EA046012B1 - VACCINES AGAINST THE FLU VIRUS AND WAYS OF THEIR APPLICATION - Google Patents

Description

Introduction

The present invention relates to the field of medicine. Presented herein are influenza A virus hemagglutinin (HA) stem domain polypeptides, nucleic acids encoding said polypeptides, pharmaceutical compositions containing them, and methods of using them.

The present invention was made, at least in part, with government support under agreement HHS0100201700018C issued by HHS. The Government has certain rights to the present invention.

BACKGROUND OF THE INVENTION

Influenza viruses are major human pathogens that cause respiratory illness (commonly called influenza), the severity of which ranges from subclinical infection to primary viral pneumonia that can lead to death. The clinical effects of infection vary depending on the virulence of the influenza virus strain and exposure, medical history, age, and immune status of the host. It is estimated that approximately 1 billion people worldwide are infected with influenza virus each year, resulting in 3 to 5 million severe illnesses and an estimated 300,000 to 500,000 influenza-related deaths. The majority 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 therefore their representatives are usually included in the seasonal vaccine. Current immunization practices rely on early identification of circulating influenza viruses to ensure timely receipt 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 virus strain originating from animal reservoirs and reassorted with increased human-to-human spread still poses a significant and present threat to global health.

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 non-structural proteins (NS1, NS2 and PB1 -F2) and two external glycoproteins hemagglutinin (HA) and neuraminidase (NA).

Influenza A viruses are widespread in nature and can infect many birds and mammals. Viruses are classified based on differences in the antigenic structure of the HA and NA proteins, with their different combinations 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 of influenza A viruses has demonstrated the division of hemagglutinins into two main so-called phylogenetic groups: among others, subtypes H1, H2, H5 and H9 in phylogenetic group 1 (group 1 viruses), and, among others, subtypes H3, H4 and H7 in phylogenetic group 2 (group 2 viruses).

Influenza B virus strains are exclusively human. The antigenic variability of NA in influenza B virus strains is less than that observed in type A strains. The two genetically and antigenically distinct influenza B virus lineages circulating in humans are the B/Yamagata/16/88 (also called B/Yamagata) lineages and B/Victoria/2/87 (B/Victoria). Although the spectrum of illness caused by influenza B viruses is generally milder than that caused by influenza A viruses, severe illness requiring hospitalization is common when influenza B infection occurs.

It is known that antibodies that neutralize the influenza virus are directed primarily to hemagglutinin (HA). Hemagglutinin or HA is a trimeric glycoprotein that is anchored in the viral membrane 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 membrane, resulting in the release of viral RNA into the cytosol target cells. HA contains a large head domain and a smaller stem domain. The stem domain is anchored in the viral membrane by a C-terminal transmembrane domain sequence. The protein undergoes post-translational cleavage to produce two polypeptides HA-HA1 and HA2 (the complete sequence is called HA0) (Fig. 1A, 1B). The membrane-distal head domain is mainly formed from HA1, and the membrane-proximal stem domain is mainly formed from HA2. Cleavage of the HA precursor molecule HA0 is required to activate virus infectivity, and the distribution of activating proteases in the host is one of the determinants of influenza virus pathogenicity. The NAs of mammalian viruses and non-pathogenic avian viruses are cleaved extracellularly, which limits their spread in hosts to tissues where the corresponding proteases are found. At the same time, the NAs of pathogenic viruses are cleaved intracellularly by ubiquitous proteases and therefore have the ability to infect various types of cells and cause systemic infections.

- 1 046012 tion.

The reason the seasonal influenza vaccine must be updated every year is because the virus is highly variable. In the HA protein, such variability is especially evident in the head domain, where antigenic drift and shift have led to the formation of large numbers of different variants. Because it also represents an area that is immunodominant, most neutralizing antibodies are directed to this domain, and such antibodies act by interfering with receptor binding. The combination of immunodominance and significant variability in the head domain explains that infection with one particular strain does not lead to the formation of immunity to other strains: antibodies produced as a result of the first infection recognize only a limited number of strains that are closely related to the virus that caused the primary infection.

Recently, influenza virus hemagglutinin stem domain polypeptides lacking all or a significant portion of the influenza virus hemagglutinin globular head domain have been described and used to generate an immune response to one or more conserved stem domain polypeptide epitopes. It is believed that stem domain polypeptide epitopes are less immunogenic than the highly immunogenic regions of the globular head domain, and therefore the absence of a globular head domain in the stem domain polypeptide may allow the development of an immune response to one or more epitopes of the stem domain polypeptide (Steel et al., 2010) . Steel et al. thus created an influenza virus HA stem domain polypeptide by deleting amino acid residues 53-276 from the HA1 domain of strains A/Puerto Rico/8/1934 (H1N1) and A/Hong Kong/1/1968 (H3N2) and replacing the deleted sequence with a short flexible linker sequence: GGGG. Vaccination of mice with the H3/HK68 construct did not result in the production of antisera that cross-reacted with group 1 HA. In addition, as explained in WO 2013/079473, the stem domain polypeptides were unstable and did not adopt the correct conformation, as evidenced by the lack of binding of antibodies that, were previously shown to bind to conserved epitopes in the full-length stem region of wild-type HA.

Bommakanti et al. (2010) described a HA2-based polypeptide containing amino acid residues 330-501 (HA2), a 7-amino acid linker (GSAGSAG), amino acid residues 16-55 from HA1, a 6-amino acid linker GSAGSA followed by residues 290-321 from HA1, with the V297T mutations , I300E, Y302T and C305T in HA1. This developed structure was based on the H3 HA sequence (A/Hong Kong/1/1968). The polypeptide provided cross-protection only against another influenza virus strain within the H3 subtype (A/PI^^), but not against the H1 subtype (A/PR/8/34). In an earlier paper, Bommakanti et al. (2012) described an HA-based stem domain polypeptide from H1N1 A/Puerto Rico/8/1934 (H1HA0HA6). In this polypeptide, the equivalent of amino acid residues 48-288 was deleted and the mutations I297T, V300T, I302N, C305S, F392D, F395T and L402D were produced. Both H3 and H1 based polypeptides were expressed in E. coli and therefore lacked glycans that are part of naturally occurring HA proteins.

Corbett et al. (2019) described trimers of the H3 and H7 HA stem domains of influenza A virus presented on self-assembled ferritin nanoparticles that induce the production of protective homosubtypic antibodies in mice. Despite their immunogenicity, HA antigens fused to ferritin can also induce unwanted responses against nanoparticle carriers, which can lead to a decrease in the immune response directed to HAs and an increase in their lifespan after booster immunization. In addition, expression levels and purification issues of NAferritin fusion proteins may prevent the production of large numbers of vaccine doses. In addition, the availability of HA epitopes (such as the CR8020 binding site) near the surface of such nanoparticles may reduce the immune response to such favorable conserved HA surfaces.

To this day, influenza remains a major burden on the global health system, even though vaccine technology based on egg-grown whole inactivated influenza virus was developed more than 70 years ago. The constant antigenic drift of the influenza virus hemagglutinin (HA), coupled with immunodominant strain-specific antibody responses that target the variable head domain of the influenza virus, results in traditional vaccine efficacy ranging from 10 to 60% and the need for seasonal updates of virus strains included in licensed vaccines. Moreover, current approaches to creating vaccines provide minimal protection against pandemic strains of the influenza virus.

The need for an improved group 2 vaccine is particularly acute as vaccine efficacy against H3N2 has averaged only 33% over the past decade (Belongia et al. (2016)), recent H3N2 strains have demonstrated increased virulence (Garten et al. (2017)), and H7 viruses represent one of the greatest pandemic threats posed by non-seasonal strains.

Thus, there is a need for a safe and effective universal vaccine that

- 2 046012 which stimulates the formation of a sustained response with the formation of broad-spectrum neutralizing antibodies and which provides protection against a wide range of existing and future strains of influenza virus (both seasonal and pandemic), in particular, in a vaccine that provides protection against one or more influenza A virus subtypes within phylogenetic group 2 for effective influenza prevention.

Short description

The present invention provides novel monomeric and multimeric, particularly trimeric, polypeptides derived from influenza virus group 2 hemagglutinin (HA), wherein the polypeptides contain the influenza virus HA stem domain and do not contain the globular head region and are referred to herein as hemagglutinin stem domain polypeptides (NA) influenza virus or mini-NA. The polypeptides induce a cellular and/or humoral immune response against at least group 2 influenza viruses when administered to a subject, in particular a human subject. The polypeptides of the present invention are thermostable and present conserved epitopes of the membrane-proximal stem domain of the group 2 HA molecule to the immune system in the absence of dominant epitopes present in the membrane-distal head domain.

Thus, in the HA stem domain polypeptides of the present invention, part of the primary sequence of the HA0 protein, i.e. the portion constituting the head domain has been deleted and the remaining amino acid sequence has been rejoined directly or, in some embodiments, by introducing a short flexible linker sequence to restore continuity of the polypeptide chain. The resulting amino acid sequence is further modified by introducing specific modifications that stabilize the native 3-dimensional structure of the remainder of the HA molecule.

In a first aspect, the present invention provides monomeric influenza A virus hemagglutinin (HA) stem domain polypeptides comprising the HA1 and HA2 domains of influenza A virus group 2 HA, wherein said HA stem domain polypeptides contain an amino acid sequence characterized by the following:

(i) deletion of the head region from the HA1 domain;

(ii) modification of the trimerization region in the HA2 domain;

(iii) at least two cysteine residues capable of forming at least one intramonomer cysteine bridge;

and wherein in the amino acid sequence, the amino acid at position 355 is W;

where the numbering of amino acid positions in the amino acid sequence of the HA stem domain polypeptide corresponds to the numbering of H3 in accordance with the HA nomenclature of Winter et al. (1981), which corresponds to the numbering in the full-length HA of the reference strain A/Aichi/2/68 H3N2 (SEQ ID NO: 1).

In certain embodiments, the present invention provides influenza A virus group 2 hemagglutinin (HA) stem domain polypeptides comprising HA1 and HA2 domains, wherein the HA stem polypeptides comprise an amino acid sequence that is characterized by:

(i) deletion of the head region in the HA1 domain, wherein the deletion spans at least the amino acid sequence from the amino acid corresponding to the amino acid at position 50 to the amino acid corresponding to the amino acid at position 302, inclusive;

(ii) modifying a trimerization region in the HA2 domain, preferably modifying a trimerization region in the C-helix, said trimerization region comprising an amino acid sequence from an amino acid corresponding to the amino acid at position 405 to an amino acid corresponding to the amino acid at position 419, inclusive;

(iii) a cysteine at an amino acid position corresponding to position 310, in combination with a cysteine at a position corresponding to position 422; or a cysteine at the amino acid position corresponding to position 311, in combination with a cysteine at a position corresponding to position 422; or a cysteine at the amino acid position corresponding to position 308, in combination with a cysteine at a position corresponding to position 418, which form (are capable of forming) an intramonomer disulfide bridge;

and wherein in the amino acid sequence, the amino acid at position 355 is W;

wherein the numbering of amino acid positions in the amino acid sequence of the HA stem domain polypeptide corresponds to the numbering of H3 according to the HA nomenclature of Winter et al., above, which corresponds to the numbering in the full-length HA of the reference strain A/Aichi/2/68 H3N2 (SEQ ID NO: 1) .

In accordance with the present invention, it has surprisingly been demonstrated that the novel group 2 influenza virus HA stem domain polypeptides of the present invention can be expressed recombinantly at high expression levels, are trimeric in cell culture supernatant in the absence of additional artificial C-terminal trimerization domains, and/ or have an elevated melting point, indicating pain

- 3 046012 high thermal stability. In addition, the group 2 HA stem domain polypeptides of the present invention mimic the stem domain of full-length group 2 HA through stable epitope presentation of HA stem domain-binding antibodies that bind to group 2 HA, such as CR9114 (which is described in WO 2013/007770) and/ or CR8020 (which is described in WO 2010/130636).

In a second aspect, the present invention provides a multimeric influenza A hemagglutinin (HA) stem domain polypeptide comprising at least two HA stem domain polypeptide monomers as described herein.

In a further aspect, the present invention provides nucleic acid molecules encoding group 2 influenza virus HA stem domain polypeptides.

In yet another aspect, the present invention provides vectors, particularly recombinant adenoviral vectors, comprising nucleic acids encoding influenza virus HA stem domain polypeptides.

In a further aspect, the present invention provides methods for inducing an immune response to group 2 influenza virus HA in a subject in need thereof, the method comprising administering to the subject an influenza virus HA stem domain polypeptide, nucleic acid molecule and/or vector in accordance with the present invention.

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

In a further aspect, the present invention provides group 2 influenza virus HA stem domain polypeptides, nucleic acid molecules encoding said influenza virus HA stem domain polypeptides, and/or vectors containing said nucleic acid molecules, for use in inducing an immune response against an influenza virus, in particular, for use as a vaccine to prevent a disease or pathological condition caused by strain A of the influenza virus from phylogenetic group 2.

Brief description of graphic materials

Fig. 1. A. Schematic overview of the polypeptides of the present invention (lower figure); B. Removal of the HA head region results in the stem domain polypeptides of the present invention (mini-HA); C. Three-dimensional representation of a stem domain-based polypeptide monomer (mini-HA) of the present invention; D. Schematic representation of a polypeptide of the present invention (specifically, the UFV180088 polypeptide).

Fig. 2. A. Protein expression levels in EXPI-CHO culture supernatants determined by OCTET (anti-C-tag antibody); B. Results of SEC analysis of EXPI-CHO cell culture supernatants expressing construct 180088 (left panel) and SEC-MALS analysis of purified 180088; C. Binding of mAb CR9114 to purified polypeptides by ELISA (EC ₅₀ values); D. Results of analysis of the temperature stability of purified polypeptides by differential scanning fluorimetry.

Fig. 3. SEC profiles and elution analysis results of EXPI-293 cell culture supernatants expressing several stem domain polypeptides of the present invention. A. SEC profiles of polypeptides with recurrent mutations to wild-type (WT) residues; the dotted line shows the stabilized mini-HA reference without the head domain (UFV180141), the black line shows the sequence of the WT mutant mini-HA; B. Elution time (left) and trimer peak height (right) of SEC profiles in panel A; C. SEC profiles of minimal structure polypeptides and staged introduction of selected mutations from UFV180088; the dotted line shows the reference minimal mini-HA structure (UFV180647), the black line shows the mutant mini-HA; D. Elution time and peak height of SEC profiles in panel C.

Fig. 4. Stability of a trimeric stem domain polypeptide with and without an interprotomer disulfide bridge. A. Results of SEC analysis of Expi293F cell culture supernatants (left panel) during collection of production and supernatants of EXPICHO cell cultures expressing purified polypeptides after one week incubation at 4°C without (UFV180192) and with (UFV180141) introduced cysteine residues at positions 398 and 408 (as indicated in the mini-HA structural model at the top of this figure); B. Temperature stability of purified polypeptides determined by differential scanning fluorimetry; C. Results of SDS-PAGE analysis of protein purity under non-reducing and reducing conditions. As can be seen, the introduced cysteine residues form interprotomer disulfide bridges and increase temperature stability.

Fig. 5. A. Protein expression levels and antibody binding, as determined by AlphaLISA culture supernatants, for EXPI-293-expressed polypeptides that differ at positions 355 and 482. Values determined by AlphaLISA and normalized to reference (UFV161333) ; B. Protein expression, trimer content, and antibody binding, as determined by AlphaLISA culture supernatants, of EXPI-293-expressed polypeptides mutant at positions 380 and 432. Values

- 4 046012 normalized to the standard (UFV170991); C. I. Protein expression, trimer content, and antibody binding, as determined by AlphaLISA culture supernatants, of EXPI-293-expressed polypeptides mutant at position 435. Values normalized to reference (UFV170611), C. P. Protein expression levels of the UFV171004 (435N) and UFV171197 (435R) constructs expressed in EXPI-CHO culture supernatants as determined by OCTET (left panel) and SEC analysis (right panel). In vitro characterization studies of purified polypeptides (bottom panel): binding to mAb CR9114 and mAb CT149 (ELISA, EC ₅₀ values) and temperature stability (differential scanning fluorimetry, Tm ₅₀ values in °C); D. Protein expression levels of polypeptides mutant at position 388 as determined by OCTET and SEC analysis of EXPI-CHO culture supernatants.

Fig. 6. Schematic representation of the removal of the HA head domain (HA1). Expression levels, trimer content, and mAb binding determined by AlphaLISA of cell culture supernatants of polypeptides expressed by EXPI-293 cells. All data are normalized to the reference structures UFV161908 (A), UFV160653 (B), and UFV160321 (C). A. In the reference structure (UFV161908), the portion of the head domain from amino acid position 46 to amino acid position 306, inclusive, was removed and the two ends of HA1 were connected with an artificial GPGS linker. Various alternative cutting positions for removing the HA head domain (the top strand of HA1) and direct joining of the ends of HA1 (i.e., joining the N-terminal segment of HA1 to the C-terminal segment of HA1 after removal of the head domain) are shown. In construct UFV170637 (dark gray), the head domain starting from amino acid position 47 to amino acid position 306, inclusive, was deleted similarly to the preferred constructs UFV180088, UFV180089, and UFV180090); B. Direct connection of the top and/or bottom strands of HA1 after removal of the HA head domain; C. Connection of the N-terminus and the C-terminus of HA1 via a homologous linker sequence derived from the head domain. All constructs are characterized by a deletion of the amino acid sequence from amino acid position 46 to amino acid position 306, inclusive.

Fig. 7. Expression level and mAb binding, as determined by AlphaLISA culture supernatants, of EXPI-293-expressed trimeric stem domain polypeptides with B-loop stabilization mutations. All data are normalized to the reference structures UFV161686 (A), UFV161333 (B, C), and UFV171187 (D);

A. Optimization and protection of the B-loop by introducing a glycosylation motif at position 401-403 for N-linked glycosylation at position 401;

B. Introduction of proline residues by point mutations to stabilize the B-loop; C. Introduction of a second glycosylation motif (for N-linked glycosylation at position 393) to further protect the B-loop; D. Combination of added N-linked glycosylation motifs (at position 401 and 392 or 393) with proline substitutions. Protein expression levels determined by OCTET (anti-His2 antibody); E. SEC profiles of EXPI-293 cell culture supernatants expressing polypeptides show that the introduction of one glycan (UFV180208) or two glycans and two proline residues (UFV180217) is quite acceptable. The retention time and height of the peak corresponding to the trimeric polypeptide are shown in gray.

Fig. 8. Expression level and mAb binding of EXPI-CHO-expressed trimeric stem domain polypeptides with and without the N-linked glycan motif at position 38. A. Expression level determined by OCTET (anti-C tag antibody) and antibody binding determined by ELISA (EC ₅₀ values); B. ELISA dilution curves for UFV170282 (solid line) and UFV170278 (dashed line).

Fig. 9. Results of analysis of supernatants from EXPI-293 cultures expressing polypeptides with variations in the position of the introduced intraprotomer disulfide bridge.

Protein expression and antibody binding as determined by AlphaLISA, normalized to the UFV160595 reference. A. Alternative cysteine residues introduced near the position present in the reference construct UFV160595; B. An alternative second disulfide bridge introduced into the region below the first disulfide bridge.

Fig. 10. Results of analysis of supernatants from EXPI-293 cultures expressing polypeptides with variations in the position of the introduced interprotomer disulfide bridge. Protein expression and antibody binding as determined by AlphaLISA, normalized to the UFV170051 reference structure.

Fig. 11. Results of analysis of EXPI-293 culture supernatants expressing soluble trimeric polypeptide variants with alternative C-terminal truncations (at position 515 in UFV171272 and stepwise to position 499 in UFV171280). A. SEC profiles with retention time and trimer peak height indicated in gray; B. Binding of polypeptides to the broad spectrum neutralizing antibodies CR9114 and CT149 determined by OCTET, showing the relative K _ON values of the polypeptides compared to the reference UFV170991 (in black).

- 5 046012

Fig. 12. Results of in vitro characterization studies of polypeptides of the present invention with substitutions of residues in the A-helix from wild type (WT) H3 towards H1. A. Protein expression levels and antibody binding as determined by AlphaLISA of EXPI-293 cell culture supernatants containing expressed polypeptides. The values are normalized to the standard (UFV161454); B. Protein expression levels in three independent EXPI-CHO culture supernatants determined by OCTET (anti-His2 antibody, left panel) and SEC-MALS analysis (right panel). Results of in vitro characterization of purified polypeptides (lower panel): binding to mAbs CR9114 and CT149 (ELISA, EC ₅₀ values) and temperature stability (differential scanning fluorimetry, Tm ₅₀ values).

Fig. 13. Results of analysis of the supernatant fluid of the EXPI-293 cell culture expressing trimeric polypeptides of the stem domain with surface mutations in the stem domain in the direction of HA H7. Trimer content and antibody binding determined by AlphaLISA. All data are normalized to the UFV172561 reference structure containing the 379 and 381 A-helix mutations in the H1 direction (A) or the UFV172562 reference structure containing wild-type H3 A-helix residues at positions 379 and 381 (B). In SEC profiles, standards are indicated by a dotted line.

Fig. 14. Results of SEC analysis of EXPI-293 cell culture supernatants expressing polypeptides derived from various group 2 H3 strains containing the appropriate structural elements to produce soluble trimeric stem domain polypeptides. A. SEC profiles of mini-HA polypeptides containing set I structural elements and based on A/Hong Kong/1/1968, A/Wisconsin/67/05 or A/Singapore/INFIMH/16/0019/2016 (trimer peak indicated icon T); B. SEC profiles of polypeptides containing additional stabilizing mutations in the B-loop: structure I (dashed line), structure II (gray) and structure III (black).

Fig. 15. Numbering of amino acid positions in wild-type A/Hong Kong/1/1968 H3 (wt A/HK/1/1968) and in H3-derived mini-HA structures UFV180088, UFV180089 and UFV180090 according to H3 numbering from Winter et al . (1981).

Fig. 16. Adenovirus (Ad26.FLU.004)-driven expression and folding of UFV180480 (UFV18088 with native transmembrane domain). Results of FACS analysis of MRC-5 cells transduced with A) Ad26.Empty (10,000 vp/cell, negative control) and B) Ad26.FLU.004 (5,000 vp/cell). Transduced MRC-5 cells were stained with antibody CR9114; C. Western blot analysis of MRC-5 cell lysates transduced with either Ad26.FLU.004 (5000 vp/cell) or Ad26.Empty (5000 vp/cell). UFV180088 (200 ng/line) was loaded as a positive control. All samples were run under non-reducing (lane 1-3) or reducing (lane 4-6) conditions. Antibody CR9114 was used to detect expressed mini-HA.

Fig. 17. Results of a study of the in vivo characteristics of the polypeptides UFV170278, a polypeptide that contains the wild-type 38-NAT-40 motif for N-linked glycosylation, and UFV170282, a polypeptide in which this glycan motif was knocked out using a point mutation of the T40I substitution type. A. Titers of antibodies specific to the stem domain of HA FL H3 A/Hong Kong/1/1968, 4 weeks after the third immunization of mice with the polypeptides of the present invention or PBS. The horizontal line in each group represents the median value within the group. B. Left panels: proportion of mice surviving during the follow-up period after challenge with A/Hong Kong/1/1968 H3N2 immunized with the indicated polypeptides of the present invention or PBS; top panel - UFV170278, bottom panel - UFV170282. Right panels: relative body weight of mice during the follow-up period after challenge with A/Hong Kong/1/1968 H3N2 immunized with the indicated polypeptides of the present invention or PBS; top panel -UFV170278, bottom panel 1 UFV170282. Change in relative body weight was expressed relative to day 0. Cumulative weight loss during the follow-up period was determined by calculating the area under the curve (AUC). Error bars indicate 95% confidence intervals.

Fig. 18. Results of studying the in vivo characteristics of the immunogenicity of the polypeptides UFV180088, UFV180089 and UFV180090 according to the present invention in an intact mouse model. A. Titers of antibodies specific to the stem domain of HA A/Hong Kong/1/1968 H3 FL after one (1 x), two (2 x) or three (3 x) immunization of mice with the polypeptides of the present invention or PBS. The horizontal line in each group represents the median value within the group. B Titers of antibodies to HA A/Hong Kong/1/1968 H3 FL, A/Texas/50/2012 H3 and A/Netherlands/219/2003 H7 after one (1 x), two (2 x) or three (3 x ) immunizing mice with the polypeptides of the present invention or PBS. The horizontal line in each group represents the median value within the group. The dotted line represents LLOQ, with open symbols indicating values in LLOQ.

Fig. 19. Results of an in vivo characterization study of the UFV180088, UFV180089 and UFV180090 polypeptides of the present invention in an intact mouse model of lethal H3N2 infection. Left panels: proportion of mice immunized with the indicated polypeptides that survived the follow-up period after challenge with A/Hong Kong/1/1968 H3N2

- 6 046012 according to the present invention or PBS; the top panel is UFV180088, the middle panel is UFV180089 and the bottom panel is UFVI80090. Right panels: relative body weight of mice during the follow-up period after challenge with A/Hong Kong/1/1968 H3N2 immunized with the indicated polypeptides of the present invention or PBS. Change in relative body weight was expressed relative to day 0. Cumulative weight loss during the follow-up period was determined by calculating the area under the curve (AUC). Error bars indicate 95% confidence intervals. Intact mouse model of lethal H7N9 infection.

Definitions

The following are definitions of terms used in the present invention.

The amino acid of the present invention may be any of 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 naturally found in proteins , such as, for example, norleucine. 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 protein structure and protein-protein interactions. Some amino acids have special properties, such as cysteine, which can form 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 7 shows the abbreviations and properties of standard amino acids.

The term included or including as used herein is intended to be followed by the words without limitation.

As used herein, the term infection means the invasion, multiplication 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 replicates in a cell or in the body of a subject. 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, infection refers to a pathological condition caused by the presence of a virus in a cell or body of a subject or invasion of a virus into a cell or body of a 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 virus surface proteins hemagglutinin (H) and neuraminidase (N). In accordance with the present invention, influenza virus subtypes may be named by their H number, such as an H3 HA-containing influenza virus, an H3 subtype influenza virus, or an H3 influenza virus, or by a combination of an H number and an N number, such as, subtype of influenza virus H3N2 or H3N2. The term subtype includes, but is not limited to, all of the individual strains within each subtype that are generally formed by mutation and exhibit different pathogenic profiles, including natural isolates as well as artificial mutants or reassortants, and the like. Such strains may also be referred to as different virus subtype isolates. Accordingly, as used herein, the terms strains and isolates may be used interchangeably. The existing nomenclature of human influenza virus strains or isolates includes the type (genus) of the virus, i.e. A, B or C, geographical location of first isolation, strain number and year of isolation, usually with a description of the HA and NA antigens given in parentheses, e.g. A/Moscow/10/00 (H3N2). Non-human strains also include in their nomenclature the host from which they originate.

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

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 virus, in a subject. As used herein, the terms disease and disorder are used interchangeably. In specific embodiments, the term refers to a respiratory disease 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 DNA molecules (eg, cDNA or genomic DNA) and RNA molecules (eg, mRNA), as well as DNA and RNA analogs made using nucleotide analogs. Nucleic acid can 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 apparent to those skilled in the art. Such modifications include, for example, tags, methylation, substitution of one or more

- 7 046012 naturally occurring nucleotide analogs, modifications of internucleotide bonds, such as uncharged bonds (for example, methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.), charged bonds (for example, phosphorothioate, phosphodithioate, etc.), pendant fragments (eg, polypeptides), intercalators (eg, acridine, psoralen, etc.), chelating agents, alkylating agents, and modified linkages (eg, alpha-anomeric nucleic acids, etc.). References to a nucleic acid sequence include 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 useful, for example, for antisense therapeutics, hybridization probes, and PCR primers.

The HA amino acid numbering used herein is based on the H3 numbering described by Winter et al. (1981). The numbering of amino acid residues or amino acid positions thus refers to the numbering in full-length H3 HA (specifically, the numbering of amino acid positions in A/Aichi/2/68) described and shown in FIG. 2 in Winter et al. (1981). Thus, the numbering is based on the numbering in the full-length HA of the reference strain A/Aichi/2/68 H3N2 (SEQ ID NO: 1). Numbering specifically refers to the numbering of amino acid positions in SEQ ID NO: 1. For example, the expressions amino acid at position 392 or amino acid corresponding to amino acid at position 392 (which are used interchangeably throughout the present invention) refer to the amino acid residue located at position 392 according to H3 numbering from Winter et al. (1981). It should be noted that since in the polypeptides of the present invention a portion of the HA1 domain (head domain) has been deleted, the numbering used herein does not necessarily refer to the actual amino acid positions in the HA stem domain polypeptides of the present invention, but instead refers to the position of the specified amino acid in the full-length HA molecule (i.e., without deletion in the head domain). Moreover, one skilled in the art will appreciate that equivalent amino acids (i.e., amino acids corresponding to the amino acid at a particular position in SEQ ID NO: 1) in other influenza virus strains and/or subtypes and in the stem domain polypeptides of the present invention can be determined using sequence alignment.

A polypeptide refers to a polymer of amino acids linked by amide bonds, as known to those skilled in the art. This term, as used herein, may refer to a single polypeptide chain linked by covalent amide bonds. The term can also refer to multiple polypeptide chains linked through 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 (eg S-palmitoylation).

An HA stem domain polypeptide refers to a polypeptide derived from HA that does not contain a naturally occurring (or wild-type) hemagglutinin (HA) head domain.

As used herein, the term wild type refers to HA from influenza viruses that circulate in nature.

Detailed description

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 or quadrivalent influenza vaccines produce a strong neutralizing antibody response to vaccine strains and closely related isolates, but rarely extends to more divergent strains within a subtype or to other subtypes. In addition, the selection of appropriate vaccine strains presents many challenges and often results in insufficient protection. Moreover, predicting the subtype of the next pandemic virus, including when and where it will appear, is currently still impossible.

Hemagglutinin (HA) is the major envelope glycoprotein of influenza viruses and represents the primary target of neutralizing antibodies. Hemagglutinin has two main functions during the penetration process. First, hemagglutinin mediates viral attachment to the surface of target cells through interaction with sialic acid-containing receptors. Second, after endocytosis of the virus, hemagglutinin subsequently initiates the fusion of the viral and endosomal membranes, releasing the viral genome into the cytoplasm of the target cell. HA contains a large ectodomain of ~500 amino acids, which is cleaved by host enzymes to form 2 polypeptides (HA1 and HA2), which remain connected by a disulfide bond. Most of the N-terminal fragment (HA1 domain, ~320-330 amino acids) forms the membrane-distal globular head domain, which contains the receptor-binding region and most of the determinants recognized by antibodies that neutralize the virus. Smaller C-terminal part (HA2 domain, ~180

- 8046012 amino acids) forms a stalk-like structure (stem domain) that anchors the globular domain to the cellular or viral membrane. The degree of sequence identity between subtypes is less for HA1 polypeptides (34% - 59% identity between subtypes) than for HA2 polypeptides (51% - 80% identity). The most conserved region is the sequence near the protease cleavage site, specifically the N-terminal 23 amino acids of HA2, whose sequence is conserved across all influenza A virus subtypes (Lorieau et al., 2010). Part of this region is accessible as a surface loop in the HA precursor molecule (HA0), but becomes inaccessible after HA0 is cleaved into HA1 and HA2.

Most neutralizing antibodies bind to loops that surround the receptor-binding site and thereby interfere with receptor binding and attachment. Because these loops are highly variable, most antibodies targeting these regions are strain-specific, which explains why current vaccines induce such limited strain-specific immunity. Fully human monoclonal antibodies to influenza virus hemagglutinin with a broad spectrum of cross-neutralizing activity have been created, such as, for example, CR6261 (WO 2008/028946). Functional and structural analysis showed that these antibodies interfere with the process of membrane fusion and are directed to highly conserved epitopes in the stem domain of the influenza virus group 1 HA protein (Throsby et al., 2008; Ekiert et al. 2009, WO 2008/028946). Following the identification of CR9114 (as described in WO 2013/007770), which cross-reacts with many group 1 and 2 HA molecules, it became clear that the human immune system can be induced to produce very broad-spectrum neutralizing antibodies to influenza viruses. However, given the requirement for an annual vaccination schedule, the production of such antibodies following infection or vaccination with (seasonal) influenza viruses of subtypes H1 and/or H3 does not appear to always be elicited or reach protective levels.

The present invention provides novel HA stem domain polypeptides that mimic specific epitopes, for example, antibodies CR9114 (containing the heavy chain variable region of SEQ ID NO: 7 and the light chain variable region of SEQ ID NO: 8), and/or CR8020 (comprising the heavy chain variable region of SEQ ID NO: 5 and the light chain variable region of SEQ ID NO: 6). The polypeptides of the present invention can be used to generate influenza virus-binding and/or influenza virus-neutralizing antibodies, preferably cross-binding and/or cross-neutralizing antibodies, when administered in vivo alone or in combination with other prophylactic and/or therapeutic treatments. By cross-binding and/or cross-neutralizing antibodies are meant antibodies that are capable of binding and/or neutralizing at least two, preferably at least three, four or five different subtypes of influenza A viruses from phylogenetic group 2, or antibodies that are capable of binding and /or neutralize at least one group 1 influenza virus and at least one group 2 influenza virus.

Influenza virus HA stem domain polypeptides stably presenting antibody epitopes CR6261 and/or CR9114 were previously described in WO 2013/079473. At least some of these HA stem domain polypeptides were able to stably present an epitope for CR6261 and/or CR9114 and were shown to be immunogenic in mice. Additional HA stem domain polypeptides capable of stably presenting an epitope for CR6261 and/or CR9114 have been described in WO 2014/191435, WO 2016/005480 and WO 2016/005482. Such stem domain polypeptides are based on the group 1 HA of influenza A viruses and induce an immune response only against group 1 influenza A viruses.

In the study that led to the present invention, it was shown that modifications that were introduced into group 1 HA stem domain polypeptides did not result in stable and trimeric stem domain polypeptides when using the HA of group 2 influenza viruses.

The present invention now provides group 2 influenza virus HA stem domain polypeptides containing novel modifications, such polypeptides can be expressed well in mammalian cells, are trimeric (e.g., as measured by AlphaLISA and SEC), and thermostable (e.g., when measured by for example, dynamic scanning fluorimetry/differential scanning calorimetry (DSF/DSC)). In addition, the group 2 influenza virus stem domain polypeptides of the present invention have been shown to induce the production of neutralizing antibodies in vivo. In addition, all broad-spectrum neutralizing antibodies (bnAbs) tested for the polypeptides of the present invention are less than 1 nM (as measured by Octet and ELISA), which is similar to the affinity of antibodies to full-length HA, clearly demonstrating that the polypeptides mimic stem domain of native full-length HA. In addition, novel HA stem domain polypeptides may, but are not required to, contain any artificial linkers, tags, or N-, C-terminal trimerization domains.

Thus, in a first aspect, the present invention provides monomeric polypeptides

- 9 046012 hemagglutinin (HA) stem domain polypeptides of the influenza A virus, containing the HA1 and HA2 domains of the HA influenza A virus group 2, wherein said HA stem domain polypeptides contain an amino acid sequence characterized by the following:

(i) deletion of the head region from the HA1 domain;

(ii) modification of the trimerization region in the HA2 domain;

(iii) at least two cysteine residues capable of forming at least one intramonomer cysteine bridge;

and wherein in the amino acid sequence, the amino acid at position 355 is W;

wherein the numbering of amino acid positions in the amino acid sequence of the HA stem domain polypeptides corresponds to the numbering of H3 in accordance with the HA nomenclature of Winter et al., which corresponds to the numbering in the full-length HA of the reference strain A/Aichi/2/68 H3N2 (SEQ ID NO: 1).

Thus, the present invention provides HA stem domain polypeptides (i.e., HA polypeptides without a head domain) containing a trimerization region modification in the HA2 domain, preferably a modification in the C-helix, and at least 2 cysteine residues forming an intramonomer disulfide bridge ; where in the amino acid sequence, the amino acid at position 355 is W; and wherein the numbering of amino acid positions in the amino acid sequence of the HA stem domain polypeptides corresponds to the numbering of H3 in accordance with the HA nomenclature of Winter et al. based on the full-length HA numbering of the reference strain A/Aichi/2/68 H3N2 (SEQ ID NO: 1).

In certain embodiments, the present invention provides monomeric influenza A virus hemagglutinin (HA) stem domain polypeptides comprising the HA1 and HA2 domains of influenza A virus group 2 HA, wherein the HA stem domain polypeptides contain an amino acid sequence that is characterized by the following:

(i) deletion of the head region from the HA1 domain;

(ii) modification of the trimerization region in the HA2 domain;

(iii) at least two cysteine residues capable of forming at least one intramonomer cysteine bridge;

and where the polypeptides contain a mutation such as replacing an amino acid at position 355 with W;

wherein the numbering of amino acid positions in the amino acid sequence of the HA stem domain polypeptides corresponds to the numbering of H3 in accordance with the HA nomenclature of Winter et al., which corresponds to the numbering in the full-length HA of the reference strain A/Aichi/2/68 H3N2 (SEQ ID NO: 1).

In certain embodiments, the amino acid at position 355 is W and the amino acid at position 432 is I, or the amino acid at position 355 is W and the amino acid at position 432 is I, and the amino acid at position 380 is I. In certain embodiments implementation, the polypeptides contain a mutation replacing the amino acid at position 355 with W and a mutation replacing the amino acid at position 432 with I, or a mutation replacing the amino acid at position 355 with W, and a mutation replacing the amino acid at position 432 with I, and a mutation by replacing the amino acid at position 380 with I. According to the present invention, the presence of these amino acids has been shown to increase the levels of trimers of the polypeptides of the present invention.

In certain other embodiments, the amino acid at position 355 is (mutation to) W and the amino acid at position 378 is (mutation to) T, the amino acid at position 379 is (mutation to) N , and/or the amino acid at position 381 is (mutated to) V. The presence of these amino acids has been shown to increase the expression and binding of broad-spectrum neutralizing antibodies.

In certain embodiments, the polypeptides further comprise an introduced glycosylation motif (NxT) for N-linked glycosylation at position 401 to protect potential neo-epitopes in the B-loop. Thus, according to the present invention, the polypeptides contain a glycosylation motif (NxT) at positions 401-403 for N-linked glycosylation at position 401.

In specific embodiments, the present invention provides influenza A virus group 2 hemagglutinin (HA) stem domain polypeptides comprising HA1 and HA2 domains, wherein said HA stem domain polypeptides contain an amino acid sequence that is characterized by:

(i) deletion of the head region in the HA1 domain, wherein the deletion spans at least the amino acid sequence from the amino acid corresponding to the amino acid at position 50 to the amino acid corresponding to the amino acid at position 302, inclusive;

(ii) modification of a trimerization region in the HA2 domain, preferably modification of a trimerization region in the C-helix, wherein said trimerization region contains an amino

- 10 046012 acid sequence from the amino acid corresponding to the amino acid at position 405 to the amino acid corresponding to the amino acid at position 419, inclusive;

(iii) a cysteine at an amino acid position corresponding to position 310, in combination with a cysteine at a position corresponding to position 422; or a cysteine at an amino acid position corresponding to position 311, in combination with a cysteine at a position corresponding to position 422; or a cysteine at the amino acid position corresponding to position 308, in combination with a cysteine at a position corresponding to position 418, wherein said cysteine residues form (are capable of forming) an intramonomer disulfide bridge; where in the amino acid sequence, the amino acid at position 355 is W; and wherein the numbering of amino acid positions in the amino acid sequence of the HA stem domain polypeptide corresponds to the numbering of H3 in accordance with the HA nomenclature of Winter et al. (see above) based on the full-length HA numbering of the reference strain A/Aichi/2/68 H3N2 (SEQ ID NO: 1).

According to the present invention, it was unexpectedly discovered that influenza virus group 2 HA stem domain polypeptides having an amino acid sequence where the amino acid at position 355 is W had high levels of expression in mammalian cells, had an increased tendency to trimerize and/or increased thermal stability according to compared to previously prepared group 2 HA stem domain polypeptides. In addition, the HA stem domain polypeptides of the present invention induce a humoral and/or cellular immune response against group 2 influenza virus in vivo.

As is known to those skilled in the art, full-length influenza virus hemagglutinin (HAE) typically contains a HA1 domain and a HA2 domain. In addition, full-length influenza virus hemagglutinin (HAG) typically contains a stem domain and a head domain. The stem domain is formed by two segments of the HA1 domain and most or all of the HA2 domain. The two segments of the HA1 domain are separated in the primary sequence by the globular head domain. As described herein, the HA stem domain polypeptides of the present invention contain an amino acid sequence that contains several modifications in the HA1 and/or HA2 domain compared to the wild-type amino acid sequence of a full-length HA polypeptide (HAO), particularly the group 2 HA amino acid sequence In the context of this invention, the numbering of amino acid positions in the amino acid sequence of the HA stem domain polypeptides corresponds to the numbering of the HA in accordance with the HA nomenclature from Winter et al., see above (i.e., corresponds to the numbering of the full-length HA of the reference strain A/Aichi/2/68 H3N2 (SEQ ID NO: 1)).

According to the present invention, at least a portion of the highly variable and immunodominant head region in the HA1 domain of an influenza virus HA polypeptide, said portion comprising at least the amino acid sequence beginning at amino acid position 50 and extending to amino acid position 302, inclusive, has been subjected to domain deletion HA1 full-length HA protein (HAO) to create a stem domain polypeptide, also called mini-HA. The remaining parts of the HA1 domain (ie, the N-terminal segment of the HA1 domain and the C-terminal segment of the HA1 domain) are linked either directly (ie, without a linker) or through a linker of 1-10 amino acids. Thus, for example, when deleting an amino acid sequence from amino acid at position 50 to amino acid at position 302, inclusive, amino acid at position 49 (the last amino acid of the N-terminal segment of HA1) is joined to the amino acid at position 303 (the first amino acid of the C-terminal segment of HA1) either directly or due to the replacement of the deleted head region with a linker of 1-10 amino acids. A deletion of the amino acid sequence from amino acid position 50 to amino acid position 302, inclusive, is a minimal deletion in the HA1 domain. In accordance with the present invention, a larger portion of the HA1 domain may also be deleted, for example, the amino acid sequence starting at amino acid position 47 and continuing up to and including amino acid position 306, as shown in FIG. 1A in case of lower design.

In a preferred embodiment, the deletion in the HA1 domain spans at least the amino acid sequence from amino acid position 47 to amino acid position 306, inclusive. In such an embodiment, the stem domain polypeptide thus comprises an N-terminal HA1 segment up to and including amino acid position 46 and a C-terminal HA1 segment beginning at amino acid position 307 (dark gray portions in FIG. 1A).

In a preferred embodiment, the deletion in the HA1 domain comprises the amino acid sequence from amino acid position 47 to amino acid position 306, inclusive.

In some embodiments, the deleted region of the HA1 domain has been replaced with a linker sequence of 1-10 amino acids.

In addition, the HA stem domain polypeptides of the present invention described herein contain a modification of the trimerization region in the HA2 domain, preferably a modification in the C-helix, to improve the trimerization of the HA stem domain polypeptides after deletion of the head region. In certain preferred embodiments, said may

- 11 046012 modification in the HA2 domain is a modification that enhances the trimerization of the HA stem domain polypeptide.

In certain embodiments, the modification includes introducing a heterologous trimerization domain into the C-helix. The C-helix is generally considered to contain the amino acid sequence from the amino acid at position 405 to the amino acid at position 434, inclusive (H3 numbering). In a preferred embodiment, said heterologous trimerization domain was introduced at a position corresponding to the amino acid sequence from amino acid position 405 to amino acid position 419, inclusive (Fig. 1A). Thus, in certain embodiments, the original (wt) amino acid sequence in the HA2 domain from position 405 to position 419 has been replaced by a heterologous trimerization sequence of the same length, i.e. with the same number of amino acids.

In certain embodiments, the heterologous trimerization domain is the sequence GCN4.

In certain preferred embodiments, the modified trimerization region (ie, comprising a heterologous trimerization domain) comprises an amino acid sequence selected from the group consisting of ₄₀₅ RMKQIEDKIEEIESK ₄₁₉ (SEQ ID NO: 9) and ₄₀₅ PMKQIEDKIEEIESK ₄₁₉ (SEQ ID NO: 10).

In some embodiments, at least one of the amino acids of the heterologous trimerization sequence has been mutated to C to form an intermonomer cysteine bridge (as described below). Thus, in certain embodiments, the trimerization region thus comprises an amino acid sequence selected from the group consisting of ₄₀₅ RMKCIEDKIEEIESK ₄₁₉ (SEQ ID NO: 11) and ₄₀₅ PMKCIEDKIEEIESK ₄₁₉ (SEQ ID NO: 12). In a preferred embodiment, the trimerization region consists of amino acid sequence ₄₀₅ PMKCIEDKIEEIESK ₄₁₉ (SEQ ID NO: 12).

In certain embodiments, the modification includes changing, preferably optimizing, the heptad repeat sequence in the C-helix, preferably in the trimerization region comprising the amino acid sequence from the amino acid at position 405 to the amino acid at position 419, inclusive. The heptad repeat, designated [abcdefg] _n , typically has hydrophobic residues at a and d and polar/charged residues at e and g. These motifs are the basis for most supercoil structures, which are a structural motif in proteins in which alpha helices are twisted together like strands in a rope (dimers and trimers are the most common types) (Ciani et al., 2010).

As a further modification, the HA stem domain polypeptides of the present invention contain at least two cysteine residues that form (are capable of forming) an intramonomeric (or intraprotomer) cysteine (or disulfide) bridge. An engineered cysteine bridge can be introduced by mutation of at least one residue (if the other is already a cysteine), but usually by mutation of two residues that are spatially close to each other, as a cysteine substitution, resulting spontaneously or through active oxidation a covalent bond is formed between the sulfur atoms in these residues. In a preferred embodiment, the polypeptides comprise a cysteine at position 310 and a cysteine at position 422, or a cysteine at amino acid position corresponding to position 311 in combination with a cysteine at position 422, or a cysteine at amino acid position corresponding to position 308 in combination with cysteine at a position corresponding to position 418, which makes possible the formation of an intramonomer cysteine bridge. In certain embodiments, the polypeptides contain an amino acid substitution mutation at positions 310 and/or 422 to C, or an amino acid substitution mutation at positions 311 and/or 422 at C, or an amino acid substitution mutation at amino acid position , corresponding to position 308 and/or 418 on C, wherein said cysteine residues form said intramonomer cysteine bridge. Such cysteine residues thus form an intramonomer (or intraprotomer) cysteine (or disulfide) bridge that stabilizes the protein. In a preferred embodiment, the polypeptides contain a (mutation to) cysteine at position 310 and a (mutation to) cysteine at position 422 to form at least one intramonomer cysteine bridge.

The polypeptides of the present invention typically contain at least 4 native (i.e., naturally occurring) glycosylation motifs (or glycan motifs) (NxT) for N-linked glycosylation, for example, the glycan motif at positions 8-10 (8NSTj ₀ ), positions 2224 ( ₂₂ NGT ₂₄ ), provisions 38-40 ( ₃₈ NAT ₄₀ ) and 483-485 ( ₄₈₃ NGT ₄₈₅ ). In certain embodiments, the polypeptides contain at least one introduced glycan motif at positions 401-403 for N-linked glycosylation at position 401, as described above. In certain embodiments, the polypeptides contain at least one additional introduced glyco motif

- 12 046012 zylation. Thus, in certain embodiments, at least one additional N-linked glycosylation motif is present and/or introduced at positions 392-394 for N-linked glycosylation at position 392 and/or at positions 393-395 for N-linked glycosylation at position 393. In a preferred embodiment, the polypeptides contain a glycosylation motif at positions 401-403 for N-linked glycosylation at position 401 and a glycosylation motif at positions 393-395 for N-linked glycosylation at position 393.

In additional embodiments, the amino acid at the position corresponding to position 388 is M. In certain embodiments, the amino acid at the position corresponding to position 388 is mutated to M. However, other amino acids are also possible at this position, including without restrictions T, V, I, L, F, Y, W, H, K and R.

Moreover, in certain embodiments, the polypeptides comprise an amino acid sequence where:

the amino acid at position 31 is E and the amino acid at position 34 is V;

the amino acid at position 392 is S or P;

the amino acid at position 395 is T or P;

the amino acid at position 399 is S or P;

the amino acid at position 435 is N or R; and/or the amino acid at position 439 is Y.

Thus, in certain embodiments, the amino acid at position 31 is E and the amino acid at position 34 is V. In certain embodiments, the polypeptides comprise an amino acid sequence comprising an amino acid substitution mutation at position 31 to E and an amino acid substitution mutation at position 34 on V. According to the present invention, it has been found that due to the presence of such amino acid residues (ie 31E and 34V) there is an optimization of the hydrogen bonding network, which is an important factor in the stability of the polypeptides of the present invention. The polypeptides may further comprise an amino acid sequence wherein the amino acid at position 392 is (mutated to) S or P; the amino acid at position 395 is (mutation to) T or P; and/or the amino acid at position 399 is a (substitution mutation to) S or P. Thus, the polypeptides of the present invention may contain one or more mutations in the so-called B-loop, wherein the B-loop contains the amino acid sequence ranging from amino acids at position 385 to amino acids at position 404, inclusive (see Fig. 1C). B-loop mutations increase the solubility of polypeptides by reducing hydrophobicity. In certain preferred embodiments, the polypeptides, relative to the wild-type HA polypeptide, thus contain at least one additional B-loop mutation selected from the group consisting of:

mutations by replacing the amino acid corresponding to the amino acid at position 392 with S or P, preferably with S;

mutations by replacing the amino acid corresponding to the amino acid at position 395 with T or P; preferably on T; and mutations by replacing the amino acid corresponding to the amino acid at position 399 with S or P, preferably P.

The polypeptides may further comprise a mutation such as replacing the amino acid corresponding to the amino acid at position 435 with N or R, preferably N; and/or an amino acid substitution mutation corresponding to the amino acid at position 439 to Y. Such mutations are believed to optimize the trimer interface, promoting stability of the trimers in solution.

It should again be noted that the amino acid position numbering used herein is based on the H3 numbering according to Winter et al. (1981). It should also be noted again that the numbering of amino acid positions used herein is based on the numbering of positions in the full-length H3 HA polypeptide (HA0). Thus, as used herein, the term amino acid at position 434 refers to the amino acid at position 434 in HA0 H3. The numbering thus does not refer to the actual amino acid positions in the HA stem domain polypeptides of the present invention due to the deletion of the head domain (see FIG. 15).

In accordance with the present invention, the HA stem domain polypeptide is a group 2 HA polypeptide. Thus, in accordance with the present invention, the modifications described herein were introduced into the HA of an influenza virus from phylogenetic group 2, such as an influenza virus containing HA subtype H3, H7 or H10, resulting in the HA stem domain polypeptides of the present invention. In certain embodiments, the HA stem domain polypeptide is an H3 HA polypeptide. Thus, in certain embodiments, the HA stem domain polypeptide is derived from an influenza A virus HA containing an H3 subtype HA, such as from an influenza A/Hong Kong/1/68 virus with an amino acid after

- 13 046012 with SEQ ID NO: 2, or A/Wisconsin/67/2005 with amino acid sequence SEQ ID NO: 13, or A/Singapore/INFMH/16/0019/2016 with amino acid sequence SEQ ID NO: 14. To a person skilled in the art It will be appreciated in the art that the polypeptides of the present invention may also be derived from the HA of other influenza A H3 virus strains, including but not limited to A/Perth/16/2009 (SEQ ID NO: 15), A/Brisbane/10/ 2007 (SEQ ID NO: 16) or A/Panama/2007/1999 (SEQ ID NO: 17).

As described above, stem domain polypeptides may or may not contain a linker sequence of 1-10 amino acid residues replacing the deleted HA1 head domain sequence and thereby connecting the two remaining HA1 portions. In certain embodiments, the linker sequence contains from 1 to 5 amino acids. In certain embodiments, the linker sequence contains 2, 3, or 4 amino acids. The linker sequence may be a heterologous linker sequence, i.e. an amino acid sequence that is not found in naturally occurring or wild type HA, such as, but not limited to, GGGG and GPSG.

In certain embodiments, the linker sequence is a homologous linker sequence, i.e. an amino acid sequence derived from a deleted corresponding head region, such as, but not limited to, NPHR, GDPH, NGGS, GGSN, GSNA, GPGS, GSGF, GSG, GG, GGS, SGS, HPST, IPNI, GLSS, KPGD, DAPI, TPN, and TPNG .

In preferred embodiments, the polypeptides do not contain a linker sequence.

As described above, cleavage of the influenza virus HA0 protein (into HA1 and HA2) is required for its activity, which facilitates 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 contain 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 polypeptides do not contain a protease cleavage site. Thus, in certain preferred embodiments, the protease cleavage site has been removed by mutating the amino acid residue at position 329 to 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 contain a mutation such as replacing the amino acid at position 329 with glutamine (Q). Thus, in certain preferred embodiments, the polypeptides of the present invention contain an R329Q knockout mutation at the cleavage site to prevent the expected cleavage of the molecule during in vitro or in vivo production following administration.

In other embodiments, the polypeptides contain a polybasic cleavage site, such as a furin cleavage site. Thus, the polypeptides can be cleaved by furin-like proteases in the cell to produce cleaved mini-HA in a manner similar to natively folded and processed HA.

In certain embodiments, the polypeptides do not contain a signal sequence. A signal sequence (sometimes called a signal peptide, targeting signal, localization signal, localization sequence, transit peptide, leader sequence, or leader peptide) is a short peptide (usually 16–30 amino acids long) that is present at the N-terminus of most newly synthesized proteins that set the direction of movement towards the secretory pathway. Signal sequences function by causing a protein to move within a cell, usually to the cell membrane. In many cases, the amino acids that make up the signal peptide are cleaved from the protein after it reaches its final destination. In influenza virus HA, the signal sequences typically contain the first 16 amino acids of the full-length HA0 amino acid sequence (corresponding to amino acids from position -6 to position 10 according to H3 numbering, see FIG. 15).

In certain embodiments, the polypeptides contain a signal sequence (part of a signal sequence). Polypeptides may contain a wild-type signal sequence (part of a wild-type signal sequence) or may contain alternative signal sequences (part of alternative signal sequences), such as, without limitation, a signal sequence selected from the group:

MKTIIALSYIFCLALG (SEQ Ш NO: 18);

MKTIIALSYILCLVFA (SEQ Ш NO: 19);

MKTIIALSYILCLVFT (SEQ Ш NO: 20); And

MKTIVALSYILCLVFA (SEQ Ш NO: 21).

In preferred embodiments, the (soluble) polypeptides do not contain a signal

- 14 046012 new sequence.

In a preferred embodiment, the polypeptides of the present invention are characterized by (i) a deletion of the head region from the HA1 domain, wherein said deletion spans the amino acid sequence from the amino acid at position 47 to the amino acid at position 306, inclusive, where the amino acid at position 46 is directly linked to the amino acid at position 307 ;

(ii) introducing a heterologous trimerization domain containing the amino acid sequence ₄₀₅ PMKCIEDKIEEIESK ₄₁₉ (SEQ ID NO: 12) replacing the original amino acid sequence from the amino acid at position 405 to the amino acid at position 419, inclusive;

(iii) a (cysteine mutation) cysteine at amino acid position corresponding to position 310 in combination with a (cysteine mutation) cysteine at amino acid position 422;

(iv) introduced glycosylation motifs at positions _: 393-395 (ie _393NQT395 ) and 401-403 (ie 401NAT403);

and where, moreover, in the amino acid sequence:

(a) the amino acid at position 355 is (substitution mutation to) W;

(b) the amino acid at position 432 is (mutation to) I, and the amino acid at position 380 is (mutation to) I;

(c) the amino acid at position 378 is (mutation to) T and the amino acid at position 379 is (mutation to) N, and/or the amino acid at position 381 is (mutation to) V;

(d) the amino acid at position 388 is (mutation to) M;

(e) the amino acid at position 31 is (mutation to) E, and the amino acid at position 34 is (mutation to) V;

(f) the amino acid at position 392 is (mutation to) S;

(g) the amino acid at position 395 is (mutation to) T;

(h) the amino acid at position 398 is (mutation to) C;

(i) the amino acid at position 399 is (substitution mutation to) P;

(j) the amino acid at position 408 is (mutation to) C;

(k) the amino acid at position 435 is (mutation to) N;

(l) the amino acid at position 439 is (mutation to) Y and (m) the amino acid at position 329 is (mutation to) Q;

where the numbering of amino acid positions in the amino acid sequence of the HA stem domain polypeptide corresponds to the numbering of H3, corresponding to the numbering in the full-length HA of the reference strain A/Aichi/2/68 H3N2 (SEQ ID NO: 1).

In a specific embodiment, the HA1 and HA2 domains are derived from an influenza virus containing HA subtype H3, preferably from an influenza A/Hong Kong/1/68 virus.

In certain preferred embodiments, the HA1 and HA2 domains are derived from an influenza virus containing an H3 HA subtype, preferably an influenza A/Hong Kong/1/68 virus, wherein one or more amino acids in said H3 HA1 and HA2 domains have been mutated to corresponding amino acids HA H7.

Thus, in certain embodiments, the HA1 and HA2 domains are derived from an influenza virus containing HA subtype H3, preferably from the influenza A/Hong Kong/1/68 virus of claim 19, wherein:

(a) the amino acid at position 25 is (substitution mutation to) K;

(b) the amino acid at position 367 is (mutation to) Y;

(c) the amino acid at position 378 is (mutation to) T;

(d) the amino acid at position 475 is (mutation to) D;

(e) the amino acid at position 476 is (mutation to) D; and/or (f) the amino acid at position 479 is (substitution mutation to) A.

Without wishing to be bound by any theory, it is believed that by reconstituting the surface of an H3-derived stabilized stem domain polypeptide with the desired characteristics, such as expression, folding, and thermostability, toward the H7 HA, an antibody response can be induced, which may confer greater protection against distant H7 viruses, without having to completely switch to an H7-derived stem domain polypeptide with less favorable characteristics, i.e. which is more difficult to produce, which has lower expression levels and lower stability.

In certain embodiments, the polypeptides comprise a HA2 domain comprising transmembrane (TM) and cytoplasmic (CD) domains (wherein said TM and CD domains comprise an amino acid sequence corresponding to the amino acid sequence starting with the amino acid corresponding to the amino acid at position 514 and continuing to the amino acid , corresponding to the amino acid at position 550 inclusive (numbering H3)). So

- 15 046012 in this way, membrane-bound mini-HA polypeptides are obtained.

To produce secreted soluble stem domain polypeptides, in certain embodiments, the polypeptides do not contain transmembrane and cytoplasmic domains. Thus, in certain embodiments, the polypeptides contain a truncated HA2 domain, particularly a HA2 domain that is C-terminally truncated. The truncated HA2 domain of the present invention is thus shorter than the full-length HA2 sequence due to the deletion of one or more amino acid residues at the C-terminus of the HA2 domain.

In certain embodiments, the C-terminal portion of the HA2 domain beginning at the amino acid corresponding to amino acid position 514 has been deleted, thereby removing substantially the entire transmembrane and cytoplasmic domain.

In certain embodiments, a portion of the C-terminal helix has also been deleted. In accordance with the present invention, it has been discovered that even by deleting most of the HA2 domain, stable soluble HA stem domain polypeptides can be obtained. Thus, in certain embodiments, the C-terminal portion of the HA2 domain beginning at amino acid position 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, or 514 has been subjected to deletion (in this case also numbered according to the H3 numbering described by Winter et al., see above) to obtain a soluble polypeptide after expression in cells.

In a preferred embodiment, the C-terminal portion of the HA2 domain, starting at position corresponding to 506, was deleted.

A heterologous amino acid sequence (ie, an amino acid sequence that does not naturally occur in the influenza virus HA) may optionally be joined to the (truncated) HA2 domain.

Thus, in certain embodiments, the His tag sequence, for example, HNNNNNN (SEQ ID NO: 22) or HNNNNNN (SEQ ID NO: 23), or the FLAG tag DYKDDDDK (SEQ ID NO: 24), or the EPEA C tag (SEQ ID NO: 25), or a combination thereof, were fused to the C-terminal amino acid of the (optionally truncated) HA2 domain for detection and/or purification purposes. In certain embodiments, a heterologous amino acid sequence, such as a His tag sequence, can be attached to the (truncated) HA2 domain using a linker. In certain embodiments, the linker may comprise a proteolytic cleavage site (part of a proteolytic cleavage site), such as the amino acid sequence IEGR (SEQ ID NO: 26) or LVPRGS (SEQ ID NO: 27), to enzymatically remove the His tag sequence after purification.

In certain embodiments, the heterologous amino acid sequence fused to the C-terminal amino acid of the (truncated) HA2 domain comprises an amino acid sequence selected from the group consisting of:

GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ Ш NO: 28),

AAADYKDDDDKLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH (SEQIDNO: 29 ),

AAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH (FLAG - GS linker - His tag), (SEQIDNO: 30),

EGRAAAGGSGGGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVT PIQRIVLSGENGLKIDIHVnPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVI DGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGV TGWRLCERILAAAAWSHP QFEKGAAWSHPQFEKGAAWSHPQFEK (NanoLuc - Streptag), (SEQ ID NO: 31),

EGRAAAGGSGGGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVT PIQRIVLSGENGLKIDIHVnPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVI DGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGV TGWRLCERILAGAAEPEA (NanoLuc - C-label), (SEQ ID NO: 32),

- 16 046012

EGRAAAWSHPQFEKGAAWSHPQFEKGAAWSHPQFEK (Strep-tag), SEQ Ш NO: 33),

EGRAAALPETGGGAAEPEA (Sortase - C-tag), (SEQ Ш NO: 34),

SGRDYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSWSHPQFEK GAAWSHPQFEKGAAWSHPQFEK (FLAG - GS linker - Strep tag), (SEQ Ш NO: 35), and

EGRAAAEQKLISEEDLGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSWSHP QFEKGAAWSHPQFEKGAAWSHPQFEK (Myc tag - GS linker - Strep tag), (SEQ ID NO: 36).

In certain embodiments, a heterologous trimerization domain has been fused to the C-terminal amino acid of an (optionally truncated) HA2 domain, such as, but not limited to, a foldon trimerization domain (as described in Letarov et al. (1993); S-Guthe et al. (2004) )).

In certain embodiments, the HA stem domain polypeptides of the present invention comprise an amino acid sequence selected from SEQ ID NOs: 40-44, 46-64, 66, 67, 69-97, 156-164, 169-181, and 189-212.

In a preferred embodiment, the polypeptide contains an amino acid sequence selected from SEQ ID NO: 40-42, 207 and 210-212, preferably an amino acid sequence selected from 210-212, more preferably SEQ ID NO: 210.

In certain embodiments, the polypeptides are glycosylated when expressed in suitable cells (eg, mammalian cells). The polypeptides of the present invention typically contain 4 native glycosylation motifs (NxT), as described above. As also described above, according to the present invention, in certain embodiments, the polypeptides contain at least one introduced glycosylation motif at positions 401-403 for N-linked glycosylation at position 401. The polypeptides preferably contain an additional introduced glycosylation motif at positions 393-395 for N -linked glycosylation at position 393.

In a further aspect, the present invention provides multimeric, preferably trimeric, HA stem domain polypeptides. To produce stable trimeric HA stem domain polypeptides, the polypeptides of the present invention preferably contain at least two cysteine residues forming (capable of forming) an intermonomeric (also called interprotomer) cysteine bridge. Thus, in certain embodiments, the polypeptides comprise a cysteine at position 396 in combination with a cysteine at position 408, or a cysteine at position 397, in combination with a cysteine at position 408, or a cysteine at a position corresponding to position 398, in combination with a cysteine at a position corresponding to position 408, or a cysteine at a position corresponding to position 398, in combination with a cysteine at a position corresponding to position 405.

In certain embodiments, the polypeptides comprise a mutation at amino acid position 396 to C and a mutation at amino acid position 408 to C; or a mutation by replacing the amino acid at position 397 with C and a mutation by replacing the amino acid at position 408 with C; or a mutation replacing an amino acid at position 398 with C and a mutation replacing an amino acid at position 408 with C, or a mutation replacing an amino acid at position 398 with C and a mutation replacing an amino acid at position 405 with C; resulting in the formation of an intermonomer cysteine bridge between the cysteine at position 396 of the first monomer and the cysteine at position 408 of the second monomer; or between the cysteine at position 397 of the first monomer and the cysteine at position 408 of the second monomer; or between the cysteine at position 398 of the first monomer and the cysteine at position 408 of the second monomer; or between the cysteine at position 398 of the first monomer and the cysteine at position 405 of the second monomer. It should be noted that in some embodiments, the amino acids at positions 405 or 408 are in a heterologous trimerization sequence.

In a preferred embodiment, the polypeptides contain a cysteine at position 398 and a cysteine at position 408, resulting in an intermonomer cysteine bridge between the cysteine at position 398 of the first monomer and the amino acid at position 408 of the second monomer.

In addition, the present invention provides nucleic acid molecules encoding the HA stem domain polypeptides of the influenza virus of the present invention. One skilled in the art will appreciate that several different nucleic acid molecules can encode the same polypeptide due to the degeneracy of the genetic code. It is also understood that those skilled in the art can, using standard techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the described polynucleotides to reflect

- 17 046012 changes in the codon usage frequency of any particular host organism in which the polypeptides are to be expressed. Therefore, unless otherwise indicated, the expression nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that are degenerate variants of each other and that encode the same amino acid sequence.

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

In certain embodiments, the nucleic acid molecules encoding the influenza virus HA stem domain polypeptide comprise a nucleic acid sequence selected from SEQ ID NO: 208 and SEQ ID NO: 209.

Influenza virus hemagglutinin stem domain polypeptides can be prepared according to any technique deemed appropriate by one of ordinary skill in the art, including those described below. Thus, the polypeptides of the present invention can be synthesized as DNA sequences using standard methods known in the art and cloned and then expressed in vitro or in vivo using suitable restriction enzymes and methods known in the art.

In addition, the present invention relates to vectors containing a nucleic acid molecule encoding a polypeptide of the present invention. In certain embodiments, the nucleic acid molecule of the present invention is thus part of a vector, such as a plasmid. Such vectors can be easily manipulated using methods well known to one skilled in the art and are, for example, designed to be capable of replication in prokaryotic and/or eukaryotic cells. The vector used may be any vector that is suitable for DNA cloning and that can be used to provide transcription of the nucleic acid of interest. When using host cells, it is preferred that the vector is an integrating vector. Alternatively, the vector may be an episomal replicating vector. One skilled in the art will be able to select suitable expression vectors and insert the nucleic acid sequences of the present invention in a functional manner. It is well known to those skilled in the art that to achieve expression of nucleic acid sequences encoding polypeptides, sequences capable of driving expression can be operably linked to nucleic acid sequences encoding the polypeptide to produce recombinant nucleic acid molecules encoding the protein or polypeptide in an expressible format. Expression control sequences may include promoters, enhancers, etc., as well as combinations thereof. They must be capable of functioning in the host cell, thereby controlling the expression of nucleic acid sequences that are operably linked to them. One skilled in the art will be aware that various promoters can be used to achieve gene expression in host cells. Promoters can be constitutive or regulated and can be obtained from a variety of sources, including viruses, prokaryotic or eukaryotic sources, or engineered artificially. Expression of nucleic acids of interest can occur under the control of a natural promoter or derivative thereof or under the control of a completely heterologous promoter (Kaufman, 2000). Some well known and most commonly used promoters for expression in eukaryotic cells include promoters derived from viruses such as adenovirus, such as the E1 A promoter, promoters derived from cytomegalovirus (CMV), such as the immediate early (IE) promoter. CMV (referred to herein as the CMV promoter) (derived, for example, from pcDNA, Invitrogen), promoters derived from simian virus 40 (SV40) (Das et al, 1985), and the like. Suitable promoters can also be obtained from eukaryotic cells, such as metallothionein (MT) gene promoters, elongation factor 1α (EF-1a) gene promoter (Gill et al., 2001), ubiquitin C or UB6 gene promoter (Gill et al., 2001 ), actin gene promoter, immunoglobulin gene promoter, heat shock protein gene promoters, etc. Testing promoter function and promoter strength is standard practice for one skilled in the art and may typically, for example, involve cloning the gene being tested, such as the lacZ gene, luciferase, GFP, etc., behind the promoter sequence and testing the expression of the gene being tested . Of course, promoters can be modified by deletion, addition, mutation of sequences therein and tested for functionality to discover new, weakened, or improved promoter sequences. In accordance with the present invention, strong promoters that provide high levels of transcription in selected eukaryotic cells are preferred.

The constructs can be introduced by transfection into eukaryotic cells (eg plant, fungal, yeast or animal cells) or suitable prokaryotic expression systems such as

- 18 046012 as E. coli, using methods that are well known to those skilled in the art. In some cases, a suitable sequence of a tag (such as, for example, but not limited to, a His, Myc, Strep tag, sortase, or FLAG tag) or a complete protein (such as, for example, but not limited to, maltose binding protein or glutathione B-transferase ) can be added to the sequences of the present invention described above to provide purification and/or identification of polypeptides from cells or supernatant. Optionally, a sequence containing a specific proteolysis site can be included for subsequent removal of the tag by proteolytic cleavage.

In preferred embodiments, the polypeptides are produced in mammalian cells.

Purified polypeptides can be analyzed using spectroscopic methods known in the art (eg, 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-pleated sheets. ELISA, AlphaLISA, label-free biolayer interferometry (Octet) and FACS, etc. can be used to study the binding of the polypeptides of the present invention to broad-spectrum neutralizing antibodies such as CR8020 and/or CR9114. Thus, polypeptides of the present invention can be selected having the desired conformation. Trimer content can be analyzed, for example, using SDS gel electrophoresis under non-reducing conditions, size exclusion chromatography in the presence of Fab antibody fragments from broad-spectrum neutralizing antibodies such as CR8020 and/or CR9114, and AlphaLISA using variously labeled antibodies . The stability of polypeptides can be assessed as described above after temperature stress, freeze-thaw cycles, increased protein concentration, or agitation. The melting point of the polypeptide can be further assessed using differential scanning fluorimetry (DSF) and/or differential scanning calorimetry (DSC).

In certain embodiments, the nucleic acid is inserted into a recombinant vector that can be used as a vaccine component. Preferably, the recombinant vector is a human adenovirus, for example human adenovirus serotype 26 (Ad26). Thus, the present invention also provides recombinant adenoviral vectors containing a nucleic acid molecule encoding the HA stem domain polypeptide of the present invention. In a preferred embodiment, the nucleic acid molecule encoding the stem domain polypeptide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 208 and SEQ ID NO: 209.

The production of recombinant adenoviral vectors is well known in the art. The term recombinant, as used herein in relation to an adenovirus, implies that it has been modified by humans, for example, it has altered terminal regions actively cloned into it, and/or it contains a heterologous gene, i.e. it is not a naturally occurring wild-type adenovirus. In certain embodiments, the adenoviral vector of the present invention is deficient in at least one essential functional E1 region gene, such as the E1a region and/or the E1b region, of the adenoviral genome, which is required for viral replication. In certain embodiments, the 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 have multiple deficiencies, which means that a given adenoviral vector is deficient in one or more essential functional genes in each of two or more regions of the adenoviral genome. For example, in the aforementioned E1-deficient or E1 and E3-deficient adenoviral vectors, there may additionally be a deficiency of at least one essential E4 region gene and/or at least one essential E2 region gene (e.g., the E2A region and/or the E2B). Adenoviral vectors, methods for their construction and methods for their propagation are well known in the art and are described, for example, in US patents No. 5559099, 5837511, 5846782, 5851806, 5994106, 5994128, 5965541, 5981225, 6040174, 6020191 and 6113913.

In certain embodiments, the adenovirus is a human adenovirus serotype 26 or 35.

In addition, the present invention provides a pharmaceutical composition containing a polypeptide, nucleic acid and/or vector in accordance with the present invention, as well as a pharmaceutically acceptable carrier. In particular, the present invention relates to pharmaceutical compositions containing a therapeutically effective amount of the polypeptides, nucleic acids and/or vectors of the present invention. The pharmaceutical compositions further contain a pharmaceutically acceptable carrier. In the context of the present invention, the term pharmaceutical

- 19 046012 commercially 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, for example, Remington: The Science and Practice of Pharmacy - 22nd edition, Loyd V. Ed. Allen, Pharmaceutical Press [2013]; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds, Taylor & Francis [2000]; Remington: Essentials of Pharmaceutics, Linda Felton, Pharmaceutical Press [2013] and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press [ 2000]). The term carrier refers to the diluent, excipient or medium with which polypeptides, nucleic acids and/or vectors are administered. Saline solutions and aqueous solutions of dextrose and glycerol can, for example, be used as liquid carriers, in particular for injection solutions.

The polypeptides or nucleic acid molecules of the present invention can also be administered in combination with nanoparticles or conjugated to nanoparticles, such as, for example, polymers, liposomes, virosomes, virus-like particles. Polypeptides or nucleic acid molecules can be combined with, encapsidated within, or conjugated with (eg, covalently bound to or adsorbed on) nanoparticles.

In addition, the present invention relates to polypeptides, nucleic acids and/or vectors described herein for use as a drug.

In particular, the present invention relates to polypeptides, nucleic acids and/or vectors described herein for use in inducing an immune response against an influenza virus, preferably a group 2 influenza virus.

The present invention also provides methods for inducing an immune response against influenza A virus in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of a polypeptide, nucleic acid molecule and/or vector described herein. The subject in accordance with the present invention is preferably a mammal that can be infected with influenza virus or can otherwise benefit from the induction of an immune response, such subject being, for example, a rodent, such as a mouse, a ferret, or a domestic or farm animal, or a primate , different from a person, or a person. The subject is preferably a human subject.

In certain embodiments, the present invention provides methods for inducing an immune response against a group 2 influenza A virus. The immune response may include a humoral (ie, inducing the production of antibodies that neutralize the influenza virus) and/or a cellular immune response. In certain embodiments, the present invention provides methods for inducing an immune response against at least two, three, four, five, or six subtypes of group 2 influenza viruses. In certain embodiments, the present invention provides methods for inducing an immune response against an influenza virus containing an HA subtype H3.

In certain embodiments, the induced immune response is effective in preventing influenza virus infection caused by a group 2 influenza A virus, such as an influenza A virus containing H3 HA subtype and/or an influenza A virus containing H7 subtype HA. In certain embodiments, the induced immune response is effective in preventing influenza virus infection caused by influenza A virus containing H3 subtype HA. In certain embodiments, the induced immune response is effective in preventing influenza virus infection caused by influenza A virus containing HA subtypes H3 and H7.

The present invention further relates to the polypeptides, nucleic acids and/or vectors described herein for use as an influenza vaccine, particularly for use as a vaccine against influenza caused by a group 2 strain of influenza virus.

In certain embodiments, the polypeptides, nucleic acid molecules and/or vectors of the present invention are administered in combination with an adjuvant. The adjuvant can be administered before, simultaneously with, or after administration of the polypeptides, nucleic acid molecules and/or vectors of the present invention. Examples of suitable adjuvants include aluminum salts such as aluminum hydroxide and/or aluminum phosphate; oil emulsion compositions (or oil-in-water compositions), including squalene-in-water emulsions such as MF59 (see, for example, WO 90/14837); saponin-based compositions such as, for example, QS21 and immunostimulating complexes (ISCOM) (see, for example, US 5057540; WO 90/03184, WO 96/11711, WO 2004/004762, WO 2005/002620); derivatives of bacteria or microorganisms, examples of which are monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), optionally formulated in liposome form, oligonucleotides containing a CpG motif, ADP-ribosylated bacterial toxins or mutant forms thereof, such as heat-labile enterotoxin LT E. coli, 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 fusion of the polypeptides of the present invention with proteins known in the art to enhance the immune response (for example, with

- 20 046012 tetanus toxoid, CRM197, rCTB, bacterial flagellins or others), or the incorporation of polypeptides into virosomes, or combinations thereof.

Administration of the polypeptides, nucleic acid molecules and/or vectors of the present invention can be accomplished using standard routes of administration. Non-limiting examples include parenteral administration, such as intravenous, intradermal, transdermal, intramuscular, subcutaneous, etc., or transmucosal administration, such as intranasal, oral, and the like. One skilled in the art will be able to identify various possibilities for administering polypeptides, nucleic acid molecules and/or vectors in accordance with the present invention for the purpose of inducing an immune response.

In certain embodiments, the polypeptide, nucleic acid molecule, and/or vector is administered more than once, i.e. according to the so-called homologous priming-stimulating scheme. The second dose can be administered, for example, one week after the first dose, two weeks after the first dose, three weeks after the first dose, one month after the first dose, six weeks after the first dose, two months after the first dose, 3 months after the first dose, or 4 months or more after the first dose, etc., up to several years after the first dose of the polypeptide, nucleic acid molecule and/or vector of the present invention. It is also possible to administer polypeptides, nucleic acid molecules, and/or vectors more than twice, eg, three times, four times, etc., such that the first priming administration is followed by more than one priming administration.

Polypeptides, nucleic acid molecules and/or vectors can also be administered as a priming or boosting vaccination according to a heterologous priming-boosting schedule.

The present invention further provides methods for preventing disease caused by influenza virus in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a polypeptide, nucleic acid molecule and/or vector described herein. A therapeutically effective amount refers to an amount of polypeptide, nucleic acid and/or vector that is effective to prevent, alleviate and/or treat a disease or condition caused by influenza virus infection. Prevention includes inhibiting or reducing the spread of influenza virus or inhibiting or reducing the onset, development or progression of one or more symptoms associated with influenza virus infection. The expression relief as used herein may refer to the reduction of visible or tangible symptoms of disease, viremia, or any other measurable manifestation of an influenza virus infection.

The subject in need of treatment includes subjects who already have a condition due to influenza virus infection, as well as those in whom influenza virus infection needs to be prevented. The polypeptides, nucleic acids and/or vectors of the present invention can thus be administered to a subject who has not previously received treatment, i.e. a subject who does not have illness due to influenza virus infection, or who has not been and is not currently infected with influenza virus infection, or subjects who have already been infected with influenza virus infection.

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

The polypeptides, nucleic acid molecules and/or vectors of the present invention can be administered to a subject in combination with one or more other active agents, such as alternative influenza vaccines, monoclonal antibodies, antivirals, antibacterial agents and/or immunomodulatory agents. One or more other active agents may be useful in treating and/or preventing disease caused by influenza virus, or may alleviate a symptom or condition associated with disease caused by influenza virus. In some embodiments, the one or more other active agents are analgesics, antipyretics, or therapeutic agents that facilitate or facilitate breathing.

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.

- 21 046012

Examples

Example 1. HA stem domain polypeptides - structure and structural elements of the preferred polypeptides of the present invention: UFV180088, UFV180089 and UFV180090.

The UFV180088 polypeptide, which is the stem domain (or stem domain) of the uncleaved influenza virus hemagglutinin (HAo) from the H3 influenza virus A/Hong Kong/1/68, was obtained by deleting (at least part of) the head domain from HA ₁ , in particular, region containing amino acids starting from position 47 up to and including amino acid position 306 (Figures 1A and 1B). It should be noted that for the numbering of amino acid positions in the present invention, the H3 numbering according to Winter et al., see above, is used. The main structural elements of the polypeptide of the present invention (mini-HA), including the A-helix, B-loop and C-, D- and E-helices, are indicated in FIG. 1C.

When expressed as a soluble ectodomain, the polypeptides of the present invention are truncated at the C-terminus after the last helix (which ends at position 499). UFV180088 was truncated at position 506, i.e. The C-terminal portion of the HA sequence, starting at amino acid position 506, was deleted.

The polypeptide of the present invention, UFV 180088, described in this example, was made resistant to protease cleavage by mutation by substitution of the amino acid arginine (R), which is a natural monobasic cleavage site, at position 329 (i.e., the C-terminal amino acid of the domain HA1, see Fig. 1), for example, glutamine (Q). Unlike native full-length HA, polypeptides of the present invention containing the R329Q mutation can no longer be cleaved and cannot undergo the corresponding conformational change in which the hydrophobic fusion peptide is embedded in the interior of the protein.

Removal of the head domain leaves exposed a portion of the HA molecule that was previously protected from the aqueous solvent. For this reason, several amino acid residues in the B-loop, i.e. in the region containing amino acids 385-404 (Fig. 1C) was mutated relative to the parental full-length wild-type HA from A/Hong Kong/1/1968 to stabilize the stem domain polypeptide. Specifically, the amino acid at position 388 was mutated to M, and the amino acid at position 392 was mutated to S.

Next, proline was introduced into the B-loop to reduce the propensity for helicalization, specifically proline at position 399. Finally, to protect potential neo-epitopes in the B-loop, one or two N-linked glycosylation motifs (e.g., NxT) were introduced into the B-loop ), in particular the glycosylation motif at positions 393-395 for N-linked glycosylation at position 393 and the glycosylation motif at positions 401-403 for N-linked glycosylation at position 401.

Moreover, to facilitate stable trimerization of the soluble polypeptide derived from the HA stem domain, a trimerization domain sequence derived from GCN4, ₄₀₅ PMKCIEDKIEEIESK ₄₁ 9 (SEQ ID NO: 12), was introduced into the HA ₂ domain, particularly the C-helix, replacing the original amino acid sequence (i.e., wild type) from amino acid at position 405 to amino acid at position 419, inclusive.

In addition, cysteine residues (if not already present) were introduced at positions 398 and 408 (it should be noted that position 408 is in the input sequence GCN4) to form interprotomer disulfide bridges between the cysteine at position 398 of the first monomer and the cysteine. at position 408 of an adjacent monomer to covalently link the monomers into a trimeric stem domain polypeptide.

To further stabilize and increase the expression of the UFV180088 polypeptide, as well as to ensure correct folding, similar to the stem domain of the full-length wild-type HA, additional substitution mutations were introduced into the polypeptide, in particular, at positions 31 (D31E), 34 (I34V), 310 ( K310C), 355 (H355W), 378 (N378T), 379 (379N), 380 (K380I), 381 (L381V), 422 (S422C), 432 (E432I), 435 (H435R) and 439 (L439Y) (Fig. 1D).

Variants of the UFV180088 polypeptide were obtained, i.e. UFV180089 and UFV180090. These polypeptides contained additional mutations compared to UFV180088.

Thus, UFV180089 contained additional mutations (compared to UFV180088) L367Y, N475D, A476D and E479A. UFV180090 contained additional (compared to UFV 180088) mutations L25K, L367Y, A476D and E479A and did not contain the G379N and L381V mutations.

Example 2 Expression, purification and in vitro characterization of trimeric polypeptides of the present invention.

Protein expression in mammalian cells.

DNA fragments encoding the polypeptides of the present invention UFV180088, UFV180089 and UFV180090 (which are described in example 1) were synthesized (GenScript) and cloned into the expression vector pcDNA2004 (plasmid pcDNA3 modified in our own laboratory with an enhanced CMV promoter). Polypeptides were produced in a suspension of ExpiCHO cells cultured in ExpiCHO™ expression medium by transient transfection with appropriate commercial grade DNA using ExpiFectamine™ transfection reagent (Gibco, ThermoFisher Scien

- 22 046012 tific) in accordance with the manufacturer's protocol. ExpiFectamine CHO stimulant and ExpiCHO supplement (Gibco, ThermoFisher Scientific) were added to cell cultures 1 day after transfection according to the manufacturer's protocol. Culture supernatant samples containing secreted polypeptides were collected on days 7–11 and clarified by centrifugation followed by filtration through a 0.2-μm bottle filter attachment (Corning).

Protein purification.

Polypeptides were purified through a two-step protocol. First, the collected and clarified culture supernatant was loaded onto a Hi Scale 16/20 column (GE Healthcare) packed with an affinity resin (Capture Select), which consists of a single-domain HA-specific antibody immobilized on agarose beads (ThermoFisher Scientific). This resin is highly specific for Smetka, a four-residue acidic peptide (E-P-E-A (SEQ ID NO: 25)) that has been fused to C-terminal polypeptides. The amount of added polypeptide in the collected culture supernatant was determined by OCTET prior to purification (see .paragraph; Analysis of culture supernatant and purified protein. C-tagged proteins were eluted using TRIS buffer containing 2 M MgCl _2. Based on the UV signal (A280), the eluted fractions were combined and filtered through a Millex-GV filter membrane with a pore size of 0.22 μm (Merck Millipore).The collected elution peak was then applied to a Superdex 200 pg 26/60 column (GE Healthcare) equilibrated with running buffer (20 mM Tris, 150 mM NaCl, pH 7.8) to remove Potential multimeric and/or monomeric protein contaminants Trimer fractions were pooled and purity assessed by analytical SEC-MALS.

Analysis of culture supernatant and purified protein.

As described above, the level of expressed stem domain polypeptide in the collected culture supernatant was assessed prior to purification by biolayer interferometry using the OCTET platform (ForteBio). Briefly, Capture Select™ biotin-C-tag antibody conjugate (ThermoFisher Scientific) was immobilized onto streptavidin (SA) biosensors (ForteBio), and a standard curve was generated by assessing the binding shift in a dilution series from a well-defined reference batch of purified homologous polypeptide. The binding shift of prediluted harvested culture supernatants (10- and 30-fold diluted in kinetic assay buffer (ForteBio)) containing the polypeptides of the present invention was then measured, and the concentration of the polypeptides was determined using a standard curve generated.

Polypeptide trimer content of the culture supernatant and purified polypeptides were assessed by size exclusion chromatography-multiangle light scattering (SEC-MALS) analysis using an Infinity 1260 series high-performance liquid chromatography (HPLC) system (Agilent). 40 μg of each purified polypeptide was run (1 ml/min) through a TSKgel G3000SWxl column (Sigma-Aldrich), and the molar mass of the eluted material was measured using a miniDAWN Treos multi-angle light scattering detector and an Optilab T-rEX differential refractometer (Wyatt Technology). Data were analyzed using the Astra 6 software package (Wyatt Technology), and molecular weight calculations were made from the refractive index signal.

Proper folding of the purified polypeptides of the present invention was assessed by ELISA (antibody binding EC ₅₀ values).

To do this, stem domain polypeptides were coated at a concentration of 10 nM and incubated with a series of dilutions of the monoclonal antibody (mAb) CR9114 (which is described in WO2013/007770) 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 addition of POD substrate. Reading was performed using an EnSight™ multi-mode plate reader (PerkinElmer). EC ₅₀ values were calculated using the Spotfire software package (Tibco Software Inc.).

Thermal stability of the purified polypeptides was determined by differential scanning fluorimetry (DSF) by monitoring the fluorescence emission of Sypro orange dye (ThermoFisher Scientific) added to a 6 μg polypeptide solution. When the temperature is gradually increased from 25 to 95°C (60°C per hour), the polypeptides unfold and the fluorescent dye binds to the exposed hydrophobic residues, resulting in a characteristic change in emission. Melting curves were measured using a ViiA7 real-time PCR device (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 has unfolded and are thus a measure of the temperature stability of the polypeptides.

Results and conclusion.

Polypeptide expression levels and trimer content were determined for two independent 70-ml ExpiCHO transfection media on day 9 posttransfection (Fig. 2A). All polypeptides

- 23 046012 were well expressed. The H3N2 A/Hong Kong/1/68-derived polypeptide UFV180088 was expressed at ∼700 mg/L culture supernatant. Polypeptides UFV180089 and UFV180090, similar in structure to the UFV180088 polypeptide and containing additional changes in surface amino acids (i.e. changes on the surface to more closely resemble H7 HA): L367Y, N475D, A476D and E479A, as well as changes in L25K, L367Y, N379G, V381L, A476D and E479A were expressed at levels of ~500 mg/L and ~350 mg/L, respectively.

Analysis of the crude cell culture supernatant by analytical SEC (Fig. 2B, left panel) indicated the presence of a well-defined population of soluble trimeric polypeptides (retention time ∼8.3 minutes). Similar analysis also indicated that the two-step purification protocol provided a very pure trimeric polypeptide (Fig. 2B, right panel). Moreover, the trimeric polypeptides were correctly folded and exposed the broad-spectrum neutralizing monoclonal antibody epitope CR9114. This is evident from the ELISA results demonstrating strong binding to CR9114 with EC ₅₀ values below 1 nM (Fig. 2C). In addition, the temperature at which 50% of the polypeptide unfolds was determined using DSF. All polypeptides were thermostable and had Tm ₅₀ values of 66.6°C, 64.6°C, and 60.9°C for UFV180088, UFV180089, and UFV180090, respectively (Figure 2D).

In conclusion, the polypeptides of the present invention described in this example were well expressed and purified from cell culture supernatants as properly folded trimeric polypeptides.

Example 3. Characterization of single-point mutations in polypeptides of the present invention (SEC profiles).

Developed structures.

To evaluate the contribution of mutations introduced into the trimeric polypeptides of the present invention (schematically shown in Fig. 1), amino acids were subjected to backmutation by replacement of the original amino acids in the backbone of strain A/Hong Kong/1/1968 (Table 1, Fig. 3A ), starting with the UFV 180141 polypeptide (characterized by all the features of UFV180088, i.e., deletion of the head region starting from amino acid position 47 to amino acid position 306 inclusive (i.e., deletion 47306), introduced by a trimerization region in the HA2 domain at positions 405-419 (it should be noted that the introduced sequence of GCN4 is slightly different from UFV180088: i.e. ₄₀₅ RMKCIEDKIEEIESK ₄₁₉ (SEQ ID NO: 11)), containing a cysteine at the amino acid position corresponding to position 310, in combination with a cysteine at a position corresponding to position 422 (forming an intraprotomer disulfide bridge) containing Q at position 329 (resistance to protease cleavage), and the amino acid at position 355 is W; and the amino acid at position 378 is T, and the amino acid at position 379 is N, and the amino acid at position 381 is V; and containing a glycan motif at positions 401-403) and containing a cysteine at position corresponding to position 398, in combination with a cysteine at position corresponding to position 408 (in the GCN4 sequence), to form an interprotomer disulfide bridge, and containing M at position 388, E at position 31 and V at position 34, I at positions 380 and 432, S at position 392, T at position 395, S at position 399 and N at position 435 and Y at position 439). Like UFV180088, the UFV180141 polypeptide was truncated after amino acid position 506. However, UFV180141 does not contain an additional glycosylation motif at positions 393-395, a B-loop stabilizing proline at position 405, and carries 399S instead of 399P. In addition, UFV180088 contained a C-terminal EPEA tag (SEQ ID NO: 25), whereas UFV180141 contained a different C-terminal tag.

The exception is the C408Q mutation, which is not a throwback mutation in wild-type H3, but which was produced as a throwback mutation to the introduced GCN4 trimerization domain sequence (inserted at positions 405-419). The impact of the absence of specific mutations was assessed using analytical SEC.

An alternative approach to assessing the positive effect of selected mutations is their stepwise introduction into a polypeptide of minimal structure, i.e. UFV180647, characterized by the following features: deletion of the head region, starting from amino acid position 47 to amino acid position 306 inclusive (i.e. deletion 47-306), introduced by the trimerization region in the HA2 domain, i.e. ₄₀₅ RMKCIEDKIEEIESK ₄₁₉ (SEQ ID NO:.11), introduced in provisions 405-419; a cysteine at the amino acid position corresponding to position 310, in combination with a cysteine at the position corresponding to position 422 (to form an intraprotomer disulfide bridge), containing a Q at position 329 (resistant to cleavage by proteases), and where the amino acid at position 355 is W; the amino acid at position 378 is T, the amino acid at position 379 is N, and the amino acid at position 381 is V; and containing a glycan motif at positions 401-403 and containing a cysteine at a position corresponding to position 398, in combination with a cysteine at a position corresponding to position 408 (with the formation of an interprotomer disulfide bridge), and containing M at position 388. Constructs with added mutations were analyzed

- 24 046012 was analyzed using analytical SEC and compared with the minimal structure polypeptide (Table 2, Fig. 3C).

Table 1

Reversal of selected mutations back to wild-type A/Hong Kong/1968 residues in the stabilized trimeric polypeptide UFV180141

No. UFV Mutation(s) UFV180141 As described above UFV180192 C398E, C408Q (interprotomer disulfide bridge) UFV180193 W355H UFV180194 GCN4 deletion UFV180195 C310K, C422S (intramolecular disulfide bridge) UFV180196 T378N, N379G, V381L (A-helix mutations) UFV180197 I380K, I432E UFV180198 I380K UFV180199 I432E UFV180200 M388T UFV180201 N401E, A402V, T403E (B-loop glycan) UFV180202 T395I UFV180203 N435H UFV180204 Y439L UFV180205 E31D, V34I UFV180206 S392F UFV180207 S399F

table 2

Step-by-step introduction of selected mutations into the minimal structure polypeptide UFV180647

No. UFV Mutations UFV180647 As described above UFV181034 KZ801, E432I UFV181036 KZ801, E432I, H435N UFV181038 K380I, E432I, H435N, L439Y UFV181040 Stabilized B-loop (F392S, H393N, I395T, F399P, R405P) UFV181042 K380I, E432I, stabilized B-loop (F392S, H393N, I395T, F399P, R405P)

Protein expression in mammalian Expi293F cells.

DNA fragments encoding polypeptides given in table. 1 and table. 2 were synthesized as described in Example 2. Polypeptides including a C-terminal FLAG linker-His tag for screening and purification were produced in the eukaryotic suspension cell line Expi293F on a microscale (200 μl). Briefly, cells were transiently transfected with commercial-grade DNA in 96-well plates with half-well depth (System Duetz) at a cell density of 2.5E+06. h/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 400 x g) followed by filtration (96-well filter plates, 0.22 µm PVDF membrane, Corning) .

Analysis of culture supernatant.

The content of the polypeptides of the present invention in the collected Expi-293 cell culture samples was then assessed by SEC analysis on an Infinity 1260 series high performance liquid chromatography (HPLC) system (Agilent). An injection volume equal to 100 μl of culture supernatant was driven (1 ml/min) through a TSK G3000SWxl gel column (Sigma-Aldrich) and elution was monitored by UV detection (Fig. 3A). Alternatively, samples were analyzed by ultra-high performance liquid chromatography (UHPLC) using a Vanquish system (ThermoFisher Scientific) with a BEN 200A column (Waters, 40 μL injection volume, 0.35 mL/min flow) and eluted fractions were monitored using a scatter detector Helios light (Wyatt Technologies, Fig. 3C). SEC profiles were analyzed using the Astra 6 software package (Wyatt Technology). The elution time and trimer peak (height and shape) of the SEC profiles were visualized in FIG. 3B and FIG. 3D.

- 25 046012

Results and conclusion.

A subset of recurrent mutations appears to be deleterious to the expression of stem domain polypeptides, as has been shown, for example, in UFV180193 (W355H recurrent mutation), UFV180194 (GCN4 deletion), UFV180195 (intramolecular disulfide bridge deletion), and UFV180199 (1432E recurrent mutation). , while other recurrent mutations were quite acceptable (Figures 3A and 3B).

Polypeptides without the stabilizing mutations introduced in UFV180088 (Table 2) showed significant improvements in the expression level of the minimal trimeric stem domain polypeptide with the additional addition of mutations of the present invention (to the structure of UFV180088). For example, as shown in FIG. 3C, the trimeric peak of the UFV1801034 polypeptide shifted in elution time and increased in height with the introduction of mutations such as the K380I, E432I substitution (Fig. 3D). Gradual addition of a stabilizing mutation in the B-loop (UFV181042: F392S, H393N, I395T, F399P, R405P) further increased the expression of the trimeric polypeptide.

This example demonstrates that at least the following amino acid positions (eg, mutations): i.e. (mutation by substitution to) W at position 355 and/or (mutation by substitution to) T at position 378, (mutation by substitution to) N at position 379 and (mutation by substitution to) V at position 381 and /or (mutation to) I at position 432 or (mutation to) I at positions 432 and 380, as well as the glycosylation motif at positions 401-403 for N-linked glycosylation at position 401, are useful for obtaining high levels of the required soluble trimeric polypeptides. The addition of a stabilizing mutation in the B-loop (eg, F392S, H393N, I395T, and F399P) further increased the expression of the trimeric polypeptide.

Example 4. Interprotomer disulfide bridge; stability of the polypeptide of the present invention.

Developed structures.

To evaluate the contribution of introduced cysteine residues to the B-loop and C-helix that form the interprotomer disulfide bridges in the polypeptides of the present invention (schematically shown in Fig. 4), the cysteine residues were backmutated by substitution into their corresponding wild-type residue (glutamine acid), present in the backbone of strain A/Hong Kong/1/1968 (position 398), and in glutamine (residue 408), present in the introduced GCN4 trimerization domain sequence (405-419). The effect of the absence of cysteine residues and the subsequent disulfide bridge was assessed using analytical SEC, DSF and SDS-PAGE.

Expression, purification and characterization of proteins.

DNA fragments encoding the UFV180192 and UFV180141 polypeptides were obtained on a microscale in Expi293F cells as described in Example 3, and on a medium scale in ExpiCHO cells as described in Example 2 (~60 ml, harvest on day 8). The culture supernatant was analyzed by analytical SEC as shown in Example 2, on the day of harvest (microscale) or after a 1-week incubation period of the collected culture supernatant at 4°C (medium scale). From collected culture supernatants (medium scale), His-tagged polypeptides were purified by a two-step protocol using the ACT Avant 25 system (GE Healthcare Life Sciences). First, immobilized metal affinity chromatography was performed using a cOmplete His-tag purification preloaded column (Roche), washed with 1 mM imidazole, and eluted with 300 mM imidazole. Second, size exclusion chromatography was performed using an SRT-10C SEC-300 column (Sepax Technologies) and peak trimer fractions were collected. Thermal stability of purified proteins was determined by DSF (as in Example 2) and protein purity was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing and reducing conditions by running through a 10% Bis-Trnis gel using the system Bolt and according to the manufacturer's instructions (Invitrogen).

Results and conclusion.

Based on the results of assessing the cell culture supernatant using analytical SEC, it was clear that both polypeptides with introduced cysteine residues (UFV180141) and without (UFV180192) introduced cysteine residues in the B-loop (at position 398) and the C-helix (at position 408) expressed the trimeric polypeptide in solution (Fig. 4A; left panel). The UFV180192 polypeptide was more abundant compared to the UFV180141 peptide on the day of production 3 days after small-scale transfection. However, at a higher scale, when production was collected on day 8 and after storage of the supernatant containing UFV180192 for one week at 4°C, a second non-trimeric peak appeared (retention time ~8.5 minutes), indicating about the structural instability of the construct without an interprotomer disulfide bridge, (Fig. 4A; right panel). Differences in stability were confirmed by DSF evaluation of the purified polypeptides. The polypeptide without an interprotomer disulfide bridge (UFV180192) unfolded at 50.8°C, while the polypeptide with introduced cysteine residues at positions 398 and

- 26 046012

408 unfolded at 67.7°C (Fig. 4B). SDS-PAGE analysis of the purified polypeptides revealed that the polypeptide lacking cysteine residues at positions 398 and 408 was driven as expected to a monomer height of ~40 kDa under both non-reducing and reducing conditions. In contrast, the major band for the polypeptide with two introduced cysteine residues was observed under non-reducing conditions at ∼120 kDa, as expected for a covalently linked trimer, and under reducing conditions to the expected height of a ∼40-kDa monomer (Figure 4C ).

The data presented in this example demonstrate that covalently linking monomers by introducing two cysteine residues at position 398 and position 408 to form interprotomer disulfide bonds resulted in a significantly more stable soluble trimeric polypeptide.

Example 5: Alternative replacements for provisions 355, 380 and 342, 435 and 388.

Developed structures.

Alternative substitutions to optimize the polypeptides of the present invention were tested at position 355 (Fig. 5A), at positions 380 and 432 (Fig. 5B), at position 435 (Fig. 5C) and at position 388 (Fig. 5D). Polypeptide expression and folding were assessed in Expi293F cell culture supernatant.

Protein expression in mammalian cells.

DNA fragments encoding polypeptides were synthesized as described in example 2. Polypeptides including a C-terminal FLAG linker-His tag for screening were prepared in eukaryotic Expi293F cells on a microscale (200 μl) as described in example 3, for excluding the polypeptides described in FIG. 5C II and FIG. 5D, which were produced in the eukaryotic cell line ExpiCHO as described in example 2 (medium scale, 50 ml and 30 ml, respectively).

Analysis of culture supernatant.

Expression, folding and trimer content of the polypeptides of the present invention were assessed using a homogeneous proximity enhanced luminescence assay (AlphaLISA, Figures 5A, 5B, 5CI) according to the manufacturer's instructions (PerkinElmer). This solution and binding equilibrium assay is based on the successful binding of both the donor and acceptor bead to the polypeptide. In close proximity, laser irradiation of a donor bead at 680 nm produces a stream of singlet oxygen, initiating chemical events in a nearby acceptor bead, resulting in chemiluminescent emission at 615 nm. Expression levels were measured using the Expression-AlphaLISA system by simultaneously adding nickel donor beads (with anti-His tag antibodies) and acceptor beads with anti-FLAG tag antibodies to the cell culture supernatant. This Expression-AlphaLISA system allowed recognition of the C-terminal Flag linker-His tag regardless of polypeptide folding. Correct folding of polypeptides was assessed in Binding-AlphaLISA by simultaneously adding nickel donor beads, human IgG CR9114 (2 nM) or CT149 (1 nM), and acceptor beads with anti-human IgG antibodies to the cell culture supernatant. The signal could only be obtained if the polypeptide folded correctly and allowed binding to IgG specific to the influenza virus HA.

The trimer-AlphaLISA system was used to determine the content of trimeric polypeptides present in the culture supernatant. It is based on human IgG, such as CT149 or CR9114, which specifically binds to monomeric HA. When variably labeled CT149 or CR9114 IgG is added to a 1:1 HA mixture, the AlphaLISA signal can only be detected if a multimer is present that allows binding to at least two antibodies, but not a monomer that allows binding to only one antibody. Trimer-AlphaLISA was performed by simultaneously adding streptavidin donor beads and anti-DIG IgG acceptor beads to the culture supernatant in the presence of biotinylated and DIG-tagged IgG CT149 or CR9114 (0.5 nM in each case, 1:1 ratio).

In all cases of AlphaLISA systems, detector beads were added at a concentration of 10 μg/ml. To avoid high dose effects, culture supernatants were tested at various dilutions according to the manufacturer's instructions. Reading was performed 2 hours after incubation at room temperature in the dark using an EnSight™ multi-mode plate reader (PerkinElmer). All data were normalized to their respective reference constructs, which were set to 100%.

The level of expressed polypeptide in cell culture supernatant was also assessed by biolayer interferometry using the OCTET platform according to the manufacturer's instructions (ForteBio). Briefly, a standard curve was generated using anti-HIS (HIS2) antibody biosensors (ForteBio) by assessing the binding shift in a series of dilutions of a strictly defined reference batch of purified homologous polypeptide. The binding shift of prediluted (in kinetic assay buffer, ForteBio) was then measured.

- 27046012 samples of cell culture supernatant containing the polypeptides of the present invention, and the concentration of the polypeptides was calculated using the standard curve generated (Figures 5C II, 5D). The content of the polypeptides of the present invention in the culture harvests was further characterized by analytical SEC in HPLC (Figure 5C II) and UHPLC (Figure 5D, injection volume: 10 μl), as described in example 4. After purification of the selected polypeptides, UFV171004 and UFV171197 binding to monoclonal antibodies CR9114 and CT149 were assessed using ELISA. Finally, the thermostability of these purified polypeptides was determined using DSF (both methods described in Example 2, Fig. 5C II).

Results and conclusion.

Introduction of tryptophan (W) at position 355 in the UFV161739 polypeptide resulted in a strong increase in expression and binding to antibodies CR9114 and CT149 compared to the construct with a wild-type histidine at position 355 (UFV161333). Co-introduction of 355W and isoleucine(I) at position 482 (UFV161800) further increased expression and antibody binding compared to the 355W mutation alone construct. Coadministration of phenylalanine (F) at positions 355 and 4821 was well tolerated but resulted in a smaller increase in expression and antibody binding compared with the control construct with the corresponding wild-type residues at positions 355 and 482 (Fig. 5A).

For positions 380 and 432, multiple amino acid residues and their combinations were evaluated. Overall, the amino acid substitutions tested were well tolerated with minimal effects on expression levels, trimer content, and antibody binding (Fig. 5B). Simultaneous introduction of isoleucine at positions 380 and 432 in the polypeptide (UFV171004) gave the highest yield of trimers (215%) compared to the standard.

It was demonstrated that four different amino acid substitutions (K, N, Q, R) were well tolerated at position 435. The corresponding polypeptides and the reference polypeptide UFV170611, containing a wild-type histidine at position 435, showed comparable levels in AlphaLISA for expression, trimer content, and binding to monoclonal antibodies CR9114 and CT149 (Figure 5C I). Additional study of the characteristics of polypeptides with substitutions 435N (UFV171004) and 435R (UFV171197) confirmed similar expression levels (OCTET), SEC profiles and binding strength with mAbs CR9114 and CT149 (ELISA). However, the polypeptide with an asparagine (N) at position 435 was characterized by higher thermostability at ~4°C compared to the polypeptide with an arginine (R) substitution at this position (Figure 5C II).

Polypeptides with amino acid substitutions at position 388, this position is located in the upper part of the A-helix (M, V, I, L, F, Y, W, H, K and R), were assessed in terms of expression level (Octet) and trimer content using analytical SEC. Expression levels were ranged from a methionine substitution (UFV180088) with 475 mg/L to a less favorable arginine substitution with an expression level of 192 mg/L. A trimeric polypeptide was visible in all SEC profiles, indicating that all residues evaluated at position 388 were transferable and did not contribute to the overall structure (Fig. 5D). All polypeptides with amino acid substitutions at position 388 eluted with a shorter retention time compared to the reference polypeptide (UFV180088) due to the difference in the length of the C-terminal tag: Flag-linker-His tag and C-tag, respectively.

Taken together, single alternative amino acid substitutions in various optimization regions of the polypeptides of the present invention were possible and well tolerated; expression level, trimer content, and protein folding were only minimally affected.

Example 6. Determination of deletion of the HA1 head domain.

Developed structures.

The stem domain polypeptides of the present invention are preferably characterized by a deletion of the head region from position 47 to amino acid position 306, inclusive (shown schematically in FIG. 1). In this example, we examined alternative deletions and linkers derived from the head domain between the ends of HA1, which arise after introduction of the deletion (schematically shown in Fig. 6). First, alternative deletion positions for the HA1 top strand were assessed (Table 3). The ends of HA1 present in the reference structure (UFV161908, with a deletion from amino acid position 47 to amino acid position 306, inclusive, replaced by a GPGS linker) are shown in gray.

- 28 046012

Table 3

End joining of HA ₁ after head domain deletion

#<? UFV Upper thread HA1 Compound Bottom thread HA1 A/Hong Kong/1/68 E L V Q S S S T G TO TO Y V To Position No. AA 41 42 43 44 45 46 47 48 49 50 307 308 309 310 Amino acid WT E L V Q S S S T G TO TO Y V To UFV161908* E L V Q S G R G S - To Y V To UFV170636 E L V Q S - - - - - To Y V To UFV170637 E L V Q S S - - - - To Y V To UFV170638 E L V Q S S S - - - To Y V To UFV170639 E L V Q S S S T - - To Y V To UFV170640 E L V Q S S S T G - To Y V To

* Standard

Second, alternative deletion positions for the upper HA ₁ strand were combined with alternative deletion positions for the lower HAi strands (Table 4).

Table 4

Alternative connection between the ends of HA1 after removal of the head domain

No. UFV Upper thread HA1 Compound Bottom thread HA1 A/Hong Kong/1/68 V Q S S S T G G A WITH R TO Y V Position No. AA 43 44 45 46 47 48 49 303 304 305 306 307 308 309 Amino acid WT V Q S S S T G G A WITH R TO Y V UFV160653* V Q S G R G S - - - - To Y V UFV160764 V Q S - - - - - - - R To Y V UFV160765 V Q S - - - - - - With R To Y V UFV160766 V Q S - - - - - A With R To Y V UFV160674 V Q S - - - - G A G* R To Y V UFV160675 V Q S - - - - G A WITH R To Y V UFV160767 V Q S S - - - - A WITH R To Y V UFV160768 V Q S S - - - - WITH R To Y V UFV160769 V Q S S S - - - A WITH R To Y V UFV160770 V Q S S S - - - - WITH R To Y V UFV160771 V Q S S - - - - - R To Y V UFV160772 V Q S S S - - - - - R To Y V

* Standard, ** Point mutation by substitution of cysteine for glycine

Third, the use of homologous linkers containing amino acid sequences from the deleted head domain to join the ends of HA ₁ was explored (Table 5).

Table 5

Alternative compounds derived from strand residues from the deleted head domain

No. UFV Upper thread HA1 Compound Bottom thread HA1 A/Hong Kong/1/68 E L V Q S S S T G TO TO Y V To Position No. AA 41 42 43 44 45 46 47 48 49 50 307 308 309 310 Amino acid WT E L V Q S S S T G TO TO Y V To UFV160321* E L V Q S G G G G - To Y V To UFV160403 E L V Q S N R N R - To Y V To UFV160404 E L V Q S G D R N - To Y V To UFV160405 E L V Q S N G G S - To Y V To UFV160406 E L V Q S G G S N - To Y V To UFV160407 E L V Q S G S N A - To Y V To UFV160408** E L V Q S G R G S - To Y V To UFV160409 E L V Q S G S G F - To Y V To UFV160410 E L V Q S G S G - - To Y V To UFV160411 E L V Q S G G - - - To Y V To UFV160412 E L V Q S G G S - - To Y V To UFV160413 E L V Q S S G S - To Y V To UFV160414 E L V Q S n R S T - To Y V To UFV160415 E L V Q S I R N I - To Y V To UFV160416 E L V Q S G L S S - To Y V To UFV160417 E L V Q S TO R G D - To Y V To UFV160418 E L V Q S D A R I - To Y V To UFV160419 E L V Q S T R N - - To Y V To UFV160420 E L V Q S T R N G - To Y V To

* Reference, ** Connection used as reference design for designs from Table 1 and Table 2

Protein expression in mammalian cells and culture supernatant analysis.

DNA fragments encoding the polypeptides of the present invention were synthesized as op.

- 29 046012 sano in example 2. Polypeptides containing C-terminal FL-AG-.inHKep-His-MeTKa for screening were produced on a microscale in eukaryotic Expi293F cells (200 μl) as described in example 3. Expression, trimer content and folding (binding to mAb either CR9114 or CT149) polypeptides were assessed using AlphaLISA as described in Example 5. All data were normalized to their respective reference constructs UFV161908 (Figure 6A), UFV160653 (Figure 6B) and UFV160321 (Figure 6C) , which were taken as 100%.

Results and conclusion.

Varying the position of the head domain deletion in the top strand of HA ₁ had only minimal effects on expression level, trimer yield, and protein folding (ie, antibody binding) (Fig. 6A). In contrast, expression of polypeptides that included an alternative deletion position in both the top and bottom strands of HA1 showed up to ∼50% reduction in expression levels and up to ∼70% reduction in binding to the CT149 antibody (Fig. 6B). This indicated that within the range of positions tested, in particular the position at which the HA1 downstream strand was deleted, both expression and folding of the polypeptides were affected.

An alternative approach to directly joining the remaining ends of HA ₁ (eg, the N-terminal fragment of HA1 and the C-terminal fragment of HA1) after removal of the head domain is to join via a homologous linker sequence. As shown in table. 5, for this purpose the upper thread HA| (residue 45) was connected to the bottom strand of HA ₁ (residue 307) via a short sequence derived from the corresponding head domain of H3 HA. This resulted in polypeptides with expression levels ranging from 33 to 223% of the reference (Fig. 6C). Similarly, binding to the CT149 antibody was highly variable, with values ranging from 57 to 350% of the reference observed. Polypeptides that were well expressed were, in most cases, also highly bound to antibodies.

Overall, the formation of trimeric and correctly folded stem domain polypeptides by deletion of the HA head domain was not dependent on one precise position of the deletion. Direct joining of HA ₁ ends and variations in deletion position in both the top and bottom strands of HA ₁ were successfully obtained. Alternatively, it was also possible to join the end of HA ₁ by introducing a homologous amino acid linker derived from the head domain, and to rejoin the ends of HA ₁ after removal of the head domain, a variety of peptides with different sequence composition and different lengths (from at least 2 to 5 amino acids).

Example 7 Optimization of the B-loop in the polypeptides of the present invention.

Developed structures.

After deletion of the HA1 head domain, the B-loop (Fig. 1B and C, comprising amino acids 385-404) of the HA stem domain polypeptide is exposed. To partially protect this zone from the immune system and further stabilize the loop, glycosylation motifs and proline residues were introduced (Fig. 7). Glycation motifs (NxT) were introduced at positions 401-403 using the E401N and E403T mutations for N-linked glycosylation at position 401 and tested in combination with N-linked glycan motifs at positions 398-400 by mutation S398N (resulting in the NxS motif), or at position 392-394 by mutation S392N and Q394T, or at position 393-395 by mutation H393N and I395T (in both cases resulting in an NxT motif). Proline substitutions, which can reduce the propensity for helicalization in the B-loop sequence, were introduced by single point mutations at any of positions ranging from 385 to 406 and double point mutations at 392 along with any of 396 or 398.

Protein expression in mammalian cells and culture supernatant analysis.

DNA fragments encoding the polypeptides of the present invention were synthesized as described in Example 2. Polypeptides containing a C-terminal FLAG linker-His tag for screening were produced on a microscale in the eukaryotic cell line Expi293F (200 μl) as described in Example 3. Expression, trimer content and folding (binding to mAbs CR9114, CT149 (which is described in Wu et al. (2015)) and SD15013 (containing the amino acid sequence of SEQ ID NO: 39) of the polypeptides were assessed using AlphaLISA as described in the example 5 (binding to SD15013 was assessed using acceptor beads with anti-His antibodies and donor beads with streptavidin in the presence of SD15013 at a concentration of 2 nM.) Cell culture supernatants were analyzed using analytical SEC as described in example 2, on the day of production.

All data were normalized to their respective reference constructs UFV161686 (Fig. 8A), UFV161333 (Fig. 7B and C), and UFV171187 (Fig. 7D), which were set to 100%. Results and conclusion

Introduction of a single glycosylation motif into the B-loop by mutation of residues at positions 401, 402, and 403 to N, A, and T, respectively, for N-linked glycosylation at position 401 resulted in a twofold increase in the expression level of the polypeptide. Similarly, a huge increase in binding to antibodies CR9114 and CT149, respectively 5- and 7-fold, and single-domain SD15013 (10-fold) was observed (Fig. 7A).

The introduction of prolines into the B-loop did not affect the level of polypeptide expression; the values varied from 94 to 128% relative to the reference design (Fig. 7B). In contrast, the addition of proline residues at positions 386, 387, 388, and 389 had a negative effect on antibody binding. Minimal binding to CR9114, CT149 and SD15013 indicated that polypeptide folding was negatively affected by the introduction of proline at the N-terminus of the B-loop. Introduction of one proline at any of positions 390-405 or two prolines at positions 392 and 396 or positions 392 and 398 resulted in a ~40% increase in binding to CR9114, while binding to CT149 remained relatively similar or decreased (~65% compared to standard). SD15013 had the greatest variation among these constructs, with relative binding ranging from ~50 to ~150% compared to the reference. Administration of two proline residues was well tolerated, and binding to CR9114, CT149, and SD15013 was generally found to be an average of antibody binding values for single proline administrations. An exception was observed in the case of binding of SD15013 to UFV161708, which was characterized by 147% binding, while in the case of single mutations at positions 392 and 396, a decrease in binding was observed (64% and 75% relative to the reference).

Introduction of a second glycosylation motif into the B-loop was well tolerated (Fig. 7C); both polypeptides with an additional motif at position 393 (UFV161715) or 398 (UFV161721) showed relatively similar levels of expression compared to the reference, increased binding to CR9114 (~145%), unaffected binding to CT149 (~90%) and a slight decrease in binding with SD15013 (~60%).

Simultaneous introduction of proline residues and/or a second glycosylation motif (at positions 392-395) resulted in polypeptides whose expression was approximately 2-fold lower than in the case of the standard, which contained a single glycosylation motif at positions 401403 and did not contain proline (Fig. .7D). Binding to antibodies (CR9114 and CT149) was practically unaffected, while a decrease was observed in the case of SD15013 (~48-88% relative to the standard). Since simultaneous glycosylation of N398 and N401 was unlikely due to their proximity, an additional glycan at N393 or N392 was preferred.

SEC-MALS analysis of EXPI-293 cell culture supernatants containing polypeptides of the present invention with one N-linked glycan motif (UFV180208) or two N-linked glycan motifs and two proline residues (UFV180217) showed a clear peak, corresponding to the corresponding trimeric polypeptide (Fig. 7E).

Mutations in the B-loop in the form of stabilizing proline residues and additional N-linked glycosylation motifs were well tolerated. Although differences in expression and antibody binding were observed (especially in the case of binding to SD15013), introduction of proline(s) other than the N-terminal B-loop region (positions 392-389) and the second N-linked glycosylation motif was possible without affecting protein folding and trimerization.

Example 8 N-linked glycosylation motif at position 38 of the polypeptides of the present invention.

Developed structures.

To evaluate the effect of a conserved glycosylation motif near the CR9114 epitope (position 38) on expression and folding, a comparison was made of a polypeptide containing the wild-type 38NAT-40 motif for N-linked glycosylation (UFV170282) with a polypeptide in which the motif was knocked out using a point T40I mutation (UFV170278).

Protein expression in mammalian cells and analysis, purification and characterization of culture supernatant.

DNA fragments encoding the polypeptides of the present invention were synthesized, expressed and purified as described in Example 2. The level of expressed polypeptide in the culture supernatant was assessed by biolayer interferometry using the OCTET platform as described in Example 2 using immobilized mAb CT149 and a 25-fold diluted cell culture supernatant containing the polypeptide of the present invention. The strength of antibody binding to the purified polypeptides was assessed using ELISA (EC ₅₀ ), as described in example 2.

Results and conclusion.

When the N-linked glycosylation motif at position 38 (UFV170278) was removed, the expression level was reduced by ~50% compared to the polypeptide with the motif present (UFV170282), however, both polypeptides remained well expressed polypeptides with values above 255 mg per liter of supernatant culture. As determined by ELISA, the strength of antibody binding was not significantly different between the two polypeptides, as evidenced by EC ₅₀ values of ~1 nM for CR9114 and CT149 (Fig. 8A and B).

In conclusion, deletion of the N-linked glycosylation motif at position 38, although a decrease in expression levels was observed, was well tolerated and did not appear to affect polypeptide folding.

Example 9. Alternative positions of the intramolecular disulfide bridge between HA1 HA2.

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Developed structures.

The protomers of the trimeric stem domain polypeptides of the present invention are preferably stabilized by an introduced disulfide bridge covalently linking the HA1 downstream strand (position 310) to the HA2 strand C-helix (position 422). Alternative options for a given intraprotomer disulfide bridge were assessed by creating small shifts in the exact position of the corresponding cysteine residues (positions 311/422 and 308/418). In addition to the 310/422 disulfide bridge, a second pair of cysteine residues was assessed to connect HAi (position 26) to the C-terminal part of the HA2 C-helix (position 433).

Protein expression in mammalian cells and culture supernatant analysis.

DNA fragments encoding the polypeptides of the present invention were synthesized as described in Example 2. Polypeptides were expressed in Expi-293 cells as described in Example 2, except that instead of a microscale (200 μl) experiment, cultures were grown at a medium scale (30 ml). The expression level and folding (binding to mAbs CR9114 and CT149) of the polypeptides of the present invention were assessed using AlphaLISA as described in Example 5, using CR9114 and CT149 at concentrations of 2.5 nM and 1.25 nM, respectively.

Results and conclusion.

Relocation of cysteine residues from residue 310 to 311 or 308 (in combination with cysteine relocation at position 422 to 418) was well tolerated and had only a minimal effect on expression levels and polypeptide folding as evidenced by antibody binding (Fig. 9A). The introduction of a second disulfide bridge into the region downstream of the 310-422 disulfide bridge was in principle possible, as was evident from the unaffected expression level, however, a sharp decrease in binding to CR9114 and CT149 (2% and 48% relative to the standard, respectively) indicated that folding required conserved stem domain epitopes had a negative effect (Fig. 9B).

Example 10. Alternative positions of interprotomer disulfide bridges.

Developed structures.

Protomers in HA stem domain polypeptides are preferentially covalently linked through an interprotomer disulfide bridge at the top of the trimeric HA protein (Figure 4A). Two cysteine residues were introduced: one in the B-loop (position 398) and one in the C-helix (position 408), both of which pair with sterically similar cysteines in the adjacent protomer in the trimer; those. cysteine 398 from protomer 1 forms a disulfide bond with cysteine 408 from protomer 2, cysteine 398 from protomer 2 forms a disulfide bond with cysteine 408 from protomer 3, and cysteine 398 from protomer 3 forms a disulfide bond with cysteine 408 from protomer 1. Alternative variants of this interprotomer The disulfide bond was examined by creating small shifts up or down in the exact position of point mutations of cysteine residues (Fig. 10).

Protein expression in mammalian cells and culture supernatant analysis.

DNA fragments encoding the polypeptides of the present invention were synthesized as described in Example 2. Polypeptides containing a C-terminal FLAG linker-His tag for screening were produced on a microscale in the eukaryotic cell line Expi293F (200 μl) as described in Example 3. Expression, trimer content and folding (binding to mAb CR9114 or CT149) of the polypeptides of the present invention were assessed using AlphaLISA as described in Example 5.

Results and conclusion.

Polypeptides with introduced interprotomer disulfides were expressed ~1.8 times lower compared to polypeptides without these cysteines. However, the difference in trimerization is significant (Fig. 10). Polypeptides with interprotomer disulfides were expressed at the same level and all exhibited similarly high levels of trimers and binding affinities to antibodies CR9114 and CT149. This demonstrates the critical importance of the interprotomer disulfide bridge for trimerization and proper folding of conserved stem domain epitopes. Moreover, small changes in the positioning of the interprotomer disulfide bridge were well tolerated.

Example 11. Alternative truncations at the C-terminus.

Developed structures.

Hemagglutinin (HA) of the influenza virus is a membrane protein that is located on the surface of the viral particle, with the C-terminal part of the protein immersed in the viral membrane. In the case of soluble versions of the polypeptides of the present invention, the ectodomain can be truncated at various positions in the natural linker sequence (positions 500-513) that connects the C-terminal alpha helix of the ectodomain to the transmembrane (TM) and cytoplasmic domain.

Alternative positions of the C-terminal truncation were assessed (Table 6).

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Table 6

Alternative C-terminal truncations of polypeptides of the present invention derived from the HA ectodomain from A/Hong Kong/1/68

Analysis of cell culture supernatant.

DNA fragments encoding the polypeptides listed in table. 6 were synthesized as described in example 3, and expressed in EXPI-293 suspension cell cultures as described in example 4.

The collected cell culture supernatants were analyzed for trimeric polypeptide levels by analytical SEC using HPLC as described in Example 4A. Proper folding of expressed polypeptides of the present invention was assessed in cell culture supernatants by biolayer interferometry using the OCTET platform (ForteBio) as described in Example 6. Briefly, supernatants diluted fivefold in kinetic assay buffer (ForteBio) were assessed for binding to biotinylated human monoclonal antibodies CR9114 or CT149 (10 μg/ml) loaded onto streptavidin biosensors (ForteBio). A curve was plotted during the first 100 seconds of the binding stage to calculate K _ON values, and the curves themselves were plotted using a 1:1 model. A mock sample was included as a negative control.

Results and conclusion.

C-terminal truncations between residues 501 and 513 were well tolerated with only minor effects on trimer and expression levels and antibody binding. The truncated polypeptides showed comparable trimeric peak patterns in the SEC assay (Fig. 11A) and good or improved K _ON for binding to CR9114 and CT149 in the Octet assay (Fig. 11B). Truncations (after position 499 in UFV171280) reaching the C-terminal helix of the ectodomain resulted in a clear drop in levels of trimer expression and antibody binding, as observed by SEC and OSTET analysis.

Example 12 Alternative mutations in the A-helix of the polypeptides of the present invention.

Developed structures.

The positioning and folding of the C-terminal portion of the A-helix of the polypeptides of the present invention is critical for proper representation of conserved stem domain epitopes. In the search for the optimal conformation, three residues of the A-helix (378, 379 and 381) were subjected to backmutation by substitution into residues originating at this position from group 1 HA (H1 A/Brisbane/59/07) or group 2 HA (H3 A /Hong Kong/1/1968). In addition, the hypothetical A-helix stabilizing mutation G379A was evaluated.

Protein expression in mammalian cells and analysis, purification and characterization of culture supernatant.

DNA fragments encoding the polypeptides of the present invention were synthesized as described in Example 2. Polypeptides containing a C-terminal FLAG linker-His tag for screening were produced on a microscale in the eukaryotic cell line Expi293F (200 μl) as described in Example 3. Expression, trimer content and folding (by binding to 2.5 nM mAb CR9114 or CT149) of the polypeptides were assessed using AlphaLISA as described in Example 5. In addition, the polypeptides were expressed on a medium scale (50 ml) in EXPI-CHO cells. as described in Example 4, and expression levels were determined by biolayer interferometry as described in Example 5, and crude cell culture supernatant was analyzed by SEC-MALS by high performance liquid chromatography (HPLC) as described in Example 3. Polypeptides were purified by two-step protocol using affinity chromatography and size exclusion chromatography as described in example 4. The antigenicity of the purified polypeptides was assessed using ELISA (EC ₅₀ values for binding to antibodies CR9114 and CT149), and the temperature at which 50% of the polypeptide unfolded was determined using DSF , both cases are described in example 2.

Results and conclusion.

A gradual increase in the number of H1-derived residues at positions 378, 379 and 381 affected the expression level of the polypeptide. The polypeptide UFV161448, containing all three H1-like residues, was the least expressed polypeptide (84%), while UFV161451, containing H1

- 33 046012 similar residues at positions 379 and 381, was the most expressed polypeptide (163%). Polypeptides containing the hypothetical A-helix stabilizing mutation 379A (UFV161459 and UFV161458) were the least expressed (42% and 75%, respectively). Correct folding of polypeptides was assessed in AlphaLISA. The relative signal for binding to both CR9114 and CT149 was characterized by a large scatter. For polypeptide UFV161453 (379N), the lowest binding was observed: 61% and 24% with CR9114 and CT149, respectively. The polypeptide UFV161448, which contained all mutations in the H1 direction (378T, 379N and 381V), showed the highest degree of binding: 1706% and 841% to CR9114 and CT149, respectively (Fig. 12A).

The effect of A-helix mutations was further studied in the more stabilized trimeric stem domain polypeptides UFV171004 and (UFV171116), which included, among others, the H355W mutation and an alternative interprotomer disulfide bridge (397/408). In three independent medium-scale ExpiCHO products, UFV171004 (which included A-helix H1 residues at positions 378, 379, and 381) was expressed at a slightly higher level than the UFV171116 polypeptide (which contained H3 residues at these positions).

Similarly, minimal differences were observed in the results of SEC-MALS analysis of culture supernatants; For both designs, the peak corresponding to the trimer fraction (retention time ~8 minutes) overlapped in shape and height. Binding to antibodies CR9114 and CT149 as an indicator of proper protein folding was determined using ELISA and the results indicated strong binding (EC ₅₀ <0.01 nM). Temperature stability determined by DSF showed a significant difference between both polypeptides. While 50% of the UFV171116 polypeptide unfolded at 66.2°C, the UFV171004 polypeptide had a Tm ₅₀ value of 68.0°C (Fig. 12B).

Taken together, the introduction of H1 residues into the A-helix of H3-based stem domain polypeptides resulted in polypeptides that were more thermostable and exhibited increased binding to CR9114 and CT149. Differences in antibody binding were less significant in the UFV171004 and UFV171116 constructs, which included, among others, the H355W stabilizing mutation.

Example 13. Introduction of surface mutations into H7.

Developed structures.

The trimeric stem domain polypeptides of the present invention, which are described in the previous examples, are based on the HA from the influenza virus H3 A/Hong Kong/1/1968. Despite the high degree of conservation in the stem domain of group 2 hemagglutinins H3 and H7, several surface residues in the region of conserved stem domain epitopes differ. In this example, selected residues located on the surface of the polypeptide were stepwise subjected to substitution mutation from H3 residues (which are present in the reference UFV172561 and UFV172562) to the corresponding H7 residues. These residues included positions in the 2φ3 loop (residues 25 and 27), residues in the A-helix (residue 367), and residues at the bottom of the polypeptide (residues 475, 476, and 479).

Protein expression in mammalian cells and culture supernatant analysis.

DNA fragments encoding the polypeptides of the present invention were synthesized as described in Example 2. Polypeptides containing a C-terminal FLAG linker-His tag for screening were produced on a microscale in the eukaryotic cell line Expi293F (200 μl) as described in Example 3. Expression, trimer content and folding (by binding to mAbs CR9114 and CT149) of polypeptides of the present invention were assessed using AlphaLISA as described in Example 5. All AlphaLISA data were normalized to their respective reference constructs UFV172561 and UFVI72562, which were set to 100% . The first reference construct included mutations of H1 residues at positions 379 and 381, the second reference construct included wild-type H3 residues at positions 379 and 381. Additionally, culture supernatants were analyzed by analytical SEC on the day of harvest as described in Example 2.

Results and conclusion.

Introduction of H7-like residues into the B2φ3-loop, A-helix (including H1-like residues at positions 379 and 381) and into the lower part of the stem domain led to a slight decrease in expression and trimer yield and a relatively small change in antibody binding (±20%) with trimeric stem polypeptides as determined by AlphaLISA. Similarly, a decrease in the expression level of polypeptides involving surface mutations in H7 was observed in SEC analysis of crude cell culture supernatants (FIG. 13A).

A similar effect was observed when surface mutations were introduced into a variant backbone that included H3-like residues at positions 379 and 381 (Fig. 13B).

Taken together, it is possible to modify the surface of the HA H3-derived stem domain polypeptides of the present invention towards H7, especially in the presence of H1-like residues at the top of the A-helix.

Example 14. Basic principle of application for mini-NA of group 2.

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Developed structures.

The structural elements required to produce the trimeric stem domain polypeptides of the present invention were expressed in the H3 HA backbone (A/Hong Kong/1/1968) and also transferred to two alternative H3 backbones; those. A/Wisconsin/67/2005 and A/Singapore/INFIMH/16/0019/2016. Structural elements were introduced gradually; set I of the polypeptides contained a minimal set of mutations, set II additionally included partial mutations for B-loop stabilization, and set III included all additional mutations for B-loop stabilization.

Protein expression in mammalian cells and culture supernatant analysis.

DNA fragments encoding the polypeptides of the present invention were synthesized as described in Example 2. Polypeptides containing a C-terminal FLAG linker-His tag for screening were produced on a microscale in the eukaryotic cell line Expi293F (200 μl) and crude cell supernatant Cultures were analyzed by analytical SEC on the day of harvest as described in Example 3.

Results and conclusion.

As seen in SEC-MALS analysis, the transfer of set I structural elements resulted in the formation of trimeric stem domain polypeptides for all 3 backbones, but the peak of trimers was most obvious for the polypeptide obtained from A/Wisconsin/67/2005 and least for polypeptide obtained from A/Hong Kong/1/1968 (Fig. 14A). Additional stabilizing mutations in the B-loop (set II and set III) resulted in a significant increase in polypeptide expression levels and trimer content (Fig. 14B). Taken together, the results confirmed that transfer of modifications of the polypeptides of the present invention to other group 2 backbones resulted in soluble trimeric mini-HA.

Example 15. Adenovirus-driven in vitro expression of correctly folded trimeric group 2 miniHA on the cell membrane of human lung fibroblast cells.

In this example, adenovirus 26 (Ad26.FLU.004)-driven expression and folding of trimeric UFV180480 (UFV18088 with a native transmembrane domain) on the cell surface of human lung fibroblasts (MRC-5) was assessed. MRC-5 cells were transduced (5000 i.p./cell) in culture medium. Two days later, cells were either lysed in lysis buffer to assess expression of trimeric UFV180480 by Western blotting, or cells were harvested by trypsinization to assess cell surface expression of properly folded UFV180480 by flow citrometry. In both cases, Ad26.Empty, which lacks the transgene encoding UFV180480, was included as a negative control.

Protein lysates from cells transduced with Ad26.FLU.004, as well as the UFV180088 protein, which served as a positive control, were processed for SDS-PAGE under reducing conditions (ensuring complete unfolding of trimeric group 2 mini-HA proteins) or non-reducing conditions (ensuring preservation of the trimeric nature of the mini-HA protein) and the proteins were transferred to a nitrocellulose blot. Expression was tested by probing the blot with biotinylated antibody CR9114, specific for the group 2 mini-HA conformation, and expression was visualized using HRP-conjugated streptavidin.

To test cell membrane-associated expression of UFV180480 by flow nitrometry, cells transduced with Ad26.FLU.004 were trypsinized and resuspended in flow cytometry buffer. Non-permeabilized cells were probed with CR9114 and, after extensive washing, with PE-conjugated anti-human antibody to visualize UFV180480.

Results and conclusion.

Cell surface expression of UFV180480 was analyzed using flow cytometry analysis. Compared to cells transduced with Ad26.Empty (Fig. 16A), the majority of MRC-5 cells transduced with Ad26.FLU.004 (~96.3%) exhibited high levels of UFV180480 at the cell surface (Fig. 16B).

Analysis of UFV180480 expression in MRC-5 by flow cytometry did not differentiate the expression of monomeric or trimeric UFV180480. Therefore, Western blotting was performed. Based on molecular weight and by comparison with trimeric UFV180088, Ad26.FLU.004 was demonstrated to drive the expression of trimeric UFV180480 in MRC-5 cells (Fig. 16C). Based on the amino acid sequence of monomeric UFV180480, the molecular mass was estimated to be 34.4 kDa, giving the size of trimeric UFV180480 about 103.2 kDa. Lysate from cells transduced with Ad26.FLU.004 (5000 vp/cell), but not from cells transduced with Ad26.Empty, produced a band at approximately 103.2 kDa and was slightly higher compared to trimeric UFV180088 ( expected MW 89.1 kDa) (see Fig. 16C, lanes 1-3). No bands were observed at about 34 kDa, indicating that the bulk of UFV180480 was present in its trimeric form (Fig. 16C, lane 1). No specific band was observed in any of the samples when treated under reducing (ie, full unfolding) conditions (FIG. 16C, lanes 4-6).

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Thus, according to the present invention, UFV180480 was confirmed to be expressed in vitro by transduction with Ad26.FLU.004. The presence of a trimeric protein was also confirmed on the surface of human cells.

Example 16 The UFV170278 and UFV170282 polypeptides of the present invention are immunogenic and induce protection in an intact mouse model of lethal H3N2 A/Hong Kong/1/1968 challenge.

This example compared the in vivo immunogenicity and protective efficacy (based on the proportion surviving at the end of the observation period) of a 2% (v/v) dose range of UFV170278 (with the conserved N-linked glycan motif at position 38) and UFV170282 (without the conserved N-linked glycan motif at position 38). motif for N-linked glycan at position 38) with the addition of Adjuplex as an adjuvant compared to animals immunized with blank (PBS).

Groups of 10 female BALB/c mice (6-8 weeks old) were immunized intramuscularly three times at three-week intervals with a range of doses of soluble trimeric UFV170278 or UFV170282 supplemented with 2% (v/v) Adjuplex as an adjuvant. The dose range consisted of 4 10-fold dilutions, starting at 30 μg and up to 0.03 μg. As a negative control, 18 mice were immunized three times with PBS. Four weeks after the last immunization, mice were bled for immune response analysis and one day later, mice were challenged with 12.5 x LD ₅₀ mouse-adapted challenge virus H3N2 A/Hong Kong/1/1968 and monitored (survival, weight , clinical indicators) for 3 weeks. The proportion surviving at the end of the follow-up period served as the primary outcome parameter.

Results.

It was demonstrated that UFV170278 and UFV170282 were immunogenic, as all doses of UFV170278 and UFV170282 induced significantly higher titers of antibodies specific to the stem domain of the H3 A/Hong Kong/1/1968 HA (measured by competition assay with CR9114) compared to titers in the PBS group (P<0.001; ANOVA with post hoc t test, stepwise testing (highest dose starting), and 2-fold Bonferroni correction by construct), see FIG. 17A.

In addition, UFV170278 and UFV170282 adjuvanted with Adjuplex provided significant protection (P < 0.001; Fisher's exact test, stepwise testing (highest dose), and 2-fold Bonferroni adjustment by construct) at all doses compared with the group receiving PBS, see FIG. 17V. Weight loss (as measured by area under the curve) was significantly reduced (P<0.001; ANOVA, 2-fold Bonferroni correction by design and stepwise testing starting at the highest dose) at all doses compared with the PBS group, see Table 1. fig. 17V.

Conclusions.

In accordance with the present invention, UFV170278 and UFV170282 have been shown to be immunogenic and protective in a mouse model of lethal challenge with H3N2 A/Hong Kong/1/1968.

Example 17 The polypeptides UFV180088, UFV180089 and UFV180090 of the present invention are immunogenic in an intact mouse challenge model.

This example compared the in vivo immunogenicity of a 2% (v/v) dose range of UFV180088, UFV180089 and UFV180090 with the addition of Adjuplex as an adjuvant versus animals immunized with blank challenge (PBS).

Groups of 10 female BALB/c mice (6-8 weeks old) were immunized intramuscularly once, twice, or three times at 3-week intervals with 3 μg of soluble trimeric UFV180088, UFV180089, or UFV180090. Final immunizations were performed on the same day. As a negative control, 18 mice were immunized three times with PBS. All immunizations were supplemented with 2% (v/v) Adjuplex as an adjuvant. Four weeks after the last immunization, blood was drawn from the mice to analyze the immune response.

Results.

UFV180088, UFV180089 and UFV180090 were shown to be immunogenic, as all constructs induced significantly higher titers of A/Hong Kong/1/1968 H3 stem domain-specific antibodies (measured by competition assay with CR9114) after two or three immunizations compared with titers in the PBS group (P<0.001; Wilcoxcon, 3-fold Bonferroni correction for multiple comparisons and stepwise testing starting with the highest dose), see FIG. 18A. Neither construct significantly induced the production of stem domain-specific antibodies after a single immunization.

All constructs induced significantly higher titers of antibodies specific to H3 A/Hong Kong/1/1968 and H3 A/Texas/50/2012 HA (measured by FL HA binding ELISA) after two or three immunizations compared with titers in PBS group (P<0.01; Wilcoxcon, 3x Bonferroni correction for multiple comparisons and stepwise testing starting at highest dose), see FIG. 18V. In addition, one immunization with UFV180088 and

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UFV180089 induced significantly higher titers of antibodies specific to HA H3 A/Texas/50/2012 compared to titers in the PBS group (P<0.01).

All constructs induced significantly higher titers of H7 A/Netherlands/219/2003 HA-specific antibodies (measured by FL HA binding assay) after three immunizations compared with titers in the PBS group (P<0.001; Wilcoxcon, 3 -fold Bonferroni correction for multiple comparisons and stepwise testing starting at the highest dose), see FIG. 18V. Dual immunizations with UFV180089 and UFV180090 induced significantly higher titers compared to antibody titers in the PBS group (P<0.001 and P<0.01, respectively), whereas significantly higher titers were not detected after a single immunization.

Conclusions.

Thus, in accordance with the present invention, UFV180088, UFV180089 and UFV180090 were shown to be immunogenic in an intact mouse model. All constructs induced the production of significant titers of antibodies specific to the HA stem domain and antibodies that bind many phylogenetically distinct HA proteins H3 (from strains isolated in different years) and H7.

Example 18 The UFV180088, UFV180089 and UFV180090 polypeptides of the present invention induce protection against lethal challenge by H3N2 A/Hong Kong/1/1968 in an intact mouse challenge model.

This example compared the in vivo protective efficacy (based on the proportion surviving at the end of the observation period) of a 2% (v/v) dose range of UFV180088, UFV180089, and UFV180090 supplemented with Adjuplex as an adjuvant versus animals immunized with blank challenge (PBS). ).

Groups of 10 female BALB/c mice (6-8 weeks old) were immunized intramuscularly once, twice, or three times at 3-week intervals with 3 μg of soluble trimeric UFV180088, UFV180089, or UFV180090. Final immunizations were performed on the same day. As a negative control, 18 mice were immunized three times with PBS. All immunizations were supplemented with 2% (v/v) Adjuplex as an adjuvant. Four weeks after the last immunization, mice were challenged with 25 x LD ₅₀ mouse-adapted challenge virus H3N2 A/Hong Kong/1/1968 and monitored (survival, weight, clinical parameters) for 3 weeks. The proportion surviving at the end of the follow-up period served as the primary outcome parameter.

Results.

UFV180088, UFV180089, and UFV180090 were shown to provide significant protection (P < 0.001; Fisher's exact test, 2-fold Bonferroni adjustment by construct, and stepwise testing starting with the highest dose) in cases of two or three immunizations, compared with the group receiving PBS, see FIG. 19. Neither construct induced significant protection after a single immunization. Body weight loss (as measured by area under the curve) was significantly reduced (P < 0.05; ANOVA, 2-fold Bonferroni correction by design and stepwise testing starting at the highest dose) at all doses compared with the PBS group. with the exception of one immunization with UFV180089, see FIG. 19.

Conclusions.

Thus, in accordance with the present invention, UFV180088, UFV180089 and UFV180090 were shown to provide protection in a mouse model of lethal challenge with H3N2 A/Hong Kong/1/1968.

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Table 7

Standard amino acids, abbreviations and properties

Amino acid 3 letter code 1 letter code Side polarity Side chain charge (pH 7.4) Alanin 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 Serin 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

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Lorieau et al. (2010), Proc. Natl. Acad. Sci. USA, 107: 11341.

S-Guthe et al. (2004), J. Mol. Biol. 337:905-915.

Steel et al. (2010), mBio 1(1): 1-9.

Throsby et al. (2008), PLOS One 12(3): 1-15.

Winter et al. (1981) Nature 292: 72-75.

Wu et al. (2015) Nature Communications 6(7708):1–11.

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Sequences

SEQ ID NO: 1 - hemagglutinin CAA24269.1 (influenza A virus (A/Aichi/2/1968(H3N2)) (not including 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 AISCFLLCW LLGFIMWACQ RGNIRCNICI 550

SEQ ID NO: 1: NC, full size (A/Hong Kong/1/68)

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGKIC NNPHRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTLEF ITEGFTWTGVTQNGGSNACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGVHHPSTN QEQTSLYV QASGRVTVSTRRSQQTIIPNIWSRPWVRGLSSRISIYWTIVKPGDVLVINSNGNLIAPRG YFKMRTGKSSIMRSDAPIDTCISECITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLATGMRNVP EKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQINGKLNRVIEKTNEKF HQIEKEF SEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRENAE DMGNGCFKIYHKCDNACIESIRNGNYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLC VVLLGFIMWACQRGNIRCNICI

PROTEIN VH CR6261 (SEQ ID NO: 3)

EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYAPKFQGRV TITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVSS

PROTEIN VL CR6261 (SEQ ID NO: 4)

QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNDYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKS GTSATLGITGLQTGDEANYYCATWDRRPTAYVVFGGGTKLTVL

PROTEIN VH CR8020 (SEQ ID NO: 5)

QVQLQQSGAEVKTPGASVKVSCKASGYTFTSFGVSWIRQAPGQGLEWIGWISAYNGDTYYAQKFQARV TMTTDTSTTTAYMEMRSLRSDDTAVYYCAREPPLFYSSWSLDNWGQGTLVTVSS

PROTEIN VL CR8020 (SEQ ID NO: 6)

EIVLTQSPGTLLSLSPGERATLSCRASQSVSMNYLAWFQQKPGQAPRLLIYGASRRATGIPDRISGSGS GTDFTLTISRLEPADFAVYYCQQYGTSPRTFGQGAKVEIK

PROTEIN VH CR9114 (SEQ ID NO: 7)

QVQLVQSGAEVKKPGSSVKVSCKSSGGTSNNYAISWVRQAPGQGLDWMGGISPIFGSTAYAQKFQGRV TISADIFSNTAYMELNSLTSEDTAVYFCARHGNYYYYSGMDVWGQGTTVTVSS

PROTEIN VL CR9114 (SEQ ID NO: 8)

- 39 046012

SYVLTQPPAVSGTPGQRVTISCSGSDSNIGRRSVNWYQQFPGTAPKLLIYSNDQRPSVVPDRFSGSKS GTSASLAISGLQSEDEAEYYCAAWDDSLKGAVFGGGTQLTVL

FL HA OF Wisconsin/67/2005 (SEQ ID NO: 13)

MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATELVQSSSTGGIC DSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEF NDESFNWTGVTQNGTSSSCKRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEKFDKLYIWGVHHPVTD NDQIFLYAQASGRITVSTKRSQQTVIPNIGSPRIRNIPSRISIYWTIVKPGDILLINSTGNLIAPRG YFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVP EKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGIGQAADLKSTQAAINQINGKLNRLIGKTNEK F HQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFERTKKQLRENAE DMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLC VALLGFIMWACQKGNIRCNICI

FL HA OF A/Singapore/INFMH/16/0019/2016 (SEQ ID NO: 14)

MKTIIALSYILCLVFAQKIPGNDNSTATLCLGHHAVPNGTIVKTITNDRIEVTNATELVQNSSIGEIC DSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEF KNE S FNWTGVTQNGT S SACIRGS S S S F FS RLNWLT HLNY KY PALNVTMPNKEQ FDKLYI WGVHH PGTD KDQIFPYAQSSGRITVSTKRSQQAVIPNIGSRPRIRGIPSRISIYWTIVKPGDILLINSTGNLIAPRG YFKIRSGKSSIMRSDAPIGKCKSECITPNGSIPNDKPFQNVNRITYGACPRYVKHSTLKLATGMRNVP EKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGK LNRLIGKTNEKF HQIEKEFSEVEGRVQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAE DMGNGCFKIYHKCDNACIESIRNETYDHNVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLC VALLGFIMWACQKGNIRCNICI

FL HA OF A/Perth/16/2009 (SEQ ID NO: 15)

MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATELVQSSSTGEIC DSPHQILDGKNCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEF NNESFNWTGVTQNGTSSACIRRSKNSFFSRLNWLTHLNFKYPALNVTMPNNEQFDKLYIWGVLHPG TD KDQIFLYAQASGRITVSTKRSQQTVSPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRG YFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVP EKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNE KF HQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAE DMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLC VALLGFIMWACQKGNIRCNICI

FL HA OF A/Brisbane/10/2007 (SEQ ID NO: 16)

MKTIIALSYILCLVFTQKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATELVQSSSTGEIC DSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEF NNESFNWTGVTQNGTSSACIRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEKFDKLYIWGVHHPGTD NDQIFPYAQASGRITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRG YFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVP EKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGIGQAADLKSTQAAIDQINGKLNRLIGKTNE KF HQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAE DMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLC VALLGFIMWACQKGNIRCNICI

FL HA OF A/Panama/2007/1999 (SEQ ID NO: 17)

MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVSNGTLVKTITNDQIEVTNATELVQSSSTGRIC DSPHQILDGENCTLIDALLGDPHCDGFQNKEWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEF NNESFNWTGVAQNGTSSACKRRSNKSFFSRLNWLHQLKYKYPALNVTMPNNEKFDKLYIWGVH HPSTD SDQISIYAQASGRVTVSTKRSQQTVIPNIGSSPWVRGVSSRISIYWTIVKPGDILLINSTGNLIAPRG YFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVP EKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGTGQAADLKSTQAAINQINGKLNRLIE KTNEKF HQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFERTKKQLRENAE

- 40 046012

DMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLC VVLLGFIMWACQKGNIRCNICI

VH CT149 PROTEIN (SEQ ID NO: 37)

QVQLVQSGAEVKKPGASVKVSCKTSGYSFSTYGVSWVRQAPGQGPEWVGWISAYTGITDYAQKFQGRV TLTTDATTATAFLDLRSLRPDDTATYFCARDKVQGRVEVGSGGRHDYWGQGTLVIVSS

PROTEIN VL CT149 (SEQ ID NO: 38)

EVVLTQSPGTLALPPGERATLSCRASHRVGSTYIAWYQQKSGQAPRRLIYGASNRATDIPDRFSGSGS GTDFTLTIRRLEPEDSAVYYCQQFSVSPWTFGQGTRVEIK

SD15013 (SEQ ID NO: 39)

EVQLVE SGGGLVQAGDSLRISCAASGRTLSIY SMGWFRQAPGKERE FVATIGWNSGRT FY PDSLKGRF TISRDNARNTLYLQMNNLRPEDTAVYYCAAAKGPLRLSSQADYWGQGTQVTVSSAAAWSHPQFEKGAA WSHPQFEKGAAWSHPQFEK

SEQ ID NO 40: UFV180088

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKMNEKSNQTEKCPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNK LF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVEPEA

SEQ ID NO 41: UFV180089

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADYKSTQAAIDQITNI VNRVIEKMNEKSNQTEKCPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEM NKLF EKTRRQLRENAEDMGNGCFKIYHKCDDDCIASIRNGTYDHDVYRDEALNNRFQIKGVEPEA

SEQ ID NO 42: UFV180090

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTKVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADYKSTQAAIDQITGI LNRVIEKMNEKSNQTEKCPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDS EMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNDCIASIRNGTYDHDVYRDEALNNRFQIKGVEPEA

SEQ ID NO 43: UFV180141

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKMNEKSHQTEKCSSNATGRMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNK LF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 44: UFV180192

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNK LF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 45: UFV180193

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKMNEKSHQTEKCSSNATGRMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNK LF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

- 41 046012

SEQ ID NO 46: UFV180194

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKMNEKSHQTEKCSSNATGRIQCLEKYVEDTKIDLWCYNAELLVALINQNTIDYTDSEM NKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 47: UFV180195

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVKQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKMNEKSHQTEKCSSNATGRMKCIEDKIEEIESKLWSYNAELLVALINQNTIDYTDSEM NKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 48: UFV180196

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQINGI LNRVIEKMNEKSHQTEKCSSNATGRMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNK LF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 49: UFV180197

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNK VNRVIEKMNEKSHQTEKCSSNATGRMKCIEDKIEEIESKLWCYNAELLVALENQNTIDYTDSEMNK LF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 50: UFV180198

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNK VNRVIEKMNEKSHQTEKCSSNATGRMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNK LF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 51: UFV180199

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKMNEKSHQTEKCSSNATGRMKCIEDKIEEIESKLWCYNAELLVALENQNTIDYTDSEMNK LF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 52: UFV180200

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKTNEKSHQTEKCSSNATGRMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNK LF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 53: UFV180201

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKMNEKSHQTEKCSSEVEGRMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNK LF

- 42 046012

EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 54: UFV180202

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKMNEKSHQIEKCSSNATGRMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNK LF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 55: UFV180203

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKMNEKSHQTEKCSSNATGRMKCIEDKIEEIESKLWCYNAELLVALINQHTIDYTDSEMNK LF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 56: UFV180204

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKMNEKSHQTEKCSSNATGRMKCIEDKIEEIESKLWCYNAELLVALINQNTIDLTDSEMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 57: UFV180205

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKMNEKSHQTEKCSSNATGRMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEM NKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 58: UFV180206

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKMNEKFHQTEKCSSNATGRMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEM NKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 59: UFV180207

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKMNEKSHQTEKCFSNATGRMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNK LF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 60: UFV181034

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKMNEKFHQIEKCFSNATGRMKCIEDKIEEIESKLWCYNAELLVALINQHTIDLTDSEM NKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVHHHHHH

SEQ ID NO 61: UFV181036

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI

- 43 046012

VNRVIEKMNEKFHQIEKCFSNATGRMKCIEDKIEEIESKLWCYNAELLVALINQNTIDLTDSEMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVHHHHHH

SEQ ID NO 62: UFV181038

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKMNEKFHQIEKCFSNATGRMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDS EMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVHHHHHH

SEQ ID NO 63: UFV181040

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNK VNRVIEKMNEKSNQTEKCPSNATGPMKCIEDKIEEIESKLWCYNAELLVALENQHTIDLTDSEMNK LF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVHHHHHH

SEQ ID NO 64: UFV181042

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKMNEKSNQTEKCPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQHTIDLTDSEMNK LF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVHHHHHH

SEQ ID NO 65: UFV161333

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSE MN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 66: UFV161739

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSE MN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 67: UFV161800

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSE MN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIINGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 68: UFV161804

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRFQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSEMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIINGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 69: UFV170991

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNK VNRVIEKMNEKSHQTECESSNATGPMKCIEDKIEEIESKLWCYNAELLVALENQNTIDYTDSEMNKLF

- 44 046012

EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWIA AADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 70: UFV171004

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKMNEKSHQTECESSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWIA AADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 71: UFV171190

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNF VNRVIEKMNEKSHQTECESSNATGPMKCIEDKIEEIESKLWCYNAELLVALFNQNTIDYTDSEMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWIA AADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 72: UFV171191

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKMNEKSHQTECESSNATGPMKCIEDKIEEIESKLWCYNAELLVALMNQNTIDYTDSEMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWIA AADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 73: UFV171192

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNF VNRVIEKMNEKSHQTECESSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWIA AADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 74: UFV171193

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNV VNRVIEKMNEKSHQTECESSNATGPMKCIEDKIEEIESKLWCYNAELLVALLNQNTIDYTDSEMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWIA AADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 75: UFV171194

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNM VNRVIEKMNEKSHQTECESSNATGPMKCIEDKIEEIESKLWCYNAELLVALENQNTIDYTDSEMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWIA AADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 76: UFV171195

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNT VNRVIEKMNEKSHQTECESSNATGPMKCIEDKIEEIESKLWCYNAELLVALENQNTIDYTDSEMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWIA AADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

- 45 046012

SEQ ID NO 77: UFV170611

MKTIIALSYIECLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLEGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQI TNKVNRVIEKMNEKSHQTECESSNATGRMKCIEDKIEEIESKLWCYNAELLVALENQHTIDYTDSEMN KLEEKTRRQLRENAEDMGNGCEKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 78: UFV170612

MKTIIALSYIECLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLEGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQI TNKVNRVIEKMNEKSHQTECESSNATGRMKCIEDKIEEIESKLWCYNAELLVALENQKTIDYTDSE MN KLEEKTRRQLRENAEDMGNGCEKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 79: UFV170613

MKTIIALSYIECLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSGPGSKYV CQNTLKLATGMRNVPEKQTRGLEGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQI TNKVNRVIEKMNEKSHQTECESSNATGRMKCIEDKIEEIESKLWCYNAELLVALENQNTIDYTDSEMN KLEEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 80: UFV170614

MKTIIALSYIECLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLEGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQI TNKVNRVIEKMNEKSHQTECESSNATGRMKCIEDKIEEIESKLWCYNAELLVALENQQTIDYTDSE MN KLEEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 81: UFV161908

MKTIIALSYIECLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLEGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQI TNKVNRVIEKMNEKSHQTECESSNATGRMKCIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSEMN KLEEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 82: UFV171197

MKTIIALSYIECLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKMNEKSHQTECESSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQRTIDYTDSEMNKLE EKTRRQLRENAEDMGNGCEKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWIA AADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 83: UFV180666

MKTIIALSYIECLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKTNEKSNQTEKCPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNK LE EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNREQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 84: UFV180667

MKTIIALSYIECLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKVNEKSNQTEKCPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNK LF

- 46 046012

EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 85: UFV180668

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKINEKSNQTEKCPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 86: UFV180669

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKLNEKSNQTEKCPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNK LF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 87: UFV180670

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKFNEKSNQTEKCPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNK LF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 88: UFV180671

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKYNEKSNQTEKCPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNK LF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 89: UFV180672

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKWNEKSNQTEKCPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNK LF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 90: UFV180673

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKHNEKSNQTEKCPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNK LF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 91: UFV180674

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKKNEKSNQTEKCPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNK LF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

- 47 046012

SEQ ID NO 92: UFV180675

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKRNEKSNQTEKCPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNK LF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 93: UFV170636

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSKYVCQNT LKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNKV NRVIEKMNEKSHQTECESSNATGRMKCIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSEMNKLFE KTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWIAA ADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 94: UFV170637

E KTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWIA AADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 95: UFV170638

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSSSKYVCQ NTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITN KVNRVIEKMNEKSHQTECESSNATGRMKCIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSEMNKL FEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWI AAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 96: UFV170639

L FEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDW IAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 97: UFV170640

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSSSTGKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQI TNKVNRVIEKMNEKSHQTECESSNATGRMKCIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSEMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 98: UFV160653

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTD SEMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 99: UFV160764

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSPKYVCQN TLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQITNK VNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSEMNK LF

- 48 046012

EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWIA AADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 100: UFV160765

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSCPKYVCQ NTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQITN KVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSEM NKL FEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWI AAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 101: UFV160766

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSACPKYVC QNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQIT NKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSEM NK LFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDW IAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 102: UFV160674

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSGAGPKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTD SEMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 103: UFV160675

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSGACPKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTD SEMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 104: UFV160767

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSACPKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTD SEMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 105: UFV160768

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSCPKYVC QNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQIT NKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSEM NK LFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDW IAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 106: UFV160769

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSACPKY VCQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQ ITNKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDS EM NKLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYK DWIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

- 49 046012

SEQ ID NO 107: UFV160770

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSCPKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTD SEMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 108: UFV160771

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSPKYVCQ NTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQITN KVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSEM NKL FEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWI AAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 109: UFV160772

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSPKYVC QNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQIT NKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSEM NK LFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDW IAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 110: UFV160321

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSGGGGKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDLTDS EMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 111: UFV160403

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSNPHRKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDLTDS EMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 112: UFV160404

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSGDPHKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDLTDS EMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 113: UFV160405

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSNGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDLTDS EMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 114: UFV160406

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSGGSNKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDLTDSEM N

- 50 046012

KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 115: UFV160407

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSGSNAKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDLTDS EMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 116: UFV160408

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDLTDS EMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 117: UFV160409

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSGSGFKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDLTDS EMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 118: UFV160410

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSGSGKYVC QNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQIT NKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDLTDSEMNK LFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDW IAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 119: UFV160411

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSGGKYVCQ NTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQITN KVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDLTDSEMNK L FEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWI AAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 120: UFV160412

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSGGSKYVC QNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQIT NKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDLTDSEMNK LFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDW IAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 121: UFV160413

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSGSKYVC QNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQIT NKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDLTDSEMNK LFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDW IAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 122: UFV160414

- 51 046012

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSHPSTKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDLTDS EMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 123: UFV160415

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSIPNIKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDLTDS EMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 124: UFV160416

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSGLSSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDLTDS EMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 125: UFV160417

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSKPGDKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDLT DSEMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 126: UFV160418

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSDAPIKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDLTDS EMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 127: UFV160419

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSTPNKYVC QNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQIT NKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDLTDSEMNK LFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDW IAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 128: UFV160420

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSTPNGKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDLTDS EMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 129: UFV161686

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSEVEGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSE MN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

- 52 046012

SEQ ID NO 130: UFV161722

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSQATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTD SEMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 131: UFV161723

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNAQGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTD SEMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 132: UFV161688

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIPKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSE MN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 133: UFV161689

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEPMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSEMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 134: UFV161690

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKPNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSE MN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 135: UFV161691

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMPEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSE MN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 136: UFV161692

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNPKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSE MN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 137: UFV161693

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEPSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSEMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

- 53 046012

SEQ ID NO 138: UFV161694

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKPHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSE MN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 139: UFV161695

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSPQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSE MN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 140: UFV161696

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHPTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSEMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 141: UFV161697

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQPEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSE MN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 142: UFV161698

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTPKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSE MN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 143: UFV161699

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEPESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSE MN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 144: UFV161700

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKPSSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTD SEMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 145: UFV161701

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKEPSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTD SEMN

- 54 046012

KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 146: UFV161702

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESPNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTD SEMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 147: UFV161703

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSPATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSE MN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 148: UFV161704

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNPTGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSE MN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 149: UFV161705

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNAPGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSE MN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 150: UFV161706

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATPRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSE MN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 151: UFV161707

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATGPMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSE MN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 152: UFV161708

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKPHQTPKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSE MN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

- 55 046012

SEQ ID NO 153: UFV161709

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKPHQTEKPSSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTD SEMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 154: UFV161715

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSNQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSE MN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 155: UFV161721

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKNSSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTD SEMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYHDVYRDEALNNRFQIKGVELKSGYKDW IAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 156: UFV171187

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNK VNRVIEKMNEKSHQTECESSNATGRMKCIEDKIEEIESKLWCYNAELLVALENQNTIDYTDSEMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWIA AADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 157: UFV171120

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNK VNRVIEKMNEKnHTTECESSNATGPMKCIEDKIEEIESKLWCYNAELLVALENQNTIDYTDSEMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWIA AADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 158: UFV171121

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNK VNRVIEKMNEKnHTTECEPSNATGPMKCIEDKIEEIESKLWCYNAELLVALENQNTIDYTDSEMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWIA AADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 159: UFV170994

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNK VNRVIEKMNEKSNQTECESSNATGPMKCIEDKIEEIESKLWCYNAELLVALENQNTIDYTDSEMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWIA AADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 160: UFV170995

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNK VNRVIEKMNEKSNQTECEPSNATGPMKCIEDKIEEIESKLWCYNAELLVALENQNTIDYTDSEMNKLF

- 56 046012

EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWIA AADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 161: UFV180208

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKMNEKFHQTEKCFSNATGRMKCIEDKIEEIESKLWCYNAELLVALINQHTIDLTDSEM NKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 162: UFV180217

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKMNEKFNQTEKCPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQHTIDLTDSEM NKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 163: UFV170278

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQI TNKVNRVIEKMNEKSHQTEKCSSNATGRMKCIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSE MN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIGAAEPEA

SEQ ID NO 164: UFV170282

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQI TNKVNRVIEKMNEKSHQTEKCSSNATGRMKCIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSE MN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIGAAEPEA

SEQ ID NO 165: UFV160595

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTD SEMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 166: UFV161196

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNAIELVQSPGGSKGP KCNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSEMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 167: UFV161198

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNAIELVQSPGGSKCV PQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIECKLWAYNAELLVALENQRTIDYTDSE MN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

- 57 046012

SEQ ID NO 168: UFV161169

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLCKTITDDQIEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNSVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALECQRTIDYTDSE MN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 169: UFV170062

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQI TNKVNRVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSE MN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 170: UFV170051

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQI TNKVNRVIEKMNEKSHQTEKCSSNATGRMKCIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSE MN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 171: UFV170428

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQI TNKVNRVIEKMNEKSHQTCKESSNATGRMKCIEDKIEEIESKLWCYNAELLVALENQRTIDYTDSEMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 172: UFV170440

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQI TNKVNRVIEKMNEKSHQTEKCSSNATGCMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTD SEMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 173: UFV171272

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNK VNRVIEKMNEKSHQTECESSNATGPMKCIEDKIEEIESKLWCYNAELLVALENQNTIDYTDSEMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWI

SEQ ID NO 174: UFV171273

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNK VNRVIEKMNEKSHQTECESSNATGPMKCIEDKIEEIESKLWCYNAELLVALENQNTIDYTDSEMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD

SEQ ID NO 175: UFV171274

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNK VNRVIEKMNEKSHQTECESSNATGPMKCIEDKIEEIESKLWCYNAELLVALENQNTIDYTDSEMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGY

- 58 046012

SEQ ID NO 176: UFV171275

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNK VNRVIEKMNEKSHQTECESSNATGPMKCIEDKIEEIESKLWCYNAELLVALENQNTIDYTDSEMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKS

SEQ ID NO 177: UFV171276

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNK VNRVIEKMNEKSHQTECESSNATGPMKCIEDKIEEIESKLWCYNAELLVALENQNTIDYTDSEMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVEL

SEQ ID NO 178: UFV171277

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNK VNRVIEKMNEKSHQTECESSNATGPMKCIEDKIEEIESKLWCYNAELLVALENQNTIDYTDSEMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGV

SEQ ID NO 179: UFV171278

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNK VNRVIEKMNEKSHQTECESSNATGPMKCIEDKIEEIESKLWCYNAELLVALENQNTIDYTDSEMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIK

SEQ ID NO 180: UFV171279

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNK VNRVIEKMNEKSHQTECESSNATGPMKCIEDKIEEIESKLWCYNAELLVALENQNTIDYTDSEMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQ

SEQ ID NO 181: UFV171280

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNK VNRVIEKMNEKSHQTECESSNATGPMKCIEDKIEEIESKLWCYNAELLVALENQNTIDYTDSEMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNR

SEQ ID NO 182: UFV161454

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI NGKLNRVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDY TDSEMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 183: UFV161453

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI NNKLNRVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTD SEMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 184: UFV161459

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI NAKLNRVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDY TDSEMN

- 59 046012

KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 185: UFV161451

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI NNKVNRVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTD SEMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 186: UFV161458

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI NAKVNRVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDY TDSEMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 187: UFV161450

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKLNRVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTD SEMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 188: UFV161448

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNAIELVQSPGGSKYV CQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQI TNKVNRVIEKMNEKSHQTEKESSNATGRMKQIEDKIEEIESKLWCYNAELLVALENQRTIDYTD SEMN KLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD WIAAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 189: UFV171116

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQINGI LNRVIEKMNEKSHQTECESSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWIA AADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 190: UFV172561

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKMNEKSNQTECEPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQRTIDYTDSEMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 191: UFV172563

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADYKSTQAAIDQITNI VNRVIEKMNEKSNQTECEPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQRTIDYTDSEMNK LF EKTRRQLRENAEDMGNGCFKIYHKCDDDCIASIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

- 60 046012

SEQ ID NO 192: UFV172564

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTKVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADYKSTQAAIDQITNI VNRVIEKMNEKSNQTECEPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQRTIDYTDSEM NKLF EKTRRQLRENAEDMGNGCFKIYHKCDDDCIASIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 193: UFV172571

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTKVNTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADYKSTQAAIDQITNI VNRVIEKMNEKSNQTECEPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQRTIDYTDSEM NKLF EKTRRQLRENAEDMGNGCFKIYHKCDNDCIASIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 194: UFV172562

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITGI LNRVIEKMNEKSNQTECEPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQRTIDYTDSEMNKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 195: UFV172588

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTKVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADYKSTQAAIDQITGI LNRVIEKMNEKSNQTECEPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQRTIDYTDSEM NKLF EKTRRQLRENAEDMGNGCFKIYHKCDNDCIASIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 196: UFV172583

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTKVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADYKSTQAAIDQITGI LNRVIEKMNEKSNQTECEPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQRTIDYTDSEM NKLF EKTRRQLRENAEDMGNGCFKIYHKCDDDCIASIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 197: UFV172585

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTKVNTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADYKSTQAAIDQITGI LNRVIEKMNEKSNQTECEPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQRTIDYTDSEM NKLF EKTRRQLRENAEDMGNGCFKIYHKCDDDCIASIRNGTYDHDVYRDEALNNRFQIKGVAAADYKDDDDK PGGGGSGGGGSGGGGSHHHHHH

SEQ ID NO 198: UFV180642

MKTIIALSYILCLVFAQKIPGNDNSTATLCLGHHAVPNGTIVKTITNDRIEVTNATELVQNSRYVCHS TLKLATGMRNVPEKQTQGIFGAIAGFIENGWEGMVDGWYGFRWQNSEGRGQAADLKSTQAAIDQITNK VNRLIGKMNEKFHQIEKCFSNATGRMKCIEDKIEEIESKLWCYNAELLVALENQHTIDLTDSEMNKLF EKTKKQLRENAEDMGNGCFKIYHKCDNACIESIRNETYDHNVYRDEALNNRFQIKGVHHHHHH

SEQ ID NO 199: UFV180645

MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATELVQSSRYVCQN TLKLATGMRNVPEKQTQGIFGAIAGFIENGWEGMVDGWYGFRWQNSEGIGQAADLKSTQAAINQITNK VNRLIGKMNEKFHQIEKCFSNATGRMKCIEDKIEEIESKLWCYNAELLVALENQHTIDLTDSEM NKLF ERTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVHHHHHH

- 61 046012

SEQ ID NO 200: UFV180647

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNK VNRVIEKMNEKFHQIEKCFSNATGRMKCIEDKIEEIESKLWCYNAELLVALENQHTIDLTDSEM NKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVHHHHHH

SEQ ID NO 201: UFV181106

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKMNEKSNQTEKCPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEM NKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVHHHHHH

SEQ ID NO 202: UFV181107

MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATELVQSSRYVCQN TLKLATGMRNVPEKQTQGIFGAIAGFIENGWEGMVDGWYGFRWQNSEGIGQAADLKSTQAAINQITNI VNRLIGKMNEKSNQTEKCPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEM NKLF ERTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVHHHHHH

SEQ ID NO 203: UFV181109

MKTIIALSYILCLVFAQKIPGNDNSTATLCLGHHAVPNGTIVKTITNDRIEVTNATELVQNSRYVCHS TLKLATGMRNVPEKQTQGIFGAIAGFIENGWEGMVDGWYGFRWQNSEGRGQAADLKSTQAAIDQITNI VNRLIGKMNEKSNQTEKCPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNKLF EKTKKQLRENAEDMGNGCFKIYHKCDNACIESIRNETYDHNVYRDEALNNRFQIKGVHHHHHH

SEQ ID NO 204: UFV181117

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKMNEKSHQTEKCSSNATGRMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEM NKLF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVHHHHHH

SEQ ID NO 205: UFV181118

MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATELVQSSRYVCQN TLKLATGMRNVPEKQTQGIFGAIAGFIENGWEGMVDGWYGFRWQNSEGIGQAADLKSTQAAINQITNI VNRLIGKMNEKSHQTEKCSSNATGRMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEM NKLF ERTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVHHHHHH

SEQ ID NO 206: UFV181120

MKTIIALSYILCLVFAQKIPGNDNSTATLCLGHHAVPNGTIVKTITNDRIEVTNATELVQNSRYVCHS TLKLATGMRNVPEKQTQGIFGAIAGFIENGWEGMVDGWYGFRWQNSEGRGQAADLKSTQAAIDQITNI VNRLIGKMNEKSHQTEKCSSNATGRMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNKLF EKTKKQLRENAEDMGNGCFKIYHKCDNACIESIRNETYDHNVYRDEALNNRFQIKGVHHHHHH

SEQ ID NO 207: UFV180480 (UFV180088 + native transmembrane (TM) domain)

MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQN TLKLATGMRNVPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNI VNRVIEKMNEKSNQTEKCPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNK LF EKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWIL WISFAISCFLLCVVLLGFIMWACQRGNIRCNICI

SEQ ID NO 208: nucleotide sequence encoding UFV180088

ATGAAAACCATTATCGCACTGAGTTACATCTTCTGCCTGGCTCTGGGACAGGATCTGCCTGG GAACGACAACAGCACAGCAACACTGTGCCTGGGACACCATGCTGTGCCCAACGGGACCCTGG

- 62 046012

TCAAGACCATCACAGAGGATCAGGTGGAAGTGACTAATGCAACCGAACTGGTCCAGAGCTCC AAGTACGTGTGCCAGAACACCCTGAAACTGGCCACAGGCATGAGAAATGTCCCTGAGAAGCA GACCCAGGGGCTGTTCGGCGCTATCGCAGGCTTCATCGAGAACGGATGGGAAGGGATGATCG ACGGCTGGTATGGATTCCGGTGGCAGAATTCCGAGGGAACAGGGCAGGCCGCTGACCTGA AG T C TAC T GAG G GAG C CAT C GAT CAGAT TAC СAACAT C G T GAAT CGCGTCATC GAGAAGAT GAA CGAAAAAAGCAATCAGACAGAGAAGTGCCCCTCCAATGCCACTGGCCCCATGAAGTGTATTG AGGATAAGATCGAGGAAATCGAAAGTAAACTGTGGTGCTACAACGCCGAGCTGCTGGTGGCT C T GAT CAAT CAGAATACAAT C GAC T ATAC T GAT T CAGAGAT GAACAAG C T G T T C GAAAAAAC TAGGAGACAGCTGCGAGAGAACGCAGAAGACATGGGCAATGGATGTTTTAAGATCTACCACA AATGCGATAACGCCTGTATCGAGTCTATTAGGAATGGCACATACGACCATGACGTGTACCGG GACGAAGCACTGAACAATCGCTTTCAGATCAAGGGGGTGGAACCAGAGGCATAATAA

SEQ ID NO 209: nucleotide sequence encoding UFV180480 (UFV180088 + TM domain)

ATGAAGACCATCATCGCCCTGTCTTACATCTTCTGCCTGGCCCTGGGACAGGACCTGCCAGG CAACGATAATAGCACCGCCACCCTGTGCCTGGGACACCACGCAGTGCCCAACGGCACCCTGG TGAAGACCATCACAGAGGACCAGGTGGAGGTGACCAATGCCACAGAGCTGGTGCAGAGCTCC AAGTACGTGTGCCAGAACACCCTGAAGCTGGCCACAGGCATGAGAAATGTGCCCGAGA AGCA GACCCAGGGCCTGTTCGGAGCCATCGCCGGCTTTATCGAGAACGGCTGGGAGGGCATGATCG ATGGCTGGTATGGCTTTCGGTGGCAGAATAGCGAGGGAACAGGACAGGCAGCAGACCTGAAG T C CAC C CAG G CAG C CAT C GAT CAGAT CACAAAASAT C G T GAATAGAG T GAT C GAGAAGAT GAA CGAGAAGTCCAATCAGACCGAGAAGTGCCCCTC TAACGCCACAGGCCCTATGAAGTGTATCG AGGAAAGATCGAGGAGATCGAGTCCAAGCTGTGGTGCTACAACGCCGAGCTGCTGGTGGCC C T GAT C AAC C AGAAT AC CATC GAC TAT AC AGAT T C T GAGAT GAAT AAG C T G T T C GAGAAGAC CCGGAGACAGCTGAGGGAACGCCGAGGACATGGGCAATGGCTGTTTTAAGATCTACCACA AGTGCGATAACGCCTGTATCGAGAGCATCCGCAATGGCACATACGACCACGACGTGTACCGG GACGAGGCCCTGAACAATAGATTCCAGATCAAGGGCGTGGAGCTGAAGTCCGGCTATAAGGA TTGGATCCTGTGGATCTCTTTCGCCATCAGCTGCTTT CTGCTGTGCGTGGTGCTGCTGGGCT ttatcatgtgggcctgtcagaggggcaacatccgctgcaatatctgtatctgataa

SEQ ID NO: 210 (min sequence UFV180088)

QDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQNTLKLATGMRN VPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADLKSTQAAIDQITNIVNRV IEKMNEKSNQTEKCPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNK LFEKTRRQLRE NAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGV

SEQ ID NO: 211 (min sequence UFV180089)

QDLPGNDNSTATLCLGHHAVPNGTLVKTITEDQVEVTNATELVQSSKYVCQNTLKLATGMRN VPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADYKSTQAAIDQITNIVNRV IEKMNEKSNQTEKCPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNK LFEKTRRQL RENAEDMGNGCFKIYHKCDDDCIASIRNGTYDHDVYRDEALNNRFQIKGV

SEQ ID NO: 212 (min sequence UFV180090)

QDLPGNDNSTATLCLGHHAVPNGTKVKTITEDQVEVTNATELVQSSKYVCQNTLKLATGMRN VPEKQTQGLFGAIAGFIENGWEGMIDGWYGFRWQNSEGTGQAADYKSTQAAIDQITGILNRV IEKMNEKSNQTEKCPSNATGPMKCIEDKIEEIESKLWCYNAELLVALINQNTIDYTDSEMNK LFEKTRRQ LRENAEDMGNGCFKIYHKCDNDCIASIRNGTYDHDVYRDEALNNRFQIKGV

Claims (36)

1. A monomeric polypeptide of the hemagglutinin (HA) stem domain of the influenza A virus, containing the HA1 and HA2 domains of the HA influenza A virus group 2, wherein the HA stem domain polypeptide contains an amino acid sequence that includes: deletion of the head region from the HA1 domain; modification of the trimerization region in HA2, wherein said modification comprises introducing a GCN4 sequence at a position corresponding to the amino acid sequence from amino acid position 405 to amino acid position 419, inclusive, wherein said GCN4 sequence includes the amino acid sequence ₄₀₅ RMKQIEDKIEEIESK ₄₁₉ (SEQ ID NO: 9) or ₄₀₅ PMKQIEDKIEEIESK ₄₁₉ (SEQ ID NO: 10) or ₄₀₅ RMKCIEDKIEEIESK ₄₁₉ (SEQ ID NO: 11) or ₄₀₅ PMKCIEDKIEEIESK ₄₁₉ (SEQ ID NO: 12); a cysteine at an amino acid position corresponding to position 310 in combination with a cysteine at a position corresponding to position 422; or a cysteine at an amino acid position corresponding to position 311, in combination with a cysteine at a position corresponding to position 422; or a cysteine at the amino acid position corresponding to position 308, in combination with a cysteine at a position corresponding to position 418, wherein said cysteine residues are capable of forming an intramonomer disulfide bridge; and wherein in the amino acid sequence, the amino acid at position 355 is W, wherein the numbering of amino acid positions in the amino acid sequence of the HA stem domain polypeptide corresponds to the numbering of H3, corresponding to the numbering in the full-length HA of the reference strain A/Aichi/2/68 H3N2 (SEQ ID NO: 1) . 2. The polypeptide of claim 1, wherein 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. 3. The polypeptide of claim 1 or 2, wherein the amino acid at position 378 is T, the amino acid at position 379 is N, and/or the amino acid at position 381 is V. - 63 046012 4. The polypeptide according to claim 1, 2 or 3, additionally containing an introduced glycosylation motif at positions 401-403 for N-linked glycosylation at position 401. 5. The polypeptide according to any one of claims 1 to 4, wherein said deletion of the head region in the HA1 domain comprises a deletion spanning at least the amino acid sequence from the amino acid corresponding to the amino acid at position 50 to the amino acid corresponding to the amino acid at position 302, inclusive. 6. The polypeptide of claim 5, wherein the deletion of the head region in the HA1 domain spans at least the amino acid sequence from the amino acid at position 47 to the amino acid at position 306, inclusive. 7. A polypeptide according to any of the previous claims, containing a cysteine at an amino acid position corresponding to position 310, in combination with a cysteine at a position corresponding to position 422, wherein said cysteine residues form said at least one intramonomer cysteine bridge. 8. The polypeptide according to any of the preceding claims, wherein the amino acid at position 388 is M. 9. A polypeptide according to any of the preceding claims, comprising at least one additional introduced glycosylation motif, wherein at least one additional introduced glycosylation motif is present at positions 392-394 for N-linked glycosylation at position 392 or at positions 393-395 for N -linked glycosylation at position 393. 10. A polypeptide according to any of the previous paragraphs, where: the amino acid at position 31 is E and the amino acid at position 34 is V; the amino acid at position 392 is S or P; the amino acid at position 395 is T or P; the amino acid at position 399 is S or P; the amino acid at position 435 is N or R; and/or the amino acid at position 439 is Y. 11. The polypeptide of any one of the preceding claims, wherein the HA stem domain polypeptide monomer does not contain a protease cleavage site between the HA1 and HA2 domains. 12. The polypeptide of claim 11, wherein the amino acid at position 329 is not arginine (R), wherein preferably the amino acid at position 329 is glutamine (Q). 13. The polypeptide according to any one of claims 1 to 10, wherein the HA stem domain polypeptide monomer contains a natural cleavage site or a polybasic cleavage site. 14. The polypeptide according to any of the previous claims, wherein the HA1 and HA2 domains are derived from an influenza virus containing HA subtype H3, preferably the influenza A/Hong Kong/1/68 virus. 15. The polypeptide according to claim 14, where one or more amino acids in the HA1 and HA2 domains of the H3 subtype are subject to mutation by substitution with the corresponding amino acids HA H7. 16. The polypeptide according to claim 15, where the amino acid at position 25 is K; the amino acid at position 367 is Y; the amino acid at position 378 is T; the amino acid at position 475 is D; the amino acid at position 476 is D; and/or the amino acid at position 479 is A. 17. The polypeptide of any one of the preceding claims, wherein said HA stem domain polypeptide contains a signal sequence or a portion thereof. 18. The polypeptide of any one of the preceding claims, wherein at least the C-terminal portion of the HA2 domain beginning with the amino acid corresponding to the amino acid at position 516 is deleted. 19. The polypeptide according to any of the previous paragraphs, where a deletion of the C-terminal part of the HA2 domain is carried out, starting with the amino acid corresponding to the amino acid at position 506. 20. The polypeptide of any one of the preceding claims, wherein the deleted head portion in the HA1 domain has been replaced by a linker sequence of 1-10 amino acids. 21. The polypeptide according to any of the previous claims, containing a cysteine at a position corresponding to position 396, in combination with a cysteine at a position corresponding to position 408, or a cysteine at a position corresponding to position 397, in combination with a cysteine at a position corresponding to position 408; or a cysteine at a position corresponding to position 398, in combination with a cysteine at a position corresponding to position 408, or a cysteine at a position corresponding to position 398, in combination with a cysteine at a position corresponding to position 405. 22. The polypeptide according to claim 21, containing a cysteine at a position corresponding to position 398, in combination with a cysteine at a position corresponding to position 408. - 64 046012 23. A multimeric hemagglutinin (HA) stem domain polypeptide of an influenza A virus, comprising at least two monomers of the HA stem domain polypeptide of any of the preceding claims. 24. Multimeric polypeptide of the hemagglutinin (HA) stem domain of the influenza A virus, containing at least two monomers of the HA stem domain polypeptide according to claim 21, where the first monomer of the HA stem domain polypeptide is linked to the second monomer by an intermonomer disulfide bridge between cysteine at position 396, 397 or 398 of said first monomer and a cysteine at position 408 of a second monomer, or wherein the first monomer of the HA stem domain polypeptide is linked to the second monomer by an intermonomer disulfide bridge between a cysteine at position 398 of said first monomer and a cysteine at position 405 of the second monomer. 25. The multimeric hemagglutinin (HA) stem domain polypeptide of influenza A virus according to claim 24, wherein the first monomer of the HA stem domain polypeptide is linked to the second monomer by an intermonomer disulfide bridge between a cysteine at position 398 of said first monomer and a cysteine at position 408 of the second monomer. 26. Multimeric polypeptide according to any one of claims 23-25, where the polypeptide is trimeric. 27. A nucleic acid encoding a monomer of the HA stem domain polypeptide of any one of the preceding claims 1 to 22. 28. A vector containing a nucleic acid molecule according to claim 27. 29. The vector according to claim 28, where the vector is a recombinant adenoviral vector. 30. A pharmaceutical composition containing a monomeric HA stem domain polypeptide according to any one of claims 1-22, a multimeric HA stem domain polypeptide of an influenza virus according to any one of claims 23-26, a nucleic acid according to claim 27 or a vector according to claim 28 or 29 and a pharmaceutically acceptable carrier. 31. Use of the monomeric HA stem domain polypeptide according to any one of claims 1 to 22 for inducing an immune response against an influenza virus. 32. Use of the multimeric HA stem domain polypeptide of an influenza virus according to any one of claims 23 to 26 for inducing an immune response against an influenza virus. 33. Use of a nucleic acid according to claim 27 for inducing an immune response against the influenza virus. 34. Use of the monomeric HA stem domain polypeptide according to any one of claims 1 to 22 as a vaccine. 35. Use of the multimeric polypeptide of the stem domain HA of the influenza virus according to any one of claims 23-26 as a vaccine. 36. Use of the nucleic acid according to claim 27 as a vaccine.