JP2020162607A - Influenza virus vaccines and uses thereof - Google Patents

Abstract

To provide compositions for inducing an immune response to influenza virus in a subject.SOLUTION: Provided herein are influenza hemagglutinin stem domain polypeptides, methods for providing hemagglutinin stem domain polypeptides, compositions comprising the same, vaccines comprising the same and methods of their use, in particular in the detection, prevention and/or treatment of influenza.SELECTED DRAWING: None

Description

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

Influenza virus is a major human pathogen and is a respiratory disease that ranges in severity from asymptomatic infection to the potentially fatal primary virus pneumonia (generally "influenza (i)".
Causes (called "nfluenza") or "the flu"). The clinical efficacy of infection depends on the pathogenicity of the influenza strain and the exposure, history, age, and immune status of the host. Every year, about 1 billion people worldwide are infected with the influenza virus, with 3-5 million cases of serious illness and an estimated 300,000-500.
It is estimated to result in 000 influenza-related deaths. Most of these infections can result from influenza A virus carrying the H1 or H3 hemagglutinin subtype, and the contribution from influenza B virus is small, so all three representatives are included in seasonal vaccines. Current vaccination practices rely on early identification of circulating influenza virus to enable timely production of effective seasonal influenza vaccines. Apart from the inherent difficulty of predicting strains that will prevail 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 originating from pathogenic animals and caused by highly pathogenic virus strains rearranged to increase human-to-human transmission is a significant and realistic threat to global health. It becomes.

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

Strictly speaking, influenza B virus strains are human. Antigenic drift in HA within influenza B virus strains is smaller than that observed within strain A strains. The strains of influenza B virus that are genetically and antigenically distinct 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) strain (
Ferguson et al. , 2003). Although the spectrum of diseases caused by influenza B virus is generally milder than that caused by influenza A virus, serious illnesses requiring hospitalization for influenza B infection are still frequently observed.

It is known that influenza-neutralizing antibodies are primarily directed against hemagglutinin (HA). Hemagglutinin or HA is anchored to the virus coat and
A dual-function trimeric glycoprotein: it is responsible for binding to the cell surface receptor sialic acid, and after uptake, it mediates the fusion of the virus and endosomal membrane, viral RNA in the cytosol of the cell. Brings the release of. HA includes a large head domain and a smaller stem domain. Adhesion to the viral membrane is mediated by a C-terminal anchoring sequence linked to the stem domain. The protein is cleaved after translation in a particular loop to give rise to the two polypeptides HA1 and HA2 (the entire sequence is referred to as HA0).
The distal membrane region is primarily derived from HA1 and the proximal membrane region is primarily derived from HA2 (FIG. 1).

The reason why the seasonal influenza vaccine should be updated annually is due to the large mutation of the virus. In the hemagglutinin molecule, this mutation is particularly manifested in the head domain where antigen drift and shift resulted in a number of different variants. Since it is also an immunodominant region, most neutralizing antibodies are directed against this domain and act by interfering with receptor binding. The combination of immune dominance and large mutations in the head domain also explains why infection with one strain does not result in immunity against other strains:
Antibodies evoked by the first infection only recognize a limited number of strains that are closely associated with the virus of the primary infection.

In recent years, influenza hemagglutinin stem domain polypeptides lacking all or substantially all influenza hemagglutinin spherical head domains have been described and used to generate an immune response to one or more conserved epitopes of the stem domain polypeptide. ing. The epitope of a stem domain polypeptide is less immunogenic than the highly immunogenic region of the spherical head domain, and thus the absence of a spherical head domain in the stem domain polypeptide is one of the stem domain polypeptides. It is believed that the expression of an immune response to the above epitopes can be tolerated (Steel et al., 2010).
Therefore, Steel et al. Is A / Puerto Rico / 8/1934
Amino acid residues 53 to 276 of HA1 of the (H1N1) and A / Hong Kong / 1968 (H3N2) strains were deleted from the HA primary sequence, which was a short flexible binding sequence GG.
A new molecule was created by replacing with GG. Vaccination of mice with the H3HK68 construct did not induce group 1 HA and cross-reactive antisera. further,
As shown in PCT / EP2012 / 073706, the stem domain polypeptide is highly unstable and has been shown to bind to conserved epitopes in the stem region, as evidenced by the lack of binding of the antibody to the exact conformation. No structure was adopted.

In addition, Bommakanti et al. (2010) are amino acid residues 1 to 172 of HA2, 7 amino acid linkers (GSAGSAG), amino acid residues 7 to 46 of HA1.
A HA2-based polypeptide containing residues 290-321 of HA1 following the 6-amino acid linker GSAGSA with mutations V297T, I300E, Y302T and C305T in HA1 is described. The design was based on the sequence of H3HA (A / Hong Kong / 1968). This polypeptide only provided cross-protection against another influenza virus strain within the H3 subtype (provided against A / Phil / 2/82, but H
1 subtype (not provided for A / PR / 8/34). Bommakanti et
In a more recent paper by al (2012), H1N1A / Puerto Ric
HA-based stem domain sequences (H1HA0HA6) from o / 8/1934 have been described. In this polypeptide, the equivalents of residues 55-302 have been deleted and the mutations I311T, V314T, I316N, C319S, F406D, F409
T and L416D have been made. Both H3 and HA-based polypeptides
It is expressed in E. coli and therefore lacks glycans that are part of the natural HA protein. When expressed in E. coli, the polypeptide is recovered primarily as high molecular weight aggregates and trace monomer fractions. Polypeptides include 9 and 0.
It binds to CR6261 with two apparent dissociation constants of 2 μM. The authors have 10 mice
Immunization with 20 μg protein supplemented with 0 μg CpG7909 (
After 2 times, 4 week intervals), 1LD90 homologous H1N1A / Puerto Rico / 8 /
Shows that the 1934 virus challenge can survive. The authors are A / Hong
Polypeptides derived from Kong / 1/1968 are also described as cyclically rearranged polypeptides equivalent to those described above. These polypeptides are H1N1A / Pureto R
Derived from HA from ico / 8/1934, H1N1A / North Carolina / 20/99 or H1N1A / California / 07/2009, H1N1A /
It may provide partial protection for the Mild Challenge (1LD90) model in mice of Puerto Rico / 8/1934 (ie, within the same subtype). Serum from guinea pigs immunized with these polypeptides did not show detectable levels of neutralization when tested in a neutralization assay.

More recently, Lu et al (2013) has also been H1N1A / California / 0.
A soluble stem domain polypeptide derived from 5/2009 HA is described. In the final design, the equivalent of residues 54-303 (numbered by SEQ ID NO: 1) is deleted (leader sequence, residues 1-17 are also absent) and two mutations are in the protein B loop, That is, it is introduced in F407D and L413D. In addition, the polypeptide
It contained a C-terminal trimerization domain (foldon). In addition, there is one inter-monomer disulfide bridge in the area of the trimer folon domain, as well as 430 and 431.
One was introduced in the place. The polypeptide is produced in an E. coli-based cell-free system (and therefore lacks the glycans that are part of the natural HA protein) and is recovered in a denatured form that needs to be refolded before use. No immunology or protection was shown from influenza challenge data.

In a recent paper, Mallajosyla et al (2014) also reported a stem domain polypeptide. In this design, H1N1A / Pureto
Based on HA from Rico / 8/1934, not only is most of the HA1 sequence deleted (residues 42-289, numbered by SEQ ID NO: 1), but also the large N- and C-terminal sequences of HA2. The moieties are also deleted (residues 344 to 383, and 457 to 5, respectively).
65). Polypeptides lack the natural glycans on viral HA because they contain the fondon trimer domain at the C-terminus and are also produced in E. coli. The polypeptide binds to the broad neutralizing antibodies CR6261, F10 and FI6v3. The polypeptide is an influenza challenge model (1LD90 H1N1A / Pureto R)
It was also tested in ico / 8/1934) and could protect mice from death. H1N1A
/ New Caledonia / 20/1999 and H1N1A / Californi
A corresponding polypeptide derived from HA of a / 04/2009 could also be partially protected. H5N
The polypeptide derived from 1A / Vietnam Nam / 1203/2004 provided only limited protection in this challenge model. In addition, the challenge model used is mild at relatively low doses (1-2LD90).

Therefore, it stimulates the production of strong broad-spectrum neutralizing antibody responses and provides protection against a wide range of current and future influenza virus strains (both seasonal and epidemic), especially for effective prevention and treatment of influenza. Strain development group 1 and / or group 2
There is still a need for a safe and effective universal vaccine that provides protection against one or more influenza A virus subtypes.

In the present specification, influenza hemagglutinin stem domain polypeptide, a method for providing a stem domain polypeptide, a composition containing the same, a vaccine containing the same, and a method for using the same are provided.

In a first aspect, the invention provides a novel immunogenic polypeptide comprising an influenza hemagglutinin stem domain, referred to as an influenza (HA) stem domain polypeptide, and lacking a spherical head. Polypeptides can elicit an immune response when administered to subjects, especially human subjects. The polypeptides of the invention present conserved epitopes of membrane proximal stem domain HA molecules 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 constitutes the head domain is removed and the residual amino acid sequence is introduced directly or in some embodiments, a short flexible binding sequence (“linker”). By relinking, the continuity of the amino acid chain is restored. The resulting sequence is further modified by introducing a defined mutation that stabilizes the natural three-dimensional structure of the residual portion of the HA0 molecule. The immunogenic polypeptide does not contain the full-length HA1 domain of influenza virus.

The present invention is a novel influenza hemagglutinin stem domain polypeptide.
(A) Influenza hemagglutinin HA1 domain containing a HA1N terminal stem segment covalently linked to the HA1C terminal stem segment by a binding sequence of 0 to 50 amino acid residues, and the HA1C terminal segment is (b) influenza hemagglutinin HA
It is bound to two domains, and the HA1 and HA2 domains are derived from influenza A virus subtypes, including H1 subtype HA;
(C) Does not include protease cleavage sites at the junction between HA1 and HA2 domains;
(D) The HA1N-terminal segment contains amino acids 1 to x of HA1, preferably amino acids p to x of HA1, and the HA1C terminal stem segment contains amino acids y to C of HA1 and x = SEQ ID NO: 1. Amino acid on position 52 (or equivalent position in another hemagglutinin), p = position 18 of SEQ ID NO: 1 (or equivalent position in another hemagglutinin)
The amino acid above, y = the 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 to 418 is the amino acid X ₁ NTQX ₂ TAX ₃ GK.
Including EX 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.
X ₂ is an amino acid selected from the group consisting of F, I, N, T, H, L and Y, preferably I, L or Y.
X ₃ is an amino acid selected from the group consisting of V, A, G, I, R, F and S, preferably A, I or F.
X ₄ 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.
Selected from the group consisting of
Amino acid residues on position 340 (HA1 domain) are I, K, R, T, F, N, S and Y.
Selected from the group consisting of
Amino acid residues on position 352 (HA2 domain) are selected from the group consisting of D, V, Y, A, I, N, S, and T.
Amino acid residues on position 353 (HA2 domain) are selected from the group consisting of K, R, T, E, G, and V;
(G) Further comprises a disulfide bridge between the amino acid at position 324 and the amino acid at position 436;
(H) Further, the amino acid sequence RMKQIEDKIEIESK (SEQ ID NO: 20) is 419.
Introduced at position ~ 433, or sequence RMKQIEDKIEEESKQK (
The polypeptide in which SEQ ID NO: 21) is introduced at positions 417 to 433 is provided.

In certain embodiments, the polypeptide comprises a complete HA2 domain, i.e., a HA2 domain comprising a transmembrane domain and an intracellular sequence. In certain embodiments, the HA2 domain is truncated. Thus, in certain embodiments, the polypeptides of the invention do not contain the intracellular sequence of HA and the transmembrane domain. In certain embodiments, the HA2 domain 514, 515, 516, 517, 518, 519, 520, 521.
The C-terminal amino acid sequence of the HA2 domain has been removed from positions 522, 523, 524, 525, 526, 527, 526, 528, 529, or 530 (or their equivalents).

According to the present invention, the C-terminal amino acid of the HA1C-terminal stem segment is bound to the N-terminal amino acid of the HA2 domain and thus forms a link between the HA1 and HA2 domains. The polypeptides of the invention do not contain protease cleavage sites at the junction between HA1 and HA2 domains. In certain embodiments, the C-terminal amino acid residue of the HA1C-terminal stem segment (amino acid 343 of SEQ ID NO: 1) is any amino acid other than arginine (R) or lysine (K), preferably glutamine (Q).

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

The polypeptide of the present invention is a Group 1 cross-neutralizing antibody CR6261 (International Publication No. 2008).
/ 028946 (disclosed in Pamphlet No. 028946) and / or antibody CR9114 (International Publication No. 2)
Includes 013/007770 pamphlet), both Group 1 and Group 2 influenza A viruses, and conserved stem domain epitopes of antibodies that can bind and neutralize influenza B virus. Thus, it is an HA stem domain polypeptide, as indicated by the binding of the one or more antibodies to said polypeptide.
It is another aspect of the invention to provide a polypeptide that stably presents an epitope of antibody CR6261 and / or CR9114. In one embodiment, the polypeptide does not bind to the monoclonal antibodies CR8020 and CR8057 (described in WO 2010/130636) that bind only to the H3 influenza virus.

The influenza hemagglutinin stem domain polypeptide provided in the present invention is 1
Suitable for use in immunogenic compositions (eg, vaccines) capable of producing an immune response against one or more influenza virus A and / or B strains, particularly H1 subtype influenza virus. In one embodiment, the influenza hemagglutinin stem domain polypeptide is influenza A of phylogeny group 1 and / or group 2.
It can generate an immune response against viral strains, in particular both strains of strains 1 and 2. In one embodiment, the polypeptide may generate an immune response against an allogeneic influenza virus strain. In one embodiment, the polypeptide may generate an immune response against a heterologous influenza virus strain of the same and / or different subtypes. In a further embodiment, the polypeptide may generate an immune response against both strains of strain 1 and group 2 influenza virus strains and influenza B virus strains.

Polypeptides according to the invention can be, for example, stand-alone therapy and / or prevention and / or stand-alone therapy and / or prevention of diseases or conditions caused by influenza viruses, especially strain group 1 or 2 influenza A and / or influenza B viruses.
Alternatively, it can be used diagnostically or in combination with other prophylactic and / or therapeutic treatments, such as vaccines (existing or future), antiviral agents and / or monoclonal antibodies.

In a further aspect, the invention provides a nucleic acid molecule encoding an influenza HA stem domain polypeptide. In yet another aspect, the invention provides a vector containing 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 a composition comprising a polypeptide and / or nucleic acid molecule and / or vector 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 a specific embodiment, the composition is suitable for human administration. Polypeptides, nucleic acid molecules and compositions can be used in methods of preventing and / or treating influenza viral diseases and / or for diagnostic purposes. The composition may further comprise a pharmaceutically acceptable carrier or excipient. In certain embodiments, the compositions described herein contain an adjuvant or are administered in combination with an adjuvant.

In another embodiment, the invention is a polypeptide, nucleic acid and / / used as a vaccine.
Or provide a vector. The present invention particularly relates to diseases or conditions caused by influenza virus A subtypes and / or influenza B viruses of phylogenetic groups 1 and / or 2, especially diseases or conditions caused by influenza viruses including H1 subtype HA. With respect to immunogenic polypeptides, nucleic acids, and / or vectors used as vaccines in prophylaxis and / or treatment.

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

A model of the pre-fusion HA monomer present in the natural trimer is shown. HA1 is shown in light gray and HA2 is shown in dark gray. It shows Helix A (an important part of the epitope of CR6261) and Helix CD (a part of the trimer interface), and loops connect their secondary structure elements. The C-terminus of HA1 and the N-terminus of HA2 are also shown. 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. Captured and detected antibodies are shown above the graph. mini-HA refers to the soluble form of SEQ ID NO: 2 in which the equivalent of residues 519-565 has been replaced by RSLVPRGSPGHHHHH; FL-HA-FFH is the C-terminal Flag-thrombin-foldon-His sequence from position 520. Refers to the soluble form of SEQ ID NO: 1 containing (SEQ ID NO: 4); FL-HA-7 × His refers to the soluble form of SEQ ID NO: 1 containing the C-terminal sequence EGRHHHHHH from position 530. Sandwich Elisa results obtained for the supernatant of a culture expressing the polypeptide of the invention (t2 variant) comprising the GCN4-derived sequence RMKQIEDKIEIESK (SEQ ID NO: 20) at positions 419-433. Captured and detected antibodies are shown above the graph. mini-HA-t2 is derived from Mini-HA by introducing SEQ ID NO: 20 at positions 419-433; FL-HA-FFH, FL-HA-7 × His are as described above. Sandwich Elisa results obtained for the supernatant of a culture expressing the polypeptide of the invention (t3 variant) comprising the GCN4-derived sequence RMKQIEDKIEEISKQK (SEQ ID NO: 21) at positions 417-433. Captured and detected antibodies are shown above the graph. mini-HA-t3 is derived from Mini-HA by introducing SEQ ID NO: 21 at positions 417-433; FL-HA-FFH, FL-HA-7 × His are as described above. Elution profiles of s55G7-t2, s127H1-t2 and s86B4-t2 from the Superdex 200 size exclusion column, which is the final step in the purification procedure. The numbered lines below the chromatogram indicate the fractions recovered during the elution process. SDS-PAGE and Western blot analysis of the fractions recovered during the elution of the Superdex 200 size exclusion column. The numbers correspond to the fractions shown in FIG. Antibodies that recognize the C-terminal his tag were used for detection on Western blots. Size-exclusion chromatography of s127H1-t2 in the presence and absence of Fab fragments of the broad neutralizing antibodies CR9114, CR6261 and CR8020 (Tosoh G2000 analytical column). The molecular weights of the 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 polypeptides of the invention s127H1-t2 to monoclonal antibodies CR6261 and CR9114 using biolayer interferometry. The upper panel shows the individual binding curves for the immobilized monoclonal antibody exposed to varying concentrations of s127H1-t2, and the lower panel shows the steady-state analysis used to estimate _Kd . Survival (A), body weight loss (B) and clinical score (C) for the negative (PBS, 3 immunizations at 3-week intervals) and positive controls (15 mg / kg CR6261, 1 day before challenge). Mice were challenged with a lethal dose (25 x 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 (A), weight loss (A), weight loss (A), weight loss (A), weight loss (A), weight loss (A), weight loss (A), weight loss (A), weight loss (A), weight loss (A), weight loss (A) for the experimental group immunized with 10 μg s127H1-t2 (three immunizations at 3-week intervals) in the presence or absence of 10 μg Matrix-M B) and clinical score (C). Mice are challenged with a lethal dose (25 x 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 soluble form (B) of full-length HA as an antigen. The bar represents the median. Antibodies induced after immunization with the adjuvanted polypeptide s127H1-t2 of the present invention may compete with CR9114 for binding to full-length HA from H1N1A / Brisbane / 59/07 in competitive ELISA (A). For comparison, the level of competition with the unlabeled CR9114 (ie, self-competitive) and the unbound monoclonal antibodies CR8020 and CR-JB (both serially diluted from a starting concentration of 5 μg / ml) is shown in separate graphs. Survival (A), relative weight loss (B) and clinical score (C) for negative (PBS, 3 immunizations at 3 week intervals) and positive controls (15 mg / kg CR6261, 1 day before challenge) .. Mice were challenged with a lethal dose (25 x 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 rate for a group immunized once (A), twice (B) or three times (C) with 30 μg s127H1-t2-cl18long in the presence of 10 μg Matrix-M. Mice were challenged with a lethal dose (25 x 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 changes for a group immunized once (A), twice (B) or three times with 30 μg s127H1-t2-cl18long in the presence of 10 μg Matrix-M. Mice were challenged with a lethal dose (25 x 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 the 1 (A), 2 (B) or 3 times immunized group with 30 μg s127H1-t2-cl18long in the presence of 10 μg Matrix-M. Mice were challenged with a lethal dose (25 x 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 the 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 (B) of full-length HA as antigen. The bar represents the median. Antibodies induced after immunization with the Matrix-M adjuvanted polypeptide s127H1-t2-cl18long of the present invention may compete with CR9114 for binding to full-length HA from H1N1A / Brisbane / 59/07 in competitive ELISA (A). ). For comparison, the level of competition by unlabeled CR9114 (ie, self-competitive) and unbound monoclonal antibody CR8020 (both serially diluted from a starting concentration of 5 μg / ml) is shown in separate graphs (B). The bar represents the median. (A) Survival for negative (PBS, 3 immunizations at 3-week intervals) and positive controls (15 mg / kg CR6261, 1 day before challenge). Mice were challenged with a lethal dose (12.5 x LD50) of H5N1A / Hong Kong / 156/97 for 4 weeks after the last immunization. (B) Survival rate, (C) Relative body weight change and (D) Median clinical score for the group immunized three times with 30 μg s127H1-t2 in the presence of 10 μg Matrix-M. Error bars indicate 95% confidence intervals (C) or interquartile ranges (D). Mice were challenged with a lethal dose (12.5 x LD50) of H5N1A / Hong Kong / 156/97 for 4 weeks after the last immunization and monitored for 21 days. For comparison, the negative control group (PBS) is also shown in B, C, D. Immunized three times with the polypeptide of the invention s127H1-t2 according to Example 5 using full-length HA of several Group 1 (H1, H5 and H9) and Group II (H3 and H7) influenza strains as antigens. Elisa results for serum from mice. The induced antibody recognizes all FL HA tested from Group 1. (A) Survival for negative (PBS, 3 immunizations at 3-week intervals) and positive controls (15 mg / kg CR6261, 1 day before challenge). Mice were challenged with a lethal dose (12.5 x LD50) of H1N1A / Brisbane / 59/2007 4 weeks after the last immunization. (B) Survival rate, (C) Relative body weight change and (D) Median clinical score for the group immunized three times with 30 μg s127H1-t2 in the presence of 10 μg Matrix-M. Error bars indicate 95% confidence intervals (C) or interquartile ranges (D). Mice were challenged with a lethal dose (12.5 x LD50) of H1N1A / Brisbane / 59/2007 for 4 weeks after the last immunization and monitored for 21 days. For comparison, the negative control group (PBS) is also shown in B, C, D. A pseudoparticle neutralization assay using sera from mice immunized with the polypeptides of the invention s127H1-t2 or PBS. Antibody-dependent cellular cytotoxicity (ADCC) surrogate assay. Serum from mice immunized with the polypeptide s127H1-t2 of the present invention contains FL HA from H5N1A / Hong Kong / 156/97 (A) or H1N1A / Brisbane / 59/07 (B) as an antigen resource. The transfected target cells are used to show 30-40 fold induction of FcγRIV signaling activity at maximum serum concentration. Survival rate (A) and% body weight change (B) of mice after serum transfer and challenge with H5N1A / Hong Kong / 156/97 according to Example 9. Full-length HA (H1N1A / Brisbane / 59/2007) ELISA titer of donor mouse (D) on day 70 and recipient mouse (R) before serum transfer (day-4) or before challenge (day 0) .. The data were analyzed using a slope-based weighted average approach. The white circle symbol indicates the measured value in LOD. The bar indicates the median. Survival rate (A) and% body weight change (B) of mice after immunization and challenge with H1N1A / NL / 602/09 according to Example 10. (A): Full-length HA (H1N1A / Brisbane / 59/2007) ELISA titer of the immunized mouse according to Example 10. The data were analyzed using a slope-based weighted average approach. The white circle symbol indicates the measured value in LOD. The bar indicates the median. (B): Serum IgG CR9114 competitive binding obtained after immunization of the mouse according to Example 10. FL HA from H1N1A / Brisbane / 59/2007 was used as the antigen. The data shown is the median of the group and the error bars indicate the interquartile range. Data are shown for CR9114 and CR8020 diluted in the same manner as serum samples, starting from a 5 μg / ml solution. Initial screen for a total of 10472 clones (5544 and 4928 from sets 1 and 2, respectively). The data are normalized to the mean FL HA binding and expression included in the experiment. Those showing the binding signal to CR6261 (panel B) of the signals observed for the top 20% clones and FL HA in the CR9114 sandwich assay (panel A), which also shows> 50% expression of FL HA expression. Considered a hit; this procedure produced 703 hits (596 and 107 from libraries 1 and 2 respectively). CR9114 sandwich ELISA results for the 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 a Fab fragment of CR9114 (shown as CRF9114) or CR6261 (shown as CRF6261). The molecular mass derived from the multi-angle light scattering analysis is given in Example 12, which shows the formation of a complex with three Fab fragments per trimer of the polypeptide of the invention. Survival rate (A) and% body weight change (B) of mice after immunization and challenge with H1N1A / Brisbane / 59/07 as described in Example 13. (A): Full-length HA (H1N1A / Brisbane / 59/2007) ELISA titer of the immunized mouse according to Example 13. The data were analyzed using a slope-based weighted average approach. The white circle symbol indicates the measured value in LOD. The bar indicates the median. (B): Serum IgG CR9114 competitive binding obtained after immunization of the mouse according to Example 18. FL HA from H1N1A / Brisbane / 59/2007 was used as the antigen. The data shown is the median of the group and the error bars indicate the interquartile range. The levels are shown for CR9114 and CR8020 diluted in the same manner as the serum samples, starting from a 5 μg / ml solution. Survival rate (A) and% body weight change (B) of mice after immunization and challenge with H5N1A / Hon Kong / 156/97 as described in Example 14. (A): Full-length HA (H1N1A / Brisbane / 59/2007) ELISA titer of the immunized mouse according to Example 14. The data were analyzed using a slope-based weighted average approach. The white circle symbol indicates the measured value in LOD. The bar indicates the median. (B): Serum IgG CR9114 competitive binding obtained after immunization of the mouse according to Example 18. FL HA from H1N1A / Brisbane / 59/2007 was used as the antigen. The data shown is the median of the group and the error bars indicate the interquartile range. The levels are shown for CR9114 and CR8020 diluted in the same manner as the serum samples, starting from a 5 μg / ml solution. Survival rate (A) and% body weight change (B) of mice after immunization and challenge with H1N1A / Puerto Rico / 8/1934 as described in Example 15. (A): Full-length HA (H1N1A / Brisbane / 59/2007) ELISA titer of the immunized mouse according to Example 15. The data were analyzed using a slope-based weighted average approach. The white circle symbol indicates the measured value in LOD. The bar indicates the median. (B): Serum IgG CR9114 competitive binding obtained after immunization of the mouse according to Example 18. FL HA from H1N1A / Brisbane / 59/2007 was used as the antigen. The data shown is the median of the group and the error bars indicate the interquartile range. The levels are shown for CR9114 and CR8020 diluted in the same manner as the serum samples, starting from a 5 μg / ml solution.

Definitions The definitions of terms used in the present invention are listed below.

Amino acids according to the invention are 20 naturally occurring (or "standard" amino acids) or variants thereof, such as D-proline (D-enantiomer of proline), or any variant not found naturally 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 periodicity to the polypeptide backbone. However, glycine is more flexible than other amino acids. Table 5 shows the abbreviations and properties of standard amino acids.

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. The percent identity is the same (ie, the amino acid residue at a given position in the alignment is the same residue) or similar (ie, the amino acid substitution at a given position in the alignment is conservative as discussed below. The ratio of the amino acid residue in the candidate sequence (which is a substitution) to the corresponding amino acid residue in the peptide after aligning the sequence and introducing gaps as needed to achieve maximum percent sequence homology. is there. Sequence homology, eg, the proportion of sequence identity and similarity, is determined using, for example, visual inspection and mathematical calculations, using sequence alignment techniques well known in the art, or more preferably, comparisons are made by computer. This is done by comparing the sequence information programmatically. An exemplary preferred computer program is Gen
etcs Computer Group (GCG; Madison, Wis.) Wi
sconsin package version 10.0 program, "GAP" (Devereu)
x et al. (1984)).

“Conservative substitution” refers to the substitution of one class of amino acid with another amino acid of the same class. In certain embodiments, conservative substitutions do not alter the structure and / or function of the polypeptides of the invention. Classes of amino acids for the purpose of conservative substitution include hydrophobic (eg, Met, Ala, Val, Leu), neutrally hydrophilic (eg, Cys, Se).
r, Thr), acidic (eg Asp, Glu), basic (eg Asn, Gln, H)
is, Lys, Arg), conformational disruptors (eg Gly, Pro) and aromatics (eg, Gly, Pro)
For example, Trp, Tyr, Phe) are included.

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

The term "effective amount" as used herein in the context of administering a therapeutic agent to a subject is
Refers to the amount of therapeutic agent that has prophylactic and / or therapeutic effects. In certain embodiments, the "effective amount" in the context of administering a therapeutic agent to a subject is an influenza virus infection, reducing or ameliorating the severity of the associated disease or symptom, eg, but not limited to, an influenza virus infection. , Reduction of duration of associated disease or symptom, prevention of influenza virus infection, progression of associated disease or symptom, prevention of influenza virus infection, onset or onset or recurrence of associated disease or symptom, from one subject to another Prevention or reduction of influenza virus spread to, hospitalization of subjects and /
Alternatively, reduce hospital stay, increase survival of subjects with influenza virus infection or associated disease, eliminate influenza virus infection or associated disease, inhibit or reduce influenza virus replication, reduce influenza virus titer; and / or Refers to the amount of therapeutic agent sufficient to prevent or improve the therapeutic effect and / or achieve improvement of another therapeutic agent. In certain embodiments, effective amounts do not provide complete protection from influenza viral disease, but result in lower titers or reduced numbers of influenza virus compared to untreated subjects. Benefits of reducing the titer, number or total load of influenza virus include, but are not limited to, milder infectious symptoms, less infectious symptoms and reduced duration of disease associated with the infection.

As used herein, the term "host" is intended to refer to an organism or cell into which a vector, eg, a cloning vector or expression vector, has been introduced. An organism or cell can be a prokaryote or a eukaryote. Preferably, the host comprises an isolated host cell, eg, a host cell in culture. The term "host cell" merely means that the cell has been modified for (over) expression of the polypeptide of the invention. It should be understood that the term host refers not only to a particular organism or cell, but also to the progeny of such organism or cell. Such progeny cannot be virtually identical to a parent or cell, as certain modifications can occur in subsequent generations due to either mutations or environmental effects, but are still used herein. Included within the scope of the term "host".

The term "included" or "as an example" as used herein is deemed to be followed by the term "but not limited".

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

Influenza viruses are classified into influenza virus types: A, B and C genera. As used herein, the term "influenza virus subtype" refers to an influenza A virus variant characterized by a combination of hemagglutinin (H) and neuraminidase (N) virus surface proteins. According to the present invention, influenza virus subtypes include their H numbers, such as "influenza virus containing H3 subtype HA", "
H3 subtype influenza virus "or" H3 influenza "etc., or a combination of H and N numbers, such as" influenza virus subtype H3N2 "or" H
It can be referred to by "3N2" or the like. The term "subtype" specifically refers to all individual "strains" within each subtype that usually arise from mutations and exhibit different pathogenic profiles, such as natural isolates and artificial mutations. Includes body or rear mutant, etc. Such strains can also be referred to as various "isolators" of the viral subtype. Therefore, the terms "stock" and "separate stock" used herein can be used interchangeably.
Current nomenclature for human influenza virus strains or isolates is usually listed in parentheses for the type (genus) of the virus, ie, A, B or C, geographic location of the first isolation, strain number and year of isolation HA and NA antigens are described, eg, 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 groups. Phylogenetic analysis demonstrated subclassification of hemagglutinin into two major groups: H1, H2, H5 and H9 subtypes (“Group 1” influenza virus), especially in phylogenetic group 1, and H3, especially in phylogenetic group 2. H4, H7 and H10 subtypes ("Group 2" influenza virus ").

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

As used herein, the term "nucleic acid" includes DNA molecules (eg, cDNA or genomic DNA) and RNA molecules (eg, mRNA) and analogs of DNA or RNA produced using nucleotide analogs. Shall be. The nucleic acid can be single-strand or double-stranded. Nucleic acid molecules can be chemically or biochemically modified, or can contain unnatural or derivatized nucleotide bases, which will be readily recognized by those skilled in the art. Such modifications include, for example, labeling, methylation, substitution of one or more natural nucleotides with an analog, internucleotide modifications, such as uncharged bonds (eg, methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.) ), Charged bonds (eg, phosphorothioates, phosphorodithioates, etc.), Suspension moieties (eg, polypeptides), inserts (eg, aclysine, psolarene, etc.), chelating agents, alkylating agents, and modified bonds (eg, eg Alpha anomaly nucleic acids, etc.) are included. References to nucleic acid sequences include their complements unless otherwise stated. Therefore, a reference to a nucleic acid molecule having a particular sequence should be understood to include that complementary strand having that 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:
Based on amino acid numbering in HA0 of wild-type influenza virus, eg, amino acid numbering of H1N1 influenza strain A / Brisbane / 59/2007 (SEQ ID NO: 1). Therefore, the term "amino acid at the" x "position in HA" as used in the present invention refers to a particular wild-type influenza virus, such as A / Brisbane / 59.
/ 2007 (SEQ ID NO: 1; the amino acid in the HA2 domain is italicized) means the amino acid corresponding to the amino acid at position x in HA0. It will be appreciated by those skilled in the art that the equivalent amino acids in other influenza virus strains and / or subtypes can be determined by multi-sequence alignment. 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 begins, for example, at position 18 of SEQ ID NO: 1. A leader sequence (or signal sequence) that directs protein transport during production (eg, amino acid 1 of SEQ ID NO: 1)
It is generally understood by those skilled in the art that (corresponding to ~ 17) is not present, for example, in the final polypeptide used in the vaccine. Thus, in certain embodiments, the polypeptide according to the invention comprises an amino acid sequence that does not have a leader sequence, i.e. the amino acid sequence is based on the amino acid sequence of HA0 that does not have a signal sequence.

"Polypeptide" refers to a polymer of amino acids bound by an amide bond known to those skilled in the art. As used herein, the term may refer to a single polypeptide chain attached by a covalent amide bond. The term refers to non-shared interactions, such as ionic contacts,
It can also refer to multiple polypeptide chains associated by hydrogen bonds, van der Waals contacts and hydrophobic contacts. Those skilled in the art use the term, for example, post-translational processing, such as signal peptide cleavage, disulfide bond formation, glycosylation (eg, N-bond and O-bond glycosylation), protease cleavage and lipid modification (eg, S-palmitoyle). Recognize that it contains a polypeptide modified by Glycosylation.

"Stem domain polypeptide" refers to a polypeptide that comprises one or more polypeptide chains that make up the stem domain of natural (or wild-type) hemagglutinin (HA). Typically, the stem domain polypeptide is a single polypeptide chain (ie, corresponding to the stem domain of the hemagglutinin HA0 polypeptide) or two polypeptide chains (ie, ie).
Corresponds to the stem domain of the hemagglutinin HA1 polypeptide associated with the hemagglutinin HA2 polypeptide). According to the present invention, a stem domain polypeptide contains one or more mutations compared to a wild-type HA molecule, in particular one or more amino acid residues of wild-type HA correspond in a particular wild-type HA It may be replaced by other amino acids that do not occur naturally at the position. The stem domain polypeptide according to the invention may further comprise one or more of the following binding sequences:

The term "vector" refers to a nucleic acid molecule that is replicated and expressed in some cases, into which a second nucleic acid molecule can be inserted for introduction into the host. In other words, the vector can transport the nucleic acid molecule to which it is bound. Cloning and expression vectors are contemplated by the term "vector" used herein. Vectors include, but are not limited to, plasmids, cosmids, bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs).
Also included are vectors derived from bacteriophage or plant or animal (eg, human) viruses. The vector comprises a promoter and other regulatory regions recognized by the host in the case of a replication origin and expression vector recognized by the presented host. Some vectors can replicate autonomously in the host into which they are introduced (eg, vectors having a bacterial replication origin can replicate in bacteria). Other vectors can be integrated into the host's genome upon introduction into the host so that the vector replicates along the host's genome.

As used herein, the term "wild-type" in the context of a virus refers to an influenza virus that is frequent, naturally circulating, and causing the spread of typical diseases.

Detailed Description The influenza virus has a significant impact on global public health, causing millions of serious cases, thousands of deaths, and significant financial losses each year. Current trivalent influenza vaccines elicit a strong neutralizing antibody response against vaccine strains and closely related isolates, but rarely extend to more branched or other subtypes within one subtype. In addition, the selection of the appropriate vaccine strain presents many difficulties and often results in suboptimal protection. Moreover, prediction of the next epidemic virus subtype is currently impossible, including when and where it occurs.

Hemagglutinin (HA) is a major envelope glycoprotein from influenza A virus, which is a major target for neutralizing antibodies. Hemagglutinin is 2 during the influx process
It has two main functions. First, hemagglutinin mediates the attachment of the virus to the surface of target cells through interaction with sialic acid receptors. Second, after viral endocytosis, hemagglutinin subsequently produces a fusion of the virus and the endosomal membrane to release its genome into the cytoplasm of the target cell. HA contains a large ectodomain of about 500 amino acids that is cleaved by a host-derived enzyme to produce two polypeptides that remain bound by disulfide bonds. The majority of N-terminal fragments (HA1, 320-330 amino acids) form a membrane distal spherical domain containing the receptor binding site and most antigenic determinants recognized by virus neutralizing antibodies. The smaller C-terminal portion (HA2, about 180 amino acids) forms a stem-like structure that anchors globular domains to cells or viral membranes. The degree of sequence homology between subtypes is H for HA1 polypeptides (34% to 59% homology between subtypes).
It is smaller than the A2 polypeptide (51-80% homology). Most conserved regions are the sequences around the cleavage site, especially the HA2N-terminal 23 amino acids, which are all influenza A.
Conserved as a viral subtype (Lorieu 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 by HA1 and HA2.

Most neutralizing antibodies bind to the loop that surrounds the receptor binding site, preventing receptor binding and attachment. Because these loops are highly mutagenic, most antibodies that target those regions are strain-specific, and why current vaccines induce such limited strain-specific immunity. explain. However, in recent years, fully human monoclonal antibodies against the influenza virus hemagglutinin, which have broad cross-neutralizing efficacy, have been produced. Functional and structural analysis revealed that these antibodies interfered with the membrane fusion process and were directed towards highly conserved epitopes in the stem domain of influenza HA proteins (Throsby et al., 2008; Equiert). et al. 2009
, International Publication No. 2008/028946 Pamphlet, International Publication No. 2010/13063
Pamphlet No. 6, International Publication No. 2013/007770 Pamphlet).

Stem domain polypeptides that stably present the epitopes of these antibodies are described in the co-pending patent application PCT / EP2012 / 073706. At least some of the stem domain polypeptides described herein are CR6261 and / or C.
It stably presents an epitope of R9114 and is immunogenic in mice. CR626
An additional immunogenic stem domain polypeptide that stably presents an epitope of 1 and / or CR9114 is described in the co-pending patent application PCT / EP2014 / 060997.

According to the present invention, novel HA stem domain polypeptides that present these epitopes have been designed. These polypeptides can be used to create universal epitope-based vaccines that induce protection against a wide range of influenza strains. Similarly in the stem domain polypeptides already described, a highly mutated immunodominant moiety, i.e., the head domain, is first removed from the full-length HA molecule to create a stem domain polypeptide, also referred to as mini-HA. The immune response is redirected to the stem domain where the epitope for the broad neutralizing antibody is localized. The broad neutralizing antibodies described above are used to determine the exact folding of newly created molecules and to confirm the presence of neutralizing epitopes.

The novel stem domain polypeptides of the invention are the previously described stem polypeptides (PCT / EP2012 / 073706 and PCT / EP2014 / 0609).
Antibodies, especially CR6261 and / or CR9, as compared to the binding of the antibody to (No. 97).
It shows an increase in the binding of 114 and / or an increase in the tendency to multimerize and an increase in stability.

The stem domain polypeptides of the invention can present a conserved epitope of a membrane proximal stem domain HA molecule to the immune system in the absence of a dominant epitope present in the membrane distal head domain. To this end, by removing some of the primary sequences of the HA0 protein that make up the head domain and reintroducing a short flexible binding sequence (“linker”), either directly or in some embodiments. It is ligated to restore continuity of the polypeptide chain. The resulting polypeptide sequence is further modified by introducing a defined mutation that stabilizes the natural three-dimensional structure of the residual portion of the HA0 molecule.

The present invention is, in particular, an influenza hemagglutinin stem domain polypeptide.
(A) The HA1C terminal segment comprises an influenza hemagglutinin HA1 domain comprising a HA1N terminal stem segment covalently linked to the HA1C terminal stem segment by a binding sequence of 0 to 50 amino acid residues.
(B) Influenza hemagglutinin is bound to the HA2 domain and has HA1 and HA.
Two domains are derived from influenza A virus subtypes, including H1 subtype HA;
(C) Does not include protease cleavage sites at the junction between HA1 and HA2 domains;
(D) The HA1N terminal segment contains amino acids 1 to x of HA1, preferably amino acids p to x of HA1, and the HA1C terminal stem segment contains amino acids y to C of HA1 and x = SEQ ID NO: 1. Amino acid on position 52 (or equivalent position in hemagglutinin of another influenza virus strain) and p = amino acid on position 18 (or equivalent position in hemagglutinin of another influenza virus) of SEQ ID NO: 1. y = Amino acid on position 321 of SEQ ID NO: 1 (or corresponding position in another hemagglutinin);
(E) The region containing amino acid residues 402 to 418 is the amino acid X ₁ NTQX ₂ TAX ₃ GK.
Including EX 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.
X ₂ is an amino acid selected from the group consisting of F, I, N, T, H, L and Y, preferably I, L or Y.
X ₃ is an amino acid selected from the group consisting of V, A, G, I, R, F and S, preferably A, I or F.
X ₄ 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.
Selected from the group consisting of
Amino acid residues on position 340 (HA1 domain) are I, K, R, T, F, N, S and Y.
Selected from the group consisting of
Amino acid residues on position 352 (HA2 domain) are selected from the group consisting of D, V, Y, A, I, N, S, and T.
Amino acid residues on position 353 (HA2 domain) are selected from the group consisting of K, R, T, E, G, and V;
(G) Further comprises a disulfide bridge between the amino acid at position 324 and the amino acid at position 436;
(H) The amino acid sequence RMKQIEDKIEIESK (SEQ ID NO: 20) is 419 to 433.
Provided is a polypeptide that has been introduced at positions or the sequence RMKQIEDKIEIESKKQK (SEQ ID NO: 21) has been introduced at positions 417-433.

Therefore, the present invention provides a stable hemagglutinin stem domain polypeptide that mimics the three-dimensional conformation of the stem of a native hemagglutinin molecule.

The polypeptides of the invention do not contain a full-length HA1 domain.

The polypeptide is therefore substantially smaller than HA0 and preferably lacks the spherical head of all or substantially all HA. Preferably, the immunogenic polypeptide is 360
Amino acid length or less, preferably 350, 340, 330, 320, 310, 305, 30
It is 0, 295, 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 acids in length.

According to the present invention, the "HA1N 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 HA1N-terminal polypeptide segment contains amino acids at positions 1 to x of the HA1 domain, and the amino acid at position x is an amino acid residue within HA1. The term "HA1C terminal segment" refers to the polypeptide segment corresponding to the carboxy terminal portion of the influenza hemagglutinin HA1 domain. The HA1C-terminal polypeptide segment contains amino acids from the y-position to the C-terminal amino acid (including the amino acid) of the HA1 domain, and the amino acid on the y-position is an amino acid residue in HA1. According to the present invention, y is greater than x and therefore between the HA1N terminal segment and the HA1C terminal segment, i.e. x of HA1.
The segment of the HA1 domain between the amino acid on the position and the amino acid on the y position has been deleted and replaced by the binding sequence in some embodiments. Thus, in certain embodiments, the deletion of the HA1 segment comprises the amino acid sequence of the amino acid at position x + 1 to the amino acid at position y-1 (including that amino acid).

In certain embodiments, the polypeptide does not contain a signal sequence. Thus, in certain embodiments, the HA1N terminal segment comprises the amino acids p to x of HA1, where p is.
It is the first amino acid of the mature HA molecule (eg, p = 18 for SEQ ID NO: 1). Those skilled in the art have determined the equivalent amino acids in other hemagglutinins and in signal peptides up to the x-position of the HA1 domain (eg, amino acids 1-17 of SEQ ID NO: 1 or other H1 influenza virus strains (see, eg, Table 2)). The polypeptides described herein that do not have a corresponding position can be prepared.

According to the present invention, the HA1N terminal segment comprises amino acids 1-x, preferably p-x of the HA1 domain, x = 52, p = 18 in SEQ ID NO: 1, or H.
It is a corresponding amino acid position in another HA sequence of subtype 1.

According to the present invention, the HA1C-terminal polypeptide segment comprises amino acids from the y-position of the H1HA1 domain to the C-terminal amino acid (including that amino acid), where y is 321 or H1.
Corresponding amino acid position in other HA sequences of the subtype.

Therefore, according to the present invention, the HA1N terminal stem segment contains amino acid residues 1 to 52 of HA1, preferably amino acid residues 18 to 52 of HA1, and the HA1C terminal stem segment contains amino acid residues 321 to HA1. 343 is included. In certain embodiments, H
The A1N-terminal stem segment consists of HA1 amino acid residues 1-52, preferably HA1 amino acid residues 18-52, and the HA1C-terminal stem segment consists of HA1 amino acid residues 321-343.

According to the present invention, the polypeptide does not include a protease cleavage site at the junction between the HA1 and HA2 domains. Therefore, the hemagglutinin stem domain polypeptide is resistant to protease cleavage at the junction between HA1 and HA2. The Arg (R) -Gly (G) sequences spanning HA1 and HA2 (ie, amino acid positions 343 and 344 of SEQ ID NO: 1) are recognition sites for trypsin and trypsin-like proteases, typically for hemagglutinin activation. It is known to those skilled in the art that it is cleaved for this purpose. The influenza hemagglutinin stem domain polypeptides of the invention are resistant to protease cleavage, as the HA stem domain polypeptides described herein should not be activated. Therefore, according to the present 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 has a mutation in the C-terminal amino acid of the C-terminal HA1 segment and / or HA2 to obtain a sequence resistant to protease cleavage.
It has been removed by mutation of the N-terminal amino acid of the domain. In certain embodiments, removal of the cleavage site between HA1 and HA2 in some embodiments can be achieved by mutation of R (K in a few cases) to Q at the P1 position (eg, cleavage site (sequence). For an explanation of the nomenclature of No. 1 (343rd place), see Sun et al, 2010.
reference). Thus, in certain embodiments, the C-terminal amino acid residue of the HA1C-terminal stem segment is any amino acid other than arginine (R) or lysine (K). In certain embodiments, the HA1C 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 HA1C-terminal stem segment is glutamine (Q).

According to the present invention, the polypeptide is derived from or based on H1HA, that is, HA containing an amino acid sequence from the H1 subtype influenza virus. In certain embodiments, the polypeptide is, for example, the following influenza virus A / Brisb:
Includes a hemagglutinin stem domain from or based on the HA of influenza A virus, including H1 subtype HA, from ane / 59/2007 (H1N1) (SEQ ID NO: 1). It will also be appreciated by those skilled in the art that other influenza A viruses, including H1 subtype HA, can be used according to the present invention. In certain embodiments, the polypeptide comprises a hemagglutinin stem domain derived from or based on the HA of influenza AH1 virus selected from Table 2. "Derived from" or "based on" is a natural influenza H1 subtype in which the N-terminal and / or C-terminal segments of the HA1 and / or HA2 domains are known to those of skill in the art or later discovered. The corresponding N-terminus and / of the HA1 and / or HA2 domain of hemagglutinin
Or at least 70%, 75%, 80%, 85%, 90%, 95% with the C-terminal segment
, 96%, 97%, 98%, or 99% amino acid sequence identity.

According to the present invention, the HA2 domain sets the C-terminal residue of helix A to N of helix CD.
It contains one or more mutations in the HA2 amino acid sequence linked to the terminal residue (Fig. 1). The H1HA2 amino acid sequence linking the C-terminal residue of helix A and the N-terminal residue of helix CD comprises an amino acid sequence containing residues 402-418 of influenza HA (SEQ ID NO: 1).
(Numbering by), amino acid sequence MNTQFTAVGKEFN (H / K) LE (K / R)
(SEQ ID NO: 7) is included.

In certain embodiments, an amino acid sequence linking the C-terminal residue of helix A to the N-terminal residue of helix CD, ie, the amino acid residue 402 of influenza HA of serotype H1.
The region containing ~ 418 (numbered by SEQ ID NO: 1) is the amino acid sequence X ₁ NTQX ₂ TA.
Includes X ₃ GKEX ₄ N (H / K) X ₅ E (K / R) (SEQ ID NO: 8).

According to the present 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 X ₅ is mutated, i.e., containing amino acids stem polypeptide does not occur in their position of wild-type influenza viruses based.

In certain embodiments, the mutated amino acid at position 402, ie X _1, is M, E,
An amino acid selected from the group consisting of K, V, R and T.

In certain embodiments, the mutated amino acid at position 406, ie X _2, is F, I,
An amino acid selected from the group consisting of N, T, H, L and Y, preferably I, L or Y.

In certain embodiments, mutations amino acids on position 409, i.e., _{X 3} is, V, A,
An amino acid selected from the group consisting of G, I, R, F and S, preferably A, I or F.

In certain embodiments, mutations amino acids on 413-position, i.e., _{X 4} is, F, I,
An amino acid selected from the group consisting of N, S, T, Y, E, K, M, and V, preferably I, Y, M or V.

In certain embodiments, mutations amino acids on 416-position, i.e., _{X 5} is, L, H,
An amino acid selected from the group consisting of I, N, R, preferably I.

Combinations of these mutations are also possible.

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

According to the present invention, the stem polypeptide corresponds to the wild-type influenza virus HA.
It comprises one and / or HA2 domain, i.e., one or more additional mutations in the HA1 domain and / or HA2 domain as compared to the amino acid sequence of the influenza virus on which the stem polypeptide is based, i.e. amino acid substitution.

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

In certain embodiments, the polypeptides of the invention contain at least one mutation on position 353 of SEQ ID NO: 1 or on a corresponding position of another influenza virus.

In certain embodiments, the polypeptides of the invention contain at least one mutation on position 337 of SEQ ID NO: 1 or on the equivalent position of another influenza virus.

In certain embodiments, the polypeptides of the invention contain at least one mutation on position 340 of SEQ ID NO: 1 or on a corresponding position of another influenza virus.

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

In certain embodiments, the mutated amino acid residue on position 340 (HA1 domain) is I
, 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 consensus N-glycosylation in the sequence, eg, as in the case of I340N of SEQ ID NO: 6, for example N-X-T / S (where X is any natural except P). (Amino acid) is introduced.

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

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, amino acid numbering is based on amino acid numbering in H1HA0, in particular the amino acid numbering of the H1N1 influenza strain A / Brisbane / 59/2007 (SEQ ID NO: 1). To. Those skilled in the art can determine the equivalent (or corresponding) amino acids in the HA of other influenza viruses, and thus the equivalent mutations, eg, for sequence alignment of different H1 influenza viruses. See Table 2.

According to the present invention, the polypeptide further comprises a disulfide bridge between the amino acid at position 324 and the amino acid at position 436. Therefore, according to the present invention, at least one disulfide bridge is in the stem domain polypeptide, preferably H1A / Brisba.
It has been introduced between the amino acids at positions 324 and 436 (or its equivalent) in ne / 59/2007 (SEQ ID NO: 1). Therefore, in certain embodiments, the polypeptide is H.
It further comprises the mutant 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 the sequences using suitable algorithms such as Clustal, Muscle and the like. Genetically engineered disulfide bridges are at least one (if the other is already cysteine), but usually
It is created by mutating two spatially close residues to cysteine, which forms a covalent bond between the sulfur atoms of those residues, either spontaneously or by active oxidation.

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

Applicants are partly specific for Group 1 influenza virus (eg, CR6261 as described in WO 2008/028946) and partly specific for Group 2 influenza virus (eg, WO 2010). / 130
We have already identified a broad neutralizing antibody isolated from primary human B cells from a vaccinated individual that was CR8020) described in Pamphlet 636. Detailed epitope analysis of these monoclonal antibodies revealed the reason for the lack of cross-reactivity of those specific antibodies. In both cases, the presence of glycans in Group 1 or Group 2 HA molecules at different positions explained, at least in part, the fact that the antibody is group specific. Identification of CR9114-like antibodies that cross-react with many of the Group 1 and 2HA molecules described below reveals that the human immune system is capable of inducing a very widespread neutralizing antibody against influenza virus. However, given the need for an annual vaccination scheme, those antibodies are subtypes H1 and / or H3 (seasonal).
It is clearly not or only to a very low degree of induction after infection with the influenza virus or after vaccination.

According to the present invention, cross-neutralizing antibodies mimic the specific epitopes of CR6261 and / or CR9114 and, for example, when administered in vivo alone or in combination with other prophylactic and / or therapeutic therapies. Polypeptides that can be used as immunogenic polypeptides to induce are provided. The "cross-neutralizing antibody" is at least two, preferably at least three, four, or five different subtypes of influenza A virus of phylogenetic group 1, and / or at least two, preferably at least three. One, four
Different subtypes of influenza A virus in one or five outbreak groups 2 and / or different subtypes of at least two influenza B viruses, in particular CR626
1 and means an antibody capable of neutralizing at least all virus strains neutralized by CR9114.

The polypeptides of the invention contain epitopes of the stem-binding influenza neutralizing antibody CR6261 and / or CR9114. Thus, in certain embodiments, the polypeptide selectively binds to antibody CR6261 and / or CR9114. In certain embodiments, the polypeptides of the invention do not bind to antibodies CR8020 and / or CR8057. CR6261 used in the present invention includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 9 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 10; CR911.
Reference numeral 4 comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 11 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 12. CR8057 includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 13 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 14. CR8020 includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 17 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 18.

As described above, the polypeptide comprises an influenza hemagglutinin HA1 domain comprising a HA1N terminal stem segment covalently attached to the HA1C terminal stem segment by a binding sequence of 0-50 amino acid residues. The binding sequence, if present, does not occur in either natural HA or wild-type HA. In certain embodiments, the linker is one amino acid residue, two or less amino acid residues, three or less amino acid residues, four or less amino acid residues, five.
One or less amino acid residues, 10 or less amino acid residues, 15 or less amino acid residues, or 20
A peptide containing the following amino acid residues, 30 or less amino acid residues, 40 or less amino acid residues, or 50 or less amino acid residues. In a specific embodiment, the binding sequences are G, GS, GGG, GSG, GSA, GSGS, GSAG, GGGG, GSAGS, G.
It is a sequence selected from the group consisting of SGSG, GSAGSA, GSAGSAG, and GSGSGSG.

In certain embodiments, the HA1N terminal segment is directly bound to the HA1C terminal segment, i.e. the polypeptide does not contain a binding sequence.

The natural form of influenza HA exists as a trimer on cells or viral membranes.
In certain embodiments, the intracellular and transmembrane sequences have been removed such that the secretory (soluble) polypeptide is produced after expression in the cell. Methods for expressing and purifying the secretory ectodomain of HA have been described (eg, Dopheide et al 2009;
Equiert et al 2009, 2011; Stevens et al 200
4, 2006; see 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 comprises transmembrane and intracellular sequences. In other embodiments, the polypeptides of the invention do not contain the intracellular sequence of HA and the transmembrane domain. In certain embodiments, the polypeptide is
Includes Truncate 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 at the C-terminus (numbered by SEQ ID NO: 1) of the HA2 domain from position 530 (or its equivalent) has been removed such that soluble polypeptide is produced after expression in the cell.

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

In some cases, the his tag sequence (HHHHHH (SEQ ID NO: 15) or HHHHHHH (
SEQ ID NO: 16)) can be attached to the (optionally truncated) HA2 domain, optionally via a linker, for purification purposes. Optionally, the linker may contain a proteolytic cleavage site for enzymatically removing the his tag after purification.

In certain embodiments, the polypeptide has a sequence known to form a trimer structure, i.e. GYIPEAPRDGQAYVRKDGEWVLSTFL (SEQ ID NO: 3).
Further stabilization is achieved by introducing at the C-terminus of A2, optionally linked via a linker. Thus, in certain embodiments, the C-terminal portion of the HA2 domain is optionally linked via a linker to the amino acid sequence GYIPEAPRDGQAYVRK.
It has been replaced by DGEWVLSTFL (SEQ ID NO: 3). The linker may optionally contain a cleavage site for later processing according to a protocol well known to those of skill in the art. To facilitate purification of soluble forms, tag sequences such as his tags (HHHHH)
H (SEQ ID NO: 15) or HHHHHHH (SEQ ID NO: 16)) or FLAG tag (DY
KDDDDDK) (SEQ ID NO: 22) or a combination thereof can be added by linking them, optionally via a short linker. The linker may optionally be a proteolytic cleavage site (part of) for later processing according to a protocol well known to those of skill in the art, eg, I.
It 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 at positions 519-565 has been deleted (numbered by SEQ ID NO: 1) and SGRDYKDDDDKLVPRGSPGSGYI.
It has been replaced by PEAPRDGQAYVRKDGEWVLSTFLGHHHHHH (SEQ ID NO: 4).

In one embodiment, the C-terminal portion of the HA2 domain at positions 530-565 has been deleted (numbered by SEQ ID NO: 1) and SGRDYKDDDDKLVPRGSPGSGYI.
It has been replaced by PEAPRDGQAYVRKDGEWVLSTFLGHHHHHH (SEQ ID NO: 4).

Natural HA exists as a trimer on the cell surface. Most of the interactions between the individual monomers that hold the trimer together are localized in the head domain, while in the stem domain, trimerization is mediated by the formation of the trimer coiled coil motif. To. After removal of the head, tertiary structure is destabilized and therefore requires modification to increase protein stabilization. By strengthening the helical tendency of helix CD, a more stable protein can be produced.

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

Surprisingly, according to the present invention, the stability and multimerization state of a polypeptide depends on the exact localization and sequence of the GCN4-derived sequence in the primary sequence of the polypeptide of the invention. Shown.

Therefore, according to the present invention, the sequence RMKQIEDKIEIESK (SEQ ID NO: 20)
Is introduced at positions 419-433 (numbered by SEQ ID NO: 1), or the sequence RMKQIEDKIEEESKQK (SEQ ID NO: 21) is introduced at positions 417-433.

In certain embodiments, the polypeptide is glycosylated.

In the work that results in the present invention, using molecular biology techniques well known to those skilled in the art, for example, s74H9 (described in the co-pending patent application PCT / EP2014 / 060997).
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) modified to sequence RM at positions 419 to 433.
Sequences s74H9-t2 (SEQ ID NO: 93), s127H1-t2 (SEQ ID NO: 91), s71H2-t2 (SEQ ID NO: 97), which contain KQIEDKIEIESK (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: 95)
SEQ ID NO: 100), s6E12-t2 (SEQ ID NO: 94), and s181H9-t2 (SEQ ID NO: 98) were produced.

In a similar fashion, the sequence RMKQIEDKIEIESK 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), and s181H9-t3 (SEQ ID NO: 128) were produced.

The polypeptides of the invention are the previously described stem polypeptides (PCT / EP201).
Increased binding of influenza antibodies, especially CR6261 and / or CR9114, as compared to 2/073706 and PCT / EP2014 / 060997).
And / or show an increase in the tendency to multimerize and / or an increase in stability.

In certain embodiments, the polypeptide has an amino acid sequence:
Including
X ₁ is an amino acid selected from the group consisting of E, I, K, V, A, and T;
X ₂ 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;
X ₄ is an amino acid selected from the group consisting of I, K, R, T, E, G and V;
X ₅ 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;
X ₇ 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;
X ₉ is an amino acid selected from the group consisting of H, I, L, N, R, and S.

In certain embodiments, the polypeptide has an amino acid sequence:
Including
X ₁ is an amino acid selected from the group consisting of E, I, K, V, A, and T;
X ₂ 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;
X ₄ is an amino acid selected from the group consisting of I, K, R, T, E, G and V;
X ₅ 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;
X ₇ 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;
X ₉ is an amino acid selected from the group consisting of H, I, L, N, R, and S.

In certain embodiments, the polypeptide has an amino acid sequence:
Including
X ₁ is an amino acid selected from the group consisting of E, I, K, V, A, and T;
X ₂ 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;
X ₄ is an amino acid selected from the group consisting of I, K, R, T, E, G and V;
X ₅ 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;
X ₇ 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;
X ₉ is an amino acid selected from the group consisting of H, I, L, N, R, and S.

In certain embodiments, the polypeptide has an amino acid sequence:
Including
X ₁ is an amino acid selected from the group consisting of E, I, K, V, A, and T;
X ₂ 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;
X ₄ is an amino acid selected from the group consisting of I, K, R, T, E, G and V;
X ₅ 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;
X ₇ 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;
X ₉ 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, and 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 considered suitable for those skilled in the art, eg, the following techniques.

Therefore, 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, followed by in vitro or in vivo. Can be expressed in. Therefore, the present invention also relates to nucleic acid molecules encoding the above polypeptides. The present invention further relates to vectors containing nucleic acids encoding the polypeptides of the invention. In certain embodiments, the 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. In addition, many vectors can be used for transformation of eukaryotic cells, either directly or in the form of desired fragments isolated from it, and integrate into the genome of such cells in whole or in part. This results in a stable host cell containing the desired nucleic acid in the genome. The vector used can be any vector suitable for cloning DNA and can be used for transcription of the nucleic acid of interest. If a host cell is used, the vector is preferably an integrated vector. Alternatively, the vector can be an episomal replication vector.

One of ordinary skill in the art can select a suitable expression vector and functionally insert the nucleic acid sequence of the present invention. To obtain expression of a nucleic acid sequence encoding a polypeptide, a recombinant nucleic acid that functionally binds a sequence that can drive expression to the nucleic acid sequence that encodes the polypeptide and encodes the protein or polypeptide in an expressible format. It is well known to those skilled in the art that they can provide molecules. Generally, the promoter sequence is located upstream of the sequence to be expressed. Many expression vectors are available in the art, eg, Invitrog.
The pcDNA and pEF vector system of en, pMSCV and pTK-Hyg from BD Sciences, pCMV-Script from Stratagen, etc.
They can be used to obtain suitable promoters and / or transcription termination sequences, poly A sequences, and the like. If the sequence encoding the polypeptide of interest is properly inserted with respect to the sequence governing transcription and translation of the polypeptide of interest, the resulting expression cassette will be useful for producing the polypeptide of interest. Yes, it is called expression. The sequences that drive expression can include promoters, enhancers, etc., and combinations thereof. They should be able to function in the host cell and thereby drive the expression of nucleic acid sequences that are functionally bound to them. Those skilled in the art will appreciate that various promoters can be used to obtain expression of a gene in a host cell. Promoters can be constitutive or regulatory and can be obtained from a variety of resources, such as viruses, prokaryotes, or eukaryotic resources, or can be artificially designed. Expression of the nucleic acid of interest is from a native promoter or a derivative thereof, or from a completely heterologous promoter (Kaufm).
It can be from an, 2000). Some well-known promoters that are heavily used for expression in eukaryotic cells are promoters derived from viruses such as adenovirus.
For example, the E1A promoter, a promoter derived from cytomegalovirus (CMV), such as the pre-CMV early (IE) promoter (referred to as the CMV promoter in the present invention) (eg, obtained from pcDNA, Invitrogen), Simian virus 40. It includes a promoter (Das et al, 1985) derived from (SV40) and the like. Suitable promoters can also be derived from eukaryotic cells, such as metallothionein (MT).
) Promoter, extension 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, etc. Testing for promoter function and promoter strength is a standard of skill in the art and is generally defined as, for example, a test gene, eg, lacZ behind the promoter sequence.
It may include cloning of luciferase, GFP, etc., and expression testing of test genes. Of course, promoters can be altered by deletions, additions, or mutations in their sequences and tested for functionality to find new, attenuated, or improved promoter sequences.
According to the present invention, a strong promoter that imparts high transcriptional levels in optimal eukaryotic cells is preferred.

Constructs can be transfected into suitable prokaryotic expression systems such as eukaryotic cells (eg, plant, fungal, yeast or animal cells) or E. coli using methods well known to those skilled in the art. In some cases, a suitable "tag" sequence (eg, but not limited to, his)
, Myc, strept, or flag tag) or complete protein (eg, but not limited to, maltose binding protein or glutathione S transferase) added to the sequence of the invention to a polypeptide from cells or supernatant Purification and /
Or it can be identified. Optionally, the tag can be later removed by proteolytic digestion, including sequences containing specific proteolytic sites.

Purified polypeptides can be analyzed by spectroscopic methods known in the art (eg, circular dichroism, Fourier transform infrared spectroscopy and NMR spectroscopy or X-ray crystal spectroscopy) to include helix and beta sheets. The presence of the desired structure can be investigated. ELISA, Octet, FACS and the like can be used to investigate the binding of the polypeptides of the invention to the previously described broad neutralizing antibodies (CR6261, CR9114, CR8057). Therefore, a polypeptide according to the present invention having an accurate three-dimensional structure can be selected.

The invention further relates to immunogenic compositions comprising at least one of the polypeptides and / or nucleic acids of the invention in therapeutically effective amounts. The immunogenic composition preferably further comprises a pharmaceutically acceptable carrier. In this context, the term "pharmaceutically acceptable" means that at the dose and concentration in which the carrier is used, it does not cause any unwanted or adverse effects in the subject to which it is administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (Remington's Pharmaceutical Sc.
genes, 18th edition, A.I. R. Gennaro, Ed. , Mack
Publishing Company [1990]; Pharmaceutical
Formulation Development of Peptides and
Proteins, S.A. Forkjaer and L. Hovgaard, Eds.
, Taylor & Francis [2000]; and Handbook of P
harmonical Excipients, 3rd edition, A. et al. K
ibbe, Ed. , Pharmaceutical Press [2000]). The term "carrier" refers to a diluent, adjunct, excipient, or vehicle with which the composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can be used, for example, as liquid carriers, especially for injectable solutions. The exact formulation should be adapted to the mode of administration. The polypeptide and / or nucleic acid molecule is preferably formulated and administered as a sterile solution. The sterile solution is prepared by sterile filtration or by other methods known in the art. The solution can then be lyophilized or filled in a pharmaceutical administration container. The pH of the solution is generally pH 3.0 to 9.5, for example pH 5.0 to 7.
It is in the range of 5.

The present invention also relates to a method of inducing an immune response in a subject, comprising administering to the subject the above-mentioned polypeptide, nucleic acid molecule and / or immunogenic composition. The subject according to the invention is preferably a mammal that can be infected by an infectious disease inducer, particularly an influenza virus, or otherwise benefit from the induction of an immune response, such subject being eg. , Rodents, such as mice, ferrets, or domestic or farm animals, or non-human primates, or humans. Preferably, the subject is a human subject. Therefore, 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.
Subjects utilizing the polypeptides, nucleic acids and / or immunogenic compositions described herein for the immune response of influenza virus, including subtype 0 HA, and / or influenza B virus to influenza virus hemagglutinin (HA). Provide a method of inducing in. In some embodiments, the present invention describes the polypeptides described herein.
Provided is a method of inducing an immune response against an influenza virus containing H1 subtype HA in a subject using a nucleic acid and / or an immunogenic composition.

In some embodiments, the induced immune response is effective for preventing and / or treating influenza virus infections 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 a 2, 3, 4, 5 or 6 subtypes of influenza A virus and / Or is effective for preventing and / or treating influenza A and / or B virus infections caused by influenza B virus. In some embodiments, the induced immune response is effective for preventing and / or treating influenza virus infections caused by influenza viruses, including H1 subtype HA.

Since it is well known that small proteins and / or nucleic acid molecules do not always effectively induce a strong immune response, the addition of adjuvants to polypeptides and / or
Alternatively, it may be necessary to increase the immunogenicity of the nucleic acid molecule. In certain embodiments
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, at the same time as, or after administration of the composition. 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 (eg, MF59). , See International Publication No. 90/14837 Pamphlet)
Saponin formulations such as QS21 and immunostimulatory complex (ISCOM) (eg, US Pat. No. 5,057,540; WO 90/03184; WO 96/11711 ,. See International Publication Nos. 2004/004762, International Publication Nos. 2005/002620); include bacterial or microbial derivatives, examples of which are monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL). ), CpG-motif-containing oligonucleotides, ADP-ribosylated bacterial toxins or variants thereof, such as E. coli heat-labile enterotoxin LT, Vibrio cholerae CT, pertussis toxin PT, or tetanus toxoid TT, Matri
x M (Isconova). In addition, known immunopotentiating techniques, such as proteins known in the art to improve the immune response (eg, tetanus toxoid, CRM).
Fusion of the polypeptide of the invention to (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 include, for example, Coffman et al. (20
It is disclosed by 10).

In one embodiment, the influenza hemagglutinin stem domain polypeptide of the invention is incorporated into a virus-like particle (VLP) vector. VLPs generally include viral polypeptides derived from viral structural proteins. Preferably, the VLP cannot replicate. In certain embodiments, the VLP may lack the entire genome of the virus or may include part of the genome of the virus. In some embodiments, the VLP
Cannot infect cells. In some embodiments, VLPs express one or more viral (eg, viral surface glycoprotein) or non-viral (eg, antibody or protein) targeting moieties known to those of skill in the art on their surface.

In a specific embodiment, the polypeptides of the invention are incorporated into virosomes.
Virosomes containing the polypeptides according to the invention can be produced using techniques known to those of skill in the art. For example, virosome is produced by disrupting the purified virus, extracting the genome, and rearranging the particles with viral proteins (eg, influenza hemagglutinin stem domain polypeptide) and lipids to form lipid particles containing the viral proteins. be able to.

The present invention also relates to the above-mentioned polypeptides, nucleic acids and / or immunogenic compositions for inducing an immune response in a subject against influenza HA, which is used specifically as a vaccine. Thus, an influenza hemagglutinin stem domain polypeptide, a nucleic acid encoding such a polypeptide, or a vector containing such a nucleic acid or a polypeptide described herein is for influenza virus, eg, the stem region of influenza virus hemagglutinin. Can be used to induce neutralizing antibodies against. The present invention particularly relates to phylogenetic group 1 and / or phylogenetic group 2.
The above-mentioned polypeptides, nucleic acids, and / or immunogenic compositions used as vaccines in the prevention and / or treatment of diseases or conditions caused by influenza A virus and / or influenza B virus. In one embodiment
Vaccines can be used in the prevention and / or treatment of diseases caused by phylogenetic groups 1 and / or 2 2, 3, 4, 5, 6 or more different subtypes 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, including H1 subtype HA.

The polypeptides of the invention can be used post-synthesis in vitro or in suitable cell expression systems, eg, bacteria and eukaryotic cells, or in vivo where it is needed as an immunogen. It can be expressed by expressing a nucleic acid encoding a sex polypeptide. Such nucleic acid vaccines can take any form, eg, a naked DNA, plasmid, or viral vector, eg, an adenovirus vector.

Administration of polypeptides, nucleic acid molecules, and / or immunogenic compositions according to the invention can be performed using standard routes of administration. Non-limiting examples include parenteral administration, such as intravenous, intradermal, transdermal, intramuscular, subcutaneous, etc., or mucosal administration, such as intranasal, oral, etc. One of ordinary skill in the art can determine various possibilities for inducing an immune response by administering a polypeptide, nucleic acid molecule, and / or immunogenic composition according to the invention. In certain embodiments, the polypeptide, nucleic acid molecule, and / or immunogenic composition (or vaccine) is administered more than once, i.e., in a so-called homologous 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, for example, after a time interval of 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
Polypeptides such as 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 molecules, and / or can be performed up to several years after administration of the first dose of the immunogenic composition. It is also possible to administer the vaccine three or more times, for example three or four times, so that two or more boost administrations follow after the first priming administration. In other embodiments, the polypeptides, nucleic acid molecules, and / or immunogenic compositions according to the invention are administered only once.

The polypeptide, nucleic acid molecule, and / or immunogenic composition can also be administered in a heterologous prime-boost regimen as either prime or boost.

The present invention further provides methods of using the polypeptides, nucleic acids and / or compositions described herein to prevent and / or treat influenza viral diseases in a subject. In a specific embodiment, a method of preventing and / or treating an influenza viral disease in a subject is to administer an effective amount of the above-mentioned polypeptide, nucleic acid and / or immunogenic composition to the subject in which it is needed. Including that. A therapeutically effective amount is the prevention, amelioration and / or prevention and amelioration of diseases or conditions resulting from infection with group 1 or 2 influenza A virus and / or influenza B virus, especially those caused by infection with influenza A virus, including H1 subtype HA. Or refers to the amount of a therapeutically effective 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, onset or progression of one or more of the symptoms associated with influenza virus infection. The improvements used herein can refer to the reduction of visible or sensory disease symptoms, viremia, or any other measurable flu infection.

Those in need of treatment include those who are already suffering from the pathology resulting from infection with Group 1 or Group 2 influenza A virus or influenza B virus, and those who should be prevented from being infected with influenza virus. Thus, the polypeptides, nucleic acids and / or compositions of the present invention have not been or have been infected by an unmedicated subject, i.e., a disease caused by influenza virus infection, and are still infected. It can be administered to subjects who have not been infected or who have already been and / or have been infected with the influenza virus.

In one embodiment, prophylaxis and / or treatment can be targeted in a group of patients susceptible to influenza virus infection. Such patient groups are, but are not limited to, for example, older (eg, ≧ 50, ≧ 60, preferably ≧ 65), younger (eg, ≤5, ≤1) , Inpatients and treated with antiviral compounds,
Includes patients who have shown an inappropriate antiviral response.

In another embodiment, the polypeptide, nucleic acid and / or immunogenic composition is subject to one or more other active agents, such as existing or future influenza vaccines, monoclonal antibodies and / or antiviral agents, and. It can be administered in combination with / or an antibacterial agent and / or an immunomodulator. One or more other activators may be beneficial in the treatment and / or prevention of influenza viral disease, or may ameliorate the symptoms or conditions associated with influenza viral disease. In some embodiments, one or more other activators are analgesics, antipyretics, or therapeutic agents that relieve or assist breathing.

The administration regimen of the polypeptides and / or nucleic acid molecules of the invention provides the optimum desired response (
For example, it can be adjusted to provide a therapeutic response). A suitable dosage range can be, for example, 0.1 to 100 mg / kg body weight, preferably 1 to 50 mg / kg body weight, preferably 0.5 to 15 mg / kg body weight. The exact dose of polypeptide and / or nucleic acid molecule to be used depends, for example, on the route of administration and the severity of the infection or the disease caused by it, and should be determined according to the judgment of the physician and the circumstances of each subject. .. For example, the effective dose will vary depending on the patient's target site, physiological condition (eg, age, weight, health), regardless of whether the treatment is prophylactic or therapeutic. Patients are usually humans, but non-human mammals, eg transgenic mammals, can also be treated. Therapeutic doses are optimally titrated to optimize safety and efficacy.

The 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 test the immune status of an individual as a diagnostic tool, eg, by checking for the presence of antibodies in the serum of such individuals that can bind to the polypeptides of the invention. Can be used to Accordingly, the present invention is also an in vitro diagnostic method for detecting the presence of influenza infection in a patient, a) a step of contacting a biological sample obtained from said patient with a polypeptide according to the invention; and b) an antibody. -Regarding methods involving the step of detecting the presence of an antigen complex.

The 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 present invention will be further described in the following examples and drawings. The examples are by no means limiting the scope of the invention.

Example 1: Stem-based polypeptide described in PCT / EP2014 / 060997 PCT / EP2012 / 073706 describes an influenza hemagglutinin stem domain polypeptide, composition and vaccine and influenza prevention and /.
Or it discloses how to use them in the field of treatment. PCT / EP2014 / 0
60997 is obtained by site-specific mutation of H1-mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 2), CR6261 (Throsby et a).
l, 2009; Equiert et al 2010) and / or H1N1A / Brisbane / 59/20 that more stably presented the broadly neutralized epitope of CR9114.
The additional sequence of the stem domain polypeptide derived from the full length HA of 07 (SEQ ID NO: 1) is disclosed.

H1-mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 2) is H1N1
Derived from the full length HA of A / Brisbane / 59/2007 (SEQ ID NO: 1) by using the following steps:
1. 1. To remove the cleavage site of HA0. Cleavage of wild-type HA at this site results in HA1 and HA2. Removal can be achieved by mutation from R to Q at position P1 (eg, for a description of the nomenclature of the cleavage site (position 343 of SEQ ID NO: 1), see S.
See un et al, 2010).
2. Removing the head domain by deleting amino acids 53-320 from SEQ ID NO: 1. The remaining N- and C-terminal portions of the sequence were linked by a 4-residue flexible linker GGGG.
3. 3. Residues 402-41 in H1A / Brisbane / 59/2007 (SEQ ID NO: 1)
Increases the solubility of the loop (between A-helix and CD-helix) formed by (equivalent to) 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)
In, mutants F406S, V409T, F413G and L416S (numbering refers to SEQ ID NO: 1) were introduced.
4. Introducing disulfide bridges between amino acids at positions 324 and 436 in H1A / Brisbane / 59/2007; this is the mutations R324C and Y436.
This is achieved by introducing C (numbering refers to SEQ ID NO: 1).
5. Introduce the GCN4-derived sequence MKQIEDKIEEESKQ (SEQ ID NO: 5) known to be trimerized 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 secreted (soluble) polypeptides are produced after expression in the cell.
7,518,519,520,521,522,523,524,525,526,52
It has been deleted from position 6, 527, 528, 529, or 530 (or its equivalent as determined by sequence alignment) to the C-terminus of HA2 (numbered by SEQ ID NO: 1). Soluble polypeptides are sequences known to form trimer structures, i.e., earth.
The sequence GYIPEAPRDGQAYVRKDGEWVLSTFL (SEQ ID NO: 3) was further stabilized by introduction, optionally ligated via the short linker described above.
The linker may optionally contain a cleavage site for later processing according to a protocol well known to those of skill in the art. To facilitate purification and detection of soluble forms, tag sequences such as histidine tags (HHHHHH (SEQ ID NO: 15) or HHHHHHH (SEQ ID NO: 16) or FLAG tags (DYKDDDDK; SEQ ID NO: 22) or combinations thereof may optionally be used. It can optionally be added by ligation via a short linker. The linker may optionally be (part of) a proteolytic cleavage site for later processing according to a protocol well known to those skilled in the art, eg, LVPRGS (sequence). Number 23) (Thrombin)
Alternatively, it may contain 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, thrombin cleavage site, folon, and His sequence is FLAG-thrombin-foldo of SEQ ID NO: 4.
n-His. This sequence is H1-mini2-cluster1 + 5 + 6-GCN4.
A parent sequence (SEQ ID NO: 6) was created in combination with the soluble form of the (SEQ ID NO: 2) sequence, which was used to generate the novel polypeptide of the invention by mutagenesis. This sequence does not contain the leader sequence corresponding to amino acids 1-17 of SEQ ID NOs: 1 and 2.

Therefore, the stem domain polypeptide lacks part of the hemagglutinin sequence encoding the head domain of the molecule as described in PCT / 2012/073706 and above, and deletes the N- and C-terminal parts of the sequence. It was created by relinking via a linker on both sides of the. Removal of the head domain leaves some of the molecules already protected from the aqueous solvent exposed, potentially destabilizing the structure of the polypeptides of the invention. For this reason, the residues in the B loop, especially 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). ,
Mutations were made in various combinations using SEQ ID NO: 6 of the parent sequence as a starting point. SEQ ID NO: 6 removes the leader sequence from H1-mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 2) and leaves residues 520-565 in Flag-thrombin-fold.
It was created by substituting with the on-his sequence (SEQ ID NO: 4).

Similarly, in the area surrounding the fusion peptide, a large number of hydrophobic residues are exposed to the solvent, which, unlike natural full length HA, does not allow the polypeptide to be cleaved and implants the hydrophobic fusion peptide inside the protein. Caused by the fact that it undergoes related conformational changes. To address this issue, some or all of the 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), and 181H9 (SEQ ID NO: 62) were produced.

The DNA sequence encoding the polypeptide is transformed into Pichia pastoris using a protocol well known to those of skill in the art, or HEK2.
Transfected into 93F cells. The construct 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 the construct used for expression in pastoris), the HA leader sequence was replaced with a yeast alpha factor leader sequence (SEQ ID NO: 7). The expressed protein was thus directed to the cell culture medium, thus allowing binding and expression determination without further purification of the polypeptides of the invention. All sequences contained the FLAG-foldon-HIS C-terminal sequence (SEQ ID NO: 4).

Monoclonal antibody binding to polypeptide (CR6261, CR9114, CR802
0) was determined by ELISA. For this purpose, an ELISA plate of 2 μg / m
It was treated overnight at 4 ° C. with l of a monoclonal antibody solution (20 μl / well). After removal of the antibody solution, the residual surface was blocked with a 4% solution of skim milk powder in PBS at room temperature for at least 1 hour. After washing the plates, 20 μl of cell culture medium (undiluted or diluted) was added to each well and incubated at room temperature for at least 1 hour. The ELISA plate is then washed and 20 μl of anti-FLAG-HRP antibody solution (S).
igma A8952, diluted 2000-fold in 4% skim milk powder in PBS-Tween) was added. After incubation (1 hour at room temperature), the plate was washed once again and 20
A signal was expressed by adding μl of a luminescent substrate (Thermochemical C # 34078). Alternatively, a colorimetric detection method can be used to express the signal.

Expression of the polypeptides of the invention is a homogeneous time-resolved fluorescence assay (for general description,
For example, Degorce et al. , Curr. Chem. Genomics 20
It can be determined from 09 3: 22-32). Tellurium (Tb) for this purpose
) A mixture (HTRF solution) of a labeled anti-FLAG monoclonal antibody (donor) and an Alexa488-labeled anti-His monoclonal antibody (acceptor) is 210.5 μl of anti-F.
LAG-TB (stock solution 26 μg / ml) and 1.68 ml anti-HIS-488 (stock solution 50)
μg / ml) 1 of 80 ml of culture medium and 50 mM HEEPS + 0.1% BSA
Prepared by adding to a 1: 1 mixture. 19 μl of HTRF solution was added to each well of the ELISA plate and 1 μl of culture medium was added. Ratios of fluorescence emission at 520 and 665 nm were determined upon excitation and after a delay to allow attenuation of short-lived background signals resulting from other compounds (proteins, medium components, etc.).
This is a measure of total protein content in a sample and is used to normalize mAb binding signals between different experiments.

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

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

Example 2: Design and characterization of the polypeptide of the invention The polypeptide of the invention is derived from the yeast transcription activator protein GCN4 sequence RMKQIEDKIEIESK (SEQ ID NO: 20) or RMKQIEDKIEIESK.
KQK (SEQ ID NO: 21) is contained in the CD helix. This sequence has a high tendency to form helical secondary structures and thus can 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 depend on the exact localization and sequence of the GCN4-derived sequence in the primary sequence of the polypeptides of the invention. Found.

Thus, we hereby introduce the sequence RMKQIEDKIEIESK (SEQ ID NO: 20) at positions 419-433 (numbering by SEQ ID NO: 1; eg, SEQ ID NOs: 81-110) or the sequence RMKQIEDKIEIESKKQK (SEQ ID NO: 20). 21) is introduced at positions 417-433 (eg, SEQ ID NOs: 111-140).
) A novel set of polypeptides of the invention is described.

To this end, the polypeptides of Example 1 using molecular biology techniques well known to those of skill in the art, namely 74H9 (SEQ ID NO: 57), 127H1 (SEQ ID NO: 55), 71H2.
(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
SEQ ID NO: 74H9-t2 containing SEQ ID NO: RMKQIEDKIEIESK (SEQ ID NO: 20) at positions 419-433 by modifying 12 (SEQ ID NO: 58), 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 produced.

In a similar fashion, the sequence RMKQIEDKIEIESK at positions 417-433
SEQ ID NO: 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) and 181H9-t3 (SEQ ID NO: 118) were produced.

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

As mentioned above, the soluble polypeptide of the invention is, for example, residue 519 of the HA2 domain.
520, 521, 522, 523, 524, 525, 526, 527, 526, 528
H at the C-terminus of the HA2 domain (numbered by SEQ ID NO: 1) from 5,29, or 530
It can be produced by removing the C-terminal portion of the A-base sequence.

The polypeptide is a sequence known to form a trimer structure, i.e. GYIPEAP.
Further stabilization can be achieved by introducing RDGQAYVRKDGEWVLSTFL (SEQ ID NO: 3), optionally ligated via a linker. The linker may optionally contain a cleavage site for later processing according to a protocol well known to those of skill in the art. To facilitate purification of soluble forms, tag sequences such as his tags (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 can be added by linking them, optionally via a short linker. The linker may optionally be a proteolytic cleavage site (part of) for later processing according to a protocol well known to those of skill in the art, eg,
It 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 are residues 519-5.
The equivalent of 65 (numbering refers to SEQ ID NO: 1) was created by replacing with the sequence RSLVPRGSPGHHHHH containing both the modified thrombin cleavage site and the 6 histidine tag (SEQ ID NO: 15), according to a protocol well known to those of skill 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)
The soluble form of 3). Culture medium was harvested and binding to CR6261, CR9114 was performed by sandwich ELISA against CR6261 or CR9114 of coat mAb for capturing the polypeptide of the invention directly from the culture medium and the C-terminal his tag for detection purposes. Detected using a horseradish peroxidase (HRP) conjugated antibody directed to. Alternatively, biotinylated CR9114 was used in combination with HRP-conjugated streptavidin to detect the polypeptide of the invention captured by CR9114 in sandwich ELISA. This format allows the detection of the presence of multimeric forms of the polypeptides of the invention. All polypeptides of the invention tested are EL
It could bind to CR9114 (FIGS. 2A and B, FIGS. 3A and B and FIGS. 4A and B) and CR6261 (FIGS. 2C and D, FIGS. 3C and D, FIGS. 4C and D) as determined by ISA. CR9114 capture-biotinylated CR9114 detection sandwich ELI
The increase in the level of multimerization detected by SA is s55G7-t2 (SEQ ID NO: 95), s86B4- as shown in FIGS. 2E and F, FIGS. 3E and F and FIGS. 4E and F.
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)
, S74H9-t3 (SEQ ID NO: 123).

To obtain a highly pure preparation 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
The expression vector pcDNA2004 containing the gene encoding the soluble form of 2) and 55G7-t2 (SEQ ID NO: 85) was transfected. 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) that directs protein transport during production is present in the secretory final polypeptide.

For the production of the polypeptide of the invention 1.0 by spinning down HEK293F cells (Invitrogen) at 300 g for 5 minutes and resuspending in 300 mL of preheated Freestyle ™ medium via SF1000 flask ^* 10 ⁶ vc
/ ML was sown. This culture was placed in a multitron incubator at 37 ° C., 10 ° C.
Incubated at 110 rpm for 1 hour with% CO ₂ . After 1 hour, plasmid D
NA was pipetted to a concentration of 1.0 μg / mL in 300 mL culture volume in 9.9 mL Optimem medium. In parallel, 440 μL of 293fectin® was pipetted in 9.9 mL 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. Place the transfected culture in a multitron incubator at 37 ° C.
Incubated at 10% CO ₂ and 110 rpm. On day 7, cells were separated from the culture medium by centrifugation (30 min at 3000 g), while the supernatant containing the soluble polypeptide of the invention was filtered through a 0.2 μm bottle top filter for further treatment.

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

FIG. 5 and Table 8 show that the polypeptides of the invention s127H1-t2 have higher yields (about 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 what is expected for a monomer or dimer. SE to support protein aggregation
The C-MALS experiment was 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 (proven by a shift of peaks in the SEC chromatogram) in the presence of Fab fragments from CR6261 and CR9114, but 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 to HA from Group 1 while CR8020
Is not combined. The size of the complex is listed in Table 8, which is the polypeptide s12.
It is shown that 7H1-t2 binds to 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.

Complexes of their antibodies with purified proteins to further analyze the binding reaction of the polypeptides 127H1-t2 of the invention with the CR6261 and CR9114 of mAbs and to support the presence of conformational epitopes of CR6261 and CR9114. Chemistry was tested by biolayer interferometry (Optet Red ³⁸⁴ , Forte Bio). For this purpose, biotinylated CR6261, CR9114 and CR8020 are immobilized on a streptavidin-coated sensor, which is then exposed to a solution of the first purified polypeptide of the invention to measure the association rate and then wash. The dissociation rate was measured after exposure to the solution. The results are shown in FIG.

Immobilized CR6261 and CR9114 both recognize the polypeptides of the invention as evidenced by a clear response after exposure to the soluble form of 127H1-t2 (FIG. 8). Titer measurements were performed using a 2-fold dilution series to estimate the dissociation constants for binding interactions. Sensors containing immobilized CR6261 or CR9114, 40, 20
Soluble s127H1-t at concentrations of 10, 5, 2.5, 1.3 and 0.63 nM
The two solutions were exposed to each and the final response was recorded after 6600 seconds. Responses were plotted as a function of stem domain polypeptide concentration, fitted to a steady-state 1: 1 binding model, and 3.5 nM and CR for the CR6261 / stem domain polypeptide complex.
A dissociation constant K _d of 2.3 nM was generated for the 9114 complex (FIG. 8).

Taken together, the polypeptides of the invention s127H1-t2 (SEQ ID NO: 91 is produced in large quantities and can bind to the broadly neutralized monoclonal antibodies CR6261 and CR9114 with high affinity for the corresponding neutralizing epitopes in this stem domain polypeptide. Supporting its existence. Polypeptides tend to form dimeric structures.

Example 3: Evaluation of the protective efficacy of the polypeptide of the present invention in the lethal influenza challenge model The polypeptide of the present invention s127H1-t in the lethal influenza challenge model
10 female BALB / c mice (6-8) to assess the protective efficacy of 2 (SEQ ID NO: 91)
The (week-old) group was immunized three times at 3-week intervals with 10 μg of purified s127H1-t2 without adjuvant or with 10 μg of Matrix-M adjuvant. As a positive control for the challenge model, broad neutralizing antibody monoclonal antibody CR62
61 (15 mg / kg) was administered intramuscularly 1 day prior to the challenge, while immunization with PBS served as a negative control. Four weeks after the last immunization, mice were challenged with a 25 × LD50 heterologous challenge virus (H1N1A / Puerto Rico / 8/34) and monitored daily for 3 weeks (survival rate, body weight, clinical score). Binding of pre-challenge serum to the polypeptide s127H1-t2 of the invention used for immunization (to confirm accurate immunization), binding to soluble H1N1A / Brisbane / 59/07 full-length HA (full-length HA) For confirmation of recognition) and for competition with the broad-spectrum neutralizing antibody monoclonal antibody CR9114 for binding to full-length HA (to determine whether the induced antibody binds in close proximity to the broad-spectrum neutralizing CR9114 epitope). Tested in an ELISA assay. The results are shown in FIGS. 9-12.

The results show that the experiment is valid. This is because all mice in the PBS control group die from infection 7 days after the challenge, while the positive control group (15 mg / kg CR6261, 1 day before the challenge) is fully protected (Figure). 9). In contrast to PBS-treated mice, 3 out of 10 mice immunized with the adjuvant-free polypeptide of the invention s127H1-t2 (SEQ ID NO: 91) and the adjuvant-adjuvant polypeptide of the invention. Ten out of ten mice survive the lethal challenge (see Figure 10). An increase in survival rate, an increase in survival time and a decrease in clinical score are observed for the group immunized with the polypeptide s127H1-t2 of the present invention as compared to the PBS control group. The difference is most pronounced in the group that received the adjuvanted polypeptide of the invention, but is also observed in the group that received the adjuvant-free polypeptide.

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

Competitive binding ELISAs were performed to further understand the immunological response to immunization.
For this purpose, plate-bound full-length HA was incubated with serially diluted serum samples.
After that, CR9114-biotin at a predetermined titer measurement concentration is added. After further incubation, the amount of bound CR9114-biotin is quantified using streptavidin-conjugated horseradish peroxidase according to a protocol well known in the art. Data are analyzed using a linear regression of OD for logarithmic dilution expressed as "slope OD" (ΔOD / 10-fold dilution). The data show that a detectable level of antibody that can compete for binding with the broad neutralizing antibody CR9114 is induced by immunization with the adjuvanted polypeptide of the invention, and the increased level of competition observed in FIG. As indicated by. For comparison, the levels induced by the unlabeled CR9114 (ie, self-competitive) and the unbound monoclonal antibodies CR8020 and CR-JB (both serially diluted from a starting concentration of 5 μg / ml) are shown in separate graphs.

In summary, we present the polypeptides of the invention s127H1-t2 (SEQ ID NO: 91).
) Can protect mice against lethal influenza infection. Polypeptides are immunogenic and induce antibodies capable of binding full-length HA. When the polypeptides of the invention are used in combination with adjuvants, at least some of the derived detectable antibodies bind to, or close to, the epitope of the broadly neutralizing epitope of the monoclonal antibody CR9114.

Example 4: Evaluation of the protective efficacy of the polypeptide of the present invention in the lethal influenza challenge model Polypeptide s127H1-t of the present invention in the lethal influenza challenge model
To further evaluate the protective efficacy of 2 (SEQ ID NO: 91), 10 female BALB / c mice (
The 6-8 week old) group was immunized 1, 2 and 3 times at 3-week intervals with 30 μg of purified s127H1-t2 supplemented with 10 μg of Matrix-M. As a positive control for the challenge model, broad neutralizing antibody monoclonal antibody CR6261 (
15 mg / kg) was administered intravenously 1 day prior to the challenge, while immunization with PBS served as a negative control. Four weeks after the last immunization, mice were challenged with a 25 x LD50 heterologous challenge virus (H1N1A / Puerto Rico / 8/34).
Daily monitoring for 3 weeks (survival rate, body weight, clinical score). The pre-challenge serum obtained 4 weeks after the last immunization was used for immunization of the polypeptide of the present invention, s127H.
Binding to 1-t2 (to confirm accurate immunization), soluble H1N1A / Brisban
Competition with the broad-spectrum neutralizing monoclonal antibody CR9114 for binding to e / 59/07 full-length HA (to confirm recognition of full-length HA) and binding to full-length HA (induced antibody becomes broad-neutralizing antibody CR9114 epitope) (To determine if they bind in close proximity) were tested in an ELISA assay. The results are shown in FIGS. 13-18.

The results show that the experiment is valid. This is because all mice in the PBS control group die from infection 7 days after the challenge, while the positive control group (15 mg / kg CR6261, 1 day before the challenge) is fully protected (Figure). 13A). s12
Mice immunized once with 7H1-t2 (SEQ ID NO: 91) all died from infection between days 7 and 9 (FIG. 14A). In contrast, after two immunizations, eight out of ten mice survived, and after three immunizations, all mice (10 out of 10) survived the lethal challenge (FIG. 14B). , C). Weight loss was also reduced for multiple immunized groups, 3
The lowest proportions were observed for the immunized animals (FIGS. 15B, C). 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 are due to the polypeptides of the invention s127H1-t2 It was observed for the group immunized two or three times.

ELISA data from the pre-challenge point 4 weeks after the last immunization using s127H1-t2 (FIG. 17A) or soluble full length HA (FIG. 17B) as an antigen show that the polypeptide of the invention s127H1 is immunogenic. It is shown to induce antibodies capable of recognizing full-length HA even after one immunization, but the levels are significantly higher after two and three immunizations. Detectable levels of antibodies that can compete for binding with the broad neutralizing antibody CR9114 using the CR9114 competitive binding assay after two and three immunizations with the polypeptide of the invention s127H1-t2 (SEQ ID NO: 91). It was induced (Fig. 18A). For comparison, the levels induced by the unlabeled CR9114 (ie, self-competitive) and the unbound monoclonal antibodies CR8020 and CR-JB (both serially diluted from a starting concentration of 5 μg / ml) are shown in separate graphs (FIG. 18B). ..

In summary, we present the polypeptides of the invention s127H1-t2 (SEQ ID NO: 91).
It was shown that a few immunizations with) could protect the mice against lethal infection by influenza. Polypeptides are immunogenic and induce antibodies capable of binding full-length HA. At least a portion of the induced antibody binds to or near the epitope of the broadly neutralizing epitope of the monoclonal antibody CR9114.

Example 5: Evaluation of the protective efficacy of the polypeptide of the present invention in the lethal heterologous H5N1 influenza challenge model The polypeptide s127 of the present invention in the lethal H5N1 influenza challenge model
To further evaluate the protective efficacy of H1-t2- (SEQ ID NO: 91), 8-12 female BALs
A group of B / c mice (6-8 weeks old) was immunized three times at 3-week intervals with 30 μg of purified s127H1-t2 supplemented with 10 μg Matrix-M. Widespread Neutralizing Antibody Monoclonal Antibody CR6261 as a positive control for the challenge model
(15 mg / kg) was administered intravenously 1 day prior to the challenge, while immunization with PBS served as a negative control. Four weeks after the last immunization, mice were challenged with a 12.5 x LD50 heterologous subtype challenge virus (H5N1A / Hong Kong / 156/97) and monitored daily for 3 weeks (survival rate, body weight, clinical score). ..

The results show that the experiment is valid. This is because all mice in the PBS control group die from infection between 8 and 10 days after the challenge, while the positive control group (15 m).
This is because the g / kg CR6261 (1 day before the challenge) is completely 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 survivors 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). Compared to the PBS control group, statistically significant increase in survival rate, increase in survival time, decrease in body weight loss and reduction in clinical score are the polypeptides of the present invention s127H1.
It is observed for the group immunized by -t2. Taken together, we have shown that immunization with the polypeptide of the invention s127H1-t2 (SEQ ID NO: 91) can protect mice against lethal infections with the heterologous subtype H5N1 influenza strain.

Example 6: Evaluation of Serum Binding Region Induced Through Immunization with the Polypeptides of the Invention The pre-challenge serum from the triple-immunized mice described in Example 5 was used in several other groups. Binding to full-length HA from strains 1 (H1, H5 and H9) and Group 2 (H3 and H7) influenza was also tested by ELISA according to a protocol well known in the art (FIG. 20). The result is the polypeptide of the invention s127H1-
The antibody induced by t2 (SEQ ID NO: 91) efficiently recognizes the epitope present in the natural sequence of FL HA, and the epitope to which the antibody binds comprises different Group 1 influenza including H1, H5 and H9 HA. Demonstrate that it is preserved between strains.

Example 7: Evaluation of the protective efficacy of the polypeptide of the invention in the lethal H1N1A / Brisbane / 59/2007 influenza challenge model To further evaluate the protective efficacy of s127H1-t2 (SEQ ID NO: 91) in the lethal H1N1 influenza challenge model, 8 A group of ~ 18 female BALB / c mice (6-8 weeks old) was adjuvant with 10 μg of Matrix-M in 30 μg of purified s1
It was immunized 3 times with 27H1-t2 at 3-week intervals. As a positive control for the challenge model, broad neutralizing antibody monoclonal antibody CR6261 (15 mg / kg) was administered intravenously 1 day prior to the challenge, while immunization with PBS served as a negative control.
Four weeks after the last immunization, mice were subjected to a 12.5 x LD50 challenge virus (H1N).
1A / Brisbane / 59/2007) challenged and monitored daily for 3 weeks (survival rate, body weight, clinical score).

The results show that the experiment is valid. This is because all mice in the PBS control group die from infection between 7 and 10 days after the challenge, while the positive control group (15 m).
This is because the g / kg CR6261 (1 day before the challenge) is completely protected (Fig. 21A).
). Ten of the ten mice immunized with s127H1-t2 (SEQ ID NO: 91) survive the lethal challenge (FIG. 21B). In addition, body weight loss averages about 20% 5 days after infection (Fig. 21C), but the animals fully recover within a 21-day follow-up period. The median clinical score peaks at a value of 3 between 2 and 9 days after infection, but returns to baseline level (0) after 16 days after infection (FIG. 21D). PB
Statistically significant increase in survival rate, increase in survival time, decrease in body weight loss and decrease in clinical score were observed in the group immunized with the polypeptide s127H1-t2 of the present invention as compared to the S control group. To.

In summary, we present the polypeptides of the invention s127H1-t2 (SEQ ID NO: 91).
) Can protect mice against lethal infections by H1N1A / Brisbane / 59/2007.

Example 8: Assessment of the presence of influenza neutralizing antibodies in the sera of mice immunized with the polypeptides of the invention Pre-challenge sera were used to further investigate antibody-mediated effector mechanisms that play a role in protection against influenza. The following H5N1A / Vietnam / 1194/0
Pseudoparticle neutralization assay using sham particles derived from 4 (Alberini et al 2)
It was tested in 009).

Pseudoparticle Neutralization Assay Pseudoparticles expressing FL HA were generated as described above (Temperton et.
al. , 2007). As already described (Alberini et al 2009)
(Slightly modified), H5A / Vietnam encoding the luciferase reporter gene
Neutralizing antibodies were determined using a single transduction round of HEK293 cells with m / 1194/04 pseudoparticles. Briefly, heat-inactivated (30 minutes at 56 ° C.) pre-challenge serum sample in growth medium (2 mM L-glutamine (Lonza), 1% non-essential amino acid solution (Lonza), 100 U / ml penicillin / Streptomycin (Lonza)
) And E supplemented with 10% FBS (Euroclone, Pero, Italy)
MEM Eagle (Lonza, Basel, Switzerland) with BSS
)) In, in a 96-well flat-bottom culture plate, dilute in triplicate in triplicate, and titer H5.
A / Vietnam / 1194/04 false particles (following infection 10 ⁶ relative light units (RLU)
To give rise to) was added. 37 ° C., after incubation for 1 hour in 5% CO _2, was added 10 ⁴ HEK293 cells per well. After 48 hours of incubation at 37 ° C. and 5% CO ₂ , the luciferase substrate (Britellie Plus)
, PerkinElmer, Waltham, MA), and luminometer (Mi)
tras LB 940, Berthold Technologies, Germa
Luminescence was measured using ny) according to the manufacturer's instructions.

Pre-challenge sera obtained from animals immunized with the polypeptides of the invention s127H1-t2 (SEQ ID NO: 91) of Examples 5, 6 and 7 are high sera using a pseudoparticle neutralization assay. It showed detectable neutralization at the concentration (Fig. 22). This demonstrates the ability of the polypeptides of the invention to induce broad 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 cytotoxicity (ADCP) contribute substantially to protection against influenza, and stem-oriented bnAb is particularly effective in their mechanism (DiLil).
lo et al. , 2014). Polypeptide s127H1-t2l18lo of the present invention
To test whether antibodies induced after immunization with ng (SEQ ID NO: 186) can induce ADCC, we have adapted ADCC surrogate assays for mice as described below (Parek et al. , 2012; Schneuriger et al
.. , 2012; Cheng et al. , 2014) was used to test pre-challenge sera.

Antibody-dependent Cellular Injury (ADCC) Surrogate Assay Human lung cancer-derived A549 epithelial cells (ATCC CCL-185) at 37 ° C. in Dalveco-modified Eagle's Medium (DMEM) medium supplemented with 10% heat-inactivated fetal bovine serum. 1, 1
It was maintained at 0% CO2. Two days before the experiment, Opti-MEM (Invitrogen)
) In A5 using Lipofectamine 2000 (Invitrogen)
H5A / Hong Kong / 156/97 HA or H1A / Brisb in 49 cells
The plasmid DNA encoding ane / 59/2007 HA was transfected. One day before assay, transfected cells were harvested and white 96-well plates (Co) for ADCC.
Black transparent bottom 96-well plate (BD F) in star) and for imaging
It was sown in alcon). After 24 hours, sample the assay buffer (RPMI1640 (G).
It was diluted in 4% ultratrace IgG FBS (Gibco)) in ibco), heat-inactivated at 56 ° C. for 30 minutes, and then serially diluted in assay buffer. For ADCC bioassay, A549 cells were supplemented with fresh assay buffer, antibody dilutions and mouse Fc
ADCC Bioassay Jurkat effector cells (FcγRIV; Promega) expressing gamma receptor IV were added to the cells and incubated at 37 ° C. for 6 hours at a target-effector ratio of 1: 4.5. The cells were equilibrated to room temperature for 15 minutes before adding the Bio-Glo Luciferase System substrate (Promega). The luminescence was read out after 10 minutes on Synergy Neo (Biotek). The data are expressed as the induction factor of the signal in the absence of serum.

Using this assay, the polypeptides of the invention s12 according to Examples 5, 6 and 7.
Pre-challenge serum obtained from animals immunized with 7H1-t2 (SEQ ID NO: 91) can be used as an antigen resource for H5N1A / Hong Kong / 156/97 or H1N1.
FL HA from A / Brisbane / 59/07 was tested for FcγRIV signaling activity using target cells transfected (FIG. 23). In both cases, 30-fold induction was observed at the maximum serum concentration tested and ADCC / AD in mice.
Demonstrate the ability of the polypeptides of the invention to induce antibodies that activate FcγRIV signaling that exhibits CP effector function.

These results shown in Examples 5-8 show the polypeptides of the invention s127H1-
It is shown that the ability of t2 (SEQ ID NO: 91) can induce stem targeting, neutralization and ADCC-mediated antibodies and protect mice against lethal challenges by allogeneic, heterologous and heterologous subtype Group I influenza strains.

Example 9: Protection from lethal challenges by H5N1A / Hong Kong / 156/97 by passive transmission of serum from mice immunized with the polypeptide of the invention Derived by the polypeptide of the invention for observed protection. A transplant test was performed to determine the contribution of the antibody. The purpose of this study was to contain s127H1-t2 (SEQ ID NO: 91) and an additional His tag in the presence of an adjuvant (Matrix-M).
Passive transmission (multiple doses) of serum from mice immunized three times with H1-t2long (SEQ ID NO: 101) is H5N1 influenza A / Hong Kong / 156/97.
It was to evaluate whether or not to grant protection against the lethal challenge by.

A group of female BALB / c donor mice (6-8 weeks old) containing 30 μg of s127H1-t2 (SEQ ID NO: 91) adjuvanted with 10 μg of Matrix-M and s127H1-t2long (SEQ ID NO: 91) containing a C-terminal His tag. Immunized 3 times with number 101) or PBS at 3-week intervals. Four weeks after the last immunization (day 70), sera were isolated, pooled in groups and recipient mice (female BALB / c, 6-8 weeks old, n per group).
= 10) Transplanted during. Each mouse received 400 μl of serum intraperitoneally for 3 consecutive days (Days -3, -2 and -1) prior to the challenge. As a positive control for the challenge model, CR6261 (15 mg / kg) was administered 1 day prior to the challenge (n =).
8) Injection with PBS served as a negative control (n = 8). On day 0, mice were challenged with a 12.5 x LD50 challenge virus and monitored for 3 weeks (survival rate, body weight, clinical score).

A pooled serum sample of peripheral blood from donor mice to confirm the immunogenicity of the polypeptide of the invention in donor mice and to assess HA-specific antibody levels after serum transplantation into recipient mice (day 70). , Pool serum samples of naive recipient mice before serum transfer (day-4) and individual serum samples of recipient mice after 3 times serum transfer immediately before the challenge (day 0), H1N1A / Brisbane / 59 / Binding from 07 to FL HA was tested in ELISA.

Results Challenge-The experiment was successful; all mice in the PBS control group died from infection before 13 days after the challenge (median 9.5 days), while the positive control group (15 mg / kg CR6).
261 (1 day before the challenge) is fully protected (p <0.001).
− Three-time serum transfer of serum from mice immunized with the Matrix-M adjuvanted polypeptide SEQ ID NO: 91 of the present invention into naive recipient mice has a significant survival time compared to the PBS serum transfer control group. (P = 0.007) and a decrease in clinical score (p = 0.012) (Fig. 24).
− Three-time serum transfer of serum from mice immunized with the Matrix-M adjuvanted polypeptide SEQ ID NO: 101 of the present invention into naive recipient mice is PBS.
Significant increase in survival rate (p = 0.002) and increase in survival time (p = 0.002) compared to the serum transfer control group
p <0.001), reduced body weight loss (p = 0.002) and reduced clinical score (p <0)
.. 001) is brought about (Fig. 24).
− For the polypeptide of the invention, FL HA A / Br tested after 3 serum transfers.
The isbane / 59/07 specific antibody titers were similar to those obtained after active immunization (FIG. 25).

CONCLUSIONS: Polypeptide SEQ ID NOs: 91 and 101 of the invention with the addition of Matrix-M adjuvant
Serum components (most likely antibodies) induced by three immunizations with
Mice can be protected from lethal challenges by H5N1A / Hong Kong / 156/97 (survival rates are 30 and 78%, respectively).

Example 10: In vivo protective efficacy of the polypeptide of the invention in the H1N1A / NL / 602/09 challenge model in mice The invention having Matrix-M in the H1N1A / NL / 602/09 challenge model compared to the PBS control group. The protective efficacy of the polypeptide s127H1-t2 (SEQ ID NO: 91) and s127H1-t2long (SEQ ID NO: 101) containing an additional His tag was determined.

A group of 10 female BALB / c mice (6-8 weeks old) was immunized three times at 3-week intervals with 30 μg of the polypeptide of the invention with 10 μg Matrix-M. As a positive control for the challenge model, CR6261 (15 mg / kg) was administered 1 day prior to the 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 a 12.5 x LD50 challenge virus and monitored for 3 weeks (survival rate, body weight, clinical score).

In order to confirm the immunogenicity of the polypeptide of the present invention, pre-challenge serum (day -1) was subjected to ELI for binding to FL HA from H1N1A / Brisbane / 59/07.
Tested in SA assay. A CR9114 competitive ELISA was performed to determine if the induced antibody binds in close proximity to the CR9114 epitope. Conflict data,
The response was expressed using a slope OD that could quantify the response.

RESULTS-The experiment was successful; all mice in the PBS control group died of infection prior to 8 days post-challenge (median 5 days), while the positive control group (15 mg / kg CR6261).
, 1 day before the challenge) is completely protected (p <0.001).
-Three immunizations with s127H1-t2 (SEQ ID NO: 91) supplemented with Matrix-M adjuvant and s127H1-t2long (SEQ ID NO: 101) containing an additional His tag had 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) resulted in a significant weight loss (p <0.001) compared to the PBS control group (FIG. 26).
-H1N1A / Brisbane / 59/07 derived from the polypeptide of the invention
IgG antibody titers against FL HA were significantly higher (p <0.001) compared to PBS for all H1H1 mini-HA variants tested (FIG. 27A).
−H1 mini-HA variant s127H1-t2 (SEQ ID NO: 91) provides additional H.
It has an IgG antibody titer against H1N1A / Brisbane / 59/07FL HA that is significantly higher than that of s127H1-t2long (SEQ ID NO: 101) containing the is tag (p = 0.021) (FIG. 27A).
-All Matrix-M adjuvanted polypeptides of the invention tested are PB.
It has a significantly higher CR9114 competitive potency compared to S (p <0.001) (FIG. 27B).
..

Conclusion:
The s127H1-t2long (SEQ ID NO: 10) containing the Matrix-M adjuvanted polypeptide of the invention s127H1-t2 (SEQ ID NO: 91) and an additional His tag.
1) provides protection against lethal challenges by H1N1A / NL / 602/09.
Confirmed as increased survival rate, increased survival time and decreased clinical score. In addition, Matr
s127H1-t2 (SEQ ID NO: 91) with ix-M adjuvant added is H1N1A / NL.
It also resulted in a reduction in weight loss after the lethal challenge of / 602/09.

Example 11: Library Screening PCT / EP2012 / 073706, Influenza Hemagglutinin Stem Domain Polypeptides, Compositions and Vaccines and Influenza Prevention and /
Or it discloses how to use them in the field of treatment. Here, the present inventors have H.
The additional sequence of the stem domain polypeptide derived from the full length HA of 1N1A / Brisbane / 59/2007 (SEQ ID NO: 1) is described. The stem domain polypeptide is H1-
Obtained by site-specific mutation of mini2-cluster1 + 5 + 6-GCN4t2 (SEQ ID NO: 52), CR6261 (Throsby et al, 2009; Equie
rt et al 2010) and / or widespread influenza neutralizing epitopes of CR9114 are presented.

H1-mini2-cluster1 + 5 + 6-GCN4t2 (SEQ ID NO: 52) is H
Derived from the full length HA of 1N1A / Brisbane / 59/2007 (SEQ ID NO: 1) by using the following steps:
-Remove the cleavage site of HA0. Cleavage of wild-type HA at this site results in HA1 and HA2. Removal can be achieved by mutation from R to Q at position P1 (eg, for a description of the nomenclature of the cleavage site (position 343 of SEQ ID NO: 1), see S.
See un et al, 2010.
-Remove the head domain by deleting amino acids 53-320 from SEQ ID NO: 1. The remaining N- and C-terminal portions of the sequence were linked by a 4-residue flexible linker GGGG.
− Residues 402-41 in H1A / Brisbane / 59/2007 (SEQ ID NO: 1)
Increases the solubility of the loop (between A-helix and CD-helix) formed by (equivalent to) 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)
In, mutants F406S, V409T, F413G and L416S (numbering refers to SEQ ID NO: 1) were introduced.
-Introducing disulfide bridges between amino acids at positions 324 and 436 in H1A / Brisbane / 59/2007; this is the mutations R324C and Y436.
This is achieved by introducing C (numbering refers to SEQ ID NO: 1).
-Introduce the GCN4-derived sequence RMKQIEDKIEIESK (SEQ ID NO: 20) known to be trimerized at positions 419-433 (numbering refers to SEQ ID NO: 1).

In certain embodiments, the polypeptides of the invention contain the intracellular sequence of HA and the transmembrane domain. In other embodiments, the transmembrane and intracellular domain sequences are 519,520 of HA2 so that secreted (soluble) polypeptides are produced after expression in the cell.
521, 522, 523, 524, 525, 526, 526, 527, 528, 259
, Or the C of HA2 from position 530 (or its equivalent as determined from the sequence alignment)
It has been deleted up to the end (numbered by SEQ ID NO: 1). The soluble polypeptide is a sequence known to form a trimer structure, i.e. the folon sequence AYVRKDGEWVL.
Further stabilization can be achieved by introducing L (SEQ ID NO: 3), optionally via the short linker described above. The linker may optionally contain a cleavage site for later processing according to a protocol well known to those of skill 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 can optionally be added by ligation via a shorter linker. The linker may optionally be a proteolytic cleavage site (part of) for later processing according to a protocol well known to those of skill in the art, eg, LVPR.
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, thrombin cleavage site, folon, and His sequence is FLAG-thrombin-foldo of SEQ ID NO: 4.
n-His. This sequence is H1-mini2-cluster1 + 5 + 6-GCN4.
A parent sequence (SEQ ID NO: 156) was created in combination with the soluble form of the t2 (SEQ ID NO: 51) sequence, which was used to generate the novel polypeptide of the invention by mutagenesis. This sequence does not contain the leader sequence corresponding to amino acids 1-17 of SEQ ID NOs: 1 and 2.

The stem domain polypeptide lacks part of the hemagglutinin sequence encoding the head domain of the molecule as described in PCT / 2012/073706 and above.
It is created by relinking the N- and C-terminal portions of the sequence via a linker on both sides of the deletion. Removal of the head domain leaves some of the molecules already protected from the aqueous solvent exposed, potentially destabilizing the structure of the polypeptides of the invention. For this reason, residues in the B loop (especially amino acid residues 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 SEQ ID NO: 156 of the parent sequence as a starting point. SEQ ID NO: 15
In No. 6, the leader sequence was removed from H1-mini2-cluster1 + 5 + 6-GCN4t2 (SEQ ID NO: 52), and residues 520 to 565 were added to Flag-thrombin-fol.
It was created by substituting with the don--his sequence (SEQ ID NO: 4).

Similarly, in the area surrounding the fusion peptide, a large number of hydrophobic residues were exposed to the solvent, which, unlike natural full length HA, could not cleave the polypeptide of the invention and hydrophobic fusion within the protein. It is caused by the fact that it undergoes related conformational changes that embed peptides. 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 trimer polypeptides of the invention Created a library of polypeptides (set 1 and set 2) of Example 11 containing SEQ ID NO: 20 at positions 419-433. did. Single clones into screen culture medium for HEK293F cells and multimers (CR9114 sandwich ELISA), CR6261 binding (ELISA) and protein expression (HTRF assay).
Individually transfected. CR9114 sandwich assay, CR9114, CR6261
, And CR8020ELISA, as well as hits based on the HTRF assay.
I ranked them.

Cross-linking with a primary amine (present in the lysine residue) specific cross-linking agent BS3 followed by SD
Multimerization was evaluated by S-PAGE (see below). C-terminal F due to widespread multimerization
The lag-Foldon-His (FFH) tag sequence was replaced by a thrombin cleavage site and his tag sequence (TCSis). Subsequently, multimerization of TCS-his-containing sequences (CR9114 sandwich assay, BS3 cross-linking) was reconfirmed and clones were ranked and selected. Selective clones were expressed, purified and characterized.

The cross-linking assay was performed as follows:
-Add the cross-linking agent BS3 (bis (sulfosuccinimidyl) svelate) directly to the culture medium.-Incubate at room temperature for 30 minutes.
-Collect the medium and SDS-P under reduced (R, 5 mM DTT) and non-reducing (NR) conditions.
Analyzed by AGE / Western blot • Only BS3 crosslinked species remain covalently bound under reducing conditions • mini-HA via Western blotting using his tag-specific mAbs
To detect

result:
Two libraries of high quality (> 90% of corrected ORF) containing SEQ ID NO: 20 at positions 1.419-433 and predicted sequence mutations (> 97% randomization) were successfully produced. A total of 10472 clones (5544 and 4928 from sets 1 and 2 respectively)
Was evaluated on the initial screen (Fig. 28).
3. 3. > 50% expression of FL HA expression and> of signals observed for FL HA
A clone showing a binding signal to 80% CR6261 is considered a hit; this procedure is 7
5. Of the 4.703 hits that produced 03 hits (596 and 107 from libraries 1 and 2 respectively), 658 were retained after the confirmation screen. Cross-linking assays for the top 20% of hits (111) showed the presence of higher-order multimers that could potentially interfere with the purification of trimer species.
6. 7. The top 20% of confirmed hits (111) were successfully cloned to replace the FFHC ends with TCS-his sequences and then evaluated by CR9114 sandwich ELISA and cross-linking assay. The cross-linking assay yielded nine clones that were considered the most promising trimer candidates (
SEQ ID NOs: 158 to 166, Table 11). CR9114 sandwich ELISA (Fig. 29)
Based on, three candidates (two with TCS-his and one with FFHC terminus)
Was selected for expression and purification. Two of the selection candidates were not fully expressed and purification was not continued. Candidate GW1.5E2. F
FH (SEQ ID NO: 158) was uniformly purified according to the procedure described in Example 4 (7.6 mg).
Total protein; purity> 95%, HP-SEC).
9. GW1.5E2 by SEC-MALS analysis. The characterization of FFH (SEQ ID NO: 158) indicates trimer formation in solution, with three Fab fragments of CR9114 or CR6261 binding per trimer (FIG. 30 and Table 10 below). Biolayer interference measurement method (Oc)
The _Kd ^app determined from tet) is 1 nM for both CR6261 and CR9114. As expected, no CR8020 (negative control) binding could be detected by either method.

CONCLUSIONS: Non-covalent trimer polypeptides of the invention (GW1.5E2.FFH, SEQ ID NO: 1) that bind to CR6261 and CR9114 of bnAb with high affinity in 3: 1 stoichiometry.
58) was identified.

Example 13: Protective Efficacy of Polypeptide sH1 mini-HA GW1.5E2-FFH (SEQ ID NO: 158) of the Present Invention in H1N1A / Brisbane / 59/07 Mouse Model H1N1A / Brisbane / 59/07 compared to PBS control group SH1 mini-HA GW1 supplemented with Matrix-M in the challenge model
.. The protective efficacy of 5E2-FFH (SEQ ID NO: 158) was determined.

A group of 10 female BALB / c mice (6-8 weeks old) was administered at 3 week intervals with 30 μg sH1mini-HA GW1.5E2-FFH (SEQ ID NO: 158) supplemented with 10 μg Matrix-M. Immunized 3 times. As a positive control for the challenge model, CR6261 (15 mg / kg) was administered 1 day prior to the challenge (
n = 8), injection with PBS served as a negative control (n = 16). Four weeks after the last immunization, mice were challenged with a 12.5 x LD50 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 serum (day -1) was added to H1N1A / Brisbane / 59.
Binding to FL HA from / 07 was tested in an ELISA assay. To determine whether the polypeptide sH1 mini-HA GW1.5E2-FFH (SEQ ID NO: 158) -inducing antibody of the invention binds in close proximity to the CR9114 epitope, CR911
4 Competitive ELISA was performed. Visualize the contention data as "% contention" and (AP) / A
Defined as × 100), in the formula, A is CR91 to FL HA in the absence of serum.
The 14-binding maximal OD signal, P is FL HA in the presence of serum at a given dilution.
An OD signal of CR9114 binding to, or expressed using a slope OD metric that can quantify the response; CR9114 and CR8020 (starting concentration 5 mg / ml) for reference.
) The solution was included.

result:
-The experiment was successful; all mice in the PBS control group (n = 16) died from infection before 10 days after the challenge (median 8 days), while the positive control group (n = 8, 15 m).
g / kg CR6261, 1 day before challenge) is fully protected (p <0.001).
-SH1 mini-HA GW1.5E supplemented with Matrix-M
Three immunizations with 2-FFH (SEQ ID NO: 158) resulted in a significant increase in survival rate (p <0.001), survival time (p <0.001), and body weight compared to the PBS control group. It results in a reduction in loss (p <0.001) and a reduction in clinical score (p <0.001) (FIG. 31).
Pre-challenge Ig against H1N1A / Brisbane / 59/07FL HA induced by − sH1 mini-HA GW1.5E2-FFH (SEQ ID NO: 158)
The G antibody titer is significantly higher than PBS (p ≦ 0.001) (FIG. 32A).
-The IgG antibody titer against H1N1A / Brisbane / 59/07FL HA is
It becomes a plateau after two immunizations (not shown).
-Matrix-M adjuvant-added polypeptide of the present invention sH1 mini-HA
GW1.5E2-FFH (SEQ ID NO: 158) has a significantly higher CR911 compared to PBS.
4 Induces competitive titer (p <0.001) (FIG. 32B).

CONCLUSIONS: Matrix-M adjuvanted polypeptide sH1 mini-H of the present invention
A GW1.5E2-FFH (SEQ ID NO: 158) is H1N1A / Brisbane / 5
Grant protection against the lethal challenge of 9/07.

Example 14: Polypeptide sH1 mini-HA GW1.5E2-FFH of the present invention in the H5N1A / Hong Kong / 156/97 mouse model (SEQ ID NO: 158).
Protective efficacy of Matrix-M in the H5N1A / Hong Kong / 156/97 challenge model compared to the PBS control group, superior H1 mini-HA supplemented with adjuvant
The protective effect of the variant was determined.

A group of 10 female BALB / c mice (6-8 weeks old), 30 μg of the polypeptide of the invention sH1 mini-HA GW supplemented with 10 μg of Matrix-M.
Immunized with 1.5E2-FFH (SEQ ID NO: 158) three times at 3-week intervals. As a positive control for the challenge model, CR6261 (15 mg / kg) was administered 1 day prior to the 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 a 12.5 x LD50 challenge virus and monitored for 3 weeks (survival rate, body weight, clinical score).

The polypeptide of the invention sH1 mini-HA GW1.5E2-FFH (SEQ ID NO: 1)
In order to confirm the immunogenicity of 58), pre-challenge serum (day -1) was used for H1N1A / B.
Binding to FL HA from risbane / 59/07 was tested in an ELISA assay. A CR9114 competitive ELISA was performed to determine if the mini-HA-induced antibody binds in close proximity to the CR9114 epitope. "% Conflict" for competing data
Visualized as (AP) / A × 100), where A is the maximum OD signal for CR9114 binding to FL HA in the absence of serum and P is a given dilution. OD signal of CR9114 binding to FL HA in the presence of serum in, or expressed using a gradient OD metric that can quantify the response; CR9114 and CR for reference.
A 8020 (starting concentration 5 μg / ml) solution was included.

result:
-The experiment was successful; 15 of the 16 mice in the PBS control group died of infection before 9 days after challenge (median 9 days), while the positive control group (n = 8, 15 m).
g / kg CR6261, 1 day before challenge) is fully protected (p <0.001).
-SH1 mini-HA GW1.5E supplemented with Matrix-M
Three immunizations with 2-FFH (SEQ ID NO: 158) resulted in a significant increase in survival rate (p <0.001), survival time (p <0.001), and body weight compared to the PBS control group. It results in a reduction in loss (p <0.001) and a reduction in clinical score (p <0.001) (FIG. 33).
-Pre-challenge IgG antibody titers against H1N1A / Brisbane / 59/07FL HA induced by the polypeptide sH1 mini-HA GW1.5E2-FFH (SEQ ID NO: 158) of the present invention are significantly higher than PBS (PBS). p ≦ 0.001) (
FIG. 34A).
-Matrix-M adjuvant-added polypeptide of the present invention sH1 mini-HA
GW1.5E2-FFH (SEQ ID NO: 158) has a significantly higher CR911 compared to PBS.
4 Induces competitive titer (p <0.001) (Fig. 34B).

CONCLUSIONS: Matrix-M adjuvanted polypeptide sH1 mini-H of the present invention
A GW1.5E2-FFH (SEQ ID NO: 158) is H5N1A / Hong Kong /
Grants heterogeneous subtype protection against lethal challenges by 156/97.

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

A group of 10 female BALB / c mice (6-8 weeks old), 30 μg of the polypeptide of the invention sH1 mini-HA GW supplemented with 10 μg of Matrix-M.
Immunized with 1.5E2-FFH (SEQ ID NO: 158) three times at 3-week intervals. As a positive control for the challenge model, CR6261 (15 mg / kg) was administered 1 day prior to the challenge (n = 8), while three immunizations with PBS functioned as a negative control (n = 16). Four weeks after the last immunization, mice were challenged with a 25 x LD50 challenge virus and monitored for 3 weeks (survival rate, body weight, clinical score).

The polypeptide of the invention sH1 mini-HA GW1.5E2-FFH (SEQ ID NO: 1)
In order to confirm the immunogenicity of 58), pre-challenge serum (day -1) was used as H1N1A / Br.
Binding to FL HA from isbane / 59/07 was tested in an ELISA assay. A CR9114 competitive ELISA was performed to determine if the mini-HA-induced antibody binds in close proximity to the CR9114 epitope. The competition data was visualized as "% competition" and defined as (AP) / A × 100), where A is the maximum OD signal for CR9114 binding to FL HA in the absence of serum. P is the OD signal for CR9114 binding to FL HA in the presence of serum at a given dilution, or is expressed using a gradient OD metric that can quantify the response; CR9114 and CR8 for reference.
A 020 (starting concentration 5 μg / ml) solution was included.

Results-The experiment is valid; all mice in the PBS control group (n = 16) die from infection before 9 days post-challenge (median 8 days), while the positive control group (n = 8, 15 mg /).
kg CR6261, 1 day before the challenge) is fully protected (p <0.001).
− Matrix-M-adjuvant-added polypeptide of the invention sH1 mini
Three immunizations with -HA GW1.5E2-FFH (SEQ ID NO: 158) resulted in a significant increase in survival rate (p <0.001) and survival time (p <0.) Compared to the PBS control group. 00
1), resulting in reduced body weight loss (p <0.001) and reduced clinical score (p <0.001) (FIG. 35).
-Pre-challenge IgG antibody titers against H1N1A / Brisbane / 59/07FL HA induced by the polypeptide sH1 mini-HA GW1.5E2-FFH (SEQ ID NO: 158) of the present invention are significantly higher than PBS (PBS). p <0.001) (
FIG. 36A).
-Matrix-M adjuvant-added polypeptide of the present invention sH1 mini-HA
GW1.5E2-FFH (SEQ ID NO: 158) has a significantly higher CR911 compared to PBS.
4 Induces competitive titer (p <0.001) (FIG. 36B).

CONCLUSIONS: Matrix-M adjuvanted polypeptide sH1 mini-H of the present invention
A GW1.5E2-FFH (SEQ ID NO: 158) is H1N1A / Puerto Rico.
Grant protection against lethal challenges by o / 8/34.

REFERENCES

Alberini et al. (2009), Vaccine 27: 5998-60
03.
Bommakanti et al. (2010), PNAS 107 (31): 137
01-13706.
Bommakanti et al. (2012), J Virol 86: 13434
..
Cheng et al. (2014), J.M. Immunol. Methods 1-1
3. 3. (Doi: 10.016 / j. Jim. 2014.07.010)
Coffman et al. (2010), Immunoty 33: 492.
Deverex et al. (1984), Nucl. Acids Res. 12:
387.
DiLillo et al. (2014), Nat Med 20, 143.
Dopheide TA, Ward CW. (1981) J Gen Virol. 36
7-370
Equiert et al. (2009), Science 324: 246.
Equiert et al. (2011), Science 333: 844.
Ferguson et al. (2003), Nature 422: 428-443
..
Lorieu et al. 2010, Proc. Natl. Acad. Sci. US
A, 107: 11341.
Lu et al. (2013), www. pnas. org / cgi / doi / 10.
1073 / pnas. 1308701110.
Mallajosyla et al. (2014). , Www. pnas. org /
cgi / doi / 10.1073 / pnas. 1402766111.
Parek et al. (2012), mAbs 4: 310.
Schnuerger et al. (2011), Molecular immun
logy 48: 1512.
Steel et al. (2010), mBio 1 (1): 1-9.
Stephen et al. (2004) Science 303: 1866.
Stephen et al. (2006) Science 312: 404.
Temperton et al. (2007) Virus 1: 105-12.
Throsby et al. (2008), Plos One 12 (3): 1-15
..
Wilson et al (1981) Nature 289: 366.

Sequence listing
SEQ ID NO: 3: earth
GYIPEAPRDGQAYVRKDGEWVLSTFL
SEQ ID NO: 4: FLAG-thrombin-foldon-HIS
SGRDYKDDDDKLVPRGSPGSGYIPEAPRDGQAYVRKDGEW
VLLSTFLGHHHHHH
SEQ ID NO: 5:
MKQIEDKIEIESKKQ

REFERENCES
Alberini et al. (2009), Vaccine 27: 5998-6003.
Bommakanti et al. (2010), PNAS 107 (31): 13701-1706.
Bommakanti et al. (2012), J Virol 86: 13434.
Cheng et al. (2014), J.M. Immunol. Methods 1-13. (Doi: 10.016 / j. Jim. 2014.07.010)
Coffman et al. (2010), Immunoty 33: 492.
Deverex 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
Equiert et al. (2009), Science 324: 246.
Equiert et al. (2011), Science 333: 844.
Ferguson et al. (2003), Nature 422: 428-443.
Lorieu et al. 2010, Proc. Natl. Acad. Sci. USA, 107: 11341.
Lu et al. (2013), www. pnas. org / cgi / doi / 10.1073 / pnas. 1308701110.
Mallajosyla et al. (2014). , Www. pnas. org / cgi / doi / 10.1073 / pnas. 1402766111.
Parek et al. (2012), mAbs 4: 310.
Schnuerger et al. (2011), Molecular immunology 48: 1512.
Steel et al. (2010), mBio 1 (1): 1-9.
Stephen et al. (2004) Science 303: 1866.
Stephen et al. (2006) Science 312: 404.
Temperton et al. (2007) Virus 1: 105-12.
Throsby et al. (2008), Plos One 12 (3): 1-15.
Wilson et al (1981) Nature 289: 366.
The present invention also provides the following.
[1]
Influenza hemagglutinin stem domain polypeptide
(A) The HA1C terminal segment comprises an influenza hemagglutinin HA1 domain comprising a HA1N terminal stem segment covalently linked to the HA1C terminal stem segment by a binding sequence of 0 to 50 amino acid residues.
(B) Bound to influenza hemagglutinin HA2, said HA1 and HA2 domains are derived from influenza A virus subtypes, including H1 subtype HA;
(C) Does not include protease cleavage sites at the junction between the HA1 and HA2 domains;
(D) The HA1N terminal segment contains amino acids 1 to x of HA1, preferably amino acids p to x of HA1, and the HA1C terminal stem segment contains amino acids y to C of HA1 and x = SEQ ID NO. It is an amino acid on position 52 (or a corresponding position in hemagglutinin of another influenza virus strain) of 1 and p = an amino acid on position 18 (or a corresponding position in hemagglutinin of another influenza virus) of SEQ ID NO: 1. , Y = amino acid on position 320 (or corresponding position in another hemagglutinin) of SEQ ID NO: 1;
(E) The region containing 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.
X ₂ is an amino acid selected from the group consisting of F, I, N, T, H, L and Y, preferably I, L or Y.
X ₃ is an amino acid selected from the group consisting of V, A, G, I, R, F and S, preferably A, I or F.
X ₄ 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 selected from the group consisting of I, E, K, V, A, and T.
Amino acid residues on position 340 (HA1 domain) are selected from the group consisting of I, K, R, T, F, N, S and Y.
Amino acid residues on position 352 (HA2 domain) are selected from the group consisting of D, V, Y, A, I, N, S, and T.
Amino acid residues on position 353 (HA2 domain) are selected from the group consisting of K, R, T, E, G, and V;
(G) Further comprises a disulfide bridge between the amino acid at position 324 and the amino acid at position 436;
(H) Further, the amino acid sequence RMKQIEDKIEIESK (SEQ ID NO: 20) is introduced at positions 419-433, or the sequence RMKQIEDKIEEISKQK (SEQ ID NO: 21) is introduced at positions 417-433.
Polypeptide.
[2]
The polypeptide according to [1], wherein the HA2 domain is truncated.
[3]
The polypeptide according to [1] or [2], wherein the C-terminal portion of the HA2 domain from position 519 to the C-terminal amino acid is deleted.
[4]
The polypeptide according to [1] or [2], wherein the C-terminal portion of the HA2 domain from position 530 to the C-terminal amino acid is deleted.
[5]
The polypeptide according to [3] or [4], wherein the C-terminal portion of the HA2 domain is optionally replaced by the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLSTFL (SEQ ID NO: 3) linked via a linker.
[6]
The C-terminal amino acid residue of the HA1C-terminal stem segment is any amino acid other than arginine (R) or lysine (K), preferably glutamine (Q), any one of [1] to [5]. The polypeptide according to.
[7]
The polypeptide according to any one of [1] to [6], which comprises the HA1 domain and / or one or more additional mutations in the HA2 domain.
[8]
The polypeptide according to any one of [1] to [7], which selectively binds to the antibody CR6261 and / or CR9114.
[9]
The polypeptide according to any one of [1] to [8], which does not bind to the antibodies CR8020 and / or CR8057.
[10]
A nucleic acid molecule encoding the polypeptide according to any one of [1] to [9].
[11]
A vector containing the nucleic acid molecule according to [10].
[12]
A composition comprising the polypeptide according to any one of [1] to [9] and / or the nucleic acid molecule according to [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 are used as pharmaceuticals.
[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], which are used 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], which are used as a vaccine.

Claims (15)

Influenza hemagglutinin stem domain polypeptide
(A) The HA1C terminal segment comprises an influenza hemagglutinin HA1 domain comprising a HA1N terminal stem segment covalently linked to the HA1C terminal stem segment by a binding sequence of 0 to 50 amino acid residues.
(B) Bound to influenza hemagglutinin HA2, said HA1 and HA2 domains are derived from influenza A virus subtypes, including H1 subtype HA;
(C) Does not include protease cleavage sites at the junction between the HA1 and HA2 domains;
(D) The HA1N terminal segment contains amino acids 1 to x of HA1, preferably amino acids p to x of HA1, and the HA1C terminal stem segment contains amino acids y to C of HA1.
Containing terminal amino acids, x = amino acid on position 52 of SEQ ID NO: 1 (or corresponding position in hemagglutinin of another influenza virus strain), p = position 18 of SEQ ID NO: 1 (or in hemagglutinin of another influenza virus) Amino acid on (equivalent position) and y = amino acid on position 320 (or equivalent position in another hemagglutinin) of SEQ ID NO: 1;
(E) The region containing the amino acid residues 402 to 418 is the amino acid sequence X ₁ NTQX ₂ TAX ₃
Includes 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.
X ₂ is an amino acid selected from the group consisting of F, I, N, T, H, L and Y, preferably I, L or Y.
X ₃ is an amino acid selected from the group consisting of V, A, G, I, R, F and S, preferably A, I or F.
X ₄ 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.
Selected from the group consisting of
Amino acid residues on position 340 (HA1 domain) are I, K, R, T, F, N, S and Y.
Selected from the group consisting of
Amino acid residues on position 352 (HA2 domain) are selected from the group consisting of D, V, Y, A, I, N, S, and T.
Amino acid residues on position 353 (HA2 domain) are selected from the group consisting of K, R, T, E, G, and V;
(G) Further comprises a disulfide bridge between the amino acid at position 324 and the amino acid at position 436;
(H) Further, the amino acid sequence RMKQIEDKIEIESK (SEQ ID NO: 20) is 41.
Introduced at positions 9-433, or sequence RMKQIEDKIEIESKKQK
A polypeptide in which (SEQ ID NO: 21) is introduced at positions 417-433. The polypeptide of claim 1, wherein the HA2 domain is truncated. The polypeptide according to claim 1 or 2, wherein the C-terminal portion of the HA2 domain from position 519 to the C-terminal amino acid is deleted. The polypeptide according to claim 1 or 2, wherein the C-terminal portion of the HA2 domain from position 530 to the C-terminal amino acid is deleted. The polypeptide according to claim 3 or 4, wherein the C-terminal portion of the HA2 domain is optionally replaced by the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLSTFL (SEQ ID NO: 3) linked via a linker. Claims 1 to 1, wherein the C-terminal amino acid residue of the HA1C-terminal stem segment is any amino acid other than arginine (R) or lysine (K), preferably glutamine (Q).
5. The polypeptide according to any one of 5. The polypeptide of any one of claims 1-6, comprising one or more additional mutations in the HA1 domain and / or the HA2 domain. The polypeptide according to any one of claims 1 to 7, which selectively binds to the antibody CR6261 and / or CR9114. The polypeptide according to any one of claims 1 to 8, which does not bind to the antibodies CR8020 and / or CR8057. A nucleic acid molecule encoding the polypeptide according to any one of claims 1 to 9. A vector containing the nucleic acid molecule according to claim 10. A composition comprising the polypeptide according to any one of claims 1 to 9 and / or the nucleic acid molecule according to claim 10. The polypeptide according to any one of claims 1 to 9, the nucleic acid molecule according to claim 10, and / or the vector according to claim 11, which are used as pharmaceuticals. The polypeptide according to any one of claims 1 to 9, the nucleic acid molecule according to claim 10, and / or the vector according to claim 11, which is used in inducing an immune response against influenza virus. The polypeptide according to any one of claims 1 to 9, the nucleic acid molecule according to claim 10, and / or the vector according to claim 11, which is used as a vaccine.