGIHHPSSTQE KNDLYGTQSLSISVESSTYQNNFVPVVGARPQ VNGQSGRIDFHWTLVQPGDNITFSHNGGLIAPSRVSKLTGRGLGIQSEALIDNSCESKCF WRGGSINTKLPFQNLSPRTVGQCPKYVNQRSLLLATGMRNVPEVVQGRGLFGAIAGFIE NGWEGMVDGWYGFRHQNAQGTGQAADYKSTQAAIDQITGKLNRLIEKTNTEFESIESE FSETEHQIGNVINWTKDSITDIWTYQAELLVAMENQHTIDMADSEMLNLYERVRKQLR QNAEEDGKGCFEIYHTCDDSCMESIRNNTYDHSQYREEALLNRLNINSVGSHHHHHH

SEQ ID NO 28: UFV180662 (signal peptide and tag are underlined)

MYKVVVIIALLGAVKGLDRICLGHHAVANGTIVKTLTNEQEEVTNATETVESTN LNKLCMKGRSYKDLGNCHPVGMLIGTPVCDPHLTGTWDTLIERENAIAHCYPGATINEE ALRQKIMESGGISKMSTGFTYGSSINSAGTTKACMRNGGDSFYAELKWLVSKTKGQNFP QTTNTYRNTDTAEHLIIWGIHHPSSTQE KNDLYGTQSLSISVESSTYQNNFVPVVGARPQ VNGQSGRIDFHWTLVQPGDNITFSHNGGLIAPSRVSKLTGRGLGIQSEALIDNSCESKCF WRGGSINTKLPFQNLSPRTVGQCPKYVNQRSLLLATGMRNVPEVVQGRGLFGAIAGFIE NGWEGMVDGWYGFRWQNAQGTGQAADYKSTQAAIDQITGILNR LIEKMNTEFESIESE FSETEHQIGNVINWTKDSITDIWTYQAELLVAMINQHTIDMADSEMLNLYERVRKQLRQ NAEEDGKGCFEIYHTCDDSCMESIRNNTYDHSQYREEALLNRLNINSSGSLPETGGGSHH NNNNN

SEQ ID NO 29: UFV4239 (signal peptide and tag are underlined)

- 25 047998

MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKH NGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFID YEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNS YPKLSKSYINDKGKEVLVLWGIHHPSTS ADQQSLYQNADAYVFVGSSRYSKKFKPEIAIR PKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCN TTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNIPSIQSQGLFGAIAGFIEG GWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGK EFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQL KNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGRSLVPRGSG HHHHHH

SEQ ID NO 30: UFV180843 (signal peptide and tag are underlined)

MKAILVVLLYTFTTANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKH NGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETSNSDNGTCYPGDFIN YEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNS YPKLNQSYINDKGKEVLVLWGIHH PSTTADQQSLYQNADAYVFVGTSRYSKKFKPEIAT RPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFTMERNAGSGIIISDTPVHDC NTTCQTPEGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIE GGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDKITNKVNSVIEKMNTQFTAVG KEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRNQ LKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREKIDSGSLVPSGSP GSGYIPEAPRDG QAYVRKDGEWVLLSTFLGGSLPETGGGSHHHHHH

SEQ ID NO 31: UFV180436 (signal peptide and tag are underlined)

MKTIIALSYIFCLALGODLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNAT ELVQSSSTGKICNNPHRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPY DVPDYASLRSLVASSGTLEFITEGFTWTGVTQNGGSNACKRGPGSGFFSRLNWLTKSGS TYPVLNVTMPNNDNFDKLYIWGVHHPSTNQEQTSLY VQASGRVTVSTRRSQQTIIPNIGS RPWVRGLSSRISIYWTIVKPGDVLVINSNGNLIAPRGYFKMRTGKSSIMRSDAPIDTCISE CITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLATGMRNVPEKQTRGLFGAIAGFIE NGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQINGKLNRVIEKTNEKFHQIEKE FSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRE NAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQSGSLVPSGSPGSGYI PEAPRDGQAYVRK DGEWVLLSTFLGGSLPETGGGSHHHHHH

SEQ ID NO 32: UFV170466 (signal peptide and tag are underlined)

MKTIVALSYILCLVFAQKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNA TELVQSSSTGEICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYP YDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACIRRSNSSFFSRLNWLTHLNF KYPALNVTMPNNEQFDKLYIWGVHHPG TDKDQIFLYAQSSGRITVSTKRSQQAVIPNIGS RPRIRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITP NGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMRNVPEKQTRGIFGAIAGFIENGW EGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSE

- 26 047998

VEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAE DMGNGCFKIYHKCDNACIGSIRNGTYNHDVYRDEALNNRFQSGSLVPRGSGSGYIPEAP RDGQAYVRKDGEWVLLSTFLGGSEPEA

SEQ ID NO 33: UFV181099 (signal peptide and tag are underlined)

MKTIVALSYILCLVFAQKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNA TELVQSSSTGEICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYP YDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACIRRSNSSFFSRLNWLTHLNF KYPALNVTMPNNEQFDKLYIWGVHHPG TDKDQIFLYAQSSGRITVSTKRSQQAVIPNIGS RPRIRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITP NGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMRNVPEKQTRGIFGAIAGFIENGW NNN

SEQ ID NO 34: UFV181005 (signal peptide and tag are underlined)

MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKH NGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFID YEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNS YPKLSKSYINDKGKEVLVLWGIHHPSTS ADQQSLYQNADAYVFVGSSRYSKKFKPEIAIR PKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCN TTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNIPSIQSRGLFGAIAGFIEG GWTGMVDGWYGYHWQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGK EFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQL KNNAKEIGNGCFEFYHKCDNTCIESVKNGTYDYPKYSEEAKLNREEIDSGSLPETGGGS NNNNNNN

SEQ ID NO 35: UFV181007 (signal peptide and tag are underlined)

MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKH NGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFID YEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNS YPKLSKSYINDKGKEVLVLWGIHHPSTS ADQQSLYQNADAYVFVGSSRYSKKFKPEIAIR PKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCN TTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNIPSIQSRGLFGAIAGFIEG GWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNIVNSVIEKMNTQFTAVGKE FNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLINERTLDYHDSNVKNLYEKVRSQLKN NAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDSGSLPETGGGSHH NNNNN

SEQ ID NO 36: UFV181090 (signal peptide and tag are underlined)

MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENSH NGKLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGHFAD YEELREQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGESSFYRNLLWLTGKNGLY

- 27 047998

PNLSKSYANNKEKEVLVLWGVHHPPNIGDQKALYHTENAYVSVVSSHYSRKFTPEIAK RPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPRYAFALSRGFGSGIINSNAPMDKCD AKCQTPQGAINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFI EGGWTGMVDGWYGYH WQNEQGSGYAADQKSTQNAINGITNIVNSVIEKMNTQFTAV GKEFNKLERRMENLNKKVDDGFIDIWTYNAELLVLLINERTLDFHDSNVKNLYEKVKS OLKNNAKEIGNGCFEFYHKCNDECIESVKNGTYDYPKYSEESKLNREKIDSGSLPETGG GSHHHHHH

SEQ ID NO 37: UFV181135 (signal peptide and tag are underlined)

MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENSH NGKLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGHFAD YEELREQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGESSFYRNLLWLTGKNGLY PNLSKSYANNKEKEVLVLWGVHHPPNIG DQKALYHTENAYVSVVSSHYSRKFTPEIAK RPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPRYAFALSRGFGSGIINSNAPMDKCD AKCQTPQGAINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSQGLFGAIAGFI EGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAV GKEFNKLERRMENLNKKVDDGFIDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKS OLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGSHHHHHH

SEQ ID NO:38: UFV181084 (signal peptide and tag are underlined)

MEARLLVLLCAFAATNADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSH NGKLCKLKGIAPLQLGKCNIAGWLLGNPECDLLLTASSWSYIVETSNSENGTCYPGDFID YEELREQLSSVSSFEKFEIFPKTSSWPNHETTKGVTAACSYAGASSFYRNLLWLTKKGSS YPKLSKSYVNNKGKEVLVLWGVHHPPT GTDQQSLYQNADAYVSVGSSKYNRRFTPEIA ARPKVRDQAGRMNYYWTLLEPGDTITFEATGNLIAPWYAFALNRGSGSGIITSDAPVHD CNTKCQTPHGAINSSLPFQNIHPVTIGECPKYVRSTKLRMATGLRNIPSIQSRGLFGAIAGF IEGGWTGMIDGWYGYHWQNEQGSGYAADQKSTQNAIDGITNIVNSVIEKMNTQFTAV GKEFNNLERRIENLNKKVDDGFLDIWTYNAELLVLLINERTLDFHDSNVRNLYEKVKSQ LKNNAKEIGNGCFEFYHKCDDACIESVRNGTYDYPKYSEESKLNREEIDSGSLPETGGGS NNNNNNN

SEQ ID NO 39: UFV181131 (signal peptide and tag are underlined)

MEARLLVLLCAFAATNADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSH NGKLCKLKGIAPLQLGKCNIAGWLLGNPECDLLLTASSWSYIVETSNSENGTCYPGDFID YEELREQLSSVSSFEKFEIFPKTSSWPNHETTKGVTAACSYAGASSFYRNLLWLTKKGSS YPKLSKSYVNNKGKEVLVLWGVHHPPT GTDQQSLYQNADAYVSVGSSKYNRRFTPEIA ARPKVRDQAGRMNYYWTLLEPGDTITFEATGNLIAPWYAFALNRGSGSGIITSDAPVHD CNTKCQTPHGAINSSLPFQNIHPVTIGECPKYVRSTKLRMATGLRNIPSIQSRGLFGAIAGF IEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAIDGITNKVNSVIEKMNTQFTAV GKEFNNLERRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVRNLYEKVKS QLKNNAKEIGNGCFEFYHKCDDACMESVRNGTYDYPKYSEESKLNREEIDGSHHHHHH

SEQ ID NO:40: UFV181095 (signal peptide and tag are underlined)

MKTIIALSYILCLVFAOKLPGNDNSTATLCLGHHAVPNGTLVKTITNDQIEVTNAT

- 28 047998

ELVQSSSTGRICDSPHRILDGKNCTLIDALLGDPHCDGFQNKEWDLFVERSKAYSNCYPY DVPDYASLRSLVASSGTLEFINEDFNWTGVAQDGKSYTCKRGSVNSFFSRLNWLHKLEY KYPALNVTMPNNGKFDKLYIWGVHHPSTDSDQTSLYVRASGRVTVSTKRSQQTVIPNIG SRPWVRGLSSRISIYWTIV KPGDILLINSTGNLIAPRGYFKIRNGKSSIMRSDAPIGNCSSEC ITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIEN GWEGMVDGWYGFRWQNSEGTGQAADLKSTQAAIDQINGILNRLIEKTNEKFHQIEKEF SEVEGRIQDLEKYVEDTKIDLWSYNAELLVALINQHTIDLTDSEMNKLFERTRKQLREN AEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVSGSLPETGGGSH HHHHH

SEQ ID NO 41: UFV181140 (signal peptide and tag are underlined)

MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVPNGTLVKTITNDQIEVTNAT ELVQSSSTGRICDSPHRILDGKNCTLIDALLGDPHCDGFQNKEWDLFVERSKAYSNCYPY DVPDYASLRSLVASSGTLEFINEDFNWTGVAQDGKSYTCKRGSVNSFFSRLNWLHKLEY KYPALNVTMPNNGKFDKLYIWGV HHPSTDSDQTSLYVRASGRVTVSTKRSQQTVIPNIG SRPWVRGLSSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRNGKSSIMRSDAPIGNCSSEC ITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIEN GWEGMVDGWYGFRHQNSEGTGQAADLKSTQAAIDQINGKLNRLIEKTNEKFHQIEKEF SEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFERTRKQLREN AEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVGSHHHHHH

SEQ ID NO 42: UFV181093 (signal peptide and tag are underlined)

MKTIIALSYIFCOVLAONLPGNDNSTATLCLGHHAVPNGTLVKTITNDQIEVTNA TELVQSSSTGRICDSPHRILDGKNCTLIDALLGDPHCDGFQNEKWDLFVERSKAFSNCYP YDVPDYASLRSLVASSGTLEFINEGFNWTGVTQNGGSYACKRGPDKSFFFSRLNWLYESE STYPVLNVTMPNNDNFDKLYIWGVHHPSTDKEQ TNLYVQASGRVTVSTKRSQQTIIPNV GSRPWVRGLSSRISIYWTIVKPGDILLINSNGNLIAPRGYFKIRTGKSSIMRSDAPIGTCSSE CITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIE NGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQINGILNRVIEKTNEKFHQIEKEF SEVEGRIQDLEKYVEDTKIDLWSYNAELLVALINQHTIDLTDSEMNKLFEKTRRQLREN AEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVSGSLPETGGGSH NNNNN

SEQ ID NO 43: UFV181136 (signal peptide and tag are underlined)

MKTIIALSYIFCOVLAONLPGNDNSTATLCLGHHAVPNGTLVKTITNDQIEVTNA TELVQSSSTGRICDSPHRILDGKNCTLIDALLGDPHCDGFQNEKWDLFVERSKAFSNCYP YDVPDYASLRSLVASSGTLEFINEGFNWTGVTQNGGSYACKRGPDKSFFFSRLNWLYESE STYPVLNVTMPNNDNFDKLYIWGVHHPSTDKEQ TNLYVQASGRVTVSTKRSQQTIIPNV GSRPWVRGLSSRISIYWTIVKPGDILLINSNGNLIAPRGYFKIRTGKSSIMRSDAPIGTCSSE CITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIE NGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQINGKLNRVIEKTNEKFHQIEKE FSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRE NAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVGSHHHHHH

- 29 047998

SEQ ID NO 44: UFV181097 (signal peptide and tag are underlined)

MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVPNGTIVKTITNDOIEVTNAT ELVQSSSTGGICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYP YDVPDYASLRSLVASSGTLEFNDESFNWTGVTQNGTSSSCKRRSNNSFFSRLNWLTHLK FKYPALNVTMPNNEKFDKLYIWGVHHPVTD NDQIFLYAQASGRITVSTKRSQQTVIPNI GSRPRIRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSEC ITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIEN GWEGMVDGWYGFRWQNSEGIGQAADLKSTQAAINQINGILNRLIGKTNEKFHQIEKEFS EVEGRIQDLEKYVEDTKIDLWSYNAELLVALINQHTIDLTDSEMNKLFERTKKQLRENA EDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVSGSLPETGGGSHH NNNNN

SEQ ID NO 45: UFV181138 (signal peptide and tag are underlined)

MKTIIALSYILCLVFAOKLPGNDNSTATLCLGHHAVPNGTIVKTITNDOIEVTNAT ELVQSSSTGGICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYP YDVPDYASLRSLVASSGTLEFNDESFNWTGVTQNGTSSSCKRRSNNSFFSRLNWLTHLK FKYPALNVTMPNNEKFDKLYIWGVHHPVTD NDQIFLYAQASGRITVSTKRSQQTVIPNI GSRPRIRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSEC ITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIEN GWEGMVDGWYGFRHQNSEGIGQAADLKSTQAAINQINGKLNRLIGKTNEKFHQIEKEF SEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFERTKKQLREN AEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVGSHHHHHH

SEQ ID NO 46: UFV181148 (signal peptide and tag are underlined)

MLSIVILFLLVAENSSONYTGNPVICMGHHAVANGTMVKTLTDDOVEVVTAQEL VESQNLPELCPSPLRLVDGQTCDIINGALGSPGCDHLNGAEWDVFIERPNAMDTCYPFD VPDYQSLRSILANNGKFEFIAEEFQWTTVKQNGKSGACKRANVNDFFRRRLNWLVKSDR NAYPLQNLTKVNNGDYARLYIWGVHHPSTDTE QTNLYKNNPGRVTVSTKTSQTSVIPNI GSRPWVRGQSGRISFYWTIVEPGDLIVFNTIGNLIAPRGHYKLNNQKKGTILNTAIPIGSC VSKCHTDKGSLSTTKPFQNISRIAIGDCPKYVKQGSLKLATGMRNIPEKAASRGLFGAIAG FIENGWQGLIDGWYGFRHQNAEGTGTAADLKSTQAAIDQINGKLNRLIEKTNEKYHQIE KEFEQVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDVTDSEMNKLFERVRRQL RENAEDKGNGCFEIFHKCDNNCIESIRNGTYDHDIYRDEAINNRFOIQGVGSHHHHHH

SEQ ID NO 47: UFV181149 (signal peptide and tag are underlined)

MLSIVILFLLVAENSSONYTGNPVICMGHHAVANGTMVKTLTDDOVEVVTAQEL VESQNLPELCPSPLRLVDGQTCDIINGALGSPGCDHLNGAEWDVFIERPNAMDTCYPFD VPDYQSLRSILANNGKFEFIAEEFQWTTVKQNGKSGACKRANVNDFFRRRLNWLVKSDR NAYPLQNLTKVNNGDYARLYIWGVHHPSTDTE QTNLYKNNPGRVTVSTKTSQTSVIPNI GSRPWVRGQSGRISFYWTIVEPGDLIVFNTIGNLIAPRGHYKLNNQKKGTILNTAIPIGSC VSKCHTDKGSLSTTKPFQNISRIAIGDCPKYVKQGSLKLATGMRNIPEKAASRGLFGAIAG FIENGWQGLIDGWYGFRWQNAEGTGTAADLKSTQAAIDQINGILNRLIEKTNEKYHQIE KEFEQVEGRIQDLEKYVEDTKIDLWSYNAELLVALINQHTIDVTDSEMNKLFERVRRQL

- 30 047998

RENAEDKGNGCFEIFHKCDNNCIESIRNGTYDHDIYRDEAINNRFQIQGVSGSLPETGGG SHHHHHH

SEQ ID NO 50: UFV190839 (signal peptide and tag are underlined)

MKTIIALSYIFCLALGODLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNAT ELVQSSSTGKICNNPHRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPY DVPDYASLRSLVASSGTLEFITEGFTWTGVTQNGGSNACKRGPGSGFFSRLNWLTKSGS TYPVLNVTMPNNDNFDKLYIWGVHHPSTNQEQTSLY VQASGRVTVSTRRSQQTIIPNIGS RPWVRGLSSRISIYWTIVKPGDVLVINSNGNLIAPRGYFKMRTGKSSIMRSDAPIDTCISE CITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLATGMRNVPEKQTRGLFGAIAGFIE NGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQINGILNRVIEKTNEKFHQ IEKEF SEVEGRIQDLEKYVEDTKIDLWSYNAELLVALINQHTIDLTDSEMNKLFEKTRRQLREN AEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVSGSLPETGGGSH NNNNN

SEQ ID NO 51: UFV190068 (signal peptide and tag are underlined) MNTOILVFALMAIIPTNADKICLGHHAVSNGTKVNTLTERGVEVVNATETVERTNVPRI CSKGKRTVDLGQCGLLGTITGPPQCDQFLEFSADLIIERREGSDVCYPGKFVNEEALRQIL RESGGIDKETMGFTYSGIRTNGATSACR RSGSSFYAEMKWLLSNTDNAAFPQMTKSYK NTRKDPALIIWGIHHSGSTTEQTKLYGSGNKLITVGSSNYQQSFVPSPGARPQVNGQSGR IDFHWLILNPNDTVTFSFNGAFIAPDRASFLRGKSMGIQSGVQVDANCEGDCYHSGGTIIS NLPFQNINSRAVGKCPRYVKQESLLLATGMKNVPEIPKGRGLFGAIAGF IENGWEGLIDG WYGFRWQNAQGEGTAADYKSTQSAIDQITGILNRLIEKTNQQFELIDNEFTEVEKQIGNV INWTRDSMTEVWSYNAELLVAMINQHTIDLADSEMNKLYERVKRQLRENAEEDGTGCF EIFHKCDDDCMASIRNNTYDHSKYREEAMQNRIOIDPVSGSLPETGGGSHHHHHH

SEQ ID NO 52: UFV190841 (signal peptide and tag are underlined)

MYKVVVIIALLGAVKGDRICLGHHAVANGTIVKTLTNEQEEVTNATETVESTNL NKLCMKGRSYKDLGNCHPVGMLIGTPVCDPHLTGTWDTLIERENAIAHCYPGATINEEA LRQKIMESGGISKMSTGFTYGSSINSAGTTKACMRNGGDSFYAELKWLVSKTKGQNFPQ TTNTYRNTDTAEHLIIWGIHHPSSTQE KNDLYGTQSLSISVESSTYQNNFVPVVGARPQV NGQSGRIDFHWTLVQPGDNITFSHNGGLIAPSRVSKLTGRGLGIQSEALIDNSCESKCFW RGGSINTKLPFQNLSPRTVGQCPKYVNQRSLLLATGMRNVPEVVQGRGLFGAIAGFIEN NNN

Claims (21)

1. A recombinant influenza A virus hemagglutinin (HA) polypeptide comprising the HA1 and HA2 domains from influenza A virus HA and comprising an amino acid sequence where: (a) the amino acid at position 355 is W, and (b) the amino acid at position 432 is I, and the amino acid at position 380 is I, and wherein the numbering of the amino acid positions in the amino acid sequence of the HA polypeptide is presented according to the amino acid numbering in SEQ ID NO: 1. 2. The HA polypeptide according to claim 1, comprising an amino acid sequence where: (a) the amino acid at position 388 is M and/or (b) the amino acid at position 478 is I. 3. The HA polypeptide of claim 1 or 2, wherein the polypeptide does not comprise a protease cleavage site between the HA1 and HA2 domains. 4. The HA polypeptide according to claim 1, 2 or 3, wherein the HA1 and HA2 domains are derived from influenza A virus group 1 and/or group 2. 5. The HA polypeptide according to any one of claims 1 to 4, comprising a truncated HA1 and/or HA2 domain. 6. The HA polypeptide of claim 5, wherein the transmembrane and intracytoplasmic domains have been deleted from the HA2 domain. 7. The HA polypeptide of claim 5 or 6, wherein at least the C-terminal portion of the HA2 domain, beginning with the amino acid corresponding to the amino acid at position 515, has been deleted. - 31 047998 8. The HA polypeptide according to any one of claims 1 to 7, comprising a label for detection and/or purification located at the C-terminus of the (truncated) HA2 domain. 9. An immunogenic fragment of a polypeptide according to any one of claims 1-8. 10. A multimeric polypeptide comprising at least two HA polypeptides according to any one of claims 1 to 8 or an immunogenic fragment according to claim 9. 11. The multimeric polypeptide of claim 10, wherein the polypeptide is trimeric and comprises three HA polypeptides of any one of claims 1-8. 12. A nucleic acid encoding an HA polypeptide according to any one of claims 1-8, 10 or 11 or an immunogenic fragment according to claim 9. 13. A vector containing a nucleic acid molecule according to claim 12. 14. The vector of claim 13, wherein the vector is a recombinant adenoviral vector. 15. A method for producing a recombinant HA polypeptide according to any one of claims 1-8, 10 or 11 or an immunogenic fragment according to claim 9, comprising expressing a nucleic acid molecule according to claim 12 in a prokaryotic or eukaryotic cell, wherein said method comprises the step of isolating the HA polypeptide or a fragment thereof from said cell. 16. An immunogenic composition comprising an HA polypeptide according to any one of claims 1-8, 10 or 11, an immunogenic fragment according to claim 9, a nucleic acid according to claim 12 and/or a vector according to claim 13 or 14 and a pharmaceutically acceptable carrier. 17. Use of an HA polypeptide according to any one of claims 1-8, 10 or 11 in inducing an immune response against an influenza virus. 18. Use of an immunogenic fragment according to claim 9 in inducing an immune response against the influenza virus. 19. Use of a nucleic acid according to claim 12 in inducing an immune response against the influenza virus. 20. Use of the HA polypeptide according to any one of claims 1-8, 10 or 11 as a vaccine. 21. Use of the immunogenic fragment according to item 9 as a vaccine. 22. Use of the nucleic acid according to item 12 as a vaccine. A ·, , xc ' , (Chil 7/ Head region LMg . ---------------------------------------------------------------------- c' /t Stem region h Zh S '' *--- ^{,8KM 4321} —/ <____ 380» h ' 4____ 355W M * 4781 HA of influenza virus B !---=---- ^HL ----- ₌ ! j— HA, .....HA _g ---- _! 388M , 4781 “ il II 1 1 1 1 1 1 1 1 1 ! ! ⁴³²¹ 1 3801 1 355W Fig. 1 Minor mutations Mutations according to the present invention