JP7167088B2 - Influenza virus vaccine and its use - Google Patents

Description

The present invention relates to the field of medicine. Provided herein are influenza hemagglutinin stem domain polypeptides, methods of providing hemagglutinin stem domain polypeptides, compositions comprising them, vaccines comprising them and methods of their use, particularly in the detection, prevention and/or treatment of influenza. be done.

Influenza viruses are major human pathogens and cause respiratory illness (commonly called "influenza (i
nfluenza" or "the flu"). The clinical efficacy of infection varies with the pathogenicity of the influenza strain and the host's exposure, medical history, age, and immune status. Each year, approximately 1 billion people worldwide are infected with influenza viruses, with 3-5 million cases of severe illness and an estimated 300,000 to 500
,000 flu-related deaths. The majority of these infections can be attributed to influenza A viruses carrying H1 or H3 hemagglutinin subtypes, with minor contributions from influenza B viruses, and therefore representatives of all three are included in seasonal vaccines. Current vaccination practices rely on early identification of circulating influenza viruses to enable timely production of effective seasonal influenza vaccines. Apart from the inherent difficulty in predicting which strains will be dominant during the next season, antiviral resistance and immune evasion also contribute to the inability of current vaccines to prevent morbidity and mortality. In addition, the potential for epidemics caused by highly pathogenic virus strains that originate from reservoir animals and are reassorted to increase human-to-human spread is a significant and real threat to global health. becomes.

Influenza A viruses are widely distributed in nature and can infect a wide variety of birds and mammals. Influenza viruses belong to the family Orthomyxoviridae.
viridae) is an enveloped RNA virus belonging to the family. These genomes are
11 different proteins, 1 nucleoprotein (NP), 3 polymerase proteins (PA, PB1 and PB2), 2 matrix proteins (M1 and M2),
It consists of eight single-stranded RNA segments that encode three nonstructural proteins (NS1, NS2, and PB1-F2) and two external glycoproteins: hemagglutinin (HA) and neuraminidase (NA). Viruses are classified based on differences in the antigenic structure of the HA and NA proteins, and their different combinations represent unique virus subtypes that are further classified into defined influenza virus strains. All known subtypes can be found in birds, but currently circulating human influenza A subtypes are H1N1 and H3N2. Phylogenetic analysis demonstrated a subclassification of hemagglutinins into two main groups: H1, H2, H5 and H9 subtypes in phylogenetic group 1 among others and H3, H4 and H7 subtypes in phylogenetic group 2 among others.

Influenza B virus strains are strictly human. Antigenic variation in HA within influenza B virus strains is smaller than that observed within A strains. Two genetically and antigenically distinct lineages of influenza B virus are B/Yamagata/
16/88 (also known as B/Yamagata) and B/Victoria/2/8
circulating in humans as represented by the 7 (B/Victoria) lineage (
Ferguson et al. , 2003). 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 still frequently observed for influenza B infections.

Antibodies that neutralize influenza are known to be directed primarily against hemagglutinin (HA). Hemagglutinin or HA is anchored in the viral coat and
It is a trimeric glycoprotein with dual functions: it is responsible for binding to the cell surface receptor sialic acid, after uptake it mediates the fusion of viral and endosomal membranes, and viral RNA in the cytosol of the cell. resulting in the release of HA contains a large head domain and a smaller stem domain. Attachment to the viral membrane is mediated by a C-terminal anchoring sequence linked to the stem domain. The protein is post-translationally cleaved at a specific loop to produce two polypeptides HA1 and HA2 (the full sequence is designated HA0).
The membrane distal head region is primarily derived from HA1 and the membrane proximal stem region is primarily derived from HA2 (Fig. 1).

The reason seasonal influenza vaccines must be updated annually is the large variability of the virus. In the hemagglutinin molecule this mutation is particularly manifested in the head domain where antigenic drift and shift have resulted in a large number of different variants. Since this is also an immunodominant area, most neutralizing antibodies are directed against this domain and act by interfering with receptor binding. The combination of immunodominance and large mutations in the head domain also explains why infection with certain strains does not confer immunity to others:
Antibodies induced by the primary infection only recognize a limited number of strains closely related to the virus of the primary infection.

Recently, influenza hemagglutinin stem domain polypeptides lacking all or substantially all of the influenza hemagglutinin globular head domain have been described and used to generate an immune response against one or more conserved epitopes of the stem domain polypeptide. ing. The epitope of the stem domain polypeptide is less immunogenic than the highly immunogenic region of the globular head domain, thus the absence of the globular head domain in the stem domain polypeptide is one of the It is believed to be permissive for the development of an immune response against these epitopes (Steel et al., 2010).
Therefore, Steel et al. is A/Puerto Rico/8/1934
(H1N1) and A/HongKong/1968 (H3N2) strain HA1 amino acid residues 53 to 276 were deleted from the HA primary sequence and replaced with the short flexible binding sequence GG.
A new molecule was created by replacing it with GG. Vaccination of mice with the H3HK68 construct did not induce antisera cross-reactive with group 1 HA. moreover,
As shown in PCT/EP2012/073706, stem domain polypeptides are highly labile and have the correct stereochemistry as evidenced by the lack of binding of antibodies shown to bind conserved epitopes in the stem region. No structure was used.

Furthermore, Bommakanti et al. (2010), amino acid residues 1-172 of HA2, a seven amino acid linker (GSAGSAG), amino acid residues 7-46 of HA1,
A HA2-based polypeptide was described comprising residues 290-321 of HA1 followed by a 6 amino acid linker GSAGSA, along with mutations V297T, I300E, Y302T and C305T in HA1. The design was based on the sequence of H3HA (A/HongKong/1968). This polypeptide only provided cross-protection against another influenza virus strain within the H3 subtype (against A/Phil/2/82, but against H
1 subtype (not provided for A/PR/8/34). Bommakanti et
In a more recent paper by al (2012), H1N1A/Puerto Ric
The HA-based stem domain sequence (H1HA0HA6) from o/8/1934 has been described. In this polypeptide, the equivalent of residues 55 to 302 are deleted and mutations I311T, V314T, I316N, C319S, F406D, F409
T, and L416D have been made. Both H3 and HA base polypeptides
It is expressed in E. coli and thus lacks the glycans that are part of the native HA protein. When expressed in E. coli, the polypeptide is recovered primarily as high molecular weight aggregates and minor monomeric fractions. The polypeptides are 9 and 0.
Binds CR6261 with two apparent dissociation constants of 2 μM. The authors found that mice were
Immunization with 20 μg protein adjuvanted with 0 μg CpG7909 (
2 times, 4 weeks interval) followed by 1LD90 allogeneic H1N1A/Puerto Rico/8/
1934 virus can survive challenge. The authors, A/Hong
Circularly permuted polypeptides equivalent to those described above for Kong/1/1968-derived polypeptides are also described. These polypeptides are H1N1A/Puerto R
ico/8/1934, H1N1A/North Carolina/20/99 or H1N1A/California/07/2009, and H1N1A/
It may provide partial protection of the mild challenge (1LD90) model in Puerto Rico/8/1934 (ie within the same subtype) mice. Sera from guinea pigs immunized with these polypeptides did not show detectable levels of neutralization when tested in the neutralization assay.

More recently, Lu et al (2013) also found that H1N1A/California/0
5/2009 HA-derived soluble stem domain polypeptides have been described. In the final design, the equivalent of residues 54-303 (numbered according to SEQ ID NO: 1) were deleted (the leader sequence, residues 1-17 are also absent) and two mutations were added to the B loop of the protein; That is, it has been introduced into F407D and L413D. Furthermore, the polypeptide is
It contained a C-terminal trimerization domain (foldon). In addition, two intermonomer disulfide bridges, one in the region of the trimeric foldon domain and 430 and 431
introduced one in the first place. Polypeptides are produced in an E. coli-based cell-free system (thus lacking the glycans that are part of the native HA protein) and recovered in a denatured form that must be refolded prior to use. Influenza challenge data showed no immunological or protection.

In a recent paper, Mallajosyula et al (2014) also reported stem domain polypeptides. In this design, H1N1A/Puerto
Based on HA from Rico/8/1934, not only is most of the HA1 sequence deleted (residues 42 to 289, numbered according to SEQ ID NO:1), but most of the N- and C-terminal sequences of HA2 are also deleted. are also deleted (residues 344-383 and 457-5, respectively).
65). The polypeptide contains a foldon trimerization domain at the C-terminus and is also produced in E. coli, thus lacking the natural glycans on viral HA. The polypeptide binds to broadly neutralizing antibodies CR6261, F10 and FI6v3. The polypeptide was tested in an influenza challenge model (1LD90 H1N1A/Puerto R
ico/8/1934) and could protect mice from death. H1N1A
/New Caledonia/20/1999 and H1N1A/Californi
The equivalent polypeptide derived from HA of a/04/2009 could also be partially protected. H5N
1A/Viet Nam/1203/2004-derived polypeptides conferred only limited protection in this challenge model. Furthermore, the challenge model used is mild with relatively low doses (1-2 LD90).

Therefore, for effective prophylaxis and treatment of influenza in particular, stimulating the production of robust broadly neutralizing antibody responses and providing protection against a wide range of current and future influenza virus strains (both seasonal and epidemic) Phylogenetic Group 1 and/or Group 2
There remains a need for a safe and effective universal vaccine that provides protection against one or more influenza A virus subtypes within.

Provided herein are influenza hemagglutinin stem domain polypeptides, methods of providing stem domain polypeptides, compositions comprising them, vaccines comprising them and methods of use thereof.

In a first aspect, the present invention provides novel immunogenic polypeptides comprising an influenza hemagglutinin stem domain and lacking a globular head, referred to as influenza (HA) stem domain polypeptides. A polypeptide can induce an immune response when administered to a subject, particularly a human subject. The polypeptides of the invention present conserved epitopes of the membrane-proximal stem domain HA molecule to the immune system in the absence of dominant epitopes present in the membrane-distal head domain. To this end, a portion of the primary sequence of the HA0 protein that makes up the head domain is removed and the remaining amino acid sequence introduced either directly or, in some embodiments, a short flexible binding sequence (“linker”). This relinks and restores the continuity of the amino acid chain. The resulting sequence is further modified by introducing defined mutations that stabilize the native three-dimensional structure of the remaining portion of the HA0 molecule. An immunogenic polypeptide does not include the full-length HA1 domain of influenza virus.

The present invention provides novel influenza hemagglutinin stem domain polypeptides comprising
(a) an influenza hemagglutinin HA1 domain comprising an HA1 N-terminal stem segment covalently linked to the HA1 C-terminal stem segment by a linking sequence of 0-50 amino acid residues, said HA1 C-terminal segment comprising (b) influenza hemagglutinin HA
2 domains, the HA1 and HA2 domains are derived from an influenza A virus subtype containing HA of the H1 subtype;
(c) does not contain a protease cleavage site at the junction between the HA1 and HA2 domains;
(d) said HA1 N-terminal segment comprises amino acids 1-x of HA1, preferably amino acids p-x of HA1, and the HA1 C-terminal stem segment comprises amino acids y-C-terminal amino acids of HA1, x=SEQ ID NO: 1 and p=position 18 of SEQ ID NO: 1 (or the corresponding position in another hemagglutinin).
y=amino acid on position 321 of SEQ ID NO: 1 (or the corresponding position in another hemagglutinin);
(e) the region containing amino acid residues 402-418 is amino acid X ₁ NTQX ₂ TAX ₃ GK
_EX4N (H/K) _X5E (K/R) (SEQ ID NO: 8) comprising:
X ₁ is an amino acid selected from the group consisting of M, E, K, V, R and T;
X2 is an amino acid selected from the group consisting of F, _I , N, T, H, L and Y, preferably I, L or Y;
X3 is an amino acid selected from the group consisting of V _, A, G, I, R, F and S, preferably A, I or F;
_X4 is an amino acid selected from the group consisting of F, I, N, S, T, Y, E, K, M and V, preferably I, Y, M or V;
X ₅ is an amino acid selected from the group consisting of L, H, I, N, R, preferably I;
(f) Amino acid residues on position 337 (HA1 domain) are I, E, K, V, A, and T
is selected from the group consisting of
Amino acid residues on position 340 (HA1 domain) are I, K, R, T, F, N, S and Y
is selected from the group consisting of
the amino acid residue on position 352 (HA2 domain) is selected from the group consisting of D, V, Y, A, I, N, S, and T;
the amino acid residue on position 353 (HA2 domain) is selected from the group consisting of K, R, T, E, G, and V;
(g) further comprising a disulfide bridge between the amino acid on position 324 and the amino acid on position 436;
(h) In addition, the amino acid sequence RMKQIEDKIEEIESK (SEQ ID NO: 20) is 419
~433, or the sequence RMKQIEDKIEEIESKQK (
SEQ ID NO:21) is introduced at positions 417-433.

In certain embodiments, the polypeptide comprises a complete HA2 domain, ie, the HA2 domain including the transmembrane domain and intracellular sequences. In certain embodiments, the HA2 domain is truncated. Thus, in certain embodiments, the polypeptides of the invention do not contain the intracellular sequences and transmembrane domains of HA. In certain embodiments, HA2 domain 514, 515, 516, 517, 518, 519, 520, 521
, 522, 523, 524, 525, 526, 527, 526, 528, 529, or 530 (or their equivalents) at the C-terminus of the HA2 domain.

According to the invention, the C-terminal amino acid of the HA1 C-terminal stem segment is attached to the N-terminal amino acid of the HA2 domain, thus forming the junction between the HA1 and HA2 domains. Polypeptides of the invention do not contain a protease cleavage site at the junction between the HA1 and HA2 domains. In certain embodiments, the C-terminal amino acid residue (amino acid 343 of SEQ ID NO: 1) of the HA1 C-terminal stem segment is any amino acid other than arginine (R) or lysine (K), preferably glutamine (Q).

Polypeptides of the invention are substantially smaller than HA0 and preferably lack all or substantially all of the globular head of HA. Preferably, the immunogenic polypeptide is 360 amino acids or less in length, preferably 350, 340, 330, 320, 310, 305, 300, 2
95, 290, 285, 280, 275, or 270 amino acids or less in length. In certain embodiments, the immunogenic polypeptide is about 250 to about 350, preferably about 260
to about 340, preferably about 270 to about 330, preferably about 270 to about 330
Amino acid length.

The polypeptides of the invention are group 1 cross-neutralizing antibody CR6261 (WO 2008
/028946) and/or antibody CR9114 (WO 2
013/007770), which contains conserved stem domain epitopes of antibodies capable of binding and neutralizing both group 1 and group 2 influenza A viruses, as well as influenza B viruses. Thus, an HA stem domain polypeptide, as indicated by binding of said one or more antibodies to said polypeptide,
It is another aspect of the invention to provide polypeptides stably presenting epitopes of antibodies CR6261 and/or CR9114. In one embodiment, the polypeptide does not bind to monoclonal antibodies CR8020 and CR8057 (described in WO2010/130636), which bind only to H3 influenza virus.

Influenza hemagglutinin stem domain polypeptides provided by the present invention comprise 1
It is suitable for use in immunogenic compositions (eg, vaccines) capable of generating an immune response against one or more influenza virus A and/or B strains, particularly influenza viruses of the H1 subtype. In one embodiment, the influenza hemagglutinin stem domain polypeptide is phylogenetic group 1 and/or group 2 influenza A.
It can generate an immune response against virus strains, particularly both phylogenetic group 1 and group 2 influenza virus strains. In one embodiment, the polypeptide is capable of generating an immune response against a homologous influenza virus strain. In one embodiment, the polypeptides are capable of generating an immune response against heterologous influenza virus strains of the same and or different subtypes. In a further embodiment, the polypeptide is capable of generating an immune response against both phylogenetic group 1 and group 2 influenza virus strains as well as influenza B virus strains.

Polypeptides according to the invention may be used for stand-alone therapy and/or prophylaxis and/or of diseases or conditions caused by, for example, influenza viruses, particularly phylogenetic group 1 or 2 influenza A viruses and/or influenza B viruses.
Or in diagnosis, or in combination with other prophylactic and/or therapeutic treatments, such as (existing or future) vaccines, antiviral agents and/or monoclonal antibodies.

In a further aspect, the invention provides nucleic acid molecules encoding influenza HA stem domain polypeptides. In yet another aspect, the invention provides a vector comprising a nucleic acid encoding an immunogenic polypeptide.

In a further aspect, the invention provides a method of inducing an immune response in a subject, comprising administering to the subject a polypeptide and/or nucleic acid molecule and/or vector according to the invention.

In a further aspect, the invention provides compositions comprising polypeptides and/or nucleic acid molecules and/or vectors according to the invention. The composition is preferably an immunogenic composition. The compositions provided herein can be in any form that allows administration of the composition to a subject, eg, a mouse, ferret or human. In specific embodiments, the composition is suitable for human administration. The polypeptides, nucleic acid molecules and compositions can be used in methods of preventing and/or treating influenza virus disease and/or for diagnostic purposes. A composition may further comprise a pharmaceutically acceptable carrier or excipient. In certain embodiments, the compositions described herein include or are administered in combination with an adjuvant.

In another aspect, the invention provides polypeptides, nucleic acids and/or vaccines for use as vaccines.
Or provide a vector. The present invention is particularly useful in diseases or conditions caused by influenza virus A subtypes and/or influenza B viruses of phylogenetic groups 1 and/or 2, in particular diseases or conditions caused by influenza viruses containing HA of the H1 subtype. Immunogenic polypeptides, nucleic acids and/or vectors for use as vaccines in prophylaxis and/or therapy.

Various embodiments and uses of polypeptides according to the invention will become apparent from the detailed description of the invention below.

A model of HA monomers in the pre-fusion state present in the native trimer is shown. HA1 is shown in light gray and HA2 is shown in dark gray. Helix A (important part of the epitope of CR6261) and helix CD (part of the trimer interface) are shown, with loops connecting their secondary structure elements. The C-terminus of HA1 and the N-terminus of HA2 are also indicated. The fusion peptide is localized at the N-terminus of HA2. Sandwich Elisa results obtained for supernatants of cultures expressing SEQ ID NOs: 65-71 and SEQ ID NOs: 76-78 disclosed in PCT/EP2014/060997. Capture and detection antibodies are indicated above the graph. mini-HA refers to the soluble form of SEQ ID NO:2 with the equivalent of residues 519-565 replaced by RSLVPRGSPGHHHHHH; FL-HA-FFH is the C-terminal Flag-thrombin-foldon-His sequence from position 520 (SEQ ID NO:4); FL-HA-7xHis refers to the soluble form of SEQ ID NO:1 containing the C-terminal sequence EGRHHHHHHH from position 530. Sandwich Elisa results obtained for supernatants of cultures expressing a polypeptide of the invention (t2 variant) containing the GCN4-derived sequence RMKQIEDKIEEIESK (SEQ ID NO: 20) at positions 419-433. Capture and detection antibodies are indicated above the graph. mini-HA-t2 was derived from mini-HA by introducing SEQ ID NO: 20 at positions 419-433; FL-HA-FFH, FL-HA-7xHis as above. Sandwich Elisa results obtained for supernatants of cultures expressing a polypeptide of the invention (t3 variant) containing the GCN4-derived sequence RMKQIEDKIEEIESKQK (SEQ ID NO:21) at positions 417-433. Capture and detection antibodies are indicated above the graph. mini-HA-t3 was derived from mini-HA by introducing SEQ ID NO: 21 at positions 417-433; FL-HA-FFH, FL-HA-7xHis as above. Elution profiles of s55G7-t2, s127H1-t2 and s86B4-t2 from a Superdex200 size exclusion column, the final step in the purification procedure. Numbered lines below the chromatogram indicate fractions collected during the elution process. SDS-PAGE and Western blot analysis of fractions collected during elution of Superdex200 size exclusion columns. Numbers correspond to fractions shown in FIG. An antibody recognizing the C-terminal his-tag was used for detection on Western blots. Size exclusion chromatography of s127H1-t2 in the presence and absence of Fab fragments of broadly neutralizing antibodies CR9114, CR6261, and CR8020 (Tosoh G2000 analytical column). The molecular weights of individual proteins and/or complexes were determined by multi-angle light scattering during elution from the column and are listed in Table 8. Binding of the polypeptide s127H1-t2 of the invention to monoclonal antibodies CR6261 and CR9114 using biolayer interferometry. Top panels show individual binding curves for immobilized monoclonal antibodies exposed to varying concentrations of s127H1-t2, bottom panels show steady-state analysis used to estimate _Kd . Survival (A), weight loss (B) and clinical scores (C) for negative (PBS, 3 immunizations at 3-week intervals) and positive control (15 mg/kg CR6261, 1 day prior to challenge) groups. Mice were challenged with a lethal dose (25×LD50) of H1N1A/Puerto Rico/8/34 4 weeks after the last immunization and monitored for 21 days. Error bars indicate 95% confidence intervals (B) or interquartile ranges (C). Survival (A), weight loss ( B) and clinical scores (C). Mice are challenged with a lethal dose (25×LD50) of H1N1A/Puerto Rico/8/34 4 weeks after the last immunization and monitored for 21 days or a negative control group (PBS) is also shown for comparison. Error bars indicate 95% confidence intervals (B) or interquartile ranges (C). Elisa results for negative control and experimental group sera using s127H1-t2 (A) or the soluble form of full-length HA (B) as antigen. Bars represent median values. Antibodies induced after immunization with the adjuvanted polypeptide s127H1-t2 of the invention can compete with CR9114 for binding to full-length HA from H1N1A/Brisbane/59/07 in a competition ELISA (A). For comparison, levels of competition by unlabeled CR9114 (ie, self-competition) and unconjugated monoclonal antibodies CR8020 and CR-JB (both serially diluted from a starting concentration of 5 μg/ml) are shown in separate graphs. Survival (A), relative weight loss (B) and clinical scores (C) for negative (PBS, 3 immunizations at 3-week intervals) and positive control (15 mg/kg CR6261, 1 day prior to challenge) groups . Mice were challenged with a lethal dose (25×LD50) of H1N1A/Puerto Rico/8/34 4 weeks after the last immunization and monitored for 21 days. Error bars indicate 95% confidence intervals (B) or interquartile ranges (C). Survival rates for groups immunized once (A), twice (B) or three times (C) with 30 μg of s127H1-t2-cl18 long in the presence of 10 μg of Matrix-M. Mice were challenged with a lethal dose (25×LD50) of H1N1A/Puerto Rico/8/34 4 weeks after the last immunization and monitored for 21 days. A negative control group (PBS) is also shown for comparison. Relative body weight change for groups immunized once (A), twice (B) or three times with 30 μg of s127H1-t2-cl18 long in the presence of 10 μg of Matrix-M. Mice were challenged with a lethal dose (25×LD50) of H1N1A/Puerto Rico/8/34 4 weeks after the last immunization and monitored for 21 days. A negative control group (PBS) is also shown for comparison. Error bars indicate 95% confidence intervals. Clinical scores for groups immunized once (A), twice (B) or three times with 30 μg of s127H1-t2-cl18long in the presence of 10 μg of Matrix-M. Mice were challenged with a lethal dose (25×LD50) of H1N1A/Puerto Rico/8/34 4 weeks after the last immunization and monitored for 21 days. A negative control group (PBS) is also shown for comparison. Error bars indicate interquartile range. ELISA results for negative control and experimental group pre-challenge sera (4 weeks after last immunization) using s127H1-t2-cl18long (A) or soluble form of full-length HA (B) as antigen. Bars represent median values. Antibodies induced after immunization with the Matrix-M adjuvanted polypeptide s127H1-t2-cl18long of the invention can compete with CR9114 for binding to full-length HA from H1N1A/Brisbane/59/07 in a competitive ELISA (A ). For comparison, the levels of competition by unlabeled CR9114 (ie, self-competing) and unconjugated monoclonal antibody CR8020 (both serially diluted from a starting concentration of 5 μg/ml) are shown in separate graphs (B). Bars represent median values. (A) Survival rates for negative (PBS, 3 immunizations at 3-week intervals) and positive control (15 mg/kg CR6261, 1 day prior to challenge) groups. Mice were challenged with a lethal dose (12.5×LD50) of H5N1A/Hong Kong/156/97 for 4 weeks after the last immunization. (B) Survival, (C) relative body weight change and (D) median clinical score for the group immunized three times with 30 μg of s127H1-t2 in the presence of 10 μg of Matrix-M. Error bars indicate 95% confidence intervals (C) or interquartile ranges (D). Mice were challenged with a lethal dose (12.5×LD50) of H5N1A/Hong Kong/156/97 for 4 weeks after the last immunization and monitored for 21 days. A negative control group (PBS) is also shown in B, C, D for comparison. immunized three times with the polypeptide s127H1-t2 of the invention described in Example 5 using full-length HA of several Group 1 (H1, H5 and H9) and Group II (H3 and H7) influenza strains as antigen. Elisa results for sera from isolated mice. The induced antibodies recognize all tested FL HA from Group 1. (A) Survival rates for negative (PBS, 3 immunizations at 3-week intervals) and positive control (15 mg/kg CR6261, 1 day prior to challenge) groups. Mice were challenged with a lethal dose (12.5×LD50) of H1N1A/Brisbane/59/2007 4 weeks after the last immunization. (B) Survival, (C) relative body weight change and (D) median clinical score for the group immunized three times with 30 μg of s127H1-t2 in the presence of 10 μg of Matrix-M. Error bars indicate 95% confidence intervals (C) or interquartile ranges (D). Mice were challenged with a lethal dose (12.5×LD50) of H1N1A/Brisbane/59/2007 4 weeks after the last immunization and monitored for 21 days. A negative control group (PBS) is also shown in B, C, D for comparison. Pseudoparticle neutralization assay using sera from mice immunized with polypeptide s127H1-t2 of the invention or PBS. Antibody-dependent cellular cytotoxicity (ADCC) surrogate assay. Sera from mice immunized with the polypeptide s127H1-t2 of the present invention had FL HA from H5N1A/Hong Kong/156/97 (A) or H1N1A/Brisbane/59/07 (B) as antigen sources. Shows a 30-40 fold induction of FcγRIV signaling activity at maximal serum concentrations using transfected target cells. Survival (A) and % body weight change (B) of mice after serum transfer and challenge with H5N1A/Hong Kong/156/97 as described in Example 9. Full length HA (H1N1A/Brisbane/59/2007) ELISA titers of donor mice (D) at day 70 and recipient mice (R) before serum transfer (day -4) or before challenge (day 0) . Data were analyzed using a weighted average approach based on slope. The open circle symbol indicates the measured value in LOD. Bars indicate median values. Survival (A) and % body weight change (B) of mice after immunization as described in Example 10 and challenge with H1N1A/NL/602/09. (A): Full-length HA (H1N1A/Brisbane/59/2007) ELISA titers of mice immunized as described in Example 10. Data were analyzed using a weighted average approach based on slope. The open circle symbol indicates the measured value in LOD. Bars indicate median values. (B): Serum IgG CR9114 competitive binding obtained after immunization of mice as described in Example 10. FL HA from H1N1A/Brisbane/59/2007 was used as antigen. Data shown are group medians, error bars indicate interquartile range. Data are shown for CR9114 and CR8020 starting from a 5 μg/ml solution and diluted in the same manner as the serum samples. Initial screen of a total of 10472 clones (5544 and 4928 from sets 1 and 2 respectively). Data are normalized to the mean FL HA binding and expression included in the experiment. The top 20% clones in the CR9114 sandwich assay (Panel A) that also showed expression of >50% of FL HA expression and those that showed a binding signal to CR6261 (Panel B) that was >80% of the signal observed for FL HA. Treated as hits; this procedure yielded 703 hits (596 and 107 from libraries 1 and 2, respectively). CR9114 sandwich Elisa results for polypeptides of the invention. (A) SEQ ID NOs: 158 to 162 containing all C-terminal Flag-foldon-his sequences (B) SEQ ID NOs: 163 to 166 containing all C-terminal TCS-his sequences. SEC MALS results for SEQ ID NO: 158 in the presence and absence of Fab fragments of CR9114 (designated as CRF9114) or CR6261 (designated as CRF6261). Molecular masses derived from multi-angle light scattering analysis are given in Example 12 and show the formation of complexes with three Fab fragments per trimer of the polypeptide of the invention. Survival (A) and % body weight change (B) of mice after immunization as described in Example 13 and challenge with H1N1A/Brisbane/59/07. (A): Full-length HA (H1N1A/Brisbane/59/2007) ELISA titers of mice immunized as described in Example 13. Data were analyzed using a weighted average approach based on slope. The open circle symbol indicates the measured value in LOD. Bars indicate median values. (B): Serum IgG CR9114 competitive binding obtained after immunization of mice as described in Example 18. FL HA from H1N1A/Brisbane/59/2007 was used as antigen. Data shown are group medians, error bars indicate interquartile range. Levels are shown for CR9114 and CR8020 starting from a 5 μg/ml solution and diluted in the same manner as the serum samples. Survival (A) and % body weight change (B) of mice after immunization as described in Example 14 and challenge with H5N1A/Hon Kong/156/97. (A): Full-length HA (H1N1A/Brisbane/59/2007) ELISA titers of mice immunized as described in Example 14. Data were analyzed using a weighted average approach based on slope. The open circle symbol indicates the measured value in LOD. Bars indicate median values. (B): Serum IgG CR9114 competitive binding obtained after immunization of mice as described in Example 18. FL HA from H1N1A/Brisbane/59/2007 was used as antigen. Data shown are group medians, error bars indicate interquartile range. Levels are shown for CR9114 and CR8020 starting from a 5 μg/ml solution and diluted in the same manner as the serum samples. Survival (A) and % body weight change (B) of mice after immunization as described in Example 15 and challenge with H1N1A/Puerto Rico/8/1934. (A): Full-length HA (H1N1A/Brisbane/59/2007) ELISA titers of mice immunized as described in Example 15. Data were analyzed using a weighted average approach based on slope. The open circle symbol indicates the measured value in LOD. Bars indicate median values. (B): Serum IgG CR9114 competitive binding obtained after immunization of mice as described in Example 18. FL HA from H1N1A/Brisbane/59/2007 was used as antigen. Data shown are group medians, error bars indicate interquartile range. Levels are shown for CR9114 and CR8020 starting from a 5 μg/ml solution and diluted in the same manner as the serum samples.

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

Amino acids according to the invention are the 20 naturally occurring (or "standard" amino acids) or variants thereof, such as D-proline (the D-enantiomer of proline), or any variant not naturally found in proteins, such as norleucine. can be either 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, for example cysteine can form covalent disulfide bonds (or disulfide bridges) to other cysteine residues, and proline forms a periodicity to the polypeptide backbone. However, glycine is more flexible than other amino acids. Table 5 shows standard amino acid abbreviations and properties.

The term "amino acid sequence identity" refers to the degree of identity or similarity between a pair of aligned amino acid sequences, usually expressed as a percentage. Percent identity may be either identical (i.e., amino acid residues at a given position in an alignment are identical residues) or similar (i.e., amino acid substitutions at a given position in an alignment are conservative, as discussed below). substitution) to the corresponding amino acid residue in the peptide after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent sequence homology be. Sequence homology, including percent sequence identity and similarity, is determined using sequence alignment techniques well known in the art, such as by visual inspection and mathematical calculations, or, more preferably, the comparison is computer-assisted. This is done by using a program to compare the sequence information. An exemplary preferred computer program is Gen
etics Computer Group (GCG; Madison, Wis.) Wi
The sconsin package version 10.0 program, "GAP" (Devereu
x et al. (1984)).

A "conservative substitution" refers to the replacement of one class of amino acid by another amino acid of the same class. In certain embodiments, conservative substitutions do not alter the structure or function, or both, of the polypeptides of the invention. Classes of amino acids for purposes of conservative substitution include hydrophobic (e.g. Met, Ala, Val, Leu), neutral hydrophilic (e.g. Cys, Se
r, Thr), acidic (e.g. Asp, Glu), basic (e.g. Asn, Gln, H
is, Lys, Arg), conformational disruptors (e.g. Gly, Pro) and aromatics (
For example, Trp, Tyr, Phe).

As used herein, the terms "disease" and "disorder" are used interchangeably to refer to a condition in a subject. In some embodiments, the condition is viral infection, particularly influenza virus infection. In specific embodiments, the term "disease" refers to a pathological condition resulting from the presence of a virus in a cell or subject or due to invasion of a cell or subject by a virus. In certain embodiments, the condition is a disease in a subject whose severity is reduced by inducing an immune response in the subject via administration of an immunogenic composition.

The term "effective amount" as used herein in the context of administering a therapeutic to a subject is
It refers to the amount of therapeutic that has a prophylactic and/or therapeutic effect. In certain embodiments, an "effective amount" in the context of administering a therapeutic to a subject is a reduction or amelioration of the severity of influenza virus infection, disease or symptoms associated therewith, such as, but not limited to, influenza virus infection. , reducing the duration of disease or symptoms associated therewith, preventing the progression of influenza virus infection and disease or symptoms associated therewith, preventing the onset or onset or recurrence of influenza virus infection and disease or symptoms associated therewith, from one subject to another prevention or reduction of the spread of influenza virus to, hospitalization of subjects and/or
or reduce length of hospital stay, increase survival in subjects with influenza virus infection or disease associated therewith, eliminate influenza virus infection or disease associated therewith, inhibit or reduce influenza virus replication, reduce influenza virus titer; and/or Refers to an amount of a therapeutic sufficient to achieve prophylactic or therapeutic enhancement and/or amelioration of another therapeutic. In certain embodiments, an effective amount does not provide complete protection from influenza virus disease, but does provide lower titers or reduced numbers of influenza viruses compared to untreated subjects. Benefits of reducing the titer, number or total load of influenza virus include, but are not limited to, milder symptoms of infection, fewer symptoms of infection and reduced duration of illness associated with infection.

As used herein, the term "host" shall refer to an organism or cell into which a vector, eg, a cloning vector or an expression vector, has been introduced. Organisms or cells can be prokaryotic or eukaryotic. Preferably, the host comprises isolated host cells, eg, host cells in culture. The term "host cell" merely means that the cell has been modified for the (over)expression of the polypeptide of the invention. It should be understood that the term host is intended to refer not only to the particular subject organism or cell, but to the progeny of such organism or cell as well. Such progeny may not be virtually identical to the parent organism or cell, as certain modifications may occur in subsequent generations, either due to mutation or environmental influences, although they are still used herein. are included within the scope of the term "host".

As used herein, the term "including" or "as an example" is considered to be followed by the term "but not limited to".

As used herein, the term "infection" means invasion by the propagation and/or presence of a virus in a cell or subject. In one embodiment, the infection is an "active" infection, ie, the virus is replicating in the cell or subject. Such infection can spread from cells, tissues, and/or organs originally infected by the virus to other cells,
It is characterized by spread of the virus to tissues and/or organs. The infection can also be latent, ie the virus is not replicating. In certain embodiments, infection results from the presence of a virus in a cell or subject or refers to a pathological condition due to invasion of a cell or subject by a virus.

Influenza viruses are classified into influenza virus types: A, B and C genera. As used herein, the term "influenza virus subtype" refers to influenza A virus variants characterized by a combination of hemagglutinin (H) and neuraminidase (N) viral surface proteins. According to the present invention, influenza virus subtypes are defined by their H number, e.g.
H3 subtype influenza virus” or “H3 influenza”, etc., or a combination of H and N numbers, e.g., “influenza virus subtype H3N2” or “H
3N2” and the like. The term "subtype" specifically includes all individual "strains" within each subtype that usually arise from mutations and exhibit different pathogenic profiles, e.g., natural isolates and man-made mutations. Body or reassortants, etc. are included. Such strains may also be referred to as "isolates" of various viral subtypes. As such, the terms "strain" and "isolate" as used herein can be used interchangeably.
Current nomenclature for human influenza virus strains or isolates includes the virus type (genus), i.e., A, B or C, geographic location of first isolation, strain number and year of isolation, usually listed in parentheses. A/Mosco
w/10/00 (H3N2). Non-human strains also include the host of origin in the nomenclature. Influenza A virus subtypes can be further classified by reference to their phylogenetic group. Phylogenetic analysis demonstrated subdivision of hemagglutinins into two major groups: H1, H2, H5 and H9 subtypes (“group 1” influenza viruses) in phylogenetic group 1 among others and H3 in phylogenetic group 2 among others, H4, H7 and H10 subtypes ("group 2" influenza viruses").

As used herein, the term "influenza virus disease" refers to a pathological condition resulting from the presence of influenza virus, eg, influenza A or B virus, in a cell or subject or the invasion of a cell or subject by influenza virus. In specific embodiments, the term refers to respiratory illness caused by influenza virus.

The term "nucleic acid" as used herein includes DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of DNA or RNA produced using nucleotide analogs. shall be Nucleic acids can be single-stranded or double-stranded. Nucleic acid molecules can be chemically or biochemically modified, or can contain non-natural or derivatized nucleotide bases, which are readily recognized by those of ordinary skill in the art. Such modifications include, for example, labeling, methylation, substitution of one or more of the natural nucleotides by analogs, internucleotide modifications, such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.). ), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalating agents (e.g., acridine, psoralen, etc.), chelating agents, alkylating agents, and modified linkages (e.g., alpha anomeric nucleic acids, etc.). A reference to a nucleic acid sequence includes its complement unless otherwise specified. Thus, reference to a nucleic acid molecule with a particular sequence should be understood to encompass its complementary strand with its complementary sequence. Complementary strands are also useful, for example, in antisense therapy, hybridization probes and PCR primers.

In certain embodiments, the numbering of amino acids in HA as used herein is
Amino acid numbering in HA0 of wild-type influenza virus, eg, based on the amino acid numbering of H1N1 influenza strain A/Brisbane/59/2007 (SEQ ID NO: 1). Thus, the term "amino acid at position 'x' in HA" as used in the present invention refers to specific wild-type influenza viruses, such as A/Brisbane/59.
/2007 (SEQ ID NO: 1; amino acids of HA2 domain shown in italics) corresponding to the amino acid at position x in HA0. It will be appreciated by those skilled in the art that equivalent amino acids in other influenza virus strains and/or subtypes can be determined by multiple sequence alignments. Note that in the numbering system used throughout this application, 1 refers to the N-terminal amino acid of the immature HA0 protein (SEQ ID NO: 1). The mature sequence, for example, starts above position 18 of SEQ ID NO:1. A leader sequence (or signal sequence) that directs protein trafficking during production (e.g. amino acid 1 of SEQ ID NO: 1)
17) will generally not be present in the final polypeptide used, for example, in a vaccine. Thus, in certain embodiments, a polypeptide according to the invention comprises an amino acid sequence without a leader sequence, ie the amino acid sequence is based on the amino acid sequence of HA0 without a signal sequence.

A "polypeptide" refers to a polymer of amino acids joined by amide bonds as known by those skilled in the art. As used herein, the term can refer to a single polypeptide chain linked by covalent amide bonds. The term applies to non-covalent interactions, e.g. ionic contact,
It can also refer to multiple polypeptide chains that are associated by hydrogen bonds, van der Waals contacts and hydrophobic contacts. The skilled artisan recognizes that the term includes, for example, post-translational processing, such as signal peptide cleavage, disulfide bond formation, glycosylation (eg, N-linked and O-linked glycosylation), protease cleavage and lipid modification (eg, S-palmitoyl). It recognizes that polypeptides modified by modification) are included.

A "stem domain polypeptide" refers to a polypeptide comprising one or more polypeptide chains that make up the stem domain of native (or wild-type) hemagglutinin (HA). Typically, a stem domain polypeptide comprises a single polypeptide chain (i.e., corresponding to the stem domain of the hemagglutinin HA0 polypeptide) or two polypeptide chains (i.e.,
(corresponding to the stem domain of the hemagglutinin HA1 polypeptide associated with the hemagglutinin HA2 polypeptide). According to the present invention, the stem domain polypeptide comprises one or more mutations compared to a wild-type HA molecule, in particular one or more amino acid residues of wild-type HA have corresponding counterparts in a particular wild-type HA. Positions may be substituted with other non-naturally occurring amino acids. Stem domain polypeptides according to the invention may further comprise one or more of the binding sequences described below.

The term "vector" refers to a nucleic acid molecule capable of inserting a second nucleic acid molecule for introduction into a host, which is replicated and, in some cases, expressed. In other words, a vector can transport a nucleic acid molecule to which it has been linked. Cloning and expression vectors are contemplated by the term "vector" as used herein. Vectors include, but are not limited to, plasmids, cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC)
Also included are vectors derived from bacteriophages or plant or animal (eg, human) viruses. Vectors include an origin of replication recognized by the host to which it is presented and, in the case of expression vectors, promoters and other regulatory regions recognized by the host. Certain vectors are capable of autonomous replication in a host into which they are introduced (eg, vectors having a bacterial origin of replication are capable of replication in bacteria). Other vectors can integrate into the host's genome upon introduction into the host, causing the vector to replicate along the host genome.

As used herein, the term "wild-type" in the context of viruses refers to influenza viruses that circulate in high frequency, naturally circulate, and cause typical disease outbreaks.

DETAILED DESCRIPTION Influenza viruses have a significant impact on global public health, causing millions of cases of severe illness, thousands of deaths, and significant economic losses each year. Current trivalent influenza vaccines elicit potent neutralizing antibody responses against vaccine strains and closely related isolates, but rarely extend to more divergent strains within one subtype or other subtypes. Furthermore, selection of an appropriate vaccine strain presents many difficulties and frequently results in suboptimal protection. Moreover, prediction of the next epidemic virus subtype, including when and where it will occur, is currently not possible.

Hemagglutinin (HA) is the major envelope glycoprotein from influenza A virus that is the primary target of neutralizing antibodies. Hemagglutinin is 2-fold during the influx process.
It has two main functions. First, hemagglutinin mediates viral attachment to the surface of target cells through interaction with sialic acid receptors. Second, after viral endocytosis, hemagglutinin subsequently effects fusion of the viral and endosomal membranes to release its genome into the cytoplasm of the target cell. HA contains a large ectodomain of approximately 500 amino acids that is cleaved by host-derived enzymes to produce two polypeptides that remain linked by a disulfide bond. The majority of the N-terminal fragment (HA1, 320-330 amino acids) forms the membrane distal globular domain containing the receptor binding site and most antigenic determinants recognized by virus-neutralizing antibodies. The smaller C-terminal portion (HA2, approximately 180 amino acids) forms a stem-like structure that anchors the globular domain to the cell or viral membrane. The degree of sequence homology between subtypes is that the HA1 polypeptide (34%-59% homology between subtypes) is H
It is smaller than the A2 polypeptide (51-80% homology). The most conserved region is the sequence surrounding the cleavage site, especially the HA2 N-terminal 23 amino acids, which is the same in all influenza A
It is conserved among virus subtypes (Lorieau et al., 2010). Part of this region is exposed as a surface loop in the HA precursor molecule (HA0) but becomes inaccessible when HA0 is cleaved into HA1 and HA2.

Most neutralizing antibodies bind to loops surrounding the receptor binding site, preventing receptor binding and attachment. Because these loops are highly variable, most antibodies targeting those regions are strain-specific, explaining why current vaccines induce such limited strain-specific immunity. explain. Recently, however, fully human monoclonal antibodies against influenza virus hemagglutinin with broad cross-neutralizing potency have been generated. Functional and structural analyzes revealed that these antibodies interfered with the membrane fusion process and were directed against highly conserved epitopes in the stem domain of the influenza HA protein (Throsby et al., 2008; Ekiert et al.2009
, International Publication No. 2008/028946, International Publication No. 2010/13063
6 pamphlet, International Publication No. 2013/007770 pamphlet).

Stem domain polypeptides stably presenting epitopes of these antibodies are described in co-pending patent application PCT/EP2012/073706. At least a portion of the stem domain polypeptides described herein are CR6261 and/or C
It stably presents the R9114 epitope and is immunogenic in mice. CR626
Additional immunogenic stem domain polypeptides stably presenting epitopes of CR.1 and/or CR9114 are described in co-pending patent application PCT/EP2014/060997.

According to the invention, new HA stem domain polypeptides were designed that display these epitopes. These polypeptides can be used to create universal epitope-based vaccines that induce protection against a wide range of influenza strains. As in previously described stem domain polypeptides, the highly variable immunodominant portion, ie the head domain, is first removed from the full-length HA molecule to create a stem domain polypeptide, also called mini-HA. It redirects the immune response to the stem domain where epitopes for broadly neutralizing antibodies are located. The broadly neutralizing antibodies described above are used to examine the correct folding of the newly created molecule and to confirm the presence of neutralizing epitopes.

The novel stem domain polypeptides of the present invention are derived from previously described stem polypeptides (PCT/EP2012/073706 and PCT/EP2014/0609).
97), binding of antibodies, particularly CR6261 and/or CR9
114 shows increased binding and/or increased propensity to multimerize and increased stability.

The stem domain polypeptides of the invention are capable of presenting conserved epitopes of the membrane-proximal stem domain HA molecule to the immune system in the absence of dominant epitopes present in the membrane-distal head domain. For this purpose, a portion of the primary sequence of the HA0 protein that makes up the head domain is removed and replaced either directly or, in some embodiments, by introducing a short flexible binding sequence (“linker”). The ligation restores the continuity of the polypeptide chain. The resulting polypeptide sequence is further modified by introducing defined mutations that stabilize the native three-dimensional structure of the remaining portion of the HA0 molecule.

The present invention is particularly directed to influenza hemagglutinin stem domain polypeptides comprising
(a) an influenza hemagglutinin HA1 domain comprising a HA1 N-terminal stem segment covalently linked to the HA1 C-terminal stem segment by a linking sequence of 0-50 amino acid residues, said HA1 C-terminal segment comprising:
(b) bound to the influenza hemagglutinin HA2 domain, HA1 and HA
2 domains are derived from an influenza A virus subtype containing HA of the H1 subtype;
(c) does not contain a protease cleavage site at the junction between the HA1 and HA2 domains;
(d) said HA1 N-terminal segment comprises amino acids 1-x of HA1, preferably amino acids p-x of HA1, and the HA1 C-terminal stem segment comprises amino acids y-C-terminal amino acids of HA1, x=SEQ ID NO: 1 p = the amino acid on position 18 of SEQ ID NO: 1 (or the corresponding position in the hemagglutinin of another influenza virus strain), y = amino acid on position 321 of SEQ ID NO: 1 (or the equivalent position in another hemagglutinin);
(e) the region containing amino acid residues 402-418 is amino acid X ₁ NTQX ₂ TAX ₃ GK
_EX4N (H/K) _X5E (K/R) (SEQ ID NO: 8) comprising:
X ₁ is an amino acid selected from the group consisting of M, E, K, V, R and T;
X2 is an amino acid selected from the group consisting of F, _I , N, T, H, L and Y, preferably I, L or Y;
X3 is an amino acid selected from the group consisting of V _, A, G, I, R, F and S, preferably A, I or F;
_X4 is an amino acid selected from the group consisting of F, I, N, S, T, Y, E, K, M and V, preferably I, Y, M or V;
X ₅ is an amino acid selected from the group consisting of L, H, I, N, R, preferably I;
(f) Amino acid residues on position 337 (HA1 domain) are I, E, K, V, A, and T
is selected from the group consisting of
Amino acid residues on position 340 (HA1 domain) are I, K, R, T, F, N, S and Y
is selected from the group consisting of
the amino acid residue on position 352 (HA2 domain) is selected from the group consisting of D, V, Y, A, I, N, S, and T;
the amino acid residue on position 353 (HA2 domain) is selected from the group consisting of K, R, T, E, G, and V;
(g) further comprising a disulfide bridge between the amino acid on position 324 and the amino acid on position 436;
(h) the amino acid sequence RMKQIEDKIEEIESK (SEQ ID NO: 20) from 419 to 433
or the sequence RMKQIEDKIEEIESKQK (SEQ ID NO:21) is introduced at positions 417-433.

Accordingly, the present invention provides stable hemagglutinin stem domain polypeptides that mimic the three-dimensional conformation of the stem of the native hemagglutinin molecule.

Polypeptides of the invention do not include a full-length HA1 domain.

The polypeptide is therefore substantially smaller than HA0 and preferably lacks all or substantially all of the globular head of HA. Preferably, the immunogenic polypeptide is 360
Amino acid length or less, preferably 350, 340, 330, 320, 310, 305, 30
0, 295, 290, 285, 280, 275, or 270 amino acids in length or less. In certain embodiments, the immunogenic polypeptide is from about 250 to about 350, preferably from about 260 to about 340, preferably from about 270 to about 330, preferably from about 270 to about 330 amino acids long.

According to the present invention, the "HA1 N-terminal segment" is influenza hemagglutinin (H
A) Refers to the polypeptide segment corresponding to the amino-terminal portion of the HA1 domain of the molecule.
The HA1 N-terminal polypeptide segment includes amino acids 1 through x of the HA1 domain, where the amino acid above position x is an amino acid residue within HA1. The term "HA1 C-terminal segment" refers to the polypeptide segment corresponding to the carboxy-terminal portion of the influenza hemagglutinin HA1 domain. The HA1 C-terminal polypeptide segment includes amino acids from position y to and including the C-terminal amino acid of the HA1 domain, where the amino acid on position y is an amino acid residue within HA1. According to the invention, y is greater than x, thus between the HA1 N-terminal segment and the HA1 C-terminal segment, i.e. x
A segment of the HA1 domain between the amino acid on position and the amino acid on position y is deleted and, in some embodiments, replaced by a binding sequence. Thus, in certain embodiments, the deletion of the HA1 segment comprises the amino acid sequence from the amino acid at position x+1 to and including the amino acid at position y-1.

In some embodiments, the polypeptide does not contain a signal sequence. Thus, in certain embodiments, the HA1 N-terminal segment comprises amino acids px of HA1, where p is
The first amino acid of the mature HA molecule (eg p=18 for SEQ ID NO:1). One skilled in the art can determine the equivalent amino acids in other hemagglutinins and the signal peptide (eg, amino acids 1-17 of SEQ ID NO: 1 or in other H1 influenza virus strains (see, eg, Table 2) to position x of the HA1 domain). Polypeptides described herein can be prepared that do not have equivalent positions.

According to the invention, the HA1 N-terminal segment comprises amino acids 1 to x, preferably p to x, of the HA1 domain, with x=52, p=18 in SEQ ID NO: 1, or H
The corresponding amino acid positions in the other HA sequences of subtype 1.

According to the present invention, the HA1 C-terminal polypeptide segment comprises amino acids from position y to and including the C-terminal amino acids of the H1HA1 domain, where y is 321 or H1
Corresponding amino acid positions in other HA sequences of the subtype.

Thus, according to the present invention, the HA1 N-terminal stem segment comprises amino acid residues 1 to 52 of HA1, preferably amino acid residues 18 to 52 of HA1, and the HA1 C-terminal stem segment comprises amino acid residues 321 to 321 of HA1. 343 included. In some embodiments, H
The A1 N-terminal stem segment consists of amino acid residues 1-52 of HA1, preferably amino acid residues 18-52 of HA1, and the HA1 C-terminal stem segment consists of amino acid residues 321-343 of HA1.

According to the invention, the polypeptide does not contain a protease cleavage site at the junction between the HA1 and HA2 domains. Thus, hemagglutinin stem domain polypeptides are resistant to protease cleavage at the junction between HA1 and HA2. The Arg(R) to Gly(G) sequence spanning HA1 and HA2 (ie, amino acid positions 343 and 344 of SEQ ID NO: 1) is the recognition site for trypsin and trypsin-like proteases and is typically responsible for hemagglutinin activation. is known to those skilled in the art to be cleaved for Since the HA stem domain polypeptides described herein should not be activated, the influenza hemagglutinin stem domain polypeptides of the invention are resistant to protease cleavage. Thus, according to the invention, the protease cleavage sites are HA1 and H
It has been removed to prevent cleavage of the polypeptide at the cleavage site between the A2 domains. In certain embodiments, the protease cleavage site is modified by mutation of the C-terminal amino acids of the C-terminal HA1 segment and/or HA2 to obtain a sequence that is resistant to protease cleavage.
It has been removed by mutation of the N-terminal amino acids of the domain. In certain embodiments, removal of the cleavage site between HA1 and HA2 in certain embodiments can be achieved by an R (and in a few cases K) to Q mutation at the P1 position (e.g., cleavage site (sequence No. 1, position 343), see Sun et al, 2010
reference). Thus, in certain embodiments, the C-terminal amino acid residue of the HA1 C-terminal stem segment is any amino acid other than arginine (R) or lysine (K). In certain embodiments, the HA1 C-terminal amino acid is glutamine (Q), serine (S), threonine (T), asparagine (N), aspartic acid (D) or glutamic acid (E). In certain embodiments, the C-terminal amino acid residue of the HA1 C-terminal stem segment is glutamine (Q).

According to the present invention, the polypeptide is derived from or based on H1 HA, ie HA comprising amino acid sequences from an influenza virus of the H1 subtype. In certain embodiments, the polypeptide is, for example, influenza virus A/Brisb
ane/59/2007 (H1N1) (SEQ ID NO: 1) from or based on influenza A virus HA, including H1 subtype HA. It will also be appreciated by those skilled in the art that other influenza A viruses, including H1 subtype HA, can be used in accordance with the present invention. In certain embodiments, the polypeptide comprises a hemagglutinin stem domain derived from or based on HA of an influenza AH1 virus selected from Table 2. "Derived from" or "based on" means an H1 subtype of natural influenza in which the N-terminal and/or C-terminal segments of the HA1 and/or HA2 domains are known or later discovered by those skilled in the art corresponding N-terminal and/or HA1 and/or HA2 domains of hemagglutinin
or C-terminal segment and at least 70%, 75%, 80%, 85%, 90%, 95%
, having 96%, 97%, 98%, or 99% amino acid sequence identity.

According to the present invention, the HA2 domain connects the C-terminal residue of helix A to the N of helix CD.
Including one or more mutations in the HA2 amino acid sequence that connect to the terminal residues (Figure 1). The H1HA2 amino acid sequence linking the C-terminal residues of helix A and the N-terminal residues of helix CD comprises an amino acid sequence comprising residues 402-418 of influenza HA (SEQ ID NO: 1
numbering according to ), the amino acid sequence MNTQFTAVGKEFN(H/K)LE(K/R)
(SEQ ID NO: 7).

In one embodiment, the amino acid sequence linking the C-terminal residue of helix A to the N-terminal residue of helix CD, ie, amino acid residue 402 of influenza HA of serotype H1
-418 (numbered according to SEQ ID NO: 1) has the amino acid sequence X ₁ NTQX ₂ TA
_{X3GKEX4N (} H/K) _X5E (K/R) (SEQ ID NO: 8).

According to the invention, one or more of the amino acids on positions 402, 406, 409, 413 and 416 (numbering refers to SEQ ID NO: 1), namely amino acids X ₁ , X ₂ , X ₃ , X ₄ and One or more of _X5 are mutated, ie, contain amino acids that do not occur at those positions in the wild-type influenza virus on which the stem polypeptide is based.

_In certain embodiments, the mutated amino acid above position 402, i.e., X1 is M, E,
An amino acid selected from the group consisting of K, V, R and T.

In certain embodiments, the mutated amino acid above position 406, i.e., X2 is F, _I ,
N, T, H, L and Y, preferably an amino acid selected from the group consisting of I, L or Y.

In certain embodiments, the mutated amino acid above position 409, i.e. _, X3 is V, A,
G, I, R, F and S, preferably amino acids selected from the group consisting of A, I or F.

In certain embodiments, the mutated amino acid on position 413, i.e., _X4 is F, I,
N, S, T, Y, E, K, M and V, preferably an amino acid selected from the group consisting of I, Y, M or V;

In certain embodiments, the mutated amino acid on position 416, i.e., _X5 is L, H,
I, N, R, preferably an amino acid selected from the group consisting of I;

Combinations of these mutations are also possible.

In certain embodiments, X ₁ is M, X ₂ is Y, X ₃ is I, X ₄ is Y
and X ₅ is S.

According to the invention, the stem polypeptide is the corresponding wild-type influenza virus HA
1 and/or HA2 domains, ie, one or more additional mutations, ie, amino acid substitutions, in the HA1 and/or HA2 domains compared to the amino acid sequence of the influenza virus on which the stem polypeptide is based.

In certain embodiments, one or more amino acid residues near the HA0 cleavage site (residue 343 of SEQ ID NO:1) are mutated. In one embodiment, 337, 340 of SEQ ID NO: 1
, 352, or 353 or the corresponding position in other influenza viruses is mutated, i.e., in the amino acid sequence of wild-type influenza virus HA on which the stem polypeptide is based. is substituted by an amino acid that does not occur at the corresponding position of Table 6 shows natural amino acid mutations.

In certain embodiments, the polypeptides of the invention comprise at least one mutation above position 352 of SEQ ID NO: 1, or the corresponding position of other influenza viruses.

In certain embodiments, the polypeptides of the invention comprise at least one mutation above position 353 of SEQ ID NO: 1, or the corresponding position of other influenza viruses.

In certain embodiments, the polypeptides of the invention comprise at least one mutation above position 337 of SEQ ID NO: 1, or the corresponding position of other influenza viruses.

In certain embodiments, the polypeptides of the invention comprise at least one mutation above position 340 of SEQ ID NO: 1, or the corresponding position of other influenza viruses.

In certain embodiments, the mutated amino acid residue on position 337 (HA1 domain) is
, E, K, V, A, and T.

In certain embodiments, the mutated amino acid residue on position 340 (HA1 domain) is
, K, R, T, F, N, S and Y.

In certain embodiments, the mutated amino acid residue on position 352 (HA2 domain) is D
, V, Y, A, I, N, S, and T.

In certain embodiments, the mutated amino acid residue on position 353 (HA2 domain) is K
, R, T, E, G, and V.

In certain embodiments, the mutated amino acid is a consensus N-glycosylation in the sequence, eg, I340N of SEQ ID NO: 6, eg, NXT/S, where X is any naturally occurring is an amino acid).

In certain embodiments, the mutated amino acid is an amino acid that does not occur naturally in the same subtype sequence.

In certain embodiments, the mutated amino acid residue on position 337 (HA1 domain) is K
is.

In certain embodiments, the mutated amino acid residue on position 340 (HA1 domain) is K
is.

In certain embodiments, the mutated amino acid residue on position 352 (HA2 domain) is F
is.

In certain embodiments, the mutated amino acid residue on position 353 (HA2 domain) is T
is.

It is also noted here that throughout this application the numbering of amino acids is based on the numbering of amino acids in H1HA0, in particular that of H1N1 influenza strain A/Brisbane/59/2007 (SEQ ID NO: 1). be. One skilled in the art can determine the equivalent (or corresponding) amino acids in the HA of other influenza viruses, and thus the equivalent mutations, e.g., for sequence alignments of different H1 influenza viruses. See Table 2.

According to the invention, the polypeptide further comprises a disulfide bridge between the amino acid on position 324 and the amino acid on position 436. Thus, according to the invention, at least one disulfide bridge is in the stem domain polypeptide, preferably H1A/Brisba
It was introduced between amino acids at positions 324 and 436 (or its equivalent) in ne/59/2007 (SEQ ID NO: 1). Thus, in certain embodiments, the polypeptide is H
It further includes mutations R324C in the A1 domain and T436C in the HA2 domain. Corresponding positions can be readily determined by those skilled in the art by aligning sequences using suitable algorithms such as Clustal, Muscle, and the like. The engineered disulfide bridge is at least one (where the other is already a cysteine), but usually
Two residues that are spatially close are mutated to cysteine, which is created by forming a covalent bond between the sulfur atoms of those residues either spontaneously or by reactive oxidation.

In certain embodiments, the polypeptide is the HA domain from which the HA1 and HA2 domains are derived.
further comprises one or more additional mutations in the HA1 and/or HA2 domains compared to the amino acid sequence of . Therefore, the stability of the stem polypeptide is further increased.

Applicants have found that some are specific for group 1 influenza viruses (e.g. CR6261 described in WO2008/028946) and some are specific for group 2 influenza viruses (e.g. WO2010 /130
We have previously identified broadly neutralizing antibodies isolated from primary human B cells from vaccinated individuals who were CR8020 as described in 636). Detailed epitope analysis of these monoclonal antibodies revealed the reason for the lack of cross-reactivity of their specific antibodies. In both cases, the presence of glycans in group 1 or group 2 HA molecules on different locations at least partially explained the fact that the antibodies were group-specific. Identification of a CR9114-like antibody that cross-reacts with a number of group 1 and 2 HA molecules described below revealed that the human immune system is capable of eliciting highly broadly neutralizing antibodies against influenza virus. However, given the need for annual vaccination schemes, those antibodies of subtype H1 and/or H3 (seasonal)
It is not apparently induced or induced only to a very low degree after infection with influenza virus or after vaccination.

According to the present invention, cross-neutralizing antibodies that mimic specific epitopes of CR6261 and/or CR9114, e.g., when administered in vivo alone or in combination with other prophylactic and/or therapeutic treatments. Polypeptides are provided that can be used as immunogenic polypeptides to provoke. A "cross-neutralizing antibody" comprises at least 2, preferably at least 3, 4 or 5 different subtypes of influenza A virus of phylogenetic group 1 and/or at least 2, preferably at least 3 one, four
one or five different subtypes of influenza A viruses of phylogenetic group 2 and/or at least two different subtypes of influenza B viruses, in particular CR626
1 and CR9114 neutralize at least all strains of the virus.

Polypeptides of the invention comprise epitopes of stem-linked influenza neutralizing antibodies CR6261 and/or CR9114. Thus, in certain embodiments, the polypeptide selectively binds to antibodies CR6261 and/or CR9114. In certain embodiments, a polypeptide of the invention does not bind to antibodies CR8020 and/or CR8057. CR6261 used in the present invention comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:9 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:10; CR911
4 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:11 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:12. CR8057 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:14. CR8020 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:17 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:18.

As described above, the polypeptide comprises an influenza hemagglutinin HA1 domain comprising a HA1 N-terminal stem segment covalently linked to the HA1 C-terminal stem segment by a linking sequence of 0-50 amino acid residues. Binding sequences, if present, do not occur in native or wild-type HA. In certain embodiments, the linker is 1 amino acid residue, no more than 2 amino acid residues, no more than 3 amino acid residues, no more than 4 amino acid residues, 5
1 or fewer amino acid residues, 10 or fewer amino acid residues, 15 or fewer amino acid residues, or 20
A peptide comprising the following amino acid residues, or 30 or fewer amino acid residues, or 40 or fewer amino acid residues, or 50 or fewer amino acid residues. In specific embodiments, the binding sequence is G, GS, GGG, GSG, GSA, GSGS, GSAG, GGGG, GSAGS, G
A sequence selected from the group consisting of SGSG, GSAGSA, GSAGSAG, and GSGSGSG.

In certain embodiments, the HA1 N-terminal segment is directly linked to the HA1 C-terminal segment, ie, the polypeptide does not contain a linking sequence.

Influenza HA in its native form exists as a trimer on cell or viral membranes.
In certain embodiments, intracellular and transmembrane sequences are removed such that a secreted (soluble) polypeptide is produced following expression in the cell. Methods for expressing and purifying the secretory ectodomain of HA have been described (e.g. Dopheide et al 2009;
Ekiert et al 2009, 2011; Stevens et al 200
4, 2006; Wilson et al 1981). Those skilled in the art will appreciate that these methods can also be applied directly to the stem domain polypeptides of the invention to achieve expression of secreted (soluble) polypeptides. Therefore, these polypeptides are also included in the present invention.

In certain embodiments, the polypeptide comprises a full-length HA2 domain and thus includes transmembrane and intracellular sequences. In other embodiments, the polypeptides of the invention do not contain the intracellular sequences and transmembrane domains of HA. In certain embodiments, the polypeptide is
Contains a truncated HA2 domain. In certain embodiments, intracellular and transmembrane sequences,
For example, 514, 515, 516, 517, 518, 519, 520 of the HA2 domain,
521, 522, 523, 524, 525, 526, 527, 526, 528, 529,
Alternatively, the amino acid sequence from position 530 (or its equivalent) to the C-terminus of the HA2 domain (numbered according to SEQ ID NO: 1) has been removed so that after expression in cells a soluble polypeptide is produced.

In certain embodiments, the C-terminal portion of the HA2 domain from position 519 to the C-terminal amino acids is deleted. In a further embodiment, the C-terminal portion of the HA2 domain from position 530 to C-terminal amino acids is deleted.

optionally a his-tag sequence (HHHHHH (SEQ ID NO: 15) or HHHHHHH (
SEQ ID NO: 16)) can be attached, optionally linked via a linker, to the (optionally truncated) HA2 domain for purification purposes. Optionally, the linker may contain a proteolytic cleavage site for enzymatic removal of the his-tag after purification.

In certain embodiments, the polypeptide comprises a sequence known to form a trimeric structure, namely GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO:3)
It is further stabilized by introducing optionally linked via a linker at the C-terminus of A2. Thus, in certain embodiments, the C-terminal portion of the HA2 domain is optionally linked via a linker with the amino acid sequence GYIPEAPRDGQAYVRK
replaced by DGEWVLLSTFL (SEQ ID NO:3). Linkers may optionally contain cleavage sites for subsequent processing according to protocols well known to those skilled in the art. To facilitate purification of the soluble form, a tag sequence, e.g. his-tag (HHHHH
H (SEQ ID NO: 15) or HHHHHHH (SEQ ID NO: 16)) or FLAG tag (DY
KDDDDK) (SEQ ID NO: 22) or a combination thereof optionally linked via a short linker can be added. The linker optionally includes (part of) a proteolytic cleavage site for subsequent processing according to protocols well known to those skilled in the art, e.g.
May contain EGR (SEQ ID NO: 24) (factor X) or LVPRGS (SEQ ID NO: 23) (thrombin). Processed proteins are also included in the present invention.

In one embodiment, the C-terminal portion of the HA2 domain from positions 519-565 is deleted (numbering according to SEQ ID NO: 1), SGRDYKDDDDKLVPRGSPGSGYI
replaced by PEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH (SEQ ID NO: 4).

In one embodiment, the C-terminal portion of the HA2 domain from positions 530-565 is deleted (numbering according to SEQ ID NO: 1), SGRDYKDDDDKLVPRGSPGSGYI
replaced by PEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH (SEQ ID NO: 4).

Native HA exists as a trimer on the cell surface. Most of the interactions between individual monomers that hold the trimer together are localized in the head domain, whereas in the stem domain trimerization is mediated by the formation of a trimeric coiled-coil motif. be. After removal of the head, the tertiary structure is destabilized and thus modifications are required to increase protein stabilization. By enhancing the helical propensity of the helix CD, a more stable protein can be created.

In the polypeptide described in co-pending application PCT/EP2014/060997, the sequence MKQIEDKIEEIESKQ (SEQ ID NO: 5), which is derived from the yeast transcriptional activator protein GCN4 and is known to trimerize, is located at positions 419-433 ( was introduced in the CD helix in the equivalent of ). This sequence has a high propensity to form helical secondary structures and can thus improve the overall stability of the polypeptides of the invention.

Surprisingly, according to the present invention, the stability and multimerization state of polypeptides are dependent on the correct localization and sequence of GCN4-derived sequences in the primary sequence of the polypeptides of the present invention. shown.

Thus, according to the invention, the sequence RMKQIEDKIEEIESK (SEQ ID NO: 20)
was introduced at positions 419-433 (numbering according to SEQ ID NO: 1), or the sequence RMKQIEDKIEEIESKQK (SEQ ID NO: 21) was introduced at positions 417-433.

In certain embodiments, the polypeptide is glycosylated.

In the work leading up to the present invention, techniques of molecular biology well known to those skilled in the art were used, e.g.
SEQ ID NO: 65), s127H1 (SEQ ID NO: 66), s71H2 (SEQ ID NO: 71), s86
B4 (SEQ ID NO: 67), s115A1 (SEQ ID NO: 70), s2201C9 (SEQ ID NO: 77
), s55G7 (SEQ ID NO: 68), s113E7 (SEQ ID NO: 78), s6E12 (SEQ ID NO: 69), s181H9 (SEQ ID NO: 76) to modify the sequence RM at positions 419-433
Sequences s74H9-t2 (SEQ ID NO:93), s127H1-t2 (SEQ ID NO:91), s71H2-t2 (SEQ ID NO:97) containing KQIEDKIEEIESK (SEQ ID NO:20),
s86B4-t2 (SEQ ID NO:92), s115A1-t2 (SEQ ID NO:96), s220C
9-t2 (SEQ ID NO: 99), s55G7-t2 (SEQ ID NO: 95), s113E7-t2 (
SEQ ID NO: 100), s6E12-t2 (SEQ ID NO: 94), and s181H9-t2 (SEQ ID NO: 98) were generated.

In a similar manner, the sequence RMKQIEDKIEEIESK at positions 417-433
Polypeptide s74H9-t3 (SEQ ID NO: 123) containing QK (SEQ ID NO: 21), s
127H1-t3 (SEQ ID NO: 121), s71H2-t3 (SEQ ID NO: 127), s86B
4-t3 (SEQ ID NO: 122), s115A1-t3 (SEQ ID NO: 126), s2201C9
-t3 (SEQ ID NO: 129), s55G7-t3 (SEQ ID NO: 125), s113E7-t3
(SEQ ID NO: 130), s6E12-t3 (SEQ ID NO: 124), s181H9-t3 (SEQ ID NO: 128) were generated.

The polypeptides of the invention are stem polypeptides previously described (PCT/EP201
2/073706 and PCT/EP2014/060997) increased binding of influenza antibodies, in particular CR6261 and/or CR9114,
and/or an increased propensity to multimerize and/or an increased stability.

を含み、
Ｘ_１は、Ｅ、Ｉ、Ｋ、Ｖ、Ａ、およびＴからなる群から選択されるアミノ酸であり；
Ｘ_２は、Ｉ、Ｋ、Ｒ、Ｔ、Ｆ、Ｎ、ＳおよびＹからなる群から選択されるアミノ酸であり
；
Ｘ_３は、Ｄ、Ｆ、Ｖ、Ｙ、Ａ、Ｉ、Ｎ、Ｓ、およびＴからなる群から選択されるアミノ酸
であり；
Ｘ_４は、Ｉ、Ｋ、Ｒ、Ｔ、Ｅ、ＧおよびＶからなる群から選択されるアミノ酸であり；
Ｘ_５は、Ｍ、Ｅ、Ｋ、Ｖ、Ｒ、Ｔからなる群から選択されるアミノ酸であり；
Ｘ_６は、Ｆ、Ｉ、Ｎ、Ｓ、Ｔ、Ｙ、Ｈ、およびＬからなる群から選択されるアミノ酸であ
り；
Ｘ_７は、Ａ、Ｇ、Ｉ、Ｒ、Ｔ、Ｖ、Ｆ、およびＳからなる群から選択されるアミノ酸であ
り；
Ｘ_８は、Ｆ、Ｉ、Ｎ、Ｓ、Ｔ、Ｙ、Ｇ、Ｅ、Ｋ、Ｍ、およびＶからなる群から選択される
アミノ酸であり；
Ｘ_９は、Ｈ、Ｉ、Ｌ、Ｎ、Ｒ、およびＳからなる群から選択されるアミノ酸である。 In certain embodiments, the polypeptide has the amino acid sequence:

including
X ₁ is an amino acid selected from the group consisting of E, I, K, V, A, and T;
X2 is an amino acid selected from the group consisting of I, K, R, T, F, _N , S and Y;
X ₃ is an amino acid selected from the group consisting of D, F, V, Y, A, I, N, S, and T;
_X4 is an amino acid selected from the group consisting of I, K, R, T, E, G and V;
_X5 is an amino acid selected from the group consisting of M, E, K, V, R, T;
X ₆ is an amino acid selected from the group consisting of F, I, N, S, T, Y, H, and L;
_X7 is an amino acid selected from the group consisting of A, G, I, R, T, V, F, and S;
X ₈ is an amino acid selected from the group consisting of F, I, N, S, T, Y, G, E, K, M, and V;
_X9 is an amino acid selected from the group consisting of H, I, L, N, R, and S;

を含み、
Ｘ_１は、Ｅ、Ｉ、Ｋ、Ｖ、Ａ、およびＴからなる群から選択されるアミノ酸であり；
Ｘ_２は、Ｉ、Ｋ、Ｒ、Ｔ、Ｆ、Ｎ、ＳおよびＹからなる群から選択されるアミノ酸であり
；
Ｘ_３は、Ｄ、Ｆ、Ｖ、Ｙ、Ａ、Ｉ、Ｎ、Ｓ、およびＴからなる群から選択されるアミノ酸
であり；
Ｘ_４は、Ｉ、Ｋ、Ｒ、Ｔ、Ｅ、ＧおよびＶからなる群から選択されるアミノ酸であり；
Ｘ_５は、Ｍ、Ｅ、Ｋ、Ｖ、Ｒ、Ｔからなる群から選択されるアミノ酸であり；
Ｘ_６は、Ｆ、Ｉ、Ｎ、Ｓ、Ｔ、Ｙ、Ｈ、およびＬからなる群から選択されるアミノ酸であ
り；
Ｘ_７は、Ａ、Ｇ、Ｉ、Ｒ、Ｔ、Ｖ、Ｆ、およびＳからなる群から選択されるアミノ酸であ
り；
Ｘ_８は、Ｆ、Ｉ、Ｎ、Ｓ、Ｔ、Ｙ、Ｇ、Ｅ、Ｋ、Ｍ、およびＶからなる群から選択される
アミノ酸であり；
Ｘ_９は、Ｈ、Ｉ、Ｌ、Ｎ、Ｒ、およびＳからなる群から選択されるアミノ酸である。 In certain embodiments, the polypeptide has the amino acid sequence:

including
X ₁ is an amino acid selected from the group consisting of E, I, K, V, A, and T;
X2 is an amino acid selected from the group consisting of I, K, R, T, F, _N , S and Y;
X ₃ is an amino acid selected from the group consisting of D, F, V, Y, A, I, N, S, and T;
_X4 is an amino acid selected from the group consisting of I, K, R, T, E, G and V;
_X5 is an amino acid selected from the group consisting of M, E, K, V, R, T;
X ₆ is an amino acid selected from the group consisting of F, I, N, S, T, Y, H, and L;
_X7 is an amino acid selected from the group consisting of A, G, I, R, T, V, F, and S;
X ₈ is an amino acid selected from the group consisting of F, I, N, S, T, Y, G, E, K, M, and V;
_X9 is an amino acid selected from the group consisting of H, I, L, N, R, and S;

を含み、
Ｘ_１は、Ｅ、Ｉ、Ｋ、Ｖ、Ａ、およびＴからなる群から選択されるアミノ酸であり；
Ｘ_２は、Ｉ、Ｋ、Ｒ、Ｔ、Ｆ、Ｎ、ＳおよびＹからなる群から選択されるアミノ酸であり
；
Ｘ_３は、Ｄ、Ｆ、Ｖ、Ｙ、Ａ、Ｉ、Ｎ、Ｓ、およびＴからなる群から選択されるアミノ酸
であり；
Ｘ_４は、Ｉ、Ｋ、Ｒ、Ｔ、Ｅ、ＧおよびＶからなる群から選択されるアミノ酸であり；
Ｘ_５は、Ｍ、Ｅ、Ｋ、Ｖ、Ｒ、Ｔからなる群から選択されるアミノ酸であり；
Ｘ_６は、Ｆ、Ｉ、Ｎ、Ｓ、Ｔ、Ｙ、Ｈ、およびＬからなる群から選択されるアミノ酸であ
り；
Ｘ_７は、Ａ、Ｇ、Ｉ、Ｒ、Ｔ、Ｖ、Ｆ、およびＳからなる群から選択されるアミノ酸であ
り；
Ｘ_８は、Ｆ、Ｉ、Ｎ、Ｓ、Ｔ、Ｙ、Ｇ、Ｅ、Ｋ、ＭおよびＶからなる群から選択されるア
ミノ酸であり；
Ｘ_９は、Ｈ、Ｉ、Ｌ、Ｎ、Ｒ、およびＳからなる群から選択されるアミノ酸である。 In certain embodiments, the polypeptide has the amino acid sequence:

including
X ₁ is an amino acid selected from the group consisting of E, I, K, V, A, and T;
X2 is an amino acid selected from the group consisting of I, K, R, T, F, _N , S and Y;
X ₃ is an amino acid selected from the group consisting of D, F, V, Y, A, I, N, S, and T;
_X4 is an amino acid selected from the group consisting of I, K, R, T, E, G and V;
_X5 is an amino acid selected from the group consisting of M, E, K, V, R, T;
X ₆ is an amino acid selected from the group consisting of F, I, N, S, T, Y, H, and L;
_X7 is an amino acid selected from the group consisting of A, G, I, R, T, V, F, and S;
X ₈ is an amino acid selected from the group consisting of F, I, N, S, T, Y, G, E, K, M and V;
_X9 is an amino acid selected from the group consisting of H, I, L, N, R, and S;

を含み、
Ｘ_１は、Ｅ、Ｉ、Ｋ、Ｖ、Ａ、およびＴからなる群から選択されるアミノ酸であり；
Ｘ_２は、Ｉ、Ｋ、Ｒ、Ｔ、Ｆ、Ｎ、ＳおよびＹからなる群から選択されるアミノ酸であり
；
Ｘ_３は、Ｄ、Ｆ、Ｖ、Ｙ、Ａ、Ｉ、Ｎ、Ｓ、およびＴからなる群から選択されるアミノ酸
であり；
Ｘ_４は、Ｉ、Ｋ、Ｒ、Ｔ、Ｅ、ＧおよびＶからなる群から選択されるアミノ酸であり；
Ｘ_５は、Ｍ、Ｅ、Ｋ、Ｖ、Ｒ、Ｔからなる群から選択されるアミノ酸であり；
Ｘ_６は、Ｆ、Ｉ、Ｎ、Ｓ、Ｔ、Ｙ、Ｈ、およびＬからなる群から選択されるアミノ酸であ
り；
Ｘ_７は、Ａ、Ｇ、Ｉ、Ｒ、Ｔ、Ｖ、Ｆ、およびＳからなる群から選択されるアミノ酸であ
り；
Ｘ_８は、Ｆ、Ｉ、Ｎ、Ｓ、Ｔ、Ｙ、Ｇ、Ｅ、Ｋ、ＭおよびＶからなる群から選択されるア
ミノ酸であり；
Ｘ_９は、Ｈ、Ｉ、Ｌ、Ｎ、Ｒ、およびＳからなる群から選択されるアミノ酸である。 In certain embodiments, the polypeptide has the amino acid sequence:

including
X ₁ is an amino acid selected from the group consisting of E, I, K, V, A, and T;
X2 is an amino acid selected from the group consisting of I, K, R, T, F, _N , S and Y;
X ₃ is an amino acid selected from the group consisting of D, F, V, Y, A, I, N, S, and T;
_X4 is an amino acid selected from the group consisting of I, K, R, T, E, G and V;
_X5 is an amino acid selected from the group consisting of M, E, K, V, R, T;
X ₆ is an amino acid selected from the group consisting of F, I, N, S, T, Y, H, and L;
_X7 is an amino acid selected from the group consisting of A, G, I, R, T, V, F, and S;
X ₈ is an amino acid selected from the group consisting of F, I, N, S, T, Y, G, E, K, M and V;
_X9 is an amino acid selected from the group consisting of H, I, L, N, R, and S;

In certain embodiments, in SEQ ID NOs: 145-148, X ₁ is K and X ₂ is K
, X ₃ is F, X ₄ is T, X ₅ is M, X ₆ is Y, X ₇ is I
, X ₈ is Y and X ₉ is S.

Influenza hemagglutinin stem domain polypeptides can be prepared according to any technique deemed suitable by those skilled in the art, including the techniques described below.

Thus, the immunogenic polypeptides of the invention are synthesized as DNA sequences by standard methods known in the art, cloned using suitable restriction enzymes and methods known in the art, and subsequently isolated in vitro or in vivo. can be expressed in Accordingly, the invention also relates to nucleic acid molecules encoding the polypeptides described above. The invention further relates to vectors containing nucleic acids encoding the polypeptides of the invention. In one embodiment, a nucleic acid molecule according to the invention is part of a vector, eg, a plasmid. Such vectors can be readily manipulated by methods well known to those of skill in the art, and can be designed, for example, to replicate in prokaryotic and/or eukaryotic cells. Moreover, many vectors, either directly or in the form of desired fragments isolated therefrom, can be used to transform eukaryotic cells and integrate in whole or in part into the genome of such cells. resulting in stable host cells containing the desired nucleic acid in their genome. The vector used can be any vector that is suitable for DNA cloning and can be used for transcription of the nucleic acid of interest. When host cells are used, the vector is preferably an integrating vector. Alternatively, the vector can be an episomal replicating vector.

One skilled in the art can select a suitable expression vector and functionally insert the nucleic acid sequences of the present invention. To obtain expression of a nucleic acid sequence encoding a polypeptide, a recombinant nucleic acid encoding the protein or polypeptide in an expressible format is operably linked to the nucleic acid sequence encoding the polypeptide with a sequence capable of driving expression. It is well known to those skilled in the art that molecules can be produced. Generally, a promoter sequence is placed upstream of the sequence to be expressed. Many expression vectors are available in the art, e.g.
pcDNA and pEF vector systems from en, pMSCV and pTK-Hyg from BD Sciences, pCMV-Script from Stratagene, etc.
They can be used to obtain suitable promoters and/or transcription termination sequences, poly A sequences, and the like. When the sequence encoding the polypeptide of interest is inserted appropriately with respect to the sequences that govern the transcription and translation of the encoded polypeptide, the resulting expression cassette is useful for producing the polypeptide of interest. Yes, it is called expression. Sequences that drive expression can include promoters, enhancers, etc., and combinations thereof. They should be capable of functioning in the host cell and thereby driving expression of the nucleic acid sequences to which they are operably linked. Those skilled in the art will appreciate that a variety of promoters can be used to obtain gene expression in the host cell. Promoters can be constitutive or regulated, can be obtained from a variety of sources, including viral, prokaryotic, or eukaryotic sources, or can be artificially designed. Expression of the nucleic acid of interest can be from the native promoter or its derivatives, or from a completely heterologous promoter (Kaufm
an, 2000). Some well-known and often used promoters for expression in eukaryotic cells are promoters derived from viruses, such as adenoviruses,
E1A promoter, promoter from cytomegalovirus (CMV), e.g. CMV immediate early (IE) promoter (referred to herein as CMV promoter) (e.g. pcDNA, available from Invitrogen), Simian Virus 40 (SV40) (Das et al, 1985). Suitable promoters may also be derived from eukaryotic cells, for example metallothionein (MT
) promoter, elongation factor 1α (EF-1α) promoter (Gill et al.,
2001), ubiquitin C or UB6 promoter (Gill et al., 200
1), actin promoter, immunoglobulin promoter, heat shock promoter and the like. Testing for promoter function and promoter strength is routine for those skilled in the art and generally includes, for example, test genes, e.g.
Cloning of luciferase, GFP, etc., and expression testing of test genes can be included. Of course, promoters can be altered by deletion, addition, mutation of their sequences and tested for functionality to find new, attenuated or improved promoter sequences.
According to the invention, strong promoters conferring high transcription levels in optimal eukaryotic cells are preferred.

Constructs can be transfected into eukaryotic cells (eg, plant, fungal, yeast or animal cells) or suitable prokaryotic expression systems such as E. coli using methods well known to those of skill in the art. In some cases, a suitable "tag" sequence (e.g., but not limited to, his
, myc, strep, or flag tags) or complete proteins (such as, but not limited to, maltose binding protein or glutathione S transferase) are added to the sequences of the invention to produce polypeptides from cells or supernatants. and/or
or allow identification. Optionally, the tag can be removed later by proteolytic digestion by including a sequence containing a specific proteolytic site.

Purified polypeptides are analyzed by spectroscopic methods known in the art (eg, circular dichroism spectroscopy, Fourier transform infrared spectroscopy and NMR spectroscopy or X-ray crystallography) to identify helices and beta sheets, etc. The presence of desired structures can be investigated. ELISA, Octet and FACS and the like can be used to investigate the binding of polypeptides of the invention to previously described broadly neutralizing antibodies (CR6261, CR9114, CR8057). Thus, polypeptides according to the invention having the correct conformation can be selected.

The invention further relates to an immunogenic composition comprising a therapeutically effective amount of at least one of the polypeptides and/or nucleic acids of the invention. An immunogenic composition preferably further comprises a pharmaceutically acceptable carrier. In the present context, the term "pharmaceutically acceptable" means that at the dosages and concentrations employed the carrier does not cause undesired or adverse effects in subjects to whom it is administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (Remington's Pharmaceutical Sc.
iences, 18th edition, A.; R. Gennaro, Ed. , Mack
Publishing Company [1990];
Formulation Development of Peptides and
Proteins, S.; Frokjaer and L.L. Hovgaard, Eds.
, Taylor & Francis [2000];
Pharmaceutical Excipients, 3rd edition, A.P. K.
ibbe, Ed. , Pharmaceutical Press [2000]). The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the composition is administered. Saline solutions and aqueous dextrose and glycerol solutions, for example, can be employed as liquid carriers, particularly for injectable solutions. Precise formulation should suit the mode of administration. Polypeptides and/or nucleic acid molecules are preferably formulated and administered as sterile solutions. Sterile solutions are prepared by sterile filtration or by other methods known per se in the art. The solution can then be lyophilized or filled into pharmaceutical administration containers. The pH of the solution is generally pH 3.0 to 9.5, such as pH 5.0 to 7.0.
5 range.

The invention also relates to a method of inducing an immune response in a subject, comprising administering to the subject the polypeptides, nucleic acid molecules and/or immunogenic compositions described above. A subject according to the present invention is preferably a mammal that can be infected by an infectious disease-causing agent, particularly an influenza virus, or otherwise benefit from the induction of an immune response, such a subject being e.g. , rodents such as mice, ferrets, or domestic or farm animals, or non-human primates, or humans. Preferably, the subject is a human subject. Accordingly, the present invention particularly relates to group 1 and/or group 2 influenza A viruses, such as H1, H2, H3, H4, H5, H7 and/or H1.
A subject utilizing the polypeptides, nucleic acids and/or immunogenic compositions described herein to bolster the immune response against influenza virus, including subtype 0 HA, and/or influenza virus hemagglutinin (HA) of influenza B virus. To provide a method of inducing in In some embodiments, the invention provides a polypeptide described herein,
Methods are provided for inducing an immune response in a subject to an influenza virus containing HA of the H1 subtype utilizing nucleic acids and/or immunogenic compositions.

In some embodiments, the immune response induced is effective to prevent and/or treat influenza virus infection caused by Group 1 and/or Group 2 influenza A virus subtypes and/or influenza B virus. . In some embodiments, the immune response induced by the polypeptides, nucleic acids and/or immunogenic compositions described herein is 2, 3, 4, 5 or 6 subtypes of influenza A virus and It is effective for preventing and/or treating influenza A and/or B virus infections caused by/or influenza B virus. In some embodiments, the immune response induced is effective to prevent and/or treat influenza virus infection caused by influenza viruses comprising H1 subtype HA.

As it is well known that small proteins and/or nucleic acid molecules do not always effectively induce strong immune responses, adjuvants may be added to the polypeptides and/or nucleic acid molecules.
Or it may be necessary to increase the immunogenicity of the nucleic acid molecule. In one embodiment,
The immunogenic compositions described herein include or are administered in combination with an adjuvant. Adjuvants for combination administration with the compositions described herein can be administered before, concurrently with, or after administration of the compositions. Examples of suitable adjuvants include aluminum salts such as aluminum hydroxide and/or aluminum phosphate; oil-emulsion compositions (or oil-in-water compositions) such as squalene-water emulsions such as MF59 (such as , see WO 90/14837 pamphlet)
saponin formulations, such as QS21 and immunostimulating complexes (ISCOMs) (e.g., U.S. Patent No. 5,057,540; WO 90/03184, WO 96/11711, see WO 2004/004762, WO 2005/002620); bacterial or microbial derivatives, examples of which include monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL ), CpG-motif-containing oligonucleotides, ADP-ribosylating bacterial toxins or mutants thereof, such as E. coli heat-labile enterotoxin LT, cholera toxin CT, pertussis toxin PT, or tetanus toxoid TT, Matri
xM(Isconova). In addition, known immunopotentiating techniques, such as proteins known in the art to enhance immune responses (e.g., tetanus toxoid, CRM)
197, rCTB, bacterial flagellin, etc.) or inclusion of the polypeptide in virosomes, or a combination thereof can be used. Other non-limiting examples that can be used are, for example, Coffman et al. (20
10).

In one embodiment, the influenza hemagglutinin stem domain polypeptides of the invention are incorporated into virus-like particle (VLP) vectors. VLPs generally comprise a viral polypeptide, typically derived from structural proteins of the virus. Preferably, the VLP is replication incapable. In certain embodiments, the VLP may lack the entire viral genome, or may include a portion of the viral genome. In some embodiments, the VLP is
incapable of infecting cells. In some embodiments, the VLPs express on their surface one or more viral (eg, viral surface glycoproteins) or non-viral (eg, antibodies or proteins) targeting moieties known to those skilled in the art.

In specific embodiments, the polypeptides of the invention are taken up into virosomes.
Virosomes containing polypeptides according to the invention can be produced using techniques known to those of skill in the art. For example, virosomes are produced by disrupting purified virus, extracting the genome, and reassorting the particles with viral proteins (e.g., influenza hemagglutinin stem domain polypeptide) and lipids to form lipid particles containing the viral proteins. be able to.

The present invention also relates to a polypeptide, nucleic acid and/or immunogenic composition as described above for inducing an immune response in a subject against influenza HA, in particular for use as a vaccine. Thus, influenza hemagglutinin stem domain polypeptides, nucleic acids encoding such polypeptides, or vectors comprising such nucleic acids or polypeptides described herein, may be used against influenza virus, e.g., the stem region of influenza virus hemagglutinin. can be used to elicit neutralizing antibodies against The invention particularly relates to phylogenetic group 1 and/or phylogenetic group 2
The above polypeptides, nucleic acids and/or immunogenic compositions for use as vaccines in the prevention and/or treatment of diseases or conditions caused by influenza A and/or influenza B viruses. In one embodiment,
The vaccine can be used in the prevention and/or treatment of disease caused by 2, 3, 4, 5, 6 or more different subtypes of phylogenetic groups 1 and/or 2 and/or influenza B viruses. . In one embodiment, the vaccine can be used in the prevention and/or treatment of influenza infections caused by influenza viruses containing HA of the H1 subtype.

The polypeptides of the invention can be used in vitro after synthesis, or in suitable cellular expression systems such as bacteria and eukaryotic cells, or in vivo in a subject in need thereof as an immunogen. The expression can be achieved by expressing a nucleic acid encoding the sexual polypeptide. Such nucleic acid vaccines may take any form such as naked DNA, plasmids, or viral vectors such as adenoviral vectors.

Administration of polypeptides, nucleic acid molecules and/or immunogenic compositions according to the invention can be carried out using standard routes of administration. Non-limiting examples include parenteral administration, eg, intravenous, intradermal, transdermal, intramuscular, subcutaneous, etc., or mucosal administration, eg, intranasal, oral, etc. One skilled in the art can determine the various possibilities of administering polypeptides, nucleic acid molecules and/or immunogenic compositions according to the invention to induce an immune response. In certain embodiments, the polypeptide, nucleic acid molecule and/or immunogenic composition (or vaccine) is administered more than once, ie in a so-called allogeneic prime-boost regimen. In certain embodiments in which the polypeptide, nucleic acid molecule, and/or immunogenic composition is administered more than once, administration of the second dose is after a time interval of, e.g., one week or more after administration of the first dose. ,
2 weeks or more after administration of the first dose, 3 weeks or more after administration of the first dose, 1 after administration of the first dose
months or more, 6 weeks or more after administration of the first dose, 2 months or more after administration of the first dose, 3 months or more after administration of the first dose, 4 months or more after administration of the first dose, etc. , nucleic acid molecule, and/or immunogenic composition up to several years after administration of the first dose. The vaccine can also be administered three or more times, eg, three, four, etc., such that a first priming dose is followed by two or more boost doses. In other embodiments, the polypeptides, nucleic acid molecules and/or immunogenic compositions according to the invention are administered only once.

Polypeptides, nucleic acid molecules, and/or immunogenic compositions can also be administered in heterologous prime-boost regimens, either as primes or boosts.

The present invention further provides methods of preventing and/or treating influenza virus disease in a subject utilizing the polypeptides, nucleic acids and/or compositions described herein. In a specific embodiment, the method of preventing and/or treating influenza virus disease in a subject comprises administering to a subject in need thereof an effective amount of the above-described polypeptides, nucleic acids and/or immunogenic compositions. Including. A therapeutically effective amount is used to prevent, ameliorate and/or treat diseases or conditions resulting from infection by group 1 or 2 influenza A viruses and/or influenza B viruses, particularly diseases caused by infection with influenza A viruses, including H1 subtype HA. or therapeutically effective amount of a polypeptide, nucleic acid and/or composition as defined herein. Prevention includes inhibiting or reducing the spread of influenza virus or inhibiting or reducing the onset, manifestation or progression of one or more of the symptoms associated with infection by influenza virus. Improvement, as used herein, may refer to a reduction in visible or sensory disease symptoms, viremia, or any other measurable symptoms of influenza infection.

Those in need of treatment include those already afflicted with conditions resulting from infection with a Group 1 or Group 2 influenza A virus, or influenza B virus, and those in whom infection with influenza virus should be prevented. Thus, the polypeptides, nucleic acids and/or compositions of the invention may be used in naïve subjects, i.e., those who do not have disease caused by influenza virus infection or have never been infected by influenza virus infection and are currently infected. It can be administered to subjects who have not been infected, or subjects who have already been and/or have been infected with influenza virus.

In one embodiment, prevention and/or treatment can be targeted in patient populations susceptible to influenza virus infection. Such patient groups include, but are not limited to, the elderly (e.g., ≧50 years, ≧60 years, preferably ≧65 years), the young (e.g., ≦5 years, ≦1 years). , inpatients and treated with antiviral compounds,
Patients with inadequate antiviral responses are included.

In another embodiment, the polypeptides, nucleic acids and/or immunogenic compositions are administered to a subject with one or more other active agents, such as existing or future influenza vaccines, monoclonal antibodies and/or antiviral agents, and /or can be administered in combination with an antibacterial agent, and/or an immunomodulatory agent. The one or more other active agents may be beneficial in treating and/or preventing influenza virus disease, or may ameliorate symptoms or conditions associated with influenza virus disease. In some embodiments, the one or more other active agents is an analgesic, an antipyretic, or a therapeutic that relieves or aids breathing.

Dosage regimens for the polypeptides and/or nucleic acid molecules of the present invention will provide the optimal desired response (
for example, a therapeutic response). A suitable dosage range can be, for example, 0.1-100 mg/kg body weight, preferably 1-50 mg/kg body weight, preferably 0.5-15 mg/kg body weight. The precise dosage of polypeptide and/or nucleic acid molecule to be used will depend, for example, on the route of administration and the severity of the infection or disease caused thereby, and should be determined according to the judgment of the physician and the circumstances of each subject. . For example, effective doses will vary depending on the patient's target site, physiological state (eg, age, weight, health), whether treatment is prophylactic or therapeutic. Usually the patient is a human, but non-human mammals, such as transgenic mammals, can also be treated. Treatment dosages are optimally titrated to optimize safety and efficacy.

Polypeptides of the invention can also be used to confirm binding of monoclonal antibodies identified as potential therapeutic candidates. In addition, the polypeptides of the invention can be used as diagnostic tools to test the immune status of an individual, e.g., by determining the presence of antibodies in the serum of such individuals that can bind to the polypeptides of the invention. can be used to The invention therefore also provides an in vitro diagnostic method for detecting the presence of an influenza infection in a patient, comprising the steps of a) contacting a biological sample obtained from said patient with a polypeptide according to the invention; and b) an antibody - relates to a method comprising detecting the presence of an antigen complex.

Polypeptides of the invention can also be used to identify new binding molecules or to improve existing binding molecules, such as monoclonal antibodies and antiviral agents.

The invention is further illustrated in the following examples and figures. The examples in no way limit the scope of the invention.

Example 1: Stem-Based Polypeptides as described in PCT/EP2014/060997 PCT/EP2012/073706 describes influenza hemagglutinin stem domain polypeptides, compositions and vaccines and influenza prophylaxis and/or
or methods of their use in the field of therapy. PCT/EP2014/0
60997 was obtained by site-directed mutagenesis of H1-mini2-cluster1+5+6-GCN4 (SEQ ID NO: 2) and CR6261 (Throsby et al.
1, 2009; Ekiert et al 2010) and/or H1N1A/Brisbane/59/20 that more stably displayed the broadly neutralizing epitope of CR9114.
07 (SEQ ID NO: 1) discloses additional stem domain polypeptide sequences derived from full-length HA.

H1-mini2-cluster1+5+6-GCN4 (SEQ ID NO: 2) is H1N1
It was derived from the full-length HA of A/Brisbane/59/2007 (SEQ ID NO: 1) by utilizing the following steps:
1. Remove the HA0 cleavage site. Cleavage of wild-type HA at this site yields HA1 and HA2. Removal can be achieved by an R to Q mutation at position P1 (e.g., see S
See Un et al, 2010).
2. Removing the head domain by deleting amino acids 53 to 320 from SEQ ID NO:1. The remaining N- and C-terminal portions of the sequence were joined by a four-residue flexible linker GGGG.
3. Residues 402 to 41 in H1A/Brisbane/59/2007 (SEQ ID NO: 1)
Increase the solubility of the loop (between the A and CD helices) formed by (the equivalent of) 8 to increase the stability of the pre-fusion conformation and destabilize the post-fusion conformation of the modified HA. thing. H1-mini2-cluster1+5+6-GCN4 (SEQ ID NO: 2)
introduced the mutations F406S, V409T, F413G and L416S (numbering refers to SEQ ID NO: 1).
4. introducing a disulfide bridge between amino acids at positions 324 and 436 in H1A/Brisbane/59/2007;
This is achieved by introducing C (numbering refers to SEQ ID NO: 1).
5. Introducing the GCN4-derived sequence MKQIEDKIEEIESKQ (SEQ ID NO: 5), which is known to trimerize, at positions 419-433 (numbering refers to SEQ ID NO: 1).

In certain embodiments, the transmembrane and intracellular domain sequences are 514, 515, 516, 51 of HA2, such that a secreted (soluble) polypeptide is produced following expression in cells.
7, 518, 519, 520, 521, 522, 523, 524, 525, 526, 52
Deleted from positions 6, 527, 528, 529, or 530 (or their equivalents as determined from sequence alignments) to the C-terminus of HA2 (numbering according to SEQ ID NO: 1). Soluble polypeptides contain sequences known to form trimeric structures, namely foldon
The sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 3) was further stabilized by introducing it, optionally linked via a short linker as described above.
Linkers may optionally contain cleavage sites for subsequent processing according to protocols well known to those skilled in the art. A tag sequence such as a histidine tag (HHHHHH (SEQ ID NO: 15) or HHHHHHH (SEQ ID NO: 16) or a FLAG tag (DYKDDDDK; SEQ ID NO: 22) or combinations thereof is optionally included to facilitate purification and detection of soluble forms. It can optionally be added by joining via a short linker, which is optionally (part of) a proteolytic cleavage site for later processing according to protocols well known to those skilled in the art, such as LVPRGS (sequence Number 23) (thrombin)
or IEGR (SEQ ID NO: 24) (Factor X). Processed proteins are also included in the invention.

An example of such a C-terminal sequence combining a FLAG tag, a thrombin cleavage site, a foldon, and a His sequence is FLAG-thrombin-foldo of SEQ ID NO:4.
n-His. This sequence is H1-mini2-cluster1+5+6-GCN4
(SEQ ID NO:2) was combined with a soluble form of the sequence to create the parental sequence (SEQ ID NO:6), which was used by mutagenesis to create the novel polypeptides of the present invention. This sequence does not contain a leader sequence corresponding to amino acids 1-17 of SEQ ID NOS:1 and 2.

Thus, stem domain polypeptides lack a portion of the hemagglutinin sequence encoding the head domain of the molecule as described in PCT/2012/073706 and supra, and lack the N- and C-terminal portions of the sequence. was made by religating via linkers on both sides of the . Removal of the head domain leaves a portion of the molecule already protected from aqueous solvent exposed, potentially destabilizing the structure of the polypeptides of the invention. For this reason, residues in the B-loop (particularly amino acid residues 406 (F and S of SEQ ID NOs: 1 and 2, respectively), 409 (V and T), 413 (F and G) and 416 (L and S) are ,
The parental sequence SEQ ID NO: 6 was used as a starting point and mutated in various combinations. SEQ ID NO:6 is from H1-mini2-cluster1+5+6-GCN4 (SEQ ID NO:2) with the leader sequence removed and residues 520-565 Flag-thrombin-fold
It was created by replacing it with an on--his sequence (SEQ ID NO:4).

Similarly, in the area around the fusion peptide, many hydrophobic residues are exposed to solvent, which means that unlike native full-length HA, the polypeptide cannot be cleaved, burying the hydrophobic fusion peptide inside the protein. caused by the fact that it undergoes relevant conformational changes. To address this issue, some or all of residues I337, I340, F352 and I353 of SEQ ID NO:2 were also mutated.

Thus, the soluble form of HA stem polypeptide 74H9 (SEQ ID NO:57), 127
H1 (SEQ ID NO:55), 71H2 (SEQ ID NO:61), 86B4 (SEQ ID NO:56), 115
A1 (SEQ ID NO: 60), 2201C9 (SEQ ID NO: 63), 55G7 (SEQ ID NO: 59), 1
13E7 (SEQ ID NO:64), 6E12 (SEQ ID NO:58), 181H9 (SEQ ID NO:62) were generated.

DNA sequences encoding the above polypeptides are transformed into Pichia pastoris using protocols well known to those skilled in the art, or HEK2
Transfected into 93F cells. Constructs used for expression in mammalian cells contained the HA leader sequence (residues 1-17 of SEQ ID NOS: 1 and 2), while Pichia pastoris (P
. In constructs used for expression in S.pastoris, the HA leader sequence was replaced by the yeast alpha factor leader sequence (SEQ ID NO: 7). The expressed protein was thus directed to the cell culture medium, thus allowing determination of binding and expression without further purification of the polypeptide of the invention. All sequences contained the FLAG-foldon-HIS C-terminal sequence (SEQ ID NO:4).

Monoclonal antibody binding to polypeptides (CR6261, CR9114, CR802
0) was determined by ELISA. For this purpose, the ELISA plate was
1 monoclonal antibody solution (20 μl/well) overnight at 4°C. After removal of the antibody solution, the remaining surface was blocked with a 4% solution of non-fat dry milk in PBS at room temperature for at least 1 hour. After washing the plates, 20 μl of cell culture medium (neat or diluted) was added to each well and incubated at room temperature for at least 1 hour. The ELISA plate was then washed and 20 μl of anti-FLAG-HRP antibody solution (S
igma A8952, diluted 1:2000 in 4% non-fat dry milk in PBS-Tween) was added. After incubation (1 hour at room temperature), the plate was washed once again and 20
Signal was developed by adding μl of luminescent substrate (Thermoscientific C#34078). Alternatively, the signal can be developed using colorimetric detection methods.

Expression of the polypeptides of the invention can be measured using a homogenous time-resolved fluorescence assay (for general description, see
For example, Degorce et al. , Curr. Chem. Genomics 20
093:22-32). For this purpose, tellurium (Tb
)-labeled anti-FLAG monoclonal antibody (donor) and Alexa488-labeled anti-His monoclonal antibody (acceptor) mixture (HTRF solution) was added to 210.5 μl of anti-F
LAG-TB (26 μg/ml stock) and 1.68 ml anti-HIS-488 (50 μg/ml stock)
μg/ml) in 80 ml of culture medium and 50 mM HEPES + 0.1% BSA
It was prepared by adding to a pair 1 mixture. 19 μl of HTRF solution was added to each well of the ELISA plate and 1 μl of culture medium was added. The ratio of fluorescence emissions at 520 and 665 nm was determined upon excitation and after a delay to allow decay of short-lived background signals arising from other compounds (proteins, media components, etc.).
This is a measure of total protein content in a sample and is used to normalize mAb binding signals between different experiments.

Polypeptides listed in Tables 3 and 4 were prepared from Pichia according to protocols well known to those skilled in the art.
It was expressed in P. Pastoris. Culture medium was collected and stem domain polypeptide binding to CR6261 binding and expression was determined as described above. Since the response in the binding assay corresponds to the concentration of expressed protein, ELISA binding signals were normalized for protein expression by comparing the ratio of binding signal to that in the HTRF assay for each expressed sequence. All expressed polypeptides show a high HTRF signal to CR6261 binding ratio compared to the parental sequence of SEQ ID NO:6.

In addition, the ratio of CR6261 binding to HTRF signal was calculated and compared to the ratio calculated for parental sequence SEQ ID NO:6. The results are listed in Tables 3 and 4, column 5; all expressed proteins show higher ratios, indicating that the stem polypeptides show increased binding of CR6261.

Example 2 Design and Characterization of Polypeptides of the Invention Polypeptides of the invention are derived from the yeast transcriptional activator protein GCN4 with the sequence RMKQIEDKIEEIESK (SEQ ID NO: 20) or RMKQIEDKIEEIES
KQK (SEQ ID NO: 21) is contained in the CD helix. This sequence has a high propensity to form helical secondary structures and can thus improve the overall stability of the polypeptides of the invention.
Surprisingly, according to the present invention, the stability and aggregation state of the polypeptides of the invention are dependent on the correct localization and sequence of the GCN4-derived sequences in the primary sequence of the polypeptides of the invention. Found.

Thus, we now find that the sequence RMKQIEDKIEEIESK (SEQ ID NO: 20) has been introduced at positions 419-433 (numbering according to SEQ ID NO: 1; 21) are introduced at positions 417-433 (eg, SEQ ID NOs: 111-140
) describes a novel set of polypeptides of the invention.

For this purpose, the polypeptides described in Example 1, namely 74H9 (SEQ ID NO: 57), 127H1 (SEQ ID NO: 55), 71H2, were isolated using molecular biology techniques well known to those skilled in the art.
(SEQ ID NO:61), 86B4 (SEQ ID NO:56), 115A1 (SEQ ID NO:60), 2201
C9 (SEQ ID NO:63), 55G7 (SEQ ID NO:59), 113E7 (SEQ ID NO:64), 6E
12 (SEQ ID NO: 58), sequence 74H9-t2 containing the sequence RMKQIEDKIEEIESK (SEQ ID NO: 20) at positions 419-433 by modifying 181H9 (SEQ ID NO: 62)
(SEQ ID NO: 83), 127H1-t2 (SEQ ID NO: 81), 71H2-t2 (SEQ ID NO: 87
), 86B4-t2 (SEQ ID NO:82), 115A1-t2 (SEQ ID NO:86), 220C9
-t2 (SEQ ID NO:89), 55G7-t2 (SEQ ID NO:85), 113E7-t2 (SEQ ID NO:90), 6E12-t2 (SEQ ID NO:84), 181H9-t2 (SEQ ID NO:88) were generated.

In a similar fashion, the sequence RMKQIEDKIEEIESK at positions 417-433
Sequence 74H9-t3 (SEQ ID NO: 113) containing QK (SEQ ID NO: 21), 127H1-
t3 (SEQ ID NO: 111), 71H2-t3 (SEQ ID NO: 117), 86B4-t3 (SEQ ID NO: 112), 115A1-t3 (SEQ ID NO: 116), 2201C9-t3 (SEQ ID NO: 11
9), 55G7-t3 (SEQ ID NO: 115), 113E7-t3 (SEQ ID NO: 120), 6E
12-t3 (SEQ ID NO: 114), 181H9-t3 (SEQ ID NO: 118) were generated.

Polypeptides of the invention can be generated based on the sequences of HA molecules from different viral strains. For example, SEQ ID NOs: 149-155 are H1N1A/California/
Polypeptides of the invention based on the HA sequence of strain 07/09 are described.

As noted above, soluble polypeptides of the invention include, for example, residue 519 of the HA2 domain
, 520, 521, 522, 523, 524, 525, 526, 527, 526, 528
, 529, or 530 to the C-terminus of the HA2 domain (numbering according to SEQ ID NO: 1)
It can be created by removing the C-terminal portion of the A base sequence.

The polypeptide comprises sequences known to form trimeric structures, namely GYIPEAP
Further stabilization can be achieved by introducing RDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 3), optionally linked via a linker. Linkers may optionally contain cleavage sites for subsequent processing according to protocols well known to those skilled in the art. To facilitate purification of the soluble form, a tag sequence, e.g. his-tag (HHHHH
HH (SEQ ID NO: 16) or HHHHHH (SEQ ID NO: 15)) or FLAG tag (D
YKDDDDK) (SEQ ID NO: 22) or a combination thereof optionally linked via a short linker can be added. The linker optionally includes (part of) a proteolytic cleavage site for subsequent processing according to protocols well known to those skilled in the art, e.g.
May contain IEGR (SEQ ID NO: 24) (factor X) or LVPRGS (SEQ ID NO: 23) (thrombin). Processed proteins are also included in the present invention.

Soluble forms of the polypeptides of SEQ ID NOS: 55-64 and 81-90 have residues 519-5
65 (numbering refers to SEQ ID NO: 1) was generated by replacing the sequence RSLVPRGSPGHHHHHH containing both a modified thrombin cleavage site and a six histidine tag (SEQ ID NO: 15), according to protocols well known to those skilled in the art. It was expressed in HEK293F cells.

For comparison, H1-mini2-cluster1+5+6-GCN4t2 (SEQ ID NO:52) and H1-mini2-cluster1+5+6-GCN4t3 (SEQ ID NO:5
A soluble form of 3). Culture medium was harvested and binding to CR6261, CR9114 was tested by sandwich ELISA against coated mAbs CR6261 or CR9114 to capture the polypeptides of the invention directly from the culture medium and the C-terminal his-tag for detection purposes. was detected using a horseradish peroxidase (HRP)-conjugated antibody directed against Alternatively, biotinylated CR9114 was used in combination with HRP-conjugated streptavidin to detect polypeptides of the invention captured by CR9114 in a sandwich ELISA. This format allows detection of the presence of multimeric forms of the polypeptides of the invention. All polypeptides of the invention tested were EL
It was able to bind CR9114 (Figures 2A and B, Figures 3A and B and Figures 4A and B) and CR6261 (Figures 2C and D, Figures 3C and D, Figures 4C and D) as determined by ISA. CR9114 capture-biotinylated CR9114 detection sandwich ELI
Increased levels of multimerization detected by SA were associated with s55G7-t2 (SEQ ID NO: 95), s86B4-
t2 (SEQ ID NO: 92), s115A1-t2 (SEQ ID NO: 96), s127H1-t2 (SEQ ID NO: 91), s113E7-t2 (SEQ ID NO: 100), s220C9-t2 (SEQ ID NO: 99), s71H2-t3 (SEQ ID NO: 127 ), s127H1-t3 (SEQ ID NO: 121)
, was observed for s74H9-t3 (SEQ ID NO: 123).

To obtain highly pure preparations of the polypeptides of the invention for further characterization, H
127H1-t2 (SEQ ID NO: 81), 86B4-t2 (SEQ ID NO: 8) in EK293F cells
2) and the expression vector pcDNA2004 containing the gene encoding the soluble form of 55G7-t2 (SEQ ID NO:85). It will be appreciated by those skilled in the art that no leader sequence (or signal sequence) (corresponding to amino acids 1-17 of SEQ ID NO:1) directing protein trafficking during production is present in the final secreted polypeptide.

For production of the polypeptides of the present invention, HEK293F cells (Invitrogen) were spun down at 300 g for 5 minutes and resuspended in 300 mL of pre-warmed Freestyle™ medium via an SF1000 flask to achieve 1.0 ^{* 106vc}
/mL was seeded. This culture was incubated at 37° C. for 10 minutes in a multitron incubator.
% CO ₂ , 110 rpm for 1 hour. After 1 hour, plasmid D
NA was pipetted in 9.9 mL of Optimem medium to a concentration of 1.0 μg/mL in a 300 mL culture volume. In parallel, 440 μL of 293fectin® was pipetted into 9.9 mL of Optimem medium and incubated for 5 minutes at room temperature. After 5 minutes, the plasmid DNA/Optimem mixture was added to the 293fectin®/Optimem mixture and incubated for 20 minutes at room temperature.
After incubation, the plasmid DNA/293fectin® mixture was added dropwise to the cell suspension. Transfected cultures were incubated at 37°C in a multitron incubator.
, 10% CO ₂ and 110 rpm. On day 7, cells were separated from the culture medium by centrifugation (3000 g for 30 minutes) while the supernatant containing the soluble polypeptides of the invention was filtered over 0.2 μm bottle top filters for further processing.

For purification purposes, 1500 ml (s127H1_t2), 1800 ml (s86B4_
t2), and 2400 ml (s55G7_t2) of culture supernatant was washed with washing buffer (20 mM
24 ml of Ni pre-equilibrated in TRIS, 500 mM NaCl, pH 7.8)
Applied to a Sepharose HP column. After a washing step with 10 mM imidazole in wash buffer, bound polypeptide of the invention was eluted with a stepwise gradient of 300 mM imidazole in wash buffer. The elution peak was collected, concentrated and applied to a size exclusion column (Superdex 200) for further purification. The elution profile is shown in FIG. Fractions were collected for 55G7-t2 and 127H1-t2,
Molecular masses were estimated by pooling as indicated in the figure and analyzing by SDS-PAGE (FIG. 6), ELISA and combined analytical size exclusion chromatography with multi-angle light scattering (SEC-MALS). ELISA results confirmed binding of the polypeptides of the invention to CR6261 and CR9114, but not to CR8020. SEC-M
The ALS results are summarized in Table 8.

Figure 5 and Table 8 show that the polypeptide s127H1-t2 of the invention has a higher yield (approximately 30 mg protein per liter of culture supernatant) compared to s55G7-t2 and s86B4-t2. The majority of proteins exhibit a molecular weight of 62 kDa, which is between those predicted for monomers or dimers. To confirm the protein aggregation state, SE
C-MALS experiments were repeated in the presence of Fab fragments derived from CR6261, CR9114 and CR8020. The results are shown in FIG. 7 and summarized in Table 8.

The results show that the soluble form s127H1-t2 of the polypeptide of the invention forms a complex (evidenced by peak shift in the SEC chromatogram) in the presence of Fab fragments from CR6261 and CR9114, whereas CR8020 not shown in the presence of Fab fragments from . This is consistent with the specificity of the Fab fragment binding reaction. That's because CR
6261 and CR9114 bind HA from Group 1, whereas CR8020
are not combined. The sizes of the complexes are listed in Table 8, which is the size of polypeptide s12
7H1-t2 is shown to bind one to two Fab fragments, indicating that at least a portion of the population of purified polypeptide s127H1-t2 of the invention is in dimeric form.

To further analyze the binding reaction between the polypeptide 127H1-t2 of the invention and the mAbs CR6261 and CR9114, and to confirm the presence of conformational epitopes of CR6261 and CR9114, complexes of those antibodies with purified proteins. Conversion was examined by biolayer interferometry (Octet Red ³⁸⁴ , Forte Bio). For this purpose, biotinylated CR6261, CR9114 and CR8020 were immobilized on a streptavidin-coated sensor, which was then first exposed to a solution of the purified polypeptide of the invention to measure the association rate and then washed. Dissociation rates were measured upon exposure to solution. The results are shown in FIG.

Both immobilized CR6261 and CR9114 recognize the polypeptides of the invention as evidenced by a clear response following exposure to the soluble form of 127H1-t2 (Figure 8). To estimate the dissociation constant for binding interactions, titrations were performed using a 2-fold dilution series. Sensors containing immobilized CR6261 or CR9114 were
, 10, 5, 2.5, 1.3 and 0.63 nM concentrations of soluble s127H1-t
The final response was recorded after 6600 seconds of exposure to each of the two solutions. Responses were plotted as a function of stem domain polypeptide concentration and a fit to a steady-state 1:1 binding model was performed, 3.5 nM for CR6261/stem domain polypeptide complex and CR
It yielded a dissociation constant K _d of 2.3 nM for the 9114 complex (Figure 8).

In summary, the polypeptide s127H1-t2 (SEQ ID NO: 91) of the present invention is produced in high amounts and is capable of binding with high affinity to the broadly neutralizing monoclonal antibodies CR6261 and CR9114, and is capable of binding to the corresponding neutralizing epitopes in this stem domain polypeptide. Supports the existence Polypeptides have a propensity to form dimeric structures.

Example 3 Evaluation of Protective Efficacy of Polypeptides of the Invention in a Lethal Influenza Challenge Model Polypeptide s127H1-t of the Invention in a Lethal Influenza Challenge Model
2 (SEQ ID NO: 91), 10 female BALB/c mice (6-8
(weeks of age) groups were immunized three times at 3-week intervals with 10 μg of purified s127H1-t2 unadjuvanted or adjuvanted with 10 μg of Matrix-M. Broadly neutralizing antibody monoclonal antibody CR62 as a positive control for the challenge model
61 (15 mg/kg) was administered intramuscularly one day prior to challenge, while immunization with PBS served as a negative control. Four weeks after the last immunization, mice were challenged with 25×LD50 of heterologous challenge virus (H1N1A/Puerto Rico/8/34) and monitored daily for 3 weeks (survival, body weight, clinical score). Pre-challenge sera were tested for binding to the polypeptide s127H1-t2 of the invention used for immunization (to confirm correct immunization), binding to soluble H1N1A/Brisbane/59/07 full-length HA (to confirm correct immunization), to confirm recognition) and competition with broadly neutralizing antibody monoclonal antibody CR9114 for binding to full-length HA (to determine if the induced antibody binds closely to the broadly neutralizing CR9114 epitope). Test in ELISA assay. The results are shown in Figures 9-12.

The results show that the experiments are valid. This is because all mice in the PBS control group die of infection on day 7 post-challenge, whereas the positive control group (15 mg/kg CR6261, 1 day before challenge) is fully protected (Fig. 9). In contrast to PBS-treated mice, 3 out of 10 mice immunized with the unadjuvanted polypeptide s127H1-t2 of the invention (SEQ ID NO:91) and 3 out of 10 mice immunized with the adjuvanted polypeptide of the invention Ten out of ten mice survived the lethal challenge (see Figure 10). Increased survival rate, increased survival time and decreased clinical score are observed for the group immunized with the polypeptide s127H1-t2 of the invention compared to the PBS control group. Differences are most pronounced for the groups receiving the adjuvanted polypeptides of the invention, but are also observed for the groups receiving the unadjuvanted polypeptides.

ELISA data using s127H1-t2 or soluble full-length HA as antigen
We show that the polypeptide s127H1 of the invention is immunogenic and induces antibodies capable of recognizing full-length HA regardless of adjuvant use (FIGS. 11A and B).

To further understand the immunological response to immunization, a competitive binding ELISA was performed.
For this purpose, plate-bound full-length HA was incubated with serially diluted serum samples,
CR9114-biotin at a given titration concentration is then added. After further incubation, the amount of bound CR9114-biotin is quantified using streptavidin-conjugated horseradish peroxidase according to protocols well known in the art. Data are analyzed using linear regression of OD against logarithmic dilution expressed as "Slope OD" (ΔOD/10-fold dilution). The data show that detectable levels of antibodies capable of competing for binding with the broadly neutralizing antibody CR9114 are induced by immunization with an adjuvanted polypeptide of the invention, with the increased level of competition observed in Figure 12A. as indicated by As a comparison, levels induced by unlabeled CR9114 (ie, self-competition) and unconjugated monoclonal antibodies CR8020 and CR-JB (both serially diluted from a starting concentration of 5 μg/ml) are shown in separate graphs.

Taken together, we found that the polypeptide s127H1-t2 (SEQ ID NO: 91) of the invention
) can protect mice against lethal infection with influenza. The polypeptide is immunogenic and induces antibodies capable of binding full-length HA. When a polypeptide of the invention is used in combination with an adjuvant, at least a portion of the detectable antibody induced binds to or near an epitope of the broadly neutralizing epitope of monoclonal antibody CR9114.

Example 4 Evaluation of Protective Efficacy of Polypeptides of the Invention in a Lethal Influenza Challenge Model Polypeptide s127H1-t of the Invention in a Lethal Influenza Challenge Model
To further evaluate the protective efficacy of 2 (SEQ ID NO: 91), 10 female BALB/c mice (
6-8 weeks old) groups were immunized 1, 2 and 3 times at 3-week intervals with 30 μg of purified s127H1-t2 adjuvanted with 10 μg of Matrix-M. As a positive control for the challenge model, the broadly neutralizing antibody monoclonal antibody CR6261 (
15 mg/kg) was administered intravenously one day prior to challenge, while immunization with PBS served as a negative control. Four weeks after the last immunization, mice were challenged with 25×LD50 of a heterologous challenge virus (H1N1A/Puerto Rico/8/34),
Monitored daily for 3 weeks (survival rate, body weight, clinical score). Pre-challenge serum obtained 4 weeks after the last immunization was treated with the polypeptide s127H of the invention used for immunization.
Binding to 1-t2 (to confirm correct immunization), soluble H1N1A/Brisban
e/59/07 binding to full length HA (to confirm recognition of full length HA) and competition with broadly neutralizing monoclonal antibody CR9114 for binding to full length HA (to determine if it binds in close proximity) in an ELISA assay. The results are shown in Figures 13-18.

The results show that the experiments are valid. This is because all mice in the PBS control group die of infection on day 7 post-challenge, whereas the positive control group (15 mg/kg CR6261, 1 day before challenge) is fully protected (Fig. 13A). s12
Mice immunized once with 7H1-t2 (SEQ ID NO:91) all died of infection between days 7 and 9 (Fig. 14A). In contrast, 8 out of 10 mice survived after 2 immunizations and all mice (10 out of 10) survived the lethal challenge after 3 immunizations (Fig. 14B). , C). Weight loss was also reduced for the multiple immunization group, 3
A minimal percentage was observed for animals immunized twice (Fig. 15B,C). A statistically significant increase in survival rate, increase in survival time, reduction in body weight loss and reduction in clinical score (see FIGS. 16B,C) compared to the PBS control group was observed with the polypeptide s127H1-t2 of the present invention. Observed for groups immunized 2 or 3 times.

ELISA data from the pre-challenge time point 4 weeks after the last immunization using s127H1-t2 (FIG. 17A) or soluble full-length HA (FIG. 17B) as antigen show that the polypeptide s127H1 of the invention is immunogenic. , shows that even after 1 immunization, it induces antibodies capable of recognizing full-length HA, whereas levels are significantly higher after 2 and 3 immunizations. Using the CR9114 competitive binding assay described above, detectable levels of antibodies capable of competing for binding with the broadly neutralizing antibody CR9114 were observed after two and three immunizations with the polypeptide s127H1-t2 of the invention (SEQ ID NO:91). induced (Fig. 18A). As a comparison, levels induced by unlabeled CR9114 (ie, self-competing) and unconjugated monoclonal antibodies CR8020 and CR-JB (both serially diluted from a starting concentration of 5 μg/ml) are shown in separate graphs (FIG. 18B). .

Taken together, we found that the polypeptide s127H1-t2 (SEQ ID NO: 91) of the invention
) can protect mice against lethal infection with influenza. The polypeptide is immunogenic and induces antibodies capable of binding full-length HA. At least a portion of the antibodies that are induced bind to or near an epitope of the broadly neutralizing epitope of monoclonal antibody CR9114.

Example 5 Evaluation of Protective Efficacy of Polypeptides of the Invention in a Lethal Heterosubtypic H5N1 Influenza Challenge Model Polypeptide s127 of the invention in a lethal H5N1 influenza challenge model
To further evaluate the protective efficacy of H1-t2-(SEQ ID NO:91), 8-12 female BAL
Groups of B/c mice (6-8 weeks old) were immunized three times at 3-week intervals with 30 μg of purified s127H1-t2 adjuvanted with 10 μg of Matrix-M. Broadly neutralizing monoclonal antibody CR6261 as a positive control for the challenge model
(15 mg/kg) was administered intravenously one day prior to challenge, while immunization with PBS served as a negative control. Four weeks after the last immunization, mice were challenged with 12.5×LD50 heterosubtypic challenge virus (H5N1A/Hong Kong/156/97) and monitored daily for 3 weeks (survival rate, body weight, clinical score). .

The results show that the experiments are valid. This is because all mice in the PBS control group died of infection between days 8-10 after challenge, whereas the positive control group (15 m
g/kg of CR6261, 1 day before challenge) is fully protected (Fig. 19A).
). 8 out of 10 (80%) immunized with s127H1-t2 (SEQ ID NO: 91)
) mice survive the lethal challenge (FIG. 19B). Average weight loss is about 15% on day 9, but surviving animals recover and gain weight (Fig. 19C). The median clinical score for surviving mice was 1.5 on days 3-6, but no clinical symptoms were observed after day 8 (FIG. 19D). A statistically significant increase in survival rate, increase in survival time, decrease in weight loss and reduction in clinical score compared to the PBS control group was associated with polypeptide s127H1 of the present invention.
Observed for groups immunized with -t2. Taken together, we have shown that immunization with the polypeptide s127H1-t2 (SEQ ID NO:91) of the invention can protect mice against lethal infection by a heterosubtypic H5N1 influenza strain.

Example 6: Assessment of Binding Range of Sera Induced Via Immunization with Polypeptides of the Invention Binding to full-length HA from 1 (H1, H5 and H9) and Group 2 (H3 and H7) influenza strains was also tested by ELISA following protocols well known in the art (Figure 20). The results show that the polypeptide s127H1-
The antibody induced by t2 (SEQ ID NO: 91) efficiently recognizes an epitope present in the native sequence of FL HA and the epitopes to which the antibody binds are different group 1 influenza, including H1, H5 and H9 HA. Demonstrates conservation between strains.

Example 7 Evaluation of Protective Efficacy of Polypeptides of the Invention in a Lethal H1N1A/Brisbane/59/2007 Influenza Challenge Model To further evaluate the protective efficacy of s127H1-t2 (SEQ ID NO:91) in a lethal H1N1 influenza challenge model, 8 Groups of ˜18 female BALB/c mice (6-8 weeks old) were treated with 30 μg purified s1 adjuvanted with 10 μg Matrix-M.
Three immunizations were given with 27H1-t2 at three week intervals. As a positive control for the challenge model, broadly neutralizing monoclonal antibody CR6261 (15 mg/kg) was administered intravenously one day prior to challenge, while immunization with PBS served as a negative control.
Four weeks after the last immunization, mice were challenged with 12.5×LD50 of challenge virus (H1N
1A/Brisbane/59/2007) and monitored daily for 3 weeks (survival, body weight, clinical score).

The results show that the experiments are valid. This is because all mice in the PBS control group died of infection between days 7-10 after challenge, whereas the positive control group (15 m
g/kg of CR6261, 1 day before challenge) was completely protected (Fig. 21A).
). Ten out of ten mice immunized with s127H1-t2 (SEQ ID NO:91) survive the lethal challenge (FIG. 21B). Furthermore, weight loss averages about 20% at 5 days post-infection (Fig. 21C), but animals fully recover within the 21-day follow-up period. Median clinical scores peak at a value of 3 between days 2 and 9 post-infection, but return to baseline levels (0) after day 16 post-infection (Fig. 21D). PB
A statistically significant increase in survival rate, increased survival time, decreased weight loss and decreased clinical score were observed for the group immunized with the polypeptide s127H1-t2 of the invention compared to the S control group. be.

Taken together, we found that the polypeptide s127H1-t2 (SEQ ID NO: 91) of the invention
) can protect mice against lethal infection by H1N1A/Brisbane/59/2007.

Example 8 Evaluation of the Presence of Influenza Neutralizing Antibodies in Sera of Mice Immunized with Polypeptides of the Invention To further investigate the antibody-mediated effector mechanisms responsible for protection against influenza, pre-challenge sera were H5N1A/Vietnam/1194/0 below
Pseudoparticle neutralization assay using pseudoparticles derived from 4 (Alberini et al.
009).

Pseudoparticle Neutralization Assay Pseudoparticles expressing FL HA were generated as previously described (Temperton et al.
al. , 2007). As previously described (Alberini et al 2009)
(slightly modified), H5A/Vietna encoding luciferase reporter gene
Neutralizing antibodies were determined using a single transduction round of HEK293 cells with m/1194/04 pseudoparticles. Briefly, heat-inactivated (30 min at 56° C.) pre-challenged serum samples were placed in growth medium (2 mM L-glutamine (Lonza), 1% nonessential amino acids solution (Lonza), 100 U/ml penicillin/ Streptomycin (Lonza
) and E supplemented with 10% FBS (Euroclone, Pero, Italy)
MEM Eagle with BSS (Lonza, Basel, Switzerland
)) in 3-fold serial dilutions in triplicate in 96-well flat-bottom culture plates and titrated numbers of H5
A/Vietnam/1194/04 pseudoparticles (10 ⁶ relative luminescence units (RLU) after infection)
) was added. After 1 hour incubation at 37° C., 5% CO ₂ , 10 ⁴ HEK293 cells were added per well. After 48 hours of incubation at 37°C, 5% _CO2 , luciferase substrate (Britelie Plus
, Perkin Elmer, Waltham, Mass.) was added and a luminometer (Mi
thras LB 940, Berthold Technologies, Germany
ny) was used to measure luminescence according to the manufacturer's instructions.

Pre-challenged sera obtained from animals immunized with the polypeptide s127H1-t2 of the invention (SEQ ID NO:91) described in Examples 5, 6, and 7 showed high sera using the pseudoparticle neutralization assay. concentration showed detectable neutralization (Figure 22). This demonstrates the ability of the polypeptides of the invention to elicit broadly neutralizing antibodies when used as immunogens.

In addition to direct virus neutralization, Fc-mediated effector mechanisms such as antibody-dependent cellular cytotoxicity (A
DCC) and antibody-dependent cellular phagocytosis (ADCP) contribute substantially to protection against influenza, and stem-directed bnAbs are particularly effective at these mechanisms (DiLil
lo et al. , 2014). Polypeptide s127H1-t2l18lo of the invention
To test whether antibodies induced after immunization with ng (SEQ ID NO: 186) can induce ADCC, we performed an ADCC surrogate assay adapted for mice as described below (Parekh et al. , 2012;
. , 2012; Cheng et al. , 2014) was used to test pre-challenge sera.

Antibody-dependent cellular cytotoxicity (ADCC) surrogate assay Human lung cancer-derived A549 epithelial cells (ATCC CCL-185) were grown at 37°C in Dulbecco's Modified Eagle's Medium (DMEM) medium supplemented with 10% heat-inactivated fetal bovine serum. , 1
Maintained at 0% CO2. Two days before the experiment, Opti-MEM (Invitrogen
) using Lipofectamine 2000 (Invitrogen) in A5
49 cells with H5A/Hong Kong/156/97 HA or H1A/Brisb
Plasmid DNA encoding ane/59/2007 HA was transfected. One day before assay, transfected cells were harvested and plated in white 96-well plates (Co
star) and for imaging in black clear-bottom 96-well plates (BD F
alcon). After 24 hours, samples were added to assay buffer (RPMI 1640 (G
ibco), heat-inactivated at 56° C. for 30 min, then serially diluted in assay buffer. For the ADCC bioassay, A549 cells were replenished with fresh assay buffer, antibody dilutions and mouse Fc
ADCC Bioassay Jurkat effector cells expressing gamma receptor IV (FcγRIV; Promega) were added to the cells and incubated for 6 hours at 37° C. at a target-effector ratio of 1:4.5. Cells were equilibrated to room temperature for 15 minutes before adding Bio-Glo Luciferase System substrate (Promega). Luminescence was read out after 10 minutes on a Synergy Neo (Biotek). Data are expressed as fold induction of signal in the absence of serum.

Using this assay, the polypeptide s12 of the invention described in Examples 5, 6 and 7
Pre-challenged sera obtained from animals immunized with 7H1-t2 (SEQ ID NO: 91) were used with H5N1A/Hong Kong/156/97 or H1N1 as a source of antigen.
Target cells transfected with FL HA from A/Brisbane/59/07 were used to test for FcγRIV signaling activity (Figure 23). In both cases, a 30-fold induction was observed at the highest serum concentration tested, indicating that ADCC/AD
We demonstrate the ability of the polypeptides of the invention to elicit antibodies that activate FcγRIV signaling indicative of CP effector function.

These results, shown in Examples 5-8, demonstrate that the polypeptide s127H1-
We show that the ability of t2 (SEQ ID NO: 91) to elicit stem-targeting, neutralizing and ADCC-mediated antibodies can protect mice against lethal challenge by homologous, heterologous and heterosubtypic Group I influenza strains.

Example 9: Protection from lethal challenge with H5N1A/Hong Kong/156/97 by passive transfer of serum from mice immunized with polypeptides of the invention. Implantation studies were performed to determine antibody contribution. The purpose of this study was to test s127 H1-t2 (SEQ ID NO: 91) and an additional His-tag containing s127H1-t2 (SEQ ID NO: 91) in the presence of an adjuvant (Matrix-M).
Passive transfer (multiple doses) of sera from mice immunized three times with H1-t2long (SEQ ID NO: 101) resulted in H5N1 influenza A/Hong Kong/156/97
to assess whether it conferred protection against lethal challenge by

Groups of female BALB/c donor mice (6-8 weeks old) were treated with 30 μg of s127H1-t2 (SEQ ID NO: 91) adjuvanted with 10 μg of Matrix-M, s127H1-t2 long (SEQ ID NO: 91) containing a C-terminal His-tag. No. 101) or PBS immunized 3 times at 3-week intervals. Four weeks after the last immunization (day 70), sera were isolated, pooled by group and treated with recipient mice (female BALB/c, 6-8 weeks old, n per group).
= 10). Each mouse received 400 μl of serum intraperitoneally for three consecutive days (days -3, -2 and -1) prior to challenge. As a positive control for the challenge model, CR6261 (15 mg/kg) was administered 1 day before challenge (n =
8), injection with PBS served as a negative control (n=8). On day 0, mice were challenged with 12.5×LD50 of challenge virus and monitored for 3 weeks (survival rate, body weight, clinical score).

Pooled serum samples of peripheral blood of donor mice (day 70) to confirm the immunogenicity of the polypeptides of the invention in donor mice and to assess HA-specific antibody levels after transplantation of serum into recipient mice. , pooled serum samples from naïve recipient mice prior to serum transfer (day −4) and individual serum samples from recipient mice after three serum transfers immediately prior to challenge (day 0) were analyzed by H1N1A/Brisbane/59/ Binding to FL HA from 07 was tested in ELISA.

Results Challenge—The experiment was successful; all mice in the PBS control group died of infection before day 13 post-challenge (median 9.5 days), while the positive control group (15 mg/kg CR6
261, 1 day before challenge) are fully protected (p<0.001).
- Three serum transfers of serum from mice immunized with the Matrix-M adjuvanted polypeptide SEQ ID NO: 91 of the invention into naive recipient mice resulted in significant survival times compared to the PBS serum transfer control group (p=0.007) and decreased clinical score (p=0.012) (FIG. 24).
- three serum transfers of sera from mice immunized with the Matrix-M adjuvanted polypeptide SEQ ID NO: 101 of the invention into naive recipient mice, PBS
Significantly increased survival rate (p=0.002), increased survival time (
p<0.001), decreased weight loss (p=0.002) and decreased clinical score (p<0
. 001) (Fig. 24).
- FL HA A/Br tested after 3 serum transfers for the polypeptides of the invention
The isbane/59/07-specific antibody titers were similar to the levels obtained after active immunization (Figure 25).

CONCLUSION Polypeptides SEQ ID NO: 91 and 101 of the Invention Adjuvanted with Matrix-M
Serum components (most likely antibodies) induced by three immunizations with
Mice can be protected from lethal challenge with H5N1A/Hong Kong/156/97 (survival rates are 30 and 78%, respectively).

Example 10 In Vivo Protective Efficacy of Polypeptides of the Invention in the H1N1A/NL/602/09 Challenge Model in Mice The protective potency of polypeptide s127H1-t2 (SEQ ID NO:91) and s127H1-t2long (SEQ ID NO:101) containing an additional His-tag was determined.

Groups of 10 female BALB/c mice (6-8 weeks old) were immunized three times at 3-week intervals with 30 μg of polypeptide of the invention with 10 μg of Matrix-M. As a positive control for the challenge model, CR6261 (15 mg/kg) was administered 1 day prior to challenge (n=8), while injection with PBS served as a negative control (n=1
8). Four weeks after the last immunization, mice were challenged with 12.5×LD50 of challenge virus and monitored for 3 weeks (survival rate, body weight, clinical score).

To confirm the immunogenicity of the polypeptides of the invention, pre-challenge sera (day -1) were subjected to ELI for binding to FL HA from H1N1A/Brisbane/59/07.
Tested in SA assay. A CR9114 competition ELISA was performed to determine whether the induced antibodies bind closely to the CR9114 epitope. competitive data,
Responses were expressed using quantifiable slope OD.

Results—The experiment was successful; all mice in the PBS control group died of infection before 8 days post-challenge (median 5 days), whereas the positive control group (15 mg/kg CR6261
, 1 day before challenge) are fully protected (p<0.001).
- Three immunizations with s127H1-t2 (SEQ ID NO: 91) adjuvanted with Matrix-M and s127H1-t2 long (SEQ ID NO: 101) containing an additional His-tag resulted in a significant survival rate compared to the PBS control group (p<0.001), increased survival time (p<0.001) and decreased clinical score (p<0.001) (Fig. 2).
6).
- H1 mini-HA variant s127H1 with Matrix-M adjuvant
Three immunizations with -t2 (SEQ ID NO:91) result in significant weight loss (p<0.001) compared to the PBS control group (Figure 26).
- H1N1A/Brisbane/59/07 induced by a polypeptide of the invention
IgG antibody titers against FL HA are significantly higher compared to PBS for all H1H1 mini-HA variants tested (p<0.001) (FIG. 27A).
- H1 mini-HA variant s127H1-t2 (SEQ ID NO: 91) contains an additional H
It has significantly higher IgG antibody titers against H1N1A/Brisbane/59/07FL HA compared to s127H1-t2long (SEQ ID NO: 101) containing the is-tag (p=0.021) (FIG. 27A).
- all Matrix-M adjuvanted polypeptides of the invention tested were PB
have significantly higher CR9114 competitive titers compared to S (p<0.001) (Fig. 27B)
.

Conclusion:
Matrix-M adjuvanted polypeptide s127H1-t2 of the invention (SEQ ID NO: 91) and s127H1-t2 long (SEQ ID NO: 10) containing an additional His-tag
1) confers protection against lethal challenge by H1N1A/NL/602/09;
Confirmed as survival rate, increased survival time and decreased clinical score. Furthermore, Matr
ix-M adjuvanted s127H1-t2 (SEQ ID NO: 91) is H1N1A/NL
It also resulted in reduced weight loss after lethal challenge with /602/09.

Example 11: Library Screening PCT/EP2012/073706 describes influenza hemagglutinin stem domain polypeptides, compositions and vaccines and influenza prophylaxis and/or
or methods of their use in the field of therapy. Here, we find that H
Additional sequences for stem domain polypeptides derived from full-length HA of 1N1A/Brisbane/59/2007 (SEQ ID NO: 1) are described. The stem domain polypeptide is H1-
Obtained by site-directed mutagenesis of mini2-cluster1+5+6-GCN4t2 (SEQ ID NO: 52), CR6261 (Throsby et al, 2009; Ekie
rt et al 2010) and/or present broad-spectrum influenza neutralizing epitopes of CR9114.

H1-mini2-cluster1+5+6-GCN4t2 (SEQ ID NO: 52) is H
It was derived from the full-length HA of 1N1A/Brisbane/59/2007 (SEQ ID NO: 1) by utilizing the following steps:
- Removing the HAO cleavage site. Cleavage of wild-type HA at this site yields HA1 and HA2. Removal can be achieved by an R to Q mutation at position P1 (e.g., see S
See Un et al, 2010.
- Removing the head domain by deleting amino acids 53 to 320 from SEQ ID NO:1. The remaining N- and C-terminal portions of the sequence were joined by a four-residue flexible linker GGGG.
- residues 402 to 41 in H1A/Brisbane/59/2007 (SEQ ID NO: 1)
Increase the solubility of the loop (between the A and CD helices) formed by (the equivalent of) 8 to increase the stability of the pre-fusion conformation and destabilize the post-fusion conformation of the modified HA. thing. H1-mini2-cluster1+5+6-GCN4 (SEQ ID NO: 2)
introduced the mutations F406S, V409T, F413G and L416S (numbering refers to SEQ ID NO: 1).
- introducing a disulfide bridge between amino acids at positions 324 and 436 in H1A/Brisbane/59/2007;
This is achieved by introducing C (numbering refers to SEQ ID NO: 1).
- introducing the GCN4-derived sequence RMKQIEDKIEEIESK (SEQ ID NO: 20), which is known to trimerize, at positions 419-433 (numbering refers to SEQ ID NO: 1).

In certain embodiments, the polypeptides of the invention contain the intracellular sequence and transmembrane domain of HA. In other embodiments, the transmembrane and intracellular domain sequences are 519, 520 of HA2, such that a secreted (soluble) polypeptide is produced after expression in the cell.
, 521, 522, 523, 524, 525, 526, 526, 527, 528, 529
, or C of HA2 from position 530 (or its equivalent as determined from sequence alignment)
Deleted to the end (numbering according to SEQ ID NO: 1). The soluble polypeptide has sequences known to form trimeric structures, ie the foldon sequence AYVRKDGEWVL
Further stabilization can be achieved by introducing L (SEQ ID NO:3), optionally linked via a short linker as described above. Linkers may optionally contain cleavage sites for subsequent processing according to protocols well known to those skilled in the art. To facilitate purification and detection of soluble forms, tag sequences such as histidine tags (HHHHHHH (SEQ ID NO: 16) or HHHHHH (SEQ ID NO: 15) or FLAG tags (DYKDDDDK;
SEQ ID NO: 22) or a combination thereof optionally linked via a short linker can optionally be added. The linker optionally includes (part of) a proteolytic cleavage site for subsequent processing according to protocols well known to those skilled in the art, e.g.
It may contain GS (SEQ ID NO:23) (thrombin) or IEGR (SEQ ID NO:24) (factor X). Processed proteins are also included in the present invention.

An example of such a C-terminal sequence combining a FLAG tag, a thrombin cleavage site, a foldon, and a His sequence is FLAG-thrombin-foldo of SEQ ID NO:4.
n-His. This sequence is H1-mini2-cluster1+5+6-GCN4
A parental sequence (SEQ ID NO: 156) was generated in combination with a soluble form of the t2 (SEQ ID NO: 51) sequence and used to generate novel polypeptides of the invention by mutagenesis. This sequence does not contain a leader sequence corresponding to amino acids 1-17 of SEQ ID NOS:1 and 2.

Stem domain polypeptides lack a portion of the hemagglutinin sequence encoding the head domain of the molecule as described in PCT/2012/073706 and supra;
The N- and C-terminal portions of the sequence are generated by religating via linkers on either side of the deletion. Removal of the head domain leaves a portion of the molecule already protected from aqueous solvent exposed, potentially destabilizing the structure of the polypeptides of the invention. For this reason, residues in the B-loop (particularly amino acid residue 406 (F and S of SEQ ID NOs: 1 and 2, respectively), 4
09 (V and T) 413 (F and G) and 416 (L and S) were mutated in various combinations using parental sequence SEQ ID NO: 156 as a starting point. SEQ ID NO: 15
6 removes the leader sequence from H1-mini2-cluster1+5+6-GCN4t2 (SEQ ID NO: 52) and replaces residues 520-565 with Flag-thrombin-fol
It was created by replacing it with the don--his sequence (SEQ ID NO:4).

Similarly, in the area around the fusion peptide, many hydrophobic residues are exposed to solvent, which means that, unlike native full-length HA, the polypeptides of the invention cannot be cleaved, leaving the hydrophobic fusion within the protein. Caused by the fact that embedding peptides undergo related conformational changes. To address this issue, some or all of residues I337, I340, F352 and I353 of SEQ ID NO:156 were also mutated.

Two different sets of mutant polypeptides are disclosed in Table 9. In all cases
These polypeptides contain SEQ ID NO:20 at positions 419-433 (numbering refers to SEQ ID NO:1).

Example 12 Identification, Purification and Characterization of Trimeric Polypeptides of the Invention Libraries (Set 1 and Set 2) of the polypeptides described in Example 11 containing SEQ ID NO:20 at positions 419-433 were generated. did. Single clones into HEK293F cells and screen culture media for multimers (CR9114 sandwich ELISA), CR6261 binding (ELISA) and protein expression (HTRF assay)
Individually transfected. CR9114 sandwich assay, CR9114, CR6261
, and confirmed hits based on CR8020 ELISA, and HTRF assay,
ranked.

Cross-linking with primary amine (present in lysine residues) specific cross-linker BS3 followed by SD
Multimerization was assessed by S-PAGE (see below). C-terminal F for extensive multimerization
The lag-Foldon-His (FFH) tag sequence was replaced by a thrombin cleavage site and his tag sequence (TCHis). Subsequently, multimerization of TCS-his containing sequences was rechecked (CR9114 sandwich assay, BS3 cross-linking) and clones were ranked and selected. Selected clones were expressed, purified and characterized.

Crosslinking assays were performed as follows:
• Add the crosslinker BS3 (bis(sulfosuccinimidyl) suberate) directly to the culture medium • Incubate for 30 minutes at room temperature.
- Harvest the medium and perform SDS-P under reducing (R, 5 mM DTT) and non-reducing (NR) conditions
Analyze by AGE/Western blot Under reducing conditions, only BS3 crosslinked species remain covalently bound mini-HA via Western blotting using a his-tag specific mAb
to detect

result:
1. Two libraries of high quality (>90% of the corrected ORFs) containing SEQ ID NO:20 at positions 419-433 and the predicted sequence variation (>97% randomization) were successfully generated. Total 10472 clones (5544 and 4928 from sets 1 and 2 respectively)
was evaluated in the initial screen (Figure 28)
3. >50% expression of FL HA expression and >50% of the signal observed for FL HA
Clones showing 80% binding signal to CR6261 were considered hits;
658 of the 4.703 hits that gave rise to 03 hits (596 and 107 from libraries 1 and 2, respectively) were retained after the confirmation screen. Cross-linking assays of the top 20% hits (111) indicated the presence of higher order multimers that could potentially interfere with the purification of trimeric species.
6. The top 20% confirmed hits (111) were successfully cloned to replace the FFHC ends by TCS-his sequences and then evaluated by CR9114 sandwich ELISA and cross-linking assay7. The cross-linking assay yielded 9 clones that were considered the most promising trimer candidates (
SEQ ID NOS: 158-166, Table 11). CR9114 sandwich ELISA (Figure 29)
based on 3 candidates (2 with TCS-his and 1 with FFH C-terminus)
was selected for expression and purification8. Two of the selected candidates did not express well and were not further purified. Candidate GW1.5E2. F.
FH (SEQ ID NO: 158) was purified to homogeneity (7.6 mg
total protein; >95% purity, HP-SEC).
9. GW1.5E2. Characterization of FFH (SEQ ID NO: 158) shows trimerization in solution, with three Fab fragments of CR9114 or CR6261 bound per trimer (Figure 30 and Table 10 below). Biolayer interferometry (Oc
tet) is 1 nM for both _CR6261 and ^CR9114 . As expected, no binding of CR8020 (negative control) could be detected by either method.

Conclusion: The non-covalent trimeric polypeptide of the invention (GW1.5E2.FFH, SEQ ID NO: 1) binds bnAbs CR6261 and CR9114 with high affinity at a 3:1 stoichiometry.
58) were identified.

Example 13: Protective Efficacy of Polypeptide sH1 mini-HA GW1.5E2-FFH (SEQ ID NO: 158) of the Invention in H1N1A/Brisbane/59/07 Mouse Model H1N1A/Brisbane/59/07 compared to PBS control group sH1 mini-HA GW1 adjuvanted with Matrix-M in a challenge model
. The protective potency of 5E2-FFH (SEQ ID NO: 158) was determined.

Groups of 10 female BALB/c mice (6-8 weeks old) were treated with 30 μg sH1mini-HA GW1.5E2-FFH (SEQ ID NO: 158) adjuvanted with 10 μg Matrix-M at 3-week intervals. Immunized 3 times. As a positive control for the challenge model, CR6261 (15 mg/kg) was administered one day before challenge (
n=8), injection with PBS served as a negative control (n=16). Four weeks after the last immunization, mice were challenged with 12.5×LD50 of challenge virus and monitored for 3 weeks (survival rate, body weight, clinical score).

To confirm the immunogenicity of sH1 mini-HA GW1.5E2-FFH (SEQ ID NO: 158), pre-challenge sera (day -1) were administered to H1N1A/Brisbane/59
Binding to FL HA from /07 was tested in an ELISA assay. To determine whether polypeptide sH1 mini-HA GW1.5E2-FFH (SEQ ID NO: 158)-derived antibodies of the invention bind closely to the CR9114 epitope, CR911
4 competitive ELISA was performed. Competitive data is visualized as "% Competitive" and (AP)/A
× 100), where A is CR91 to FL HA in the absence of serum
is the maximum OD signal of 14 binding and P is the FL HA in the presence of serum at the given dilution
is the OD signal of CR9114 binding to or is expressed using a slope OD metric that can quantify the response; for reference CR9114 and CR8020 (starting concentration 5 mg/ml
) solution was included.

result:
- The experiment was valid; all mice in the PBS control group (n=16) died of infection before day 10 post-challenge (median 8 days), whereas the positive control group (n=8, 15 m
g/kg CR6261, 1 day prior to challenge) is fully protected (p<0.001).
- sH1 mini-HA GW1.5E adjuvanted with Matrix-M
Three immunizations with 2-FFH (SEQ ID NO: 158) significantly increased survival rate (p<0.001), increased survival time (p<0.001), increased body weight compared to the PBS control group. Resulting in reduced losses (p<0.001) and reduced clinical scores (p<0.001) (Figure 31).
- pre-challenge Ig against H1N1A/Brisbane/59/07FL HA induced by sH1 mini-HA GW1.5E2-FFH (SEQ ID NO: 158)
G antibody titers are significantly higher compared to PBS (p≦0.001) (FIG. 32A).
- IgG antibody titers against H1N1A/Brisbane/59/07FL HA are
Plateau after two immunizations (not shown).
- Matrix-M adjuvanted polypeptide sH1 mini-HA of the invention
GW1.5E2-FFH (SEQ ID NO: 158) has significantly higher CR911 compared to PBS
4 induces competitive titers (p<0.001) (FIG. 32B).

Conclusion: Matrix-M Adjuvanted Polypeptide sH1 mini-H of the Invention
A GW1.5E2-FFH (SEQ ID NO: 158) is H1N1A/Brisbane/5
Provides protection against lethal challenge on 9/07.

Example 14: Polypeptide sH1 mini-HA GW1.5E2-FFH of the Invention in the H5N1A/Hong Kong/156/97 Mouse Model (SEQ ID NO: 158)
Superior H1 mini-HA adjuvanted with Matrix-M in H5N1A/Hong Kong/156/97 challenge model compared to PBS control group
The protective potency of the variants was determined.

Groups of 10 female BALB/c mice (6-8 weeks old) were treated with 30 μg of the polypeptide of the invention sH1 mini-HA GW adjuvanted with 10 μg of Matrix-M.
1.5E2-FFH (SEQ ID NO: 158) was immunized three times at three week intervals. As a positive control for the challenge model, CR6261 (15 mg/kg) was administered 1 day prior to challenge (n=8), while injection with PBS served as a negative control (n=1
6). Four weeks after the last immunization, mice were challenged with 12.5×LD50 of challenge virus and monitored for 3 weeks (survival rate, body weight, clinical score).

Polypeptide sH1 mini-HA GW1.5E2-FFH of the invention (SEQ ID NO: 1
58), pre-challenge sera (day -1) were tested against H1N1A/B
Binding to FL HA from risbane/59/07 was tested in an ELISA assay. To determine whether the mini-HA derived antibodies bind closely to the CR9114 epitope, a CR9114 competition ELISA was performed. Competitive data as "% Competitive"
and defined as (A−P)/A×100), where A is the maximum OD signal for CR9114 binding to FL HA in the absence of serum and P is the maximum OD signal at a given dilution. is the OD signal of CR9114 binding to FL HA in the presence of serum at or expressed using a slope OD metric that can quantify the response; for reference CR9114 and CR
8020 (starting concentration 5 μg/ml) solution was included.

result:
- The experiment was successful; 15 out of 16 mice in the PBS control group died of infection before day 9 post-challenge (median 9 days), whereas the positive control group (n=8, 15 m
g/kg CR6261, 1 day prior to challenge) is fully protected (p<0.001).
- sH1 mini-HA GW1.5E adjuvanted with Matrix-M
Three immunizations with 2-FFH (SEQ ID NO: 158) significantly increased survival rate (p<0.001), increased survival time (p<0.001), increased body weight compared to the PBS control group. Resulting in reduced losses (p<0.001) and reduced clinical scores (p<0.001) (Figure 33).
- pre-challenge IgG antibody titers against H1N1A/Brisbane/59/07FL HA induced by the polypeptide sH1 mini-HA GW1.5E2-FFH of the invention (SEQ ID NO: 158) are significantly higher compared to PBS ( p≦0.001) (
Figure 34A).
- Matrix-M adjuvanted polypeptide sH1 mini-HA of the invention
GW1.5E2-FFH (SEQ ID NO: 158) has significantly higher CR911 compared to PBS
4 induces competitive titers (p<0.001) (FIG. 34B).

Conclusion: Matrix-M Adjuvanted Polypeptide sH1 mini-H of the Invention
A GW1.5E2-FFH (SEQ ID NO: 158) is H5N1A/Hong Kong/
Confer heterosubtypic protection against lethal challenge with 156/97.

Example 15: Polypeptide sH1 mini-HA GW1.5E2-FFH of the Invention in the H1N1A/Puerto Rico/8/34 Mouse Model (SEQ ID NO: 158)
Protective efficacy of Matrix-M adjuvanted polypeptides of the invention in the H1N1A/Puerto Rico/8/1934 challenge model compared to the PBS control group
The protective potency of H1 mini-HA GW1.5E2-FFH (SEQ ID NO: 158) was determined.

Groups of 10 female BALB/c mice (6-8 weeks old) were treated with 30 μg of the polypeptide of the invention sH1 mini-HA GW adjuvanted with 10 μg of Matrix-M.
1.5E2-FFH (SEQ ID NO: 158) was immunized three times at three week intervals. As a positive control for the challenge model, CR6261 (15 mg/kg) was administered 1 day prior to challenge (n=8), while 3 immunizations with PBS served as a negative control (n=16). Four weeks after the last immunization, mice were challenged with 25xLD50 of challenge virus and monitored for 3 weeks (survival rate, body weight, clinical score).

Polypeptide sH1 mini-HA GW1.5E2-FFH of the invention (SEQ ID NO: 1
58), pre-challenge sera (day -1) were challenged with H1N1A/Br
Binding to FL HA from isbane/59/07 was tested in an ELISA assay. To determine whether the mini-HA derived antibodies bind closely to the CR9114 epitope, a CR9114 competition ELISA was performed. Competition data were visualized as "% competition" and defined as (AP)/A x 100), where A is the maximum OD signal for CR9114 binding to FL HA in the absence of serum; P is the OD signal of CR9114 binding to FL HA in the presence of serum at a given dilution, or expressed using a slope OD metric that can quantify the response; CR9114 and CR8 for reference
020 (starting concentration 5 μg/ml) solution was included.

Results—The experiment is valid; all mice in the PBS control group (n=16) die of infection before day 9 post-challenge (median 8 days), whereas the positive control group (n=8, 15 mg/
kg of CR6261, 1 day prior to challenge) are fully protected (p<0.001).
- the polypeptide sH1 mini of the invention adjuvanted with Matrix-M
Three immunizations with -HA GW1.5E2-FFH (SEQ ID NO: 158) resulted in significantly increased survival rate (p<0.001), increased survival time (p<0.001) compared to PBS control group. 00
1), resulting in decreased weight loss (p<0.001) and decreased clinical score (p<0.001) (Figure 35).
- pre-challenge IgG antibody titers against H1N1A/Brisbane/59/07FL HA induced by the polypeptide sH1 mini-HA GW1.5E2-FFH of the invention (SEQ ID NO: 158) are significantly higher compared to PBS ( p<0.001) (
Figure 36A).
- Matrix-M adjuvanted polypeptide sH1 mini-HA of the invention
GW1.5E2-FFH (SEQ ID NO: 158) has significantly higher CR911 compared to PBS
4 induces a competitive titer (p<0.001) (Fig. 36B).

Conclusion: Matrix-M Adjuvanted Polypeptide sH1 mini-H of the Invention
A GW1.5E2-FFH (SEQ ID NO: 158) is H1N1A/Puerto Ric
confers protection against lethal challenge by o/8/34.

REFERENCES
Alberini et al. (2009), Vaccine 27:5998-6003.
Bommakanti et al. (2010), PNAS 107(31): 13701-13706.
Bommakanti et al. (2012), J Virol 86:13434.
Cheng et al. (2014), J. Immunol. Methods 1-13. (doi: 10.1016/j.jim.2014.07.010)
Coffman et al. (2010), Immunity 33:492.
Devereux et al. (1984), Nucl. Acids Res. 12:387.
DiLillo et al. (2014), Nat Med 20, 143.
Dopheide TA, Ward CW. (1981) J Gen Virol. 367-370
Ekiert et al. (2009), Science 324:246.
Ekiert et al. (2011), Science 333:844.
Ferguson et al. (2003), Nature 422:428-443.
Lorieau et al. 2010, Proc. Natl. Acad. Sci. USA, 107:11341.
Lu et al. (2013), www. pnas. org/cgi/doi/10.1073/pnas. 1308701110.
Mallajosyula et al. (2014). , www. pnas. org/cgi/doi/10.1073/pnas. 1402766111.
Parekh et al. (2012), mAbs 4:310.
Schnueriger et al. (2011), Molecular immunology 48:1512.
Steel et al. (2010), mBio 1(1):1-9.
Steven et al. (2004) Science 303:1866.
Steven et al. (2006) Science 312:404.
Temperton et al. (2007) Viruses 1:105-12.
Throsby et al. (2008), Plos One 12(3):1-15.
Wilson et al (1981) Nature 289:366.
In addition, the present invention provides the following.
[1]
An influenza hemagglutinin stem domain polypeptide, comprising:
(a) an influenza hemagglutinin HA1 domain comprising a HA1 N-terminal stem segment covalently linked to the HA1 C-terminal stem segment by a linking sequence of 0-50 amino acid residues, said HA1 C-terminal segment comprising:
(b) binds to influenza hemagglutinin HA2, wherein said HA1 and HA2 domains are derived from influenza A virus subtypes including HA of H1 subtype;
(c) does not contain a protease cleavage site at the junction between said HA1 and HA2 domains;
(d) said HA1 N-terminal segment comprises amino acids 1-x of HA1, preferably amino acids p-x of HA1, and said HA1 C-terminal stem segment comprises amino acids y-C-terminal amino acids of HA1, x=SEQ ID NO: p = the amino acid on position 18 of SEQ ID NO: 1 (or the corresponding position in the hemagglutinin of another influenza virus strain) of SEQ ID NO: 1 (or the corresponding position in the hemagglutinin of another influenza virus strain). , y=amino acid on position 320 of SEQ ID NO: 1 (or the equivalent position in another hemagglutinin);
(e) the region comprising amino acid residues 402-418 comprises the amino acid sequence X ₁ NTQX ₂ TAX ₃ GKEX ₄ N(H/K)X ₅ E(K/R) (SEQ ID NO: 8);
X ₁ is an amino acid selected from the group consisting of M, E, K, V, R and T;
X2 is an amino acid selected from the group consisting of F, I, N, T, H, L and Y, preferably I, L or Y _;
X3 is an amino acid selected from the group consisting of V, A, G, I, R, F and S, preferably A, I or F _;
X4 is an amino acid selected from the group consisting of F, I, N, S, T, Y, E, K, M and V, preferably I, Y, M or V _;
X ₅ is an amino acid selected from the group consisting of L, H, I, N, R, preferably I;
(f) the amino acid residue on position 337 (HA1 domain) is selected from the group consisting of I, E, K, V, A, and T;
the amino acid residue on position 340 (HA1 domain) is selected from the group consisting of I, K, R, T, F, N, S and Y;
the amino acid residue on position 352 (HA2 domain) is selected from the group consisting of D, V, Y, A, I, N, S, and T;
the amino acid residue on position 353 (HA2 domain) is selected from the group consisting of K, R, T, E, G, and V;
(g) further comprising a disulfide bridge between the amino acid on position 324 and the amino acid on position 436;
(h) In addition, the amino acid sequence RMKQIEDKIEEIESK (SEQ ID NO:20) has been introduced at positions 419-433, or the sequence RMKQIEDKIEEIESKQK (SEQ ID NO:21) has been introduced at positions 417-433.
Polypeptide.
[2]
The polypeptide of [1], wherein the HA2 domain is truncated.
[3]
The polypeptide of [1] or [2], wherein the C-terminal portion of said HA2 domain from position 519 to the C-terminal amino acid is deleted.
[4]
The polypeptide of [1] or [2], wherein the C-terminal portion of said HA2 domain from position 530 to the C-terminal amino acid is deleted.
[5]
The polypeptide of [3] or [4], wherein the C-terminal part of said HA2 domain is replaced by the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 3), optionally linked via a linker.
[6]
Any one of [1] to [5], wherein the C-terminal amino acid residue of said HA1 C-terminal stem segment is any amino acid other than arginine (R) or lysine (K), preferably glutamine (Q) The polypeptide according to .
[7]
A polypeptide according to any one of [1] to [6], comprising one or more additional mutations in said HA1 domain and/or said HA2 domain.
[8]
The polypeptide of any one of [1]-[7], which selectively binds to antibodies CR6261 and/or CR9114.
[9]
The polypeptide of any one of [1]-[8], which does not bind to antibodies CR8020 and/or CR8057.
[10]
A nucleic acid molecule encoding the polypeptide of any one of [1] to [9].
[11]
A vector comprising the nucleic acid molecule of [10].
[12]
A composition comprising the polypeptide of any one of [1] to [9] and/or the nucleic acid molecule of [10].
[13]
The polypeptide according to any one of [1] to [9], the nucleic acid molecule according to [10], and/or the vector according to [11], which is used as a pharmaceutical.
[14]
The polypeptide according to any one of [1] to [9], the nucleic acid molecule according to [10], and/or the vector according to [11], for use in inducing an immune response against influenza virus.
[15]
The polypeptide according to any one of [1] to [9], the nucleic acid molecule according to [10], and/or the vector according to [11], for use as a vaccine.

配列番号３：ｆｏｌｄｏｎ
ＧＹＩＰＥＡＰＲＤＧＱＡＹＶＲＫＤＧＥＷＶＬＬＳＴＦＬ
配列番号４：ＦＬＡＧ－ｔｈｒｏｍｂｉｎ－ｆｏｌｄｏｎ－ＨＩＳ
ＳＧＲＤＹＫＤＤＤＤＫＬＶＰＲＧＳＰＧＳＧＹＩＰＥＡＰＲＤＧＱＡＹＶＲＫＤＧＥＷ
ＶＬＬＳＴＦＬＧＨＨＨＨＨＨ
配列番号５：
ＭＫＱＩＥＤＫＩＥＥＩＥＳＫＱ

sequence listing

SEQ ID NO: 3: foldon
GYIPEAPRDGQAYVRKDGEWVLLSTFL
SEQ ID NO: 4: FLAG-thrombin-foldon-HIS
SGRDYKDDDDKLVPRGSPGSGYIPEAPRDGQAYVRKDGEW
VLLSTFLGHHHHHH
SEQ ID NO:5:
MKQIEDKIEEIESKQ

Claims (13)

Amino acid sequence:

In the sequence, the combination of ( X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ , X ₇ , X ₈ , X ₉ ) is (K, K, F, T, M, Y, I, Y , S), (K, N, Y, K, M, F, I, M, I), (K, N, F, K, M, Y, F, M, S), (K, N, Y , V, M, Y, I, M, L), (K, K, Y, T, M, I, V, Y, I), (K, N, F, K, M, L, I, V , S), (K, T, F, T, M, F, T, Y, L), (K, K, F, T, M, Y, T, I, H) and (K, I, Y , K, M, I, T, T, R) ,
an influenza hemagglutinin stem domain polypeptide. 2. The polypeptide of claim 1 , which selectively binds to antibodies CR6261 and/or CR9114. 3. The polypeptide of claim 1 or 2, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:81, 91 and 101. 4. A nucleic acid molecule encoding the polypeptide of claim 1 , 2 or 3 . A vector comprising the nucleic acid molecule of claim 4 . 4. A composition comprising the polypeptide of claim 1 , 2 or 3 . 5. A composition comprising an expression vector comprising the nucleic acid molecule of claim 4. Use of a polypeptide according to claims 1, 2 or 3 in the preparation of a medicament. 6. Use of the vector according to claim 5 in the preparation of a medicament, said vector being an expression vector. Use of a polypeptide according to claims 1 , 2 or 3 in the preparation of a medicament for inducing an immune response against influenza virus . Use of the vector according to claim 5, wherein said vector is an expression vector, in the preparation of a medicament for inducing an immune response against influenza virus . Use of a polypeptide according to claims 1, 2 or 3 in the preparation of a vaccine . Use of the vector according to claim 5, in the preparation of a vaccine, said vector being an expression vector .

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