CROSS-REFERENCE TO RELATED APPLICATION

This application is a Section 371 of International Application No. PCT/EP2019/051532, filed Jan. 22, 2019, which was published in the English language on Aug. 1, 2019, under International Publication No. WO 2019/145310 A1, which claims priority under 35 U.S.C. § 119(b) to EP Application No. 18152991.8, filed Jan. 23, 2018, the disclosures of which are incorporated herein by reference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name “Sequence Listing” and a creation date of Jun. 30, 2020 and having a size of 283 kb. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.

INTRODUCTION

The invention relates to the field of medicine. Provided herein are influenza A hemagglutinin (HA) stem domain polypeptides, nucleic acids encoding said polypeptides, pharmaceutical compositions comprising the same, and methods of their use.

BACKGROUND

Influenza viruses are major human pathogens, causing a respiratory disease (commonly referred to as “influenza” or “the flu”) that ranges in severity from sub-clinical infection to primary viral pneumonia which can result in death. The clinical effects of infection vary with the virulence of the influenza strain and the exposure, history, age, and immune status of the host. Every year it is estimated that approximately 1 billion people worldwide undergo infection with influenza virus, leading to severe illness in 3-5 million cases and an estimated 300,000 to 500,000 of influenza related deaths. The bulk of these infections can be attributed to influenza A viruses carrying H1 or H3 hemagglutinin subtypes, with a smaller contribution from Influenza B viruses, and therefore representatives of these are typically included in the seasonal vaccine. The current immunization practice relies on early identification of circulating influenza viruses to allow for timely production of an effective seasonal influenza vaccine. Apart from the inherent difficulties in predicting the strains that will be dominant during the next season, antiviral resistance and immune escape also play a role in failure of current vaccines to prevent morbidity and mortality. In addition, the possibility of a pandemic caused by a highly virulent viral strain originating from animal reservoirs and reassorted to increase human to human spread, still poses a significant and realistic threat to global health.

Influenza viruses are enveloped RNA viruses that belong to the family of Orthomyxoviridae. Their genomes consist of eight single-stranded RNA segments that code for 11 different proteins, one nucleoprotein (NP), three polymerase proteins (PA, PB1, and PB2), two matrix proteins (M1 and M2), three non-structural proteins (NS1, NS2, and PB1-F2), and two external glycoproteins: hemagglutinin (HA) and neuraminidase (NA).

Influenza A viruses are widely distributed in nature and can infect a variety of birds and mammals. The viruses are classified on the basis of differences in antigenic structure of the HA and NA proteins, with their different combinations representing unique virus subtypes that are further classified into specific influenza virus strains. Although all known subtypes can be found in birds, currently circulating human influenza A subtypes are H1N1 and H3N2. Phylogenetic analysis of influenza A viruses has demonstrated a subdivision of hemagglutinins into two main, so-called phylogenetic groups: inter alia the H1, H2, H5 and H9 subtypes in phylogenetic group 1 (the group 1 viruses) and inter alia the H3, H4 and H7 subtypes in phylogenetic group 2 (group 2 viruses).

The influenza type B virus strains are strictly human. The antigenic variation in HA within the influenza type B virus strains is smaller than those observed within the type A strains. Two genetically and antigenically distinct lineages of influenza B virus are circulating in humans, as represented by the B/Yamagata/16/88 (also referred to as B/Yamagata) and B/Victoria/2/87 (B/Victoria) lineages. Although the spectrum of disease caused by influenza B viruses is generally milder than that caused by influenza A viruses, severe illness requiring hospitalization is still frequently observed with influenza B infection.

It is known that antibodies that neutralize the influenza virus are primarily directed against hemagglutinin (HA). Hemagglutinin or HA is a trimeric glycoprotein that is anchored in the viral membrane and has a dual function: it is responsible for binding to the cell surface receptor sialic acid and, after uptake, it mediates the fusion of viral and endosomal membrane leading to the release of viral RNA into the cytosol of the target cell. HA comprises a large head domain and a smaller stem domain. The stem domain is anchored in to the viral membrane via a C-terminal transmembrane domain sequence. The protein is post-translationally cleaved to yield two HA polypeptides, HA1 and HA2 (the full sequence is referred to as HA0) (FIG. 1A). The membrane distal head region is mainly derived from HA1 and the membrane proximal stem region primarily from HA2. Cleavage of the HA precursor molecule HA0 is required to activate virus infectivity, and the distribution of activating proteases in the host is one of the determinants of pathogenicity of the influenza virus. The HAs of mammalian and nonpathogenic avian viruses are cleaved extracellularly, which limits their spread in hosts to tissues where the appropriate proteases are encountered. On the other hand, the HAs of pathogenic viruses are cleaved intracellularly by ubiquitously occurring proteases and therefore have the capacity to infect various cell types and cause systemic infections.

The reason that the seasonal influenza vaccine must be updated every year is the large variability of the virus. In the HA protein this variation is particularly manifested in the head domain where antigenic drift and shift have resulted in a large number of different variants. Since this is also the area that is immunodominant, most neutralizing antibodies are directed against this domain and act by interfering with receptor binding. The combination of immunodominance and large variation of the head domain explains why infection with a particular strain does not lead to immunity to other strains: the antibodies elicited by the first infection only recognize a limited number of strains closely related to the virus of the primary infection.

Recently, influenza hemagglutinin stem polypeptides, lacking the complete influenza hemagglutinin globular head domain or a substantial part of it, have been described and have been used to generate an immune response to one or more conserved epitopes of the stem domain polypeptide. It is believed that epitopes of the stem polypeptide are less immunogenic than the highly immunogenic regions of a globular head domain, and that the absence of a globular head domain in the stem polypeptide might allow an immune response against one or more epitopes of the stem polypeptide to develop (Steel et al., 2010). Steel et al. thus created an influenza HA stem polypeptide by deleting amino acid residue 53 to 276 from the HA1 domain of the A/Puerto Rico/8/1934 (H1N1) and A/Hong Kong/1968 (H3N2) strains and replacing the deleted sequence by a short flexible linking sequence GGGG. Vaccination of mice with the H3 HK68 construct did not elicit antisera that were cross-reactive with group 1 HAs. In addition, as shown in WO2013/079473, the stem polypeptides were unstable and did not adopt the correct conformation as proven by the lack of binding of antibodies that were shown to bind to conserved epitopes in the stem region.

Bommakanti et al. (2010) described an HA2 based polypeptide comprising amino acid residues 330-501 (HA2), a 7-amino acid linker (GSAGSAG), amino acid residues 16-55 of HA1, a 6-amino acid linker GSAGSA, followed by residues 290-321 of HA1, with the mutations V297T, 1300E, Y302T and C305T in HA1. The design was based on the sequence of H3 HA (A/Hong Kong/1968). The polypeptide did only provide cross-protection against another influenza virus strain within the H3 subtype (A/Phil/2/82 but not against an H1 subtype (A/PR/8/34). In a more recent paper by Bommakanti et al. (2012), a stem polypeptide based on HA from H1N1 A/Puerto Rico/8/1934 (H1HA0HA6) was described. In this polypeptide, the equivalent of amino acid residues 48 to 288 have been deleted and mutations 1297T, V300T, 1302N, C305S, F392D, F395T, and L402D have been made. Both the H3 and H1 based polypeptides were expressed inE. coliand therefore lack the glycans that are part of the naturally occurring HA proteins.

More recently, Lu et al. (2014) also described soluble stem polypeptides derived from the HA of H1N1 A/California/05/2009. In the final design, the amino acid residues from 52 to 277 were deleted (the leader sequence is also not present) and two mutations were introduced in the B-loop of the protein, i.e. F392D, and L402D. Furthermore, the polypeptide contained a C-terminal trimerization domain (foldon). In addition, two intermonomer disulfide bridges were introduced, one in the area of the trimeric foldon domain, and one at position 416 and 417 (i.e. G416C and F417C in H3 numbering). The polypeptide was produced in anE. colibased cell free system, (and thus lacks the glycans that are part of the naturally occurring HA proteins) and was recovered in a denatured form, which needs to be refolded prior to use. The refolded protein failed to bind the broadly neutralizing antibody (bnAb) CR6261 which is binding to a conserved conformational stem epitope. No immunological or protection data from influenza challenge were shown.

In another paper Mallajosyula et al. (2014) also described an influenza HA stem polypeptide. In this design, based on HA from H1N1 A/Puerto Rico/8/1934, not only a large part of the HA1 sequence was deleted (residue 48 to 289, H3 numbering), but also large part of the N- and C-terminal sequences of HA2 (residues 323 to 369 and 443 to end, respectively). The polypeptide contained a foldon trimerization domain at the C-terminus and was also produced inE. coli, so is lacking the naturally occurring glycans on viral HA. The polypeptide was shown to bind the bnAbs CR6261, F10 and FI6v3, and protected mice from a leathal influenza virus challenge (1LD90 of H1N1 A/Puerto Rico/8/1934). Equivalent polypeptides derived from HA of H1N1 A/New Caledonia/20/1999 and H1N1 A/California/04/2009 could also partially protect. A polypeptide derived from H5N1 A/Viet Nam/1203/2004 only gave limited protection in this challenge model. Moreover, the challenge model used was mild with a relatively low dose administered (1-2 LD90).

Lastly, Yassine et al. (2015) also described the development of a stabilized HA stem polypeptide derived from HA of H1N1 A/New Caledonia/20/1999. In this design, a large part of the HA1 sequence (residue 43 to 313, H3 numbering) and HA2 sequence (residue 504 to end) have been deleted. In addition, the design contains two stabilizing mutations (K380M and E432L) in HA2 and is genetically fused to the ferritin subunit ofH. pylorito create self-assembling nanoparticles displaying the stabilized HA-stem polypeptide. The stabilized HA-stem polypeptide seemed not soluble or functional without being fused to the ferritin subunit. The HA stem-ferritin polypeptide assembled to nanoparticles was tested in a heterosubtypic H5N1 2004 VN influenza virus challenge model (25×LD50and 1,000×TCID50in mouse and ferrets, respectively) and could protect mice from death whereas only partial protection was observed in ferrets. It is unclear how much ferritin response would be induced in humans and which effect that would have for multiple administrations.

There thus still exists a need for a safe and effective “universal” vaccine that stimulates the production of a robust, broadly neutralizing antibody response and that offers protection against a broad set of current and future influenza virus strains (both seasonal and pandemic), in particular a vaccine that provides protection against one or more influenza A virus subtypes within phylogenetic group 1 and/or group 2, for the effective prevention and/or treatment of influenza.

SUMMARY

The present invention provides novel polypeptides derived from influenza hemaggluinin (HA), which polypeptides comprise the influenza HA stem domain and lack the globular head region, herein referred to as influenza hemagglutinin (HA) stem polypeptides. The polypeptides induce an immune response against HA when administered to a subject, in particular a human subject. The polypeptides of the invention present conserved epitopes of the membrane proximal stem of the HA molecule to the immune system in the absence of dominant epitopes that are present in the membrane distal head domain. Thus, part of the primary sequence of the HA0 protein, i.e. the part making up the head domain has been deleted, and the remaining amino acid sequence has been reconnected, either directly or, in some embodiments, by introducing a short flexible linking sequence (‘linker’) to restore the continuity of the amino acid chain. The resulting amino acid sequence is further modified by introducing specific modifications that stabilize the native 3-dimensional structure of the remaining part of the HA molecule.

In a first aspect, the present invention relates to group 1 influenza A hemagglutinin (HA) stem polypeptides comprising an HA1 and an HA2 domain, said polypeptides comprising an amino acid sequence which comprises, as compared to the amino acid sequence of a full-length HA polypeptide comprising an HAT and an HA2 domain:(i) a deletion of the head region in the HA1 domain;(ii) a modification of the trimerization region in the HA2 domain, preferably a modification in the C-helix,(iii) at least 2 cysteine residues (capable of) forming an intramonomeric disulphide bridge;(iv) at least 2 cysteine residues (capable of) forming an intermonomeric disulphide bridge;
wherein the amino acid corresponding to the amino acid at position 392 is P, R or Y, preferably P or R, and the amino acid corresponding to the amino acid at position 434 is Q, and wherein the numbering of the amino acid positions is based on H3 numbering as used in Winter et al. (1981).

In certain embodiments, the present invention relates to group 1 influenza A hemagglutinin (HA) stem polypeptides comprising an HA1 and an HA2 domain, wherein said HA stem polypeptides comprise an amino acid sequence which comprises, as compared to the amino acid sequence of the full-length HA polypeptide (HA0) comprising an HA1 and an HA2 domain:

(i) a deletion of the head region in the HA1 domain, said deletion comprising at least the amino acid sequence from the amino acid corresponding to the amino acid at position 53 up to and including the amino acid corresponding to the amino acid at position 302;
(ii) a modification of the trimerization region in the HA2 domain, preferably a modification of the trimerization region in the C-helix, said trimerization region comprising the amino acid sequence from the amino acid corresponding to the amino acid at position 405 up to and including the amino acid corresponding to the amino acid at position 419;
(iii) a cysteine at the amino acid position corresponding to position 310 and a cysteine at the position corresponding to position 422 (capable of) forming an intramonomeric disulphide bridge;
(iv) a cysteine at the position corresponding to position 397 in combination with a cysteine at the position corresponding to position 405; or a cysteine at the position corresponding to position 396 in combination with a cysteine at the position corresponding to position 408; or a cysteine at the position corresponding to position 399 in combination with a cysteine at position 405;
wherein the amino acid at the position corresponding to position 392 is P, R or Y, preferably P or R, and wherein the amino acid at the position corresponding to position 434 is Q; and wherein the numbering of the amino acid positions is based on H3 numbering as used in Winter et al. (1981).

According to the present invention it has surprisingly been shown that the novel influenza HA stem polypeptides of the invention can be expressed in high levels, are overwhelmingly trimeric in cell culture supernatant, have an increased melting temperature which leads to greater stability. In addition, the HA stem polypeptides of the invention mimick the stem of the full-length HA by stably presenting the epitope of HA stem binding bnAbs, such as CR9114 and/or CR6261.

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

In yet another aspect, the invention provides vectors, in particular recombinant adenoviral vectors, comprising the nucleic acids encoding the influenza HA stem polypeptides.

In a further aspect, the invention provides methods for inducing an immune response against influenza HA in a subject in need thereof, the method comprising administering to the subject an influenza HA stem polypeptide, a nucleic acid molecule, and/or a vector according to the invention.

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

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

