NEW INFLUENZA ANTIGENS

专利摘要:
The present invention relates to novel influenza antigens, novel immunogenic or vaccine compositions, as well as uses and methods for producing said antigens and compositions. In particular, the invention relates to recombinant forms of hemagglutinin (HA) and their use in vaccine compositions for the prevention of influenza virus infections.
公开号:BE1024598B1
申请号:E2017/5126
申请日:2017-03-02
公开日:2018-04-25
发明作者:Ventzislav Vassilev
申请人:Glaxosmithkline Biologicals Sa；
IPC主号:

专利说明:

(73) Holder (s):
GLAXOSMITHKLINE BIOLOGICALS SA
1330, RIXENSART
Belgium (72) Inventor (s):
VASSILEVVentzislav 1330 RIXENSART Belgium (54) NEW INFLUENZA ANTIGENS (57) The present invention relates to new influenza antigens, new immunogenic or vaccine compositions, as well as uses and methods of production of said antigens and said compositions. In particular, the invention relates to recombinant forms of hemagglutinin (HA) and their use in vaccine compositions for the prevention of influenza virus infections.
BELGIAN INVENTION PATENT
FPS Economy, SMEs, Middle Classes & Energy
Publication number: 1024598 Deposit number: BE2017 / 5126
Intellectual Property Office
International Classification: A61K 39/12 A61K 39/145 C07K 14/005
Date of issue: 25/04/2018
The Minister of the Economy,
Having regard to the Paris Convention of March 20, 1883 for the Protection of Industrial Property;
Considering the law of March 28, 1984 on patents for invention, article 22, for patent applications introduced before September 22, 2014;
Given Title 1 “Patents for invention” of Book XI of the Code of Economic Law, article XI.24, for patent applications introduced from September 22, 2014;
Having regard to the Royal Decree of 2 December 1986 relating to the request, the issue and the maintenance in force of invention patents, article 28;
Considering the patent application received by the Intellectual Property Office on 02/03/2017.
Whereas for patent applications falling within the scope of Title 1, Book XI of the Code of Economic Law (hereinafter CDE), in accordance with article XI. 19, §4, paragraph 2, of the CDE, if the patent application has been the subject of a search report mentioning a lack of unity of invention within the meaning of the §ler of article XI.19 cited above and in the event that the applicant does not limit or file a divisional application in accordance with the results of the search report, the granted patent will be limited to the claims for which the search report has been drawn up.
Stopped :
First article. - It is issued to
GLAXOSMITHKLINE BIOLOGICALS SA, Rue de l'Institut 89, 1330 RIXENSART Belgium;
represented by
PRONOVEM - Office Van Malderen, Avenue Josse Goffin 158, 1082, BRUXELLES;
a Belgian invention patent with a duration of 20 years, subject to the payment of the annual fees referred to in article XI.48, §1 of the Code of Economic Law, for: NEW INFLUENZA ANTIGENS.
INVENTOR (S):
VASSILEV Ventzislav, c / o GlaxoSmithKline Biologicals s.a, Rue de I'lntitut 89, 1330, RIXENSART;
PRIORITY (S):
03/02/2016 GB 1603625.3;
DIVISION:
divided from the basic application: filing date of the basic application:
Article 2. - This patent is granted without prior examination of the patentability of the invention, without guarantee of the merit of the invention or of the accuracy of the description thereof and at the risk and peril of the applicant (s) ( s).
Brussels, 04/25/2018, By special delegation:
BE2017 / 5126
NEW INFLUENZA ANTIGENS
Technical area
The present invention relates to novel influenza antigens, novel immunogenic or vaccine compositions, as well as uses and methods of producing said antigens and said compositions. In particular, the invention relates to recombinant forms of hemagglutinin (HA) and their use in vaccine compositions for the prevention of influenza virus infections.
Context of the invention
Influenza viruses are among the most ubiquitous viruses in the world, affecting both humans and livestock. The flu causes significant economic burden, morbidity and even mortality. There are three types of influenza viruses: A, B and C. The influenza virus consists of two predominant surface antigens, the glycoproteins hemagglutinin (HA) and neuraminidase (NA), which appear as spikes on the surface of the particles. It is these surface proteins, particularly HA, that determine the antigenic specificity of influenza subtypes.
HA is a trimeric protein composed of an ectodomain of identical subunits, each of which contains two polypeptides, HAI and HA2, linked by a disulfide bond. Each monomer is initially expressed in the form of HAO, and is subsequently cleaved by the proteases of the host into HA1 and HA2 subunits
BE2017 / 5126 which are linked by a disulfide bond. HA can be functionally divided into two areas, the globular head and the stem. The globular head is composed of part of HAI and the stem structure is composed of parts of HAI and all of HA2 (Hai et al., J. Virol, 2012 86 (10): 5774-5781).
Vaccination plays an essential role in the fight against epidemics and pandemics of influenza. Many flu vaccines are made by methods that involve reassortment, adaptation and growth of viruses in chicken eggs. However, there are limitations with these existing methods. Not all influenza virus strains develop well in eggs and must be adapted or reassortant viruses must be constructed Changes in HA during manufacture can lead to strains that differ from circulating strains and which may offer lower rates -optimal protection. Another disadvantage is that people allergic to eggs may be hypersensitive to residual egg proteins in egg vaccines. In addition, egg-based processes rely on an uninterrupted supply of eggs, which may be susceptible to supply disruptions such as a disease in poultry. There is a need for vaccine production using methods which do not rely on the egg supply and where the production of vaccine proteins is more stringently controlled than in egg-based methods.
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Recombinant forms of HA (rHA) produced in cultured cells have been proposed as an alternative source of antigen for influenza vaccines to that of eggs. However, problems of maintaining immunogenicity and a regular quaternary structure of rHA have been encountered using these methods. Thus, there is still a need for alternative methods of supplying antigens for influenza vaccines, which address existing difficulties.
Summary of the invention
It has been discovered with previous efforts for rHA production that large aggregates of the recombinant protein are formed which are not acceptable for vaccine production purposes. In addition, the correct rosette structure, a multimeric form of the basic trimer structure of HA, has not always formed properly. The inventors have produced a recombinant hemagglutinin antigen (rHA) which incorporates a heterologous trimerization domain such as a foldon, as well as a hydrophobic signal such as the transmembrane domain of HA and the extracellular domain (ECD) or the one of its immunogenic parts. The rHA produced by the inventors is capable of forming the correct rosette structure without the large aggregates and the maintenance of immunogenicity. These functional properties make rHA potentially useful for the treatment and / or prevention of infection and / or influenza-like illness.
BE2017 / 5126
Consequently, in a first aspect of the invention, there is provided a recombinant influenza virus hemagglutinin (HA) antigen comprising the extracellular domain of HA or one of its immunogenic parts, a hydrophobic signal and a heterologous trimerization domain.
In another aspect, there is provided an HA antigen as described above, wherein the hydrophobic signal is a transmembrane domain of HA or an artificial hydrophobic signal.
In another aspect, there is provided a polynucleotide encoding a recombinant hemagglutinin antigen as described above.
In another aspect, there is provided an immunogenic composition comprising a recombinant antigen as defined above and a pharmaceutically acceptable carrier.
In another aspect, the immunogenic composition described above is provided for use in medicine.
In yet another aspect, there is provided the immunogenic composition described above for use in the prevention of and / or vaccination against influenza.
In yet another aspect, there is provided the immunogenic composition described above for use in the prevention and / or vaccination against influenza caused by a clade different from the clade to which the extracellular domain of the HA antigen described above belongs.
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In yet another aspect, there is provided a method of producing a recombinant antigen such as expression of a in a cell defined above comprising above described eukaryotic polynucleotide, such as a mammalian cell, by example, a CHO cell, or an insect cell, optionally further comprising purifying / isolating rHA from the eukaryotic cell.
In yet another aspect, there is provided a method for the prevention of and / or vaccination against influenza, comprising administering an antigen or immunogenic composition as described above to a person in need thereof , as a person identified as being at risk of being infected with the flu.
Brief description of the figures
Figure 1 - Representation of the design of the EDC-Foldon and the EDC-TMD-Foldon used in the examples. A. EDC-Foldon is the starting molecule, composed of the secretion signal gp67, the HA ectodomain (ECD), a thrombin cleavage site (TCS), the Foldon trimerization domain and a histidine tail (to facilitate purification). B. The ECDTMD-Foldon was designed by inserting the transmembrane domain of HA (TM) between the ectodomain and the Foldon domain.
Figure 2 - Representation of the amino acid sequences of ECD-Foldon and ECD-TMD-Foldon used in the examples. A. The EDC-Foldon is the starting molecule, and the ECD-TMD-Foldon was designed by inserting the
BE2017 / 5126 transmembrane domain of HA (TM) between the extracellular domain of HA and the Foldon domain of ECDFoldon. For information, the amino acid sequence of the HA molecule total length of the same strain as that which was used as a comparator in this study is also shown.
Figures 3A, 3B and 3C - Electron micrographs showing the appearance of ECD-Foldon, ECD-TMD-Foldon and recombinant full length HA, respectively, in solution.
Figure 4 - Mice were immunized on days 0 and 21 with ECD-Foldon (6 or 24 gg), ECD-TMDFoldon (1.5, 6 or 24 gg), a full length recombinant HA antigen ( 1.5, 6 or 24 gg), fractionated A / Indonesia / 05/2005 (1.5 gg), or PBS. The blood was taken on days 21 and 42 (3 weeks after the immunizations) and the levels of anti-A / Indonesia / 05/2005 antibodies were measured in the sera of the mice by an ELISA test. The numbers in parentheses indicate the number of mice in the group.
Figure 5 - Mice were immunized on days 0 and 21 with ECD-Foldon (6 or 24 gg), ECD-TMDFoldon (1.5, 6 or 24 gg), a full length recombinant HA antigen ( 1/5, 6 or 24 gg), fractionated A / Indonesia / 05/2005 (1.5 gg), or PBS. Blood was taken on days 21 and 42 (3 weeks after immunizations) and antiA / Indonesia / 05/2005 antibody levels were measured in mouse sera by a hemagglutination inhibition (HI) test ). The numbers in parentheses indicate the number of mice in the group.
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Figure 6 - Mice were immunized on days 0 and 21 with ECD-Foldon (6 or 24 gg), ECD-TMDFoldon (1.5, 6 or 24 gg), a full length recombinant HA antigen ( 1/5, 6 or 24 gg), fractionated A / Indonesia / 05/2005 (1.5 gg), or PBS. Blood was taken on days 21 and 42 (3 weeks after immunizations) and the levels of anti-A / Indonesia / 05/2005 neutralizing antibodies were measured in mouse sera by a neutralization test. The numbers in parentheses indicate the number of mice in the group.
Figure 7 - Mice were immunized on days 0 and 21 with ECD-Foldon (6 or 24 gg), ECD-TMDFoldon (1.5, 6 or 24 gg), a full length recombinant HA antigen ( 1/5, 6 or 24 gg), fractionated A / Indonesia / 05/2005 (1.5 gg), or PBS. Blood was taken on days 21 and 42 (3 weeks after immunizations) and antiA / Indonesia / 05/2005 antibody levels were measured in mouse sera by an ELISA test and a hemagglutination test. The diagram shows the relationships between the ELISA and IH values. The numbers in parentheses indicate the number of mice in the group. The asterisk (*) indicates that the ratio could not be calculated because at least one of the two values was less than the threshold value of the test.
Figure 8 - Mice were immunized on days 0 and 21 with ECD-Foldon (6 or 24 gg), ECD-TMDFoldon (1.5, 6 or 24 gg), a full length recombinant HA antigen ( 1/5, 6 or 24 gg), fractionated A / Indonesia / 05/2005 (1.5 gg), or PBS. The
BE2017 / 5126 blood was taken on days 21 and 42 (3 weeks after the immunizations) and the levels of antiA / Indonesia / 05/2005 antibodies were measured in the sera of mice by an ELISA test and the levels of the neutralizing antibodies by a neutralization test. The diagram shows the relationships between ELISA and neutralization values. The numbers in parentheses indicate the number of mice in the group. The asterisk (*) indicates that the ratio could not be calculated because at least one of the two values was less than the threshold value of the test.
detailed description
Here is provided a recombinant hemagglutinin antigen (rHA) of influenza virus comprising or consisting of the extracellular domain of HA (ectodomain, ECD) or one of its immunogenic parts, of a hydrophobic signal such as a transmembrane domain (TMD) of HA and a heterologous trimerization domain.
Recombinant influenza HA (rHA)
An rHA comprises or is encoded by one or more nucleic acids which are derived from a nucleic acid which has been constructed artificially. For example, the nucleic acid may comprise, or be encoded by, a cloned nucleic acid formed by joining heterologous nucleic acids.
RHA includes sequences derived from hemagglutinin (such as ECD and TMD) and may include other sequences not derived from
BE2017 / 5126
Hemagglutinin, for example, a heterologous trimerization domain not derived from hemagglutinin. Generally, the sequences derived from hemagglutinin are in the order in which they appear in hemagglutinin of natural origin and the trimerization domain appears towards, or at the Cterminal end, for example in the C-terminal half of the C-terminus of rHA relative to EDC. The rHA of the invention can in particular consist of or comprise the EDC of HA or one of its immunogenic parts followed by a TMD of HA, followed by a heterologous trimerization domain, in this order, when the trimerization domain is in the C-terminal half of the C-terminal end of the rHA relative to the EDC.
The rHA antigen of the invention can be fused to or contain another polypeptide other than ECD, TMD and the trimerization domain. The sequence encoding the other polypeptide optionally includes other features such as a flexible linker between the HA-derived sequences and the other heterologous amino acid sequences. The linkers can facilitate independent folding of the domains of HA and other heterologous sequences. The linker can be an amino acid sequence that is synthesized as part of a recombinant fusion protein. In other embodiments, a chemical linker is used to link subsequences produced by synthesis or recombination. Such flexible linkers are known to those skilled in the art.
In addition to the flexible linkers, or alternatively, the fusion proteins optionally include sub10
BE2017 / 5126 polypeptide sequences from proteins which are not related to hemagglutinin, for example, a sequence with affinity for an antibody known to facilitate affinity purification and / or detection. Such domains facilitating detection and purification include, but are not limited to, metal chelating peptides such as polyhistidine sequences and histidine-tryptophan modules which allow purification on immobilized metals and protein A domains which allow purification on immobilized immunoglobulins. Examples include heterologous fusion sequences encoding gD markers, c-Myc epitopes, polyhistidine markers, fluorescent proteins (e.g., GFP), beta-galactosidase protein or glutathione-S-transferase or any other useful sequence for detection or purification of the fusion protein expressed in or on a cell Preferably, the other polypeptide sequence is a polyhistidine marker, such as a marker for six histidines. The inclusion of a cleavable linker sequence between the purification domain (eg, the polyhistidine tag) and the rHA antigen may be useful to facilitate purification. For example, an enzymatic cleavage site, such as a thrombin cleavage site can be included between the other polypeptide and the rest of the rHA sequences. A cleavable linker sequence, for example, an enzymatic cleavage site such as a thrombin cleavage site may alternatively or additionally be included between the trimerization domain and the rest of the rHA sequences.
BE2017 / 5126
This can eliminate the trimerization domain in the final rHA. Consequently, the rHA antigen of the invention can consist of or comprise (in order) the EDC of HA or one of its immunogenic parts, a TMD of HA, a heterologous trimerization domain, a purification marker (for example, a polyhistidine marker) and optionally a cleavable linker sequence i) between the purification marker and the rest of the rHA and / or ii) between the trimerization domain and the TMD of the HA.
The gene or construct encoding the rHA antigen may include a signal peptide. Generally, the signal peptide is appropriate for the host cell in which rHA is expressed. In one embodiment, the natural sequence of the signal peptide in hemagglutinin is deleted and replaced by a signal peptide of baculovirus, for example, the secretion signal gp67 (Whitford et al. 1989, J. Virol. 63, 13931399) , for correct expression in insect cells. The gene containing the rHA antigen and the baculovirus signal peptide can be introduced into a baculovirus expression vector such that the baculovirus promoter directs the transcription of fusion proteins in infected insect cells. The signal peptide directs translation of the rHA antigen into the glycosylation pathway of the insect cell and is not present on the mature protein.
Therefore, the nucleic acid sequence encoding the rHA antigen of the invention can be
BE2017 / 5126 consisting of or comprising the sequence coding for the ECD of HA or one of its immunogenic parts, a TMD of HA, a heterologous trimerization domain (for example, foldon), a purification marker (by example, a polyhistidine tag) and a signal peptide (for example, a baculovirus signal peptide), as in order: a signal peptide (for example, a baculovirus signal peptide), HA EDC or one of its immunogenic parts, a TMD of HA, a heterologous trimerization domain (for example, foldon), a purification marker (for example, a polyhistidine marker). As another example, the nucleic acid coding for the rHA antigen of the invention can consist of or comprise the sequence coding (in order) for: a signal peptide (for example, a baculovirus signal peptide), l ECD of HA or one of its immunogenic parts, a TMD of HA, a cleavable linker sequence (for example, TCS), a heterologous trimerization domain (for example, foldon), a purification marker (by example, a polyhistidine marker).
The HA sequences (for example, the ECD (or one of its immunogenic parts) and the TMD) of the rHA antigen can be of any type or subtype (for example, H1 to H16) of influenza strain . In one embodiment, the HA sequence of the HA antigen comes from a strain chosen from the group consisting of: a strain of subtype H1, H2, H3, H5, H7 and H9. Preferably, the HA antigen comes from an H5 strain. The sequences for EDC (or one of its immunogenic parts) and TMD can
BE2017 / 5126 come from the same / same source / strain / type / subtype of influenza.
In some embodiments, HA has a sequence which is identical to HA from a pandemic strain. By pandemic strain is meant a new influenza virus against which the vast majority of the human population has no immunity. Generally, WHO identifies and discloses these pandemic strains. Suitable pandemic strains are, for example, H5N1, H9N2, H7N7, H7N9, H2N2, H7N1, H7N3, H10N7, H5N2 and H1N1. Alternatively, the HA sequence is identical to or derived from naturally occurring HA originating from a non-pandemic strain. For example, the non-pandemic strains may be strains identified by WHO as seasonal circulating strains of influenza virus or strains identified by WHO as having the potential to cause an epidemic for the following influenza season. Such strains can be, for example, 1) influenza A type strains H1N1, H3N2 and 2) one or two influenza type B strains (for example, from the Victoria and / or Yamagata lines).
For example, the rHA antigen of the invention can comprise i) an amino acid sequence comprising the EDC of HA (for example, SEQ ID NO: 7) or one of its immunogenic parts or one of its derivatives, ii) an amino acid sequence comprising a TMD of HA (for example, SEQ ID NO: 5) and iii) the heterologous trimerization domain (foldon) represented by SEQ ID NO: 9 or a derivative of this sequence which retains the possibility of inducing the rHA monomers to form
BE2017 / 5126 of the trimers. In particular, the EDC of HA can be derived from an H5 virus (eg, an H5N1 virus), such as that represented by SEQ ID NO: 7. Both the EDC and / or the TMD of HA can be derived from an H5 virus such as an H5N1 virus. For example, the rHA antigen can comprise i) SEQ ID NO: 7 or one of its immunogenic parts or one of its derivatives and / or ii) SEQ ID NO: 5 or one of its derivatives which retains the ability to orient the trimers of HA in rosette structures and maintain the immunogenicity of HA. In particular, the rHA antigen can comprise or consist of the amino acid sequence represented by SEQ ID NO: 1.
The nucleic acid sequence coding for the rHA antigen may include i) the nucleic acid coding for the EDC of HA (for example, SEQ ID NO: 8), or a fragment or derivative of this sequence coding for an immunogenic part of the EDC of HA, ii) the nucleic acid sequence (for example, SEQ ID NO: 6) coding for the TMD of HA or a fragment or derivative of this sequence which codes for a TMD which retains the capacity to orient the trimers of HA in rosette structures and maintains the immunogenicity of HA and iii) SEQ ID NO: 10 coding for the foldon or a derivative of this sequence which retains the capacity to induce the monomers of rHA expressed to form trimers.
The sequence of EDC and / or TMD can be derived from an H5 virus, for example, an H5N1 virus. For example, in one embodiment, the nucleic acid sequence coding for the rHA antigen comprises i) SEQ ID NO: 8 or a fragment or derivative thereof.
BE2017 / 5126 sequence coding for an immunogenic part of the EDC of HA and ii) SEQ ID NO: 6, or a fragment or derivative of this sequence which codes for a TMD which retains the capacity to direct the trimers of HA in rosette structures and maintains the immunogenicity of HA. In particular, the nucleic acid coding for the rHA antigen of the invention can consist of or comprise the nucleic acid sequence represented by SEQ ID NO: 2.
In another example, the nucleic acid coding for the rHA antigen of the invention can comprise any nucleic acid sequence coding for an ECD of HA, any sequence coding for a TMD of HA and the foldon sequence shown by SEQ ID NO: 10 or a derivative of this sequence which retains the capacity to induce the expressed rHA monomers to form trimers. In another example, the nucleic acid coding for the rHA antigen of the invention can comprise i) any nucleic acid sequence coding for an ECD of HA, ii) any nucleic acid sequence coding for a domain of heterologous trimerization and iii) SEQ ID NO: 6 coding for the TMD of HA or a fragment or derivative of this sequence which codes for a TMD which retains the capacity to orient the trimers of HA in rosette structures and maintains the immunogenicity of HA. In yet another example, the nucleic acid coding for the rHA of the invention may comprise i) SEQ ID NO: 8 coding for the EDC of HA, or a fragment or a derivative of this sequence coding for an immunogenic part ED of HA, ii) any nucleic acid sequence encoding a heterologous trimerization domain and iii) any
BE2017 / 5126 nucleic acid coding for a TMD of HA. In yet another example, the nucleic acid coding for the rHA antigen of the invention can comprise i) any sequence coding for an ECD of HA, ii) the foldon sequence represented by SEQ ID NO: 10 or a derivative of this sequence which retains the capacity to induce the expressed rHA monomers to form trimers and iii) any nucleic acid coding for a TMD of HA.
Preferably, the recombinant influenza virus hemagglutinin (HA) antigen of the invention lacks the intracellular domain of the influenza virus hemagglutinin, for example, the intracellular domain represented by SEQ ID NO: 3. It Also provided is a nucleic acid sequence encoding the recombinant influenza virus hemagglutinin (HA) antigen of the invention which lacks the sequence encoding the intracellular domain of influenza virus hemagglutinin, for example, intracellular domain represented by SEQ ID NO: 4.
Extracellular domain (ECD) or one of its immunogenic parts
The extracellular (or ectodomain, ECD) component of HA is present as a wild type HA protein on the cell surface. The rHA of the invention may comprise a full length ECD, or one of its immunogenic parts. EDC or one of its immunogenic parts, may, in certain embodiments, be a variant or a derivative of a wild type HA protein (for example
BE2017 / 5126 example, containing substitutions, deletions or additions of amino acids).
One of its immunogenic parts may include one or more regions of HA for which it is desirable to direct an immune response. Such regions may include conserved and / or variable epitopes known to hemagglutinin which trigger neutralizing antibodies upon vaccination. Preferably, one of its parts is capable of correct folding to interact in a hemagglutination test.
For example, the EDC may consist of or include the HAI and / or HA2 region of the HA. Alternatively, the EDC may consist of or comprise the region of the head and / or stem of the HA. An ECD may consist of or comprise of the HA2 subunit and a part of the HAI subunit, which together form the stem region of the HA. The EDC can be made up of the stem region of the HA, a so-called "headless" form of the HA. In particular, in certain embodiments, with the HA antigen, the head of the HA may be missing, or part of the head, such as more than 25%, for example, more than 50%, such as more than 75% amino acid residue from the head, or missing the HAI part of the head. Alternatively, at the HA sequence, the HA stem, or part of the stem, may be missing, such as more than 25%, for example, more than 50%, such as more than 75% of the acid residues. amines in the stem, or missing the HA2 portion of the stem.
The term "HAI" refers to the region of the HA protein comprising the amino acid residues
BE2017 / 5126 approximately from 1 to 330 of the extracellular domain of the HA protein. HAI includes all residues that are N-terminal to the HA1 / HA2 cleavage peptide of the HAO precursor protein, including the receptor binding domain of the HA protein.
The term "HA2" refers to the region of the HA protein comprising the amino acid residues approximately 331 to 504 of the hemagglutinin HAO polypeptide. Note that these residues within the HA2 chain are usually numbered independently of those in the HAI, so that the HA2 residues can be numbered consecutively from 1 to 174. The HA2 chain includes all the residues that are Cterminal to to the HA1 / HA2 cleavage peptide of the precursor protein HAO, including the hydrophobic peptide responsible for insertion into the membrane of the host cell during the membrane fusion process.
The term "HA stem" refers to the region of the HA protein comprising residues approximately 1 to 42 and 274 to 330 of the HA1 chain as well as residues (1 to 174) of the HA2 chain. The stem is located in the proximal region of the HA membrane, directly below the remnant esterase domain of the globular head of HAI.
The term "HA head" refers to a globular head region of the HA protein excluding the transmembrane domain and any intracellular region, which is composed of part of the HAI and which contains an acid binding pocket sialic who mediates the
BE2017 / 5126 attachment of the virus to the host cell. See, for example, Hai et al. (J. Virol, 2012 86 (10): 5774-5781).
The numbering of the amino acid sequence of the EDC is consecutive from the amino residue (N-) terminal to the carboxyl residue (C-) terminal, so that position 1 corresponds to the residue at the Nterminal end of each sub -domain in wild type HA as it is found in virions. Thus, all the additional modified residues at the N-terminal end, such as the heterologous sequences described here, and those introduced in the context of an expression strategy or for the purposes of solubilization or purification, are numbered in the reverse order (i.e., C- to N-terminus) from position 1, starting with position 0 (for example, 0, -1, -2, etc.).
The EDC sequence can be derived from any type or subtype (e.g., H1 to H16) of influenza strain. In one embodiment, the EDC sequence of HA is from a strain selected from the group consisting of: a strain of subtype H1, H2, H3, H5, H7 and H9. Preferably, the EDC comes from an H5 strain such as an H5N1 strain.
For example, the EDC of HA can comprise or consist of the amino acid sequence represented by SEQ ID NO: 7 or a fragment or derivative of this sequence which contains an immunogenic part of HA. The nucleic acid coding for EDC of HA can comprise or consist of the nucleic acid sequence represented by SEQ ID NO: 8 or a fragment
BE2017 / 5126 or a derivative of this sequence coding for an immunogenic part of the EDC of HA.
The term "hydrophobic signal" refers to a sequence of at least 5 or at least 6 hydrophobic amino acids, or refers to an overall structure where hydrophobic amino acids are exposed on the surface. Hydrophobic signals can be natural or artificial. Natural hydrophobic signals are present in cellular or viral transmembrane proteins. Any natural or artificial hydrophobic signal must retain the original function in the protein, for example, in the case of HA proteins, the hydrophobic signal must have the capacity to orient the trimers of HA in rosette structures. The hydrophobic signal naturally present in HA proteins is called the transmembrane domain.
Transmembrane domain (TMD)
The transmembrane domain can consist of or comprise a total length TMD originating from a wild type HA protein or from one of its truncated or derived forms. Any of its truncated or derived forms must retain the ability to orient the trimers of HA into rosette structures and maintain the immunogenicity of HA. The correct rosette structure can be detected using techniques well known to those skilled in the art such as electron microscopy.
The transmembrane domain can also be derived from any type or subtype (for example, H1 to H16) of influenza strain. In one embodiment, the
BE2017 / 5126 HA TMD sequence comes from a strain selected from the group consisting of: a Hl, H2, H3, H5, H7 and H9 strain. Preferably, the TMD comes from an H5 strain such as an H5N1 strain. TMD can be derived from the same strain or from a different strain of the EDC sequences. TDG can be either homologous or heterologous to EDC. For example, the HA TMD can comprise or consist of the amino acid sequence represented by SEQ ID NO: 5 or a fragment or derivative of this sequence which retains the capacity to orient the HA trimers in rosettes and maintains the immunogenicity of HA. The nucleic acid coding for the TMD of HA can comprise or consist of the nucleic acid sequence represented by SEQ ID NO: 6 or a fragment or derivative of this sequence coding for a TMD which retains the ability to orient the trimers of HA in rosette structures and maintains the immunogenicity of HA.
Trimerization area
A suitable trimerization domain is one that induces rHA monomers to form trimers. Preferably, the trimerization domain is or is derived from the natural trimerization domain of the phage T4 "foldon" fibritin. A 29 amino acid fold sequence can be used which forms a β helix structure comprising the C-terminus of the fibritin domain of bacteriophage T4. Other suitable trimerization domains include chloramphenicol acetyltransferase (CAT) and a leucine-zipper trimerization motif derived from the activator of
BE2017 / 5126 transcription of yeast GCN4. Most preferably, the trimerization domain, like the foldon, is placed at the C-terminus of the extracellular domain and the TMD of HA (for example, the stem domain). Generally, the trimerization domain is fused via a short linker region to the HA sequence. The region between the trimerization domain and the HA sequence may include a cleavable linker sequence, so it is possible to isolate the HA sequence from trimerization at later stages. Thus, the HA sequence (which includes EDC and TMD) can be linked (for example, in order), optionally via a linker sequence, to a heterologous sequence comprising a cleavage site protease, the trimerization domain and a purification marker such as a histidine marker to aid in purification. Such heterologous trimerization domains can be linked to HA sequences by techniques known in the art, such as molecular cloning.
For example, the trimerization domain can comprise or consist of the foldon amino acid sequence represented by SEQ ID NO: 9 or a derivative of this sequence which retains the capacity to induce the rHA monomers to form trimers. The nucleic acid coding for the trimerization domain can consist of or comprise SEQ ID NO: 10 or a derivative of this sequence which retains the capacity to induce the rHA monomers to
BE2017 / 5126 form trimer, for example, as evaluated by electron microscopy.
RHA preparation processes
The use of recombinant DNA technology to produce influenza vaccines has several advantages. This includes the advisability of avoiding the stages of adaptation and passage of infectious viruses in eggs and the production of more highly purified proteins under safer and more stringently controlled conditions. In addition, there is no need to include a virus inactivation step. Any suitable cloning and expression system can be used to recombinantly produce the rHA antigen.
The nucleotide sequences coding for the rHA antigens of the invention can be synthesized, and / or cloned and expressed according to techniques well known to those skilled in the art. See, for example, Sambrook, et al., Molecular Cloning, A Laboratory Manual, Vol. 13, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989). In some embodiments, the polynucleotide sequences will be optimized for codons for a particular recipient host cell using conventional methodologies. For example, a DNA construct encoding a hemagglutinin sequence can be optimized for codons for expression in other hosts, for example, bacteria, mammalian or insect cells. Suitable host cells can include bacterial cells such as EL coli, cells
BE2017 / 5126 fungal cells such as yeast cells, insect cells such as Drosophila S2, Spodoptera Sf9, Sf00 + or Hi-5 and animal cells such as CHO.
Hemagglutinin sequences can be produced by standard recombinant methods known in the art, such as polymerase chain reaction (PCR) or reverse transcriptase PCR, reverse engineering or DNA can be synthesized. For PCR, primers can be prepared using hemagglutinin nucleotide sequences which are available in publicly available databases. Polynucleotide constructs can be assembled from PCR cassettes and cloned sequentially into a vector containing a selectable marker for propagation in a host cell
A recombinant vector can then be introduced into the host cell by injection, transfection or electroporation or other methods (e.g. calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation). Commercial transfection reagents such as Lipofectamine (Invitrogen, Carlsbad, CA) are also available.
The rHA antigen can be recovered and purified from recombinant cell cultures by methods known in the art, including anion and / or cation exchange chromatography, affinity chromatography. Techniques such as SDS-PAGE can be used to analyze protein fractions eluted from these techniques.
BE2017 / 5126 separation / purification. Such methods are well known to those skilled in the art and will not be presented in detail here.
The correct folding of the rHA antigen can be determined, for example, using the erythrocyte hemagglutination test, by the ability of the protein to bind an influenza virus receptor, by the analysis of immunogenicity in an animal. host and / or determining the ability of the protein to adopt an appropriate quaternary structure such as rosette formation.
Preferably, a baculovirus expression system is used, which is described below.
Baculovirus expression system
When a baculovirus expression system is used, the rHA antigen of interest in conjunction with any heterologous sequence (e.g., HA sequences in conjunction with the trimerization domain) can be inserted into an baculovirus expression. Recombinant baculoviruses which express foreign genes can be made by homologous recombination between baculovirus DNA and plasmids containing the insert, using well known techniques. The insertion can, for example, be carried out in such a way that the insert is under the control of the transcription of the polyhedrin promoter, the baculovirus promoter.
Example of baculovirus expression vectors comprising a vector derived from the Autographs californica nuclear polyhedrosis virus (AcNPV) well
BE2017 / 5126 characterized which replicates effectively in sensitive cultured insect cells.
Any suitable insect host cell can be used to produce the recombinant HA antigen, including, but not limited to, Sf900 +, Sf9 or Hi-5 cells. Preferably, the insect host cell is Sf9 or Hi-5. The cells can be propagated in culture medium and under culture conditions known to be suitable for the chosen host cell. The cells can be propagated, for example, in a monolayer or in culture in free suspension.
The rHA protein can then be isolated from host cells using methods well known in the art. For example, the cell culture can be centrifuged, the supernatant collected and passed through anionic and / or cationic exchange columns suitable for purifying the protein. Techniques such as SDS-PAGE and / or the immunoblot technique can be used to verify the identity and integrity of proteins. A fraction of interest containing rHA can then be further purified, for example, by passage through a nickel column such that rHA displaying a histidine tag binds to nickel in the column.
The extent of trimerization and / or multimerization (e.g. rosette formation) can be tested, for example, by crosslinking the HA using an appropriate crosslinking agent and then using gel migration techniques, as is well known in the art. Other techniques
BE2017 / 5126 include techniques based on electron microscopy or spectroscopy which are well known to those skilled in the art.
Rosette formation
In one embodiment, the rHA antigen of the present invention is in the form of rosettes. The rosettes are made up of HA trimer multimers having a rosette-like structure. The rosettes are visible, for example, under the electron microscope. More quantitative techniques can also be used to measure rosette formation, including techniques based on gel filtration and spectroscopy. Rosettes generally include 20 to 100 trimers of HA / particle. The particle size of the rosette structures is in the range of 20 to 40 nanometers (nm) in length. HA sediments in the form of a rosette composed of 5 to 6 trimers over the pH range of 7.4 to 7.5 (Remeta et al. 2002, Biochem. 41, 2044-2054). The hydrophilic C-terminal portions of the HA trimers are believed to concentrate together in a central region from which the hydrophobic regions deviate like a snowflake or rosette structure.
Immunogenic composition
In another aspect, there is provided an immunogenic composition comprising an HA antigen of the invention and a pharmaceutically acceptable carrier.
In one embodiment, said composition further comprises an adjuvant. Preferably, the adjuvant
BE2017 / 5126 is an adjuvant in the form of an oil in water emulsion. Adjuvants in the form of oil-in-water emulsions, such as MF59 or AS03, are well known in the art and are described below.
In one embodiment, the composition is monovalent, that is to say that it comprises only one HA of influenza virus. In alternative embodiments, the composition is multivalent, that is to say that it comprises several influenza virus antigens. For example, the composition can be bivalent, trivalent or quadrivalent, for example, it can contain two or three seasonal strains with the rHA of the invention.
Adjuvant
In one embodiment, an immunogenic composition of the invention comprises an adjuvant. In particular, the adjuvant can be an emulsion, such as an oil-in-water emulsion. Optionally, other immunostimulants may be present in the oil-in-water emulsion. In a specific embodiment, an oil-in-water emulsion comprises a metabolizable oil, a non-toxic oil such as squalene or squalane, optionally a tocol such as tocopherol, in particular alpha-tocopherol and an emulsifier (or surfactant) such as the nonionic surfactant polyoxyethylene sorbitan monooleate (TWEEN80 ™ or polysorbate 80 ™). Mixtures of surfactants can be used as mixtures of polyoxyethylene sorbitan monooleate / sorbitan trioleate (SPAN 85 ™), or mixtures of
BE2017 / 5126 polyoxyethylene sorbitan / t-octylphenoxypolyethoxyethanol (TRITON X-100 ”).
Tocols (for example, vitamin E) are also used in adjuvants based on oily emulsion (EP 0382271 B1; US 5,667,784; WO 95/17210). The tocols used in oily emulsions (optionally oil-in-water emulsions) can be formulated as described in documents US 5,650,155 A; US 5,667,784 A; EP 0382271 B1, in that the tocols can be dispersions of tocol droplets, optionally comprising an emulsifier, with a diameter possibly less than 1 micron. Alternatively, the tocols can be used in combination with another oil, to form the oily phase of an oily emulsion. Examples of oily emulsions which can be used in combination with tocol are described here, such as the metabolizable oils described above. In an oil-in-water emulsion, the oil and the emulsifier must be in an aqueous support. The aqueous support can be, for example, phosphate buffer solution or citrate buffer. An example of an oil-in-water emulsion containing a tocol is AS03.
A preferred oil-in-water emulsion comprises a metabolizable oil, such as squalene, Tween 80 and optionally alpha-tocopherol. In addition, the oil in water emulsion may contain Span 85 ™ and / or lecithin.
In one aspect, the oil in water emulsion has one of the following compositions:
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- from 0.5 to 11 mg of squalene, from 0.05 to 5% of polyoxyethylene sorbitan monooleate (TWEEN-80 ”or POLYSORBATE 80”) and optionally, from 2 to 12% of alphatocopherol; or
- approximately 5% of squalene, approximately 0.5% of polyoxyethylene sorbitan monooleate (TWEEN-80 ”or POLYSORBATE 80”) and approximately 0.5% of sorbitan trioleate (SPAN 85 ”). This adjuvant is called MF59.
A variant adjuvant which can be used with the compositions, the vaccine or the antigen according to the present invention comprises an immunologically active saponin fraction derived from the bark of Quillaja Saponaria Molina (for example, QS21) presented in the form a liposome and a lipopolysaccharide (for example, 3D-MPL), optionally also comprising a sterol (cholesterol). In one embodiment, the adjuvant comprises or consists of a saponin (for example, QS21) presented in the form of a liposome, a derivative of lipid A such as 3D-MPL and a sterol (for example, cholesterol). Liposomes suitably contain a neutral lipid, for example, phosphatidylcholine, dioleoylphosphatidylcholine (DOPC) or dilauryl-phosphatidylcholine. Liposomes may also contain a charged lipid which increases the stability of the liposome-QS21 structure for liposomes composed of saturated lipids. An example of such an adjuvant is AS01, which includes 3D-MPL and QS21 in a form neutralized with cholesterol, and can be manufactured as described in WO96 / 33739. Either AS01B or AS01E forms of this adjuvant can be used. AS01 B admixture
BE2017 / 5126 includes liposomes, which in turn include dioleoyl-phosphatidylcholine (DOPC), cholesterol and 3D-MPL (in an amount of approximately 1000 micrograms of DOPC, 250 micrograms of cholesterol and 50 micrograms of 3D- MPL per dose of vaccine), QS21 (50 micrograms / dose), NaCl phosphate buffer and water to a volume of 0.5 ml.
AS01E adjuvant includes the same components as AS01 B but at a lower concentration in an amount of approximately 500 micrograms of DOPC, 125 micrograms of cholesterol, 25 micrograms of 3D-MPL and 25 micrograms of QS21, phosphate buffer NaCl and water to a volume of 0.5 ml.
Vaccination regimes, dosage and efficacy criteria
Suitably, the immunogenic compositions for use according to the present invention are a conventional injectable dose of 0.5 ml in most cases, and they contain 15 pg or less of hemagglutinin antigen component from a virus strain influenza, as measured by simple radial immunodiffusion (SRD) (JM Wood et al.: J. M m Biol. Stand. 5 (1977) 237-247; JM Wood et al., J. Biol.
Stand. 9 (1981) 317-330). Suitably, the volume of the vaccine dose will be from 0.25 ml to 1 ml, in particular a volume of conventional vaccine dose of 0.5 ml or 0.7 ml. A slight adjustment of the dose volume will be carried out routinely depending on the concentration of HA in the original bulk sample and also according to the route of administration with doses.
BE2017 / 5126 lower given by the intranasal or intradermal route. Suitably, said immunogenic compositions for use according to the invention contain a small amount of HA antigen - for example, any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14 pg of HA per strain of influenza virus or which do not exceed 15 pg of HA per strain. Said small amount of HA can be as low as practically feasible provided that they allow a vaccine to be formulated which meets international criteria, for example EU or FDA, for efficacy, as detailed below ( see Table 1 and specific parameters as presented). A suitable low amount of HA is 1 to 7.5 pg of HA per strain of influenza virus, suitably 3.5 to 5 pg, such as 3.75 or 3.8 pg of HA per strain of influenza virus, generally about 5 pg HA per strain of influenza virus. Another suitable amount of HA is 0.1 to 5 pg of HA per strain of influenza virus, suitably 1.0 to 2 pg of HA per strain of influenza virus, such as 1.9 pg of HA per strain of influenza influenza virus.
The influenza drug (e.g., the immunogenic composition) of the invention suitably meets certain international criteria for vaccines. Standards are applied internationally to measure the effectiveness of influenza vaccines.
Serological variables are estimated according to criteria of the European Agency for the Evaluation of
Medicines for human use (CHMP / BWP / 214/96,
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Pharmaceutical Specialties Committee (CSP). Note for Harmonization of Requirements for Influenza Vaccines, 1997. CHMP / BWP / 214/96 Circular No. 960666: 1-22) for clinical trials associated with annual approval procedures for influenza vaccines (table below) .
Table 1
CHMP criteria
18 to 60 years old > 60 years Seroconversion rate * > 40% > 30% Conversion factor ** > 2.5 > 2.0 Protection rate *** > 70% > 60%
* The seroconversion rate is defined as the proportion of subjects in each group with a protective titre after vaccination> 1/40. In short, the seroconversion rate is the% of subjects who have an IH titer before vaccination <1/10 and> 1/40 after vaccination. However, if the initial titer is> 1/10 then there must be an increase of at least four times the amount of antibody after vaccination.
** The conversion factor is defined as the proportional increase in serum of the geometric mean of the IH titers (MGT) after vaccination, for each vaccine strain.
*** The protection rate is defined as the proportion of subjects either who were HIV negative before vaccination and had an IH (protective) titer after vaccination> 1/40 or who were HIV positive
BE2017 / 5126 before vaccination and showed a significant quadruple increase in titer after vaccination; it is normally accepted as indicating protection.
The requirements are different for the adult populations (18 to 60 years) and the elderly populations (> 60 years). For interpandemic influenza vaccines, at least one of the estimates (seroconversion factor, seroconversion rate, seroprotection rate) must meet European requirements for all strains of influenza virus included in the vaccine. The proportion of titles greater than or equal to 1/40 is considered the most relevant because it is expected that these titles represent the best correlation with protection (Beyer et al. (1998) Clin Drug Invest 15: 1).
The compositions for use according to the present invention suitably satisfy at least one of these criteria for the strain of influenza virus included in the composition (one criterion is sufficient to obtain approval), suitably at least two, or generally at least the three criteria for protection. Suitably, the above answer (s) is / are obtained after one dose, or after two doses.
Processing procedures
In another embodiment, the HA antigen or the immunogenic composition comprising said antigen is for use in medicine, as for use in the prevention of, or vaccination
BE2017 / 5126 against, the flu, for example, administered to a person (for example, a subject) at risk for an influenza infection.
In yet another embodiment, the HA antigen or the immunogenic composition comprising said antigen is for use in the prevention of influenza caused by a clade different from the clade on which the HA antigen was based. For example, a H5N1 clade 1 HA antigen may be used for protection against influenza caused by a non-clade 1 virus, for example, a H5N1 clade 2 virus.
In another aspect, there is provided a method of prevention and / or treatment against influenza, comprising administering an antigen or immunogenic composition as described herein to a person in need thereof, for example a person (for example, a subject) at risk for influenza infection, for example, an elderly person (50 years of age or older, particularly 65 years of age or older).
In one embodiment of the method or use described above, less than 15 micrograms, such as 3.75 to 10 micrograms of HA are administered per dose.
In one aspect, the invention provides the rHA of the invention at a dose of less than 10 micrograms, or less than 8 micrograms, or from 1 to 7.5 micrograms, or from 1 to 5 micrograms of rHA for use in a vaccination plan for the prevention of influenza, in which the hemagglutinin sequences originate from, or are derived from, a strain of the
BE2017 / 5126 influenza identified by an international organization like the WHO which monitors influenza virus outbreaks, as being associated with a pandemic outbreak or as having the potential to be associated with a future pandemic outbreak.
Routes of administration
The composition of the invention can be administered by any suitable route of administration, such as intradermal, mucosal (for example, intranasal), oral, intramuscular or subcutaneous. Other routes of administration are well known in the art.
The intramuscular route of administration is particularly suitable for the adjuvanted influenza composition. The composition according to the invention can be presented in a single-dose container, or alternatively, a multidose container, particularly suitable for a pandemic vaccine. In this case, an antimicrobial preservative such as thiomersal may be present to prevent contamination during use. A thiomersal concentration of 5 pg / dose of 0.5 ml (i.e., 10 pg / ml) or 10 pg / dose of 0.5 ml (i.e., 20 pg / ml) is appropriately present. A suitable IM administration device can be used as a needle-less liquid jet injection device, for example, the Biojector 2000 (Bioject, Portland, OR). Alternatively, a pen injector device, as used for home administration of adrenaline, could be used to allow self-administration of the vaccine. The use of such
BE2017 / 5126 delivery devices may be particularly suitable for large-scale immunization campaigns as will be required during a pandemic.
Intradermal administration is another suitable route. Any suitable device can be used for intradermal administration, for example, short needle devices. Such devices are well known in the art. Intradermal vaccines can also be administered by devices that limit the length of effective needle penetration into the skin, such as those described in WO 99/34850 and EP 1092444, incorporated herein by reference, and their functional equivalents. Also suitable are jet injection devices which administer liquid vaccines to the dermis via a liquid jet injector or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis. Also suitable are ballistic powder / particle delivery devices which use compressed gas to accelerate the powdered vaccine through the outer layers of the skin to the dermis. In addition, traditional syringes can be used in the classic Mantoux method of intradermal administration.
Another suitable route of administration is the subcutaneous route. Any suitable device can be used for subcutaneous administration, for example, a conventional needle. Suitably, a needleless jet injector device is used. Such devices are well known in the art. In a way
BE2017 / 5126 appropriate, said device is pre-filled with the liquid vaccine formulation.
Alternatively, the vaccine is administered intranasally. Generally, the vaccine is administered locally to the nasopharyngeal area, appropriately without being inhaled into the lungs. It is desirable to use an intranasal delivery device which administers the vaccine formulation to the nasopharyngeal area, without or substantially without entering the lungs.
The devices suitable for intranasal administration of the vaccines according to the invention are spray devices. Commercially available nasal spray devices include Accuspray ™ (Becton Dickinson). The nebulizers produce a very fine spray which can be easily inhaled into the lungs and therefore does not effectively reach the nasal mucosa. Therefore, nebulizers are not preferred.
Spray devices suitable for intranasal use are devices for which the performance of the device is not dependent on the pressure applied by the user. These devices are known as pressure threshold devices. The liquid is released from the nozzle only when a threshold pressure is applied. These devices make it easier to get a spray with a regular droplet size.
Pressure threshold devices suitable for use with the present invention are known in the art.
BE2017 / 5126 art and are described for example in documents WO 91/13281 and EP 311 863 B and EP 516 636, incorporated herein by reference. Such devices are commercially available from Pfeiffer GmbH and are also described in Bommer, R. Pharmaceutical Technology Europe, Sept 1999.
As a variant, the epidermal or transdermal vaccination route is also envisaged in the present invention.
The teachings of all references in this application, including patent applications and allocated patents, are fully incorporated herein by reference. Any patent application to which priority is claimed by this application is incorporated by reference herein in its entirety as described herein for publications and references.
To avoid any ambiguity, the terms “comprising”, “include” and “includes” here are intended by the inventors as being possibly substitutable by the terms “consisting of”, “consist of”, and “consists of”, respectively , in all cases.
The invention will be further described with reference to the following nonlimiting examples.
Examples
Example 1 Design and Construction of the Hemagglutinin Antigen (HA) Plasmid ECD-TMD-Foldon
The hemagglutinin (HA) antigen was modified based on an HA protein native to the H5N1 strain
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A / Indonesia / 05/2005. At the N-terminus, the signal peptide of HA has been replaced by gp67, which is a signal sequence coded by a baculovirus which is very efficient for the secretion of proteins (Whitford et al. 1989, J. Virol. 63, 1393-1399). The transmembrane domain (TMD), necessary for the formation of rosettes, as well as the Foldon trimerization domain (derived from the fibritin of bacteriophage T4) (Meier et al. 2004, J. Mol. Biol. 344, 1051-1069), necessary for the trimerization of the HA monomers, were downstream of the HA sequence (ECD). Finally, a polyHis tag was added to the C-terminus to facilitate the purification of the recombinant antigen.
Figure 1 shows the composition of the ECD-Foldon starting construction and that of ECD-TMDFoldon. ECD-Foldon was a donation from the Center for Diseases Control (CDC, Atlanta, USA). This construct was incorporated into plasmid pAcGP67, a baculovirus transfer vector containing the signal sequence gp67 in front of ECD-Foldon. ECD-Foldon was used as a matrix to prepare the other construct and was first produced by transformation of TOP10 electrocompetent cells from Escherichia coll (Life Technologies). The ECD-Foldon plasmid DNA was purified by Maxiprep (Qiagen) according to the manufacturer's instructions. It was used to construct the ECD-TMDFoldon by insertion of the TM between the ECD and the Foldon by site-directed mutagenesis. For this, a mega-primer was first produced by PCR. The primers consisted of sequences complementary to the vector pAcGP67 together with sequences of TM, and the
BE2017 / 5126 matrix was the total length HA from the strain of A / Indonesia / 05/2005 in the plasmid TOPO. After the PCR reaction, the mega-primer was purified in agarose gel with a QIAquick gel extraction kit (Qiagen). The consecutive mutagenesis reaction was carried out under the following conditions: 7.5 μΐ of 10X reaction buffer, 2 μΐ of dNTP (10 mM), 100 ng of template DNA containing the ECD-Foldon; 150 ng of mega-primer, 2 μΐ of PfuUltra HF pol DNA (2.5 U / μΙ), for a total volume of 50 μΐ. The PCR conditions were 95 ° C for 1 min and then 5 cycles consisting of 50 s at 95 ° C, 1 min at 52 ° C, 22 min at 68 ° C, followed by 13 cycles consisting of 50 s at 95 ° C , 1 min at 55 ° C, and 22 min at 68 ° C. Then the template DNA was removed by digestion with Dpn I (Promega). The PCR product was used to transform TOP10 electrocompetent cells. The Miniprep purification procedure was carried out on positive colonies and the validity of the construction was confirmed by sequencing. Figure 2 discloses the whole amino acid sequences of ECD-Foldon and ECD-TMD-Foldon. The sequence of the full length recombinant HA molecule which has been used as a comparator, particularly in immunogenicity studies is also shown.
Example 2 - Production and purification of ECD-TMDFoldon
The construct was inserted into a baculovirus.
For this, Hi-5 cells in SF-900 II medium containing 264 g / l of NaCl were seeded in a
BE2017 / 5126 6-well plate at a concentration of x 10 ⁶ cells / well. Fifty ng of the transfer plasmid containing the ECD-TMD-Foldon sequence were mixed with 20 ng of BAC 3000 vector and 100 μΐ of Cellfectin reagent (Life technology; Catalog No.
10362-010) in SF-900 II medium and incubated for min. Then 800 μΐ of medium were added to the transfection mixture and the solution was distributed among the wells. After 5 h of incubation, the mixture was removed and replaced with 2 ml of medium. The incubation took place at 27 ° C for 4 days.
The cells and the medium were harvested from the 6-well plates, transferred to centrifuge tubes and centrifuged to separate the cells from the medium containing the virus. The supernatant was used to infect Hi-5 cells. Positive clones were selected based on the expression of the recombinant protein (verified by Western blot analysis) and used to produce the virus stock.
To purify the ECD-TMD-Foldon, a total of 5 to 10 liters of a suspension of Hi-5 cells (adjusted to x 10 ⁶ cells / ml in SF500 shake flasks) was infected with the modified baculovirus by ECD-TMDFoldon with a multiplicity of infection = 1. Harvest occurred 56 to 65 h after infection. The suspension was centrifuged at 200 xg for 30 min and the supernatant was discarded. Then 200 to 250 ml of buffer containing 1% Triton X100 were added to each pellet for a 30 min incubation at 4 ° C on a shaker plate to resuspend the pellet. The suspension was centrifuged at 10,000 xg for 25 min
BE2017 / 5126 and the supernatant containing the ECD-TMD-Foldon was kept. The recombinant ECD-TMD-Foldon was purified from the supernatant by ion exchange chromatography through an anion exchange column (Q sepharose Fast Flow, Amersham) followed by a column of exchange of cations (SP sepharose Fast Flow, Amersham). The recombinant molecule linked in the SP column was eluted by change in pH (5.9 to 7.2) and change in molarity (+150 mM NaCl). Polyacrylamide gel electrophoresis containing sodium lauryl sulfate (SDS-PAGE) was used to identify the fractions of interest containing the target recombinant molecule. Finally, a group of selected fractions of interest was passed through a nickel column to further purify the ECD-TMD-Foldon which has a poly-His marker capable of binding nickel. The equilibration buffer consisted of 50 mM Tris, pH 8.0 150 mM NaCl and 10 mM imidazole. The fractions were eluted with increasing concentrations of imidazole. The fractions of interest were selected after passing through SDS-PAGE. The imidazole was removed by dialysis and the concentration of the sample was adjusted to 1 mg / ml.
Example 3 - Characterization (including rosette formation) of ECD-TMD-Foldon
After purification, the total length HA, the ECD-Foldon and the ECD-TMD-Foldon were compared by electron microscopy (EM). The samples were prepared for analysis by EM in negative staining according to a conventional method of negative staining in
BE2017 / 5126 two steps using phosphotungstic acid as a contrast agent. A luminescent discharge was applied to the grids to improve the adsorption of the material on the grids.
Briefly, a nickel grid (400 mesh) with a carbon coated Formvar film was floated on a drop of the sample for 10 min at room temperature to allow adsorption of the material. The excess solution was removed. The grid was briefly floated on a drop of distilled water to remove excess salt and it was then transferred to a drop of dye prepared as follows: 2% (w / v) Na phosphotungstate in water supplemented with 1% trehalose (w / v). The grid was dried with a blotter after 30 s. The material was allowed to dry completely and examined by transmission electron microscopy under a LEO Zeiss ΕΜ912Ω at 100 kV.
The results are shown in Figures 3A, 3B and 3C. The EDC-Foldon appears as structures of approximately 10 nm. In contrast, micrographs of both the full-length HA and the ECD-TMD-Foldon showed components in the range of 10 to 50 nm. This indicates that the ECD-TMDFoldon can assemble into a trimer structure and can form a rosette, similar to the total length HA.
Example 4 - The ECD-TMD-Foldon is immunogenic and triggers neutralizing antibodies
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The major drawback of the ECD-Foldon construct was its very low immunogenicity, compared to the fractionated HA antigen. One of the essential factors for the immunogenicity of recombinant HA antigens is the possibility of oligomerization and of forming rosettes. Rosette formation results from the interaction of several HA molecules via their transmembrane domain. Consequently, the transmembrane domain was inserted into the ECD-Foldon with the objective of allowing the formation of rosettes and of increasing immunogenicity, leading to the construction ECD-TMD-Foldon.
To assess the immunogenicity of the different constructs, female C57B1 / 6 mice (8 to 10 weeks old; 5, 10 or 11 mice / group) were immunized on day 0 and on day 21 with ECD-Foldon (6 or 24 gg), ECD-TMD-Foldon (1.5, 6 or 24 gg), recombinant HA total length (1.5, 6 or 24 gg), the antigen of A / Indonesia / 05/2005 fractionated (1.5 gg) or the negative control of phosphate buffer solution (PBS). All antigens were adjuvanted with AS03, an adjuvant system containing α-tocopherol and squalene in an oil-in-water emulsion (Garçon et al. 2012, Expert. Rev. Vaccines 11, 349- 366). Blood was taken on days 21 (3 weeks after dose I) and 42 (3 weeks after dose II).
Anti-A / Indonesia / 05/2005 antibodies were determined by an enzyme-linked immunosorbent assay (ELISA) test. For this, microtiter plates were sensitized overnight with the A / Indonesia / 05/2005 antigen fractionated (1 gg / ml
BE2017 / 5126 in Dulbecco phosphate buffer solution (DPBS)) at 4 ° C. After saturation of the aspecific sites with a saturation buffer (without serum), serial dilutions to half the sera of the mice were added to the wells and incubated for 1.5 h at 37 ° C. Dilutions were made in the saturation buffer. After washing the wells with 0.1% PBS-Tween, an anti-mouse IgG antibody conjugated to peroxidase (Sigma, A5278) was added and incubated for 1 h at 37 ° C. Bound antibodies were revealed by the addition of the peroxidase substrate, ortho-phenylenediamine in the presence of hydrogen peroxide for 20 min at room temperature in the dark. The colorimetric reaction was stopped by the addition of 2N sulfuric acid and the plates were read in a microtiter plate reader. The results are shown in Figure 4. It has been observed that ECD-TMD-Foldon is more immunogenic than ECD-Foldon, but does not trigger as high antibody levels as the fractionated antigen corresponding.
Humoral immune responses were also assessed by measuring the titers of hemagglutination inhibitor (HI) antibodies. The day before the test, 50 μΐ of enzyme destroying the receptor (RDE; cholera filtrate; Sigma C-8772), diluted to 25% in DPBS, were added to 12.5 μΐ of mouse serum sample and the mixture was incubated for 18 h at 37 ° C. Then, 37.5 μΐ of 2.5% sodium citrate was added to the mixture and incubated for 30 min at 56 ° C, and DPBS was added for a total volume of 125 μΐ. After centrifugation, the
BE2017 / 5126 supernatant was diluted 1/10 and serial dilutions to half of it were pipetted in a microtiter plate. To each well, 25 μΐ of a virus suspension (4 UHA in DPBS) was added for a 30 min incubation at room temperature. Finally, 50 μΐ of equine erythrocytes (1% in DPBS) were added and incubated for 1.5 to 2 h at room temperature. The results are presented in FIG. 5. All the ECD-TMD-Foldon groups, recombinant full length HA and fragmented antigen were different from the two ECD-Foldon groups and not different from each other 3 weeks after the second immunization (unilateral ANOVA followed by a post-hoc Tuckey HSD test).
The functionality of antibodies induced by immunization was assessed by an in vitro influenza neutralization test. For this, the mouse serum sample was diluted in series to half and the dilutions were deposited in a microtiter plate (100 μΐ / well). To the samples, 50 μΐ of viral solution (2000 TCID ₅₀ / ml) were added and the mixture was incubated 1.5 h at room temperature. After incubation, 100 μΐ of Madin-Darby dog kidney cell suspension (MDCK) (2.4 x 10 ⁵ cells / ml of cell culture medium) were added per well. The microtiter plate was placed in an incubator (35 ° C with 5% CO2) for 5 to 7 days. After incubation, 50 μΐ of the supernatants in each well were transferred to another microtiter plate. In each well, 50 μΐ of chicken erythrocytes (0.5% in PBS) were added and incubated
BE2017 / 5126 for 1 h at room temperature. The results are presented in FIG. 6. All the ECD-TMDFoldon, recombinant full length HA and fragmented antigen groups were different from the two ECD-Foldon groups and not different from each other 3 weeks after the second immunization (unilateral ANOVA followed by 'a post-hoc test by Tuckey HSD).
Figures 7 and 8 show the HI and neutralization activities, respectively, with respect to the antibody titer. The ratios for ECD-TMD-Foldon are in the same range as the ratios obtained for the fractionated antigen.
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List of sequences
SEQ ID NO: 1 - Amino acid sequence of
H5 recombinant hemagglutinin (ECD-TMD-foldon) Indo05 DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDC
SVAGWLLGNPMCDEFINVPEWSYIVEKANPTNDLCYPGSFNDYEELKHLLSRINH
FEKIQIIPKSSWSDHEASSGBSSACPYLGSPSFFRNVVWLIKKNSTYPTIKKSYN NTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVN GQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCN TKCQT PMGAINS S PMFHNIHPLTIGECPKYBKSNRLVLATGLRNS PQRE SRRKKR GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSI IDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDF HDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESIRNGTYNYPQYSEE ARLKREEIS GVKLE SIGTYQILSIYSTVASS LALAIMMAGL LWMC S S S GRLVPRG SPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH
SEQ ID NO: 2 - Nucleic acid sequence of recombinant H5 hemagglutinin (ECD-TMD-foldon) GATCCCGGGGATCAGATTTGCATTGGTTACCATGCAAACAATTCAACAGAGCAGG
TTGACACAATCATGGAAAAGAACGTTACTGTTACACATGCCCAAGACATACTGGA
AAAGACACACAACGGGAAGCTCTGCGATCTAGATGGAGTGAAGCCTCTAATTTTA
AGAGATTGTAGTGTAGCTGGATGGCTCCTCGGGAACCCAATGTGTGACGAATTCA
TCAATGTACCGGAATGGTCTTACATAGTGGAGAAGGCCAATCCAACCAATGACCT
CTGTTACCCAGGGAGTTTCAACGACTATGAAGAACTGAAACACCTATTGAGCAGA
ATAAACCATTTTGAGAAAATTCAAATCATCCCCAAAAGTTCTTGGTCCGATCATG
AAGCCTCATCAGGAGTGAGCTCAGCATGTCCATACCTGGGAAGTCCCTCCTTTTT
TAGAAATGTGGTATGGCTTATCAAAAAGAACAGTACATACCCAACAATAAAGAAA
AGCTACAATAATACCAACCAAGAAGATCTTTTGGTACTGTGGGGAATTCACCATC
CTAATGATGCGGCAGAGCAGACAAGGCTATATCAAAACCCAACCACCTATATTTC
CATTGGGACATCAACACTAAACCAGAGATTGGTACCAAAAATAGCTACTAGATCC
AAAGTAAACGGGCAAAGTGGAAGGATGGAGTTCTTCTGGACAATTTTAAAACCTA
BE2017 / 5126
ATGATGCAATCAACTTCGAGAGTAATGGAAATTTCATTGCTCCAGAATATGCATA
CAAAATTGTCAAGAAAGGGGACTCAGCAATTATGAAAAGTGAATTGGAATATGGT
AACTGCAACACCAAGTGTCAAACTCCAATGGGGGCGATAAACTCTAGTATGCCAT
TCCACAACATACACCCTCTCACCATCGGGGAATGCCCCAAATATGTGAAATCAAA
CAGATTAGTCCTTGCAACAGGGCTCAGAAATAGCCCTCAAAGAGAGAGCAGAAGA
AAAAAGAGAGGACTATTTGGAGCTATAGCAGGTTTTATAGAGGGAGGATGGCAGG
GAATGGTAGATGGTTGGTATGGGTACCACCATAGCAATGAGCAGGGGAGTGGGTA
CGCTGCAGACAAAGAATCCACTCAAAAGGCAATAGATGGAGTCACCAATAAGGTC
AACTCAATCATTGACAAAATGAACACTCAGTTTGAGGCCGTTGGAAGGGAATTTA
ATAAC T TAGAAAGGAGAATAGAGAAT T TAAACAAGAAGAT GGAAGACGGGT T T C T
AGATGTCTGGACTTATAATGCCGAACTTCTGGTTCTCATGGAAAATGAGAGAACT
CTAGACTTTCATGACTCAAATGTTAAGAACCTCTACGACAAGGTCCGACTACAGC
TTAGGGATAATGCAAAGGAGCTGGGTAACGGTTGTTTCGAGTTCTATCACAAATG
TGATAATGAATGTATGGAAAGTATAAGAAACGGAACGTACAACTATCCGCAGTAT
TCAGAAGAAGCAAGACTAAAAAGAGAGGAAATAAGTGGGGTAAAATTGGAATCAA
TAGGAACTTACCAAATACTGTCAATTTATTCAACAGTGGCGAGTTCCCTAGCACT
GGCAATCATGATGGCTGGTCTATCTTTATGGATGTGCTCCAGCGGCCGCTTGGTC
CCTCGTGGAAGCCCAGGCTCCGGCTACATCCCCGAGGCCCCGCGCGACGGCCAGG
CCTACGTGCGCAAGGACGGCGAGTGGGTGCTGCTGTCCACCTTCCTGGGACATCA
TCATCATCATCATTGA
SEQ ID NO: 3 - Amino acid sequence of the intracellular domain of hemagglutinin H5
NGSLQCRICI
SEQ ID NO: 4 - Nucleic acid sequence of the intracellular domain of hemagglutinin H5 AATGGATCGTTACAATGCAGAATTTGCATT
SEQ ID NO: 5 - Amino acid sequence of the HA H5 transmembrane domain
BE2017 / 5126
GVKLESIGTYQILSIYSTVASSLALAIMMAGLSLWMCS
SEQ ID NO: 6 - Nucleic acid sequence of the HA H5 transmembrane domain
GGGGTAAAATTGGAATCAATAGGAACTTACCAAATACTGTCAATTTATTCAACAG
TGGCGAGTTCCCTAGCACTGGCAATCATGATGGCTGGTCTATCTTTATGGATGTG
CTCC
SEQ ID NO: 7 - Amino acid sequence of the HA H5 extracellular domain
DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDC
SVAGWLLGNPMCDEFINVPEWSYIVEKANPTNDLCYPGSFNDYEELKHLLSRINH
FEKIQIIPKSSWSDHEASSGBSSACPYLGSPSFFRNVVWLIKKNSTYPTIKKSYN NTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVN GQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCN TKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRESRRKKR GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSI IDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDF HDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESIRNGTYNYPQYSEE
ARLKREEIS
SEQ ID NO: 8 - Nucleic acid sequence of the HA H5 extracellular domain
GATCCCGGGGATCAGATTTGCATTGGTTACCATGCAAACAATTCAACAGAGCAGG
TTGACACAATCATGGAAAAGAACGTTACTGTTACACATGCCCAAGACATACTGGA
AAAGACACACAACGGGAAGCTCTGCGATCTAGATGGAGTGAAGCCTCTAATTTTA
AGAGATTGTAGTGTAGCTGGATGGCTCCTCGGGAACCCAATGTGTGACGAATTCA
TCAATGTACCGGAATGGTCTTACATAGTGGAGAAGGCCAATCCAACCAATGACCT
CTGTTACCCAGGGAGTTTCAACGACTATGAAGAACTGAAACACCTATTGAGCAGA
ATAAACCATTTTGAGAAAATTCAAATCATCCCCAAAAGTTCTTGGTCCGATCATG
AAGCCTCATCAGGAGTGAGCTCAGCATGTCCATACCTGGGAAGTCCCTCCTTTTT
BE2017 / 5126
TAGAAATGTGGTATGGCTTATCAAAAAGAACAGTACATACCCAACAATAAAGAAA
AGCTACAATAATACCAACCAAGAAGATCTTTTGGTACTGTGGGGAATTCACCATC
CTAATGATGCGGCAGAGCAGACAAGGCTATATCAAAACCCAACCACCTATATTTC
CATTGGGACATCAACACTAAACCAGAGATTGGTACCAAAAATAGCTACTAGATCC
AAAGTAAACGGGCAAAGTGGAAGGATGGAGTTCTTCTGGACAATTTTAAAACCTA
ATGATGCAATCAACTTCGAGAGTAATGGAAATTTCATTGCTCCAGAATATGCATA
CAAAATTGTCAAGAAAGGGGACTCAGCAATTATGAAAAGTGAATTGGAATATGGT
AACTGCAACACCAAGTGTCAAACTCCAATGGGGGCGATAAACTCTAGTATGCCAT
TCCACAACATACACCCTCTCACCATCGGGGAATGCCCCAAATATGTGAAATCAAA
CAGATTAGTCCTTGCAACAGGGCTCAGAAATAGCCCTCAAAGAGAGAGCAGAAGA
AAAAAGAGAGGACTATTTGGAGCTATAGCAGGTTTTATAGAGGGAGGATGGCAGG
GAATGGTAGATGGTTGGTATGGGTACCACCATAGCAATGAGCAGGGGAGTGGGTA
CGCTGCAGACAAAGAATCCACTCAAAAGGCAATAGATGGAGTCACCAATAAGGTC
AACTCAATCATTGACAAAATGAACACTCAGTTTGAGGCCGTTGGAAGGGAATTTA
ATAAC T TAGAAAGGAGAATAGAGAAT T TAAACAAGAAGAT GGAAGACGGGT T T C T
AGATGTCTGGACTTATAATGCCGAACTTCTGGTTCTCATGGAAAATGAGAGAACT
CTAGACTTTCATGACTCAAATGTTAAGAACCTCTACGACAAGGTCCGACTACAGC
TTAGGGATAATGCAAAGGAGCTGGGTAACGGTTGTTTCGAGTTCTATCACAAATG
TGATAATGAATGTATGGAAAGTATAAGAAACGGAACGTACAACTATCCGCAGTAT
TCAGAAGAAGCAAGACTAAAAAGAGAGGAAATAAGT
SEQ ID NO: 9 - Amino acid sequence of the fibrin of the bacteriophage T4 GSGYIPEAPRDGQAYVRKDGEWVLLSTFL
SEQ ID NO: 10 - Nucleic acid sequence of the bacteriophage T4 fibritin “fold” GGCTCCGGCTACATCCCCGAGGCCCCGCGCGACGGCCAGGCCTACGTGCGCAAGG
ACGGCGAGTGGGTGCTGCTGTCCACCTTCCTG
BE2017 / 5126
SEQ ID NO: 11 - Amino acid sequence of HA total length
DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDC
SVAGWLLGNPMCDEFINVPEWSYIVEKANPTNDLCYPGSFNDYEELKHLLSRINH
FEKIQIIPKSSWSDHEASSGBSSACPYLGSPSFFRNVVWLIKKNSTYPTIKKSYN NTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVN GQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCN TKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRESRRKKR GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSI
IDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDF HDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESIRNGTYNYPQYSEE ARLKREEISGVKLESIGTYQILSIYSTVASSLWMIMM
CILVPRGSHHHHHH
BE2017 / 5126

权利要求:
Claims (3)
[1]
1. Recombinant hemagglutinin (HA) antigen of influenza virus comprising the extracellular domain of HA or one of its immunogenic parts, a hydrophobic signal and a heterologous trimerization domain.
[2]
2. HA antigen according to claim 1 wherein said hydrophobic signal is a transmembrane domain of HA.
3. HA antigen according to any one of the preceding claims, in which the hemagglutinin antigen lacks the intracellular domain of the influenza virus hemagglutinin.
4. HA antigen according to any one of the preceding claims wherein the hemagglutinin is not full length hemagglutinin.
5. HA antigen according to any one of the preceding claims wherein the recombinant HA antigen forms rosette structures in vivo.
6. HA antigen according to claim 1, in which the recombinant HA antigen forms rosette structures in vitro.
Ί. HA antigen according to any one of the preceding claims which further comprises a His tag.
8. HA antigen according to any one of the preceding claims which further comprises a cleavable bond.
9. HA antigen according to any one of the preceding claims, in which the hemagglutinin comes from a strain H1, H2, H3, H5, H7 or H9.
BE2017 / 5126
10. HA antigen according to claim 1, in which the transmembrane domain of HA is heterologous to the extracellular domain of said HA.
11. HA antigen according to any one of the preceding claims, in which the hemagglutinin comes from an H5 strain.
12. HA antigen according to any one of the preceding claims, in which i) the EDC of HA consists of or comprises SEQ ID NO: 7, ii) the TMD of HA consists of or comprises SEQ ID NO: 5 or a fragment or derivative of this sequence which retains the ability to orient the trimers of HA into rosette structures and maintains the immunogenicity of the HA and / or iii) the trimerization domain consists of or comprises SEQ ID NO : 9 or a derivative of this sequence which retains the ability to induce the rHA monomers to form trimers.
13. HA antigen according to any one of the preceding claims, in which i) the EDC of HA consists of or comprises SEQ ID NO: 7, ii) the TMD of HA consists of or comprises SEQ ID NO: 5
and iii) the domain of trimerization is made of or includes SEQ ID NO : 9. 14. Antigen HA on 1 'a any of previous claims the antigen HA including the SEQ ID sequence NO: 1. 15. Antigen HA on 1 'a any of claims previous, in which, at Hemagglutinin, the HA stem is missing, or a part of the stem.
BE2017 / 5126
16. Polynucleotide coding for the hemagglutinin antigen as described in any one of the preceding claims.
17. Polynucleotide according to claim 16 which comprises i) SEQ ID NO: 8 or a fragment or a derivative of this sequence coding for an immunogenic part of the EDC of HA, ii) SEQ ID NO: 6 or a fragment or a derivative of this sequence which codes for a TMD which retains the capacity to orient the trimers of HA in rosette structures and maintains the immunogenicity of HA and iii) SEQ ID NO: 10 or a derivative of this sequence which retains the capacity to induce the expressed rHA monomers to form trimers.
18. Polynucleotide according to claim 17 which comprises i) SEQ ID NO: 8, ii) SEQ ID NO: 6 and iii) SEQ ID NO: 10.
19. Polynucleotide according to claim 16 which comprises SEQ ID NO: 2.
20. Immunogenic composition comprising an antigen as defined in any one of claims 1 to 15 and a pharmaceutically acceptable carrier.
21. The immunogenic composition according to claim 20, further comprising an adjuvant.
22. Immunogenic composition according to claim 21, in which the adjuvant is an adjuvant based on oil-in-water emulsion.
23. Immunogenic composition according to claim 20 or 21, the composition being multivalent.
BE2017 / 5126
24. Immunogenic composition according to claim 20 or 21, the composition being monovalent
25. Immunogenic composition according to any one of claims 20 to 24 for use in medicine.
26. Immunogenic composition according to any one of claims 20 to 24 for use in the prevention of or vaccination against influenza.
27. Immunogenic composition according to any one of claims 20 to 24 for use in the prevention of and / or vaccination against influenza caused by a clade different from the clade to which the extracellular domain of the HA antigen belongs.
28. A method of producing an antigen as defined in any one of claims 1 to 15 comprising expressing a polynucleotide according to any one of claims 16 to 19 in a eukaryotic cell, such as a mammalian cell , for example, a CHO cell, or an insect cell, optionally further comprising purifying / isolating the recombinant HA from the eukaryotic cell.
29. A method of preventing and / or vaccinating against influenza, comprising administering an antigen or an immunogenic composition according to any one of the preceding claims to a person in need thereof.
30. The method of claim 29, wherein less than 15 micrograms, such as 3.75 to 10 micrograms of hemagglutinin are administered per dose.
BE2017 / 5126 φ
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法律状态:
2018-06-15| FG| Patent granted|Effective date: 20180425 |

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2019-12-13| MM| Lapsed because of non-payment of the annual fee|Effective date: 20190331 |

优先权:

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申请号 | 申请日 | 专利标题

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GBGB1603625.3A|GB201603625D0|2016-03-02|2016-03-02|Novel influenza antigens|

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GB1603625.3|2016-03-02|

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