Patent Grant
10232029
The present invention relates to a composition comprising Neisseria meningitidis a combination of antigens including auto-transporters such as IgA1P, App, AusI or NalP, and TbpB (Transferrin binding protein B) and its use for immunizing or vaccinating a subject against an infection induced by N. meningitidis.
Neisseria meningitidis is one of the most important causes of bacterial meningitis and septicemia worldwide in both endemic and epidemic forms. The bacteria are classified into serogroups based on the structure of their capsular polysaccharides. Thirteen different serogroups have been identified but only five (A, B, C, W135 and Y) are responsible for the majority of infections, although epidemic meningitis due to meningococcal serogroup X is emerging in the Meningitis Belt of Africa. Two effective quadrivalent polysaccharide-protein conjugate vaccines have been developed and licensed against serogroups A, C, W135 and Y. In contrast to the other capsular polysaccharides, the group B polysaccharide is not an appropriate vaccinal antigen because of structural similarities with polysialic acid chains present in human cells. These properties of the serogroup B polysaccharide have impeded the development of a polysaccharide-based vaccine against group B Neisseria meningitidis (MenB) and led to the development of alternative vaccines.
During in vitro culture conditions and in vivo infection N. meningitidis releases outer membrane blebs which contain lipooligosaccharide (LOS) and outer membrane proteins (OMPs). These blebs are known as outer membrane vesicles (OMVs). Meningococcal OMV vaccines have been developed and shown to be successful in controlling outbreaks of MenB disease when using OMVs produced from a representative outbreak strain. Several approaches have been carried out to increase the breadth of coverage of OMV vaccines. Despite these developments and the suggestion that a vaccine including six PorA and five FetA variants would potentially provide protection against the majority of global circulating pathogenic strains, the search for a vaccine candidate that is highly conserved and expressed by all disease causing meningococci has continued.
Reverse vaccinology was used by Novartis Vaccines to develop a vaccine for serogroup B meningococcus (MenB), using the genome from the MenB strain MC58. The five most promising antigens on the basis of their ability to induce passive protection or serum bactericidal activity (SBA) were selected to be included in an investigational vaccine. These antigens include factor H binding protein (fHBP, also known as GNA (genome-derived neisserial antigen) 1870 or NMB1870), Neisseria Adhesin A (NadA, previously known as GNA1994), Neisseria Heparin-Binding Antigen (NHBA, previously known as GNA2132), GNA2091 and GNA1030. To further increase immunogenicity and stability, fHBP was fused with GNA2091, and NHBA with GNA1030. These two fusion proteins mixed together with NadA were proposed as a vaccine composition as described in Giuliani et al., PNAS (2006) 103 (29): 10834. Later on, the vaccinal composition was complemented with OMVs that contain the Porin A (PorA) protein. PorA is an immune-dominant protein that has been demonstrated to elicit antibodies during the course of meningococcal infections.
The final vaccine composition, which has progressed through clinical trials, has been recently licensed and commercialized under the trade name Bexsero®. As stated in the EPAR (European Public Assessment Report) of the EMEA (European Medicine Agency), this vaccine contains three recombinant N. meningitidis serogroup B proteins, namely NHBA (Neisseria Heparin Binding Antigen) fusion protein, NadA protein and fHBP fusion protein, together with outer membranes vesicles (OMVs) from N. meningitidis serogroup B.
One of the recombinant proteins of Bexsero®, fHBP, is also known as lipoprotein 2086 (LP2086) which has been independently discovered by Pfizer using an iterative process of immunisation following differential detergent extraction and protein purification.
fHBP Reference to N. meningitidis herein may be understood to refer to any serogroup, or may be understood to refer specifically to the B serogroup.
N. meningitidis species is genetically and antigenically highly diverse. Multilocus sequence typing (MLST) was first developed in the late 1990s for the meningococcus. It is a highly reliable and reproducible characterization method, which assesses variation at multiple genetic loci using nucleotide sequencing. More than 6751 sequence types (STs) have been assigned for N. meningitidis strains. While meningococcal diversity is extensive, it is highly structured. Studies of variation at housekeeping loci, initially by multilocus enzyme electrophoresis and more recently by MLST, had identified 37 groups of closely related meningococci at the time of writing, accounting for 61% of the meningococcal isolates represented in the PubMLST database. These groups, known as clonal complexes, have become the predominant unit of analysis in meningococcal population biology and epidemiology. A minority of clonal complexes, the so-called hyper-invasive lineages, are responsible for a disproportionate number of cases of disease worldwide and can be over-represented in collections of isolates from diseased patients by as much as two orders of magnitude, relative to their prevalence in asymptomatic carriage (see Table 1 herein after).
meningitidis (data compiled from the PubMLST database Jun. 2, 2009).
As shown in the above table, strains of e.g., serogroup B, belong to several clonal complexes. In particular, serogroup B strains are highly represented among significant invasive clonal complexes, including major clonal complexes spread worldwide i.e., ST-8, ST-18, ST-32, ST-41/44, ST162 and ST-269 clonal complexes, as well as clonal complex ST-11, remarkable for its very low rate of carriage relative to high incidence of disease.
To determine whether various protein antigens and combinations thereof are likely to give broad coverage across all strains of serogroup B, representative strains of that serogroup among major clonal complexes (6 ST complexes or groups) were selected and effectiveness in term of protection coverage of these various protein antigens and combinations thereof was tested against these strains.
It has now been found that a combination of at least two proteins of N. meningitidis other than those present in the licensed Bexsero® vaccine are able to offer a significant protection coverage over a collection of N. meningitidis strains of serogroup B, representative of the major invasive clonal complexes encountered worldwide.
This protection coverage has been evaluated by serum bactericidal activity (SBA) assay which reflects the ability of a given antigen to elicit bactericidal antibodies. The SBA assay measures functional activity of antibody through complement-mediated antibody lysis of the bacteria. Serum bactericidal activity has been accepted as a valid surrogate for predicting the clinical efficacy of serogroup B meningococcal vaccines.
Indirect evidence of SBA assay providing surrogate of protection came from studies by Goldschneider and colleagues in 1969 where an inverse correlation between the incidence of disease and the prevalence of serum bactericidal activity in human serum SBA against MenA, MenB and MenC were reported. In their prospective study, the SBA titer in serum was measured using human complement (e.g., endogenous complement or exogenous serum from a healthy adult who lacked intrinsic bactericidal activity). They demonstrated in incoming recruits to a US Army base that the presence of serum bactericidal activity strongly indicated resistance to meningococcal disease. This led to the establishment of SBA assay as the immunological surrogate of protection against meningococcal disease.
Indeed, in clinical trials of MenB vaccines, the measurement of the increase of the SBA titer after vaccination compared to the SBA basal titer (before any administration of the meningococcal vaccine) is an established clinical end point. Seroconversion is considered to be met when an SBA titer is superior or equal to 4. This approach was validated in 2005 at a World Health Organization sponsored meningococcal serology standardisation workshop and is based upon evidence from a number of efficacy studies of OMV vaccines.
Animal SBA assays achieved in upstream research are as well commonly acknowledged as a surrogate of protection for N. meningitidis vaccines. In the context of the present invention, initial SBA assays were first carried out against the homologous strain (‘homologous’ assays). Then, antigens, in particular those that gave positive results in the homologous SBA assay were taken forward into ‘heterologous’ SBA assays, in which the antigens-specific corresponding sera were tested against different strains, to give an indication of the vaccine coverage among epidemiologic relevant strains. The inventors of the present invention have determined that when the SBA titer (fold-increase compared to a negative control group) is superior or equal to 16 in homologous SBA assay, or superior or equal to 8 in heterologous SBA, protection is considered to be met.
An optimal vaccine shall give a broad coverage against a panel of representative strains of relevant invasive clonal complexes.
This is the reason why, the present invention relates to a composition, i.a. an immunogenic composition, comprising at least two Neisseria meningitidis (Nm) protein antigens selected from the group consisting of a trypsin-like serine protease auto-transporter antigen, a NalP antigen and a TbpB antigen. Preferably, the composition of the invention comprises (i) the trypsin-like serine protease auto-transporter antigen and (ii) the NalP antigen and/or TbpB antigen.
Although the polypeptides for use as antigens in the composition of the present invention are more particularly exemplified herein by reference to amino acid sequences of proteins that naturally-occur in a limited number of N. meningitidis strains (M982 and B16B6 for TbpB, MC58 for NalP and trypsin-like serine protease auto-transporters), polypeptides of any naturally-occurring/allelic variant of N. meningitidis strain M982/B16B6/MC58 are also encompassed within the scope invention as well as any variant that may result from genetic engineering. By extension, the term variant is therefore applied to amino acid sequences, proteins or fragments thereof other than M982, B16B6 or MC58 sequences, proteins or fragments thereof. Variations in amino acid sequence may be introduced by substitution, deletion or insertion of one or more codons into the nucleic acid sequence encoding the protein that results in a change in the amino acid sequence of the protein without substantially affecting the tri-dimensional structure and/or the biological and/or immunogenic properties. Typically, the variation may result for an amino acid substitution that may be conservative or non-conservative, preferably conservative. A conservative substitution is an amino acid substitution in which an amino acid is substituted for another amino acid with similar structural and/or chemical properties.
In what follows, variants and/or mutants are described by reference to an amino acid sequence of reference. Such a description by reference is based on the prerequisite of optimal sequence alignment in order to determine the amino acid in the variant sequence that corresponds to the amino acid defined as being in a specific position in the amino acid of reference.
In what follows, variants and/or mutants are also described by percent identity with a sequence of reference. Percent identity between two amino acid sequences or two nucleotide sequences is determined with standard alignment algorithms as those described below.
Sequence alignment can be achieved and percent identity can also be determined by standard local alignment algorithms such as the Smith-Waterman algorithm (Smith et al., J. Mol. Biol. (1981) 147: 195) (available on the EBI web site) or Basic Local Alignment Tools (BLASTs; described in Altschul et al., (1990) J. Mol. Biol., 215: 403) available on the National Center for the Biotechnology Information (NCBI) web site at http://www.ncbi.nlm.nih.gov/BLAST and may be used using the default parameters.
In the context of the invention, variant and mutant amino acid sequences include amino acid sequences that have at least about 80% sequence identity with an amino acid sequence defined herein. Preferably, a variant or mutant amino acid sequence will have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to a polypeptide sequence as defined herein. Amino acid sequence identity is defined as the percentage of amino acid residues in the variant sequence that are identical with the amino acid residues in the reference sequence, after aligning the sequences and if necessary, introducing gaps, to achieve the maximum percent sequence identity, and not considering any conservative substitution as part of the sequence identity. Standard alignment algorithms cited above are useful in this regard.
Serine-Like Auto-Transporters of N. meningitidis
Auto-transporters are virulence factors produced by Gram-negative bacteria. Auto-transporters are modular proteins initially expressed as a precursor consisting of an N-terminal signal sequence and a C-terminal translocator domain separated by an N-terminal passenger domain that is secreted into the extra cellular medium. The signal sequence directs the auto-transporter to the secretion machinery for transport across the internal membrane. The translocator domain mediates the transport of the passenger domain across the outer membrane. The term auto-transporter was coined because of the apparent absence of dedicated secretion machinery.
On the basis of their N- and C-terminus domains, auto-transporters can be divided into several categories. The classical auto-transporters, as typified by the IgA1 protease, contain a catalytic site in the N-terminal half of the passenger domain which often, although not always, is involved in the autocatalytic release of the passenger domain from the cell surface. The catalytic site is constituted by an amino acid triad comprising a serine residue. Accordingly, these auto-transporters are classified as serine proteases.
The very first N. meningitidis auto-transporter that was described was indeed the IgA1 protease. The amino acid sequence precursor of this protein was first described in Lomholt et al., Mol. Microb. (1995) 15 (3): 495. Since then, the N. meningitidis IgA1 protease has been extensively studied and characterized (Vitovski & Sayers, Infect. Immun. (2007) 75 (6): 2875; Ulsen & Tommassen, FEMS Microbiol. Rev. (2006) 30 (2): 292). The IgA1 protease was proposed for vaccinal use in the early nineties (WO 90/11367).
After becoming publicly available, the genomes of N. meningitidis strains MC58 (serogroup B) (Tettelin et al., Science (March 2000) 287: 1809), Z2491 (serogroup A) and FAM18 (serogroup C) have been systematically searched for the presence of genes encoding auto-transporters. BLAST searches using known auto-transporters as search leads resulted in the identification of eight genes putatively encoding proteins with auto-transporter characteristics, four of which encode serine proteases:
Trypsin-Like Serine Protease Auto-Transporter Antigen of N. meningitidis
While NalP is known to be a subtilisin-like serine protease, IgA1P, App and AusI are classified as being chymotrypsin-like serine proteases (herein after called trypsin-like serine proteases). IgA1P, App and AusI are different proteins displaying similar tridimensional structure, as they are all auto-transporters, and share some sequence homology at least in the passenger domain, including the catalytic triad.
The translocator domain of trypsin-like serine proteases is essential for the transport of the passenger domain across the membrane. It is constituted of several hydrophobic beta-sheets integrated into the outer-membrane that form a channel through which the passenger domain is secreted and is accordingly designated under the term “beta domain”. Typically, the IgA1P beta domain contains twelve beta sheets (Gripstra et al., Res. in Microbiol. (2013) 164: 562).
As described in Pohlner et al., Nature (1987) 325: 458, the ˜160 kDa passenger domain of IgA1P essentially consists of two sub-domains: (i) the N-terminal sub-domain containing the protease activity (protease sub-domain) and (ii) a ˜40 kDa alpha-peptide domain which are connected together via a small gamma-peptide. The two sub-domains can be released separately or as a single polypeptide.
For ease of description, the protease sub-domain together with the gamma peptide is referred hereinafter as a single entity under the term “protease domain”. The protease sub-domain extends from the N-terminal end of the mature IgA1P polypeptide, to the PAPISP auto-cleavage site.
As a matter of example, the amino acid sequence of the IgA1P precursor of MC58 (NMB0700) is shown in SEQ ID NO: 1. Further details are to be found in Table 2A below.
App and AusI were studied more recently [van Ulsen et al., FEMS Immunol Med. Microb. (2001) 32: 53; Serruto et al., Mol. Microb. (2003) 48 (2): 323; van Ulsen et al., Microbes & Infection (2006) 8: 2088; Turner et al., Infect. Immun. (2006) 74 (5): 2957); Ulsen & Tommassen (supra); Henderson et al., Microbiol. Mol. Biol. Rev. (2004) 68 (4): 692]. App and AusI are both trypsin-like serine proteases, with FINTL as putative auto-cleavage site. Although the boundaries of their domains and sub-domains are less characterized than those of IgA1P, there is no doubt that they share the same domain organization as is apparent from Tables 2B and 2C. Genome analysis shows that App is quite conserved in N. meningitidis, with sequence identities compared with MC58 App being from 88 to 98%.
The boundaries of the IgA1P, App and AusI domains may vary slightly, as it is not always possible to precisely define a domain to the exact amino acid. For example, the domains may be defined as slightly different depending on the methods/techniques used by different scientists to identify them and on the strain origin of the sequences. Thus, the domains indicated in Table 2A to 2C may be defined according to the locations given herein and/or in the Figures, or according to said locations +/−1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids N-terminal or C-terminal of said locations. A ‘domain’ of a trypsin-like serine protease auto-transporter protein as referred to herein may be said domain as defined in Table 2A, 2B or 2C, or may be said domain +/−1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids at the N-terminus and/or the C-terminus.
As already mentioned above, trypsin-like serine protease auto-transporters of N. meningitidis include IgA1P, App and AusI, each of them being useful within the frame of the present invention. Accordingly, the trypsin-like serine protease auto-transporter antigen may be e.g., an IgA1P, App or AusI antigen.
According to a preferred embodiment, a trypsin-like serine protease auto-transporter antigen suitable for the invention may be as follows.
For use in the composition of the present invention, the trypsin-like serine protease auto-transporter antigen may be a polypeptide, preferably an isolated polypeptide, selected from polypeptides comprising or consisting of:
(I):
a full-length mature N. meningitidis trypsin-like serine protease auto-transporter; or
a mutant of said a full-length mature N. meningitidis trypsin-like serine protease auto-transporter which lacks or has reduced trypsin-like serine protease activity and/or does not contain any cleavage site able/susceptible to be cleaved by a trypsin-like serine protease; or
(II):
(A) a fragment of a full-length mature trypsin-like serine protease auto-transporter of N. meningitidis, said fragment consisting of:
(i) a protease domain of a trypsin-like serine protease auto-transporter of N. meningitidis; or
(ii) a protease domain and all or part of an α-peptide domain of a trypsin-like serine protease auto-transporter of N. meningitidis; or
(iii) a protease domain, an α-peptide domain and a part of a β-domain, in particular a part of the β-domain comprising at least one and no more than eleven β-sheets of a trypsin-like serine protease auto-transporter of N. meningitidis; or
(B) a mutant of said fragment (A) which lacks or has reduced trypsin-like serine protease activity and/or does not contain any cleavage site able/susceptible to be cleaved by a trypsin-like serine protease;
wherein said polypeptide under (A) or (B) does not comprise the said full-length mature trypsin-like serine protease auto-transporter of N. meningitidis; or
(III):
a first fragment fused to a second fragment:
(1) said first fragment consisting of:
(2) said second fragment consisting of:
wherein the first and second trypsin-like serine protease auto-transporters are different; and
wherein the C-terminus of the first fragment is fused to the N-terminus of the second fragment,
wherein said polypeptide does not comprise the said full-length mature trypsin-like serine protease auto-transporter of N. meningitidis.
According to one embodiment, a trypsin-like serine protease auto-transporter of N. meningitidis suitable for the invention may be IgA1P, App or AusI, as above-described Polypeptides described under (II) (A) and (B) herein above are collectively referred to herein after as “Fragment Polypeptides”. Polypeptides described under (Ill) herein above are collectively referred to herein after as “Fusion Polypeptide”.
For use in the composition of the present invention, the trypsin-like serine protease auto-transporter antigen may be a trypsin-like serine protease auto-transporter polypeptide comprising or essentially consisting of:
According to one embodiment, a trypsin-like serine protease auto-transporter of N. meningitidis suitable for the invention may be IgA1P, App or AusI, as above-described.
By “full-length mature trypsin-like serine protease auto-transporter” is meant the trypsin-like serine protease auto-transporter lacking the signal peptide.
Accordingly, by “full-length mature trypsin-like serine protease auto-transporter” is meant the full-length mature trypsin-like serine protease auto-transporter comprising (having) (i) a naturally-occurring full-length mature amino acid sequence or (ii) a naturally-occurring full-length mature amino acid sequence lacking at most the first 50, 40, 30, 20, 10 or 5 N-terminus amino acids and/or at most the last 5 C-terminus amino acids or (iii) a naturally-occurring full-length mature amino acid sequence fused to the 1, 2 or 3 amino acids of the C-terminus of the signal sequence.
As a matter of example, a naturally-occurring full-length mature N. meningitidis trypsin-like serine protease auto-transporter may be:
Other useful full-length mature trypsin-like serine protease auto-transporters include variants of the MC58 full-length mature trypsin-like serine protease auto-transporters such as variants of MC58 full-length mature IgA1P, App or AusI.
Useful full-length mature IgA1P proteins include variants of MC58 IgA1P protein which may be described by reference to the MC58 amino acid sequence reported in SEQ ID NO: 1. These variants comprise (have) an amino acid sequence starting with an amino acid corresponding to the amino acid in position 28 and ending with an amino acid corresponding to the amino acid in position 1815. Other useful variants of the MC58 IgA1P protein, still described by reference to the MC58 amino acid sequence reported in SEQ ID NO: 1, comprise (have) an amino acid sequence starting with the amino acid corresponding to the amino acid in any one of the positions 28 to 78, preferably 28 to 58, more preferably 28 to 38, e.g. in position 27, 28, 29, 30, 31 or 32, in SEQ ID NO: 1, and ending with the amino acid corresponding to the amino acid in any one of positions 1810 to 1815 in SEQ ID NO: 1.
Useful full-length mature App proteins include variants of MC58 App protein which may be described by reference to the MC58 amino acid sequence reported in SEQ ID NO: 2. These variants comprise (have) an amino acid sequence starting with the amino acid corresponding to the amino acid in position 42 or 43 and ending with the amino acid corresponding to the amino acid in position 1457. Other useful variants of the MC58 App protein, still described by reference to the MC58 amino acid sequence reported in SEQ ID NO: 2, comprise (have) an amino acid sequence starting with the amino acid corresponding to the amino acid in any one of the positions 42 or 43 to 93, preferably 42 or 43 to 73, more preferably 42 or 43 to 53, e.g. in position 42, 43, 44, 45, 46 or 47, in SEQ ID NO: 2, and ending with the amino acid corresponding to the amino acid in any one of positions 1453 to 1457 in SEQ ID NO: 2.
Useful full-length mature AusI proteins include variants of MC58 AusI protein which may be described by reference to the MC58 amino acid sequence reported in SEQ ID NO: 3. These variants comprise (have) an amino acid sequence starting with the amino acid corresponding to the amino acid in position 27 and ending with the amino acid corresponding to the amino acid in position 1431. Other useful variants of the MC58 AusI protein, still described by reference to the MC58 amino acid sequence reported in SEQ ID NO: 3, comprise (have) an amino acid sequence starting with the amino acid corresponding to the amino acid in any one of the positions 27 to 77, preferably 27 to 57, more preferably 27 to 37, e.g., in position 27, 28, 29, 30, 31 or 32, in SEQ ID NO: 3, and ending with the amino acid corresponding to the amino acid in any one of positions 1426 to 1431 in SEQ ID NO: 3.
As mentioned above, for use in the composition of the present invention, the trypsin-like serine protease auto-transporter antigen suitable for the invention may also be a polypeptide comprising or consisting of a fragment of a full-length mature trypsin-like serine protease auto-transporter of N. meningitidis, such as IgA1P, App and AusI, which may be as described above.
Trypsin-like serine proteases are proteins with low solubility due to the presence of the beta-core and as a consequence of this, recombinant expression and purification of full-length trypsin-like serine proteases may be difficult to achieve. This is the reason why, in a preferred embodiment, the trypsin-like serine protease auto-transporter antigens may be produced as a truncated proteins, at least partially lacking the beta-core.
Accordingly, for use in the composition of the present invention, the trypsin-like serine protease auto-transporter antigen may accordingly be a polypeptide comprising or consisting of:
(A) a fragment of a full-length mature trypsin-like serine protease auto-transporter of N. meningitidis, said fragment consisting of:
(i) a protease domain of a trypsin-like serine protease auto-transporter of N. meningitidis; or
(ii) a protease domain and all or part of an α-peptide domain of a trypsin-like serine protease auto-transporter of N. meningitidis; or
(iii) a protease domain, an α-peptide domain and a part of a β-domain, in particular a part of the β-domain comprising at least one and no more than eleven β-sheets of a trypsin-like serine protease auto-transporter of N. meningitidis; or
(B) a mutant of said fragment (A) which lacks or has reduced trypsin-like serine protease activity and/or does not contain any cleavage site able/susceptible to be cleaved by a trypsin-like serine protease;
wherein said polypeptide under (A) or (B) does not comprise the said full-length mature trypsin-like serine protease auto-transporter of N. meningitidis.
According to one embodiment, a trypsin-like serine protease auto-transporter of N. meningitidis suitable for the invention may be IgA1P, App or AusI, as above-described.
Advantageously, when the fragment essentially consists of option (iii), part of the beta domain may comprise at least one and no more than eight, six, four or preferably, two beta-sheets. Part of the beta domain may comprise from N-ter to C-ter, at least the first beta-sheet; (ii) first and second beta-sheets; (iii) first, second and third beta-sheets; (iv) first, second, third and fourth beta-sheets; (v) first, second, third, fourth and fifth beta-sheets; (vi) first, second, third, fourth, fifth and sixth beta-sheets; (viii) first, second, third, fourth, fifth, sixth and seventh beta-sheets; or (viii) first, second, third, fourth, fifth, sixth, seventh, and eighth beta-sheets; option (ii) being preferred.
According to one embodiment, an isolated polypeptide in accordance with the invention may consist of the protease domain of the trypsin-like serine protease auto-transporter of N. meningitidis which is IgA1P, App or AusI, and preferably is IgA1P.
According to one embodiment, an isolated polypeptide may consist of a protease domain and all or part of an α-peptide domain, and preferably of a passenger domain (protease domain and ox-peptide domain), of the trypsin-like serine protease auto-transporter of N. meningitidis which is IgA1P, App or AusI.
According to one embodiment, an isolated polypeptide in accordance with the invention may consist of the protease domain, the α-peptide domain (together the passenger domain) and a part of a β-domain, preferably the two first β-sheets of the 1-domain, of the trypsin-like serine protease auto-transporter IgA1P.
According to one embodiment, an isolated polypeptide in accordance with the invention may consist of the protease domain, the α-peptide domain (together the passenger domain) and a part of α-domain, preferably the two first β-sheets of the β-domain, of the trypsin-like serine protease auto-transporter App.
According to one embodiment, an isolated polypeptide in accordance with the invention may consist of the protease domain, the α-peptide domain (together the passenger domain) and a part of a β-domain, preferably the two first β-sheets of the β-domain, of the trypsin-like serine protease auto-transporter AusI.
According to one embodiment, an isolated polypeptide in accordance with the invention may have an amino acid sequence having at least 90% identity with the amino acid sequence of the IgA1P of N. meningitidis MC58 shown in SEQ ID NO: 1 starting from position 27, 28, 29, 30, 31 or 32 and ending at a position selected from position 1008 to position 1505.
According to one embodiment, an isolated polypeptide in accordance with the invention may have an amino acid sequence having at least 90% identity with the amino acid sequence of the IgA1P of N. meningitidis MC58 shown in SEQ ID NO: 1 starting from position 27, 28, 29, 30, 31 or 32 and ending at position 1002, 1003, 1004, 1005, 1006, 1007 or 1008.
According to another embodiment, an isolated polypeptide in accordance with the invention may have an amino acid sequence having at least 90% identity with the amino acid sequence of the IgA1P of N. meningitidis MC58 shown in SEQ ID NO 1 starting from position 27, 28, 29, 30, 31 or 32 and ending at a position selected from position 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507, 1508, 1509 and 1510.
According to another embodiment, an isolated polypeptide in accordance with the invention may have an amino acid sequence having at least 90% identity with the amino acid sequence of the IgA1P of N. meningitidis MC58 shown in SEQ ID NO: 1 starting from position 27, 28, 29, 30, 31 or 32 and ending at position 1580, 1581, 1582, 1583, 1584, 1585, 1586, 1587 or 1588.
As a matter of example, an IgA1P fragment may essentially consist of the protease domain of MC58 IgA1P comprising (having) the amino acid sequence shown in SEQ ID NO: 1
(i) starting with the amino acid in position 27 or 28 and ending with amino acid in position 1005; or
(ii) starting with the amino acid in position 27, 28, 29, 30, 31 or 32 and ending with the amino acid in position 1002, 1003, 1004, 1005, 1006, 1007, or 1008; or
(iii) starting with an amino acid in any one of positions 27 or 28 to 78, preferably 27 or 28 to 58, more preferably 27 or 28 to 38, and ending with an amino acid in any one of positions 990 to 1015, preferably 1000 to 1010, e.g., in position 1005.
Another example of an IgA1P fragment may essentially consist of the protease domain of a variant of MC58 IgA1P, said fragment being described by reference to the MC58 amino acid sequence reported in SEQ ID NO: 1 as comprising (having) an amino acid sequence
(i) starting with the amino acid corresponding to the amino acid in position 27 or 28 and ending with the amino acid corresponding to the amino acid in position 1005;
(ii) starting with the amino acid corresponding to the amino acid in position 27, 28, 29, 30, 31 or 32 and ending with the amino acid corresponding to the amino acid in position 1002, 1003, 1004, 1005, 1006, 1007, or 1008; or
(iii) starting with the amino acid corresponding to the amino acid in any one of the positions 28 to 78, preferably 28 to 58, more preferably 28 to 38, and ending with the amino acid corresponding to the amino acid in any one of positions 990 to 1015, preferably 1000 to 1010, e.g., in position 1005, in SEQ ID NO: 1.
Still as a matter of example, an IgA1P fragment may also be an MC58 IgA1P fragment essentially consisting of the IgA1P protease domain and all or part of the α-peptide domain and e.g., comprising (having) the amino acid sequence shown in SEQ ID NO: 1
(i) starting with the amino acid in position 27 or 28 and ending with the amino acid in any one of positions 1005 to 1510, preferably 1500 to 1510, more preferably in position 1505;
or (ii) starting with the amino acid in any one of positions 27 or 28 to 78, preferably 27 or 28 to 58, more preferably 27 or 28 to 38, and ending with the amino acid in any one of positions 1005 to 1510, preferably 1300 or 1500 to 1510, e.g. in position 1505.
Another example of an IgA1P fragment may be a fragment of a variant of MC58 IgA1P, said fragment essentially consisting of the IgA1P protease domain and all or part of the α-peptide domain and being described by reference to the MC58 amino acid sequence reported in SEQ ID NO: 1, which comprises (has) an amino acid sequence
(i) starting with the amino acid corresponding to the amino acid in position 27 or 28 and ending with the amino acid corresponding to the amino acid in any one of positions 1005 to 1510, preferably 1500 to 1510, more preferably in position 1505; or
(ii) starting with the amino acid corresponding to the amino acid in any one of positions 27 or 28 to 78, preferably 27 or 28 to 58, more preferably 27 or 28 to 38, and ending with the amino acid corresponding to the amino acid in any one of positions 1005 to 1510, preferably 1300 or 1500 to 1510, e.g., in position 1505, in SEQ ID NO: 1.
Still as a matter of example, an IgA1P fragment may also be the MC58 IgA1P fragment essentially consisting of the IgA1P protease domain, the α-peptide domain and part of the beta-domain e.g. comprising at least one and no more than eleven beta-sheets and e.g., comprising (having) the amino acid sequence shown in SEQ ID NO: 1
(i) starting with the amino acid in position 27 or 28 and ending with the amino acid in position 1584; or
(ii) starting with the amino acid in any one of positions 27 or 28 to 78, preferably 27 or 28 to 58, more preferably 27 or 28 to 38, and ending with the amino acid in any one of positions 1505 to 1600, preferably 1550 to 1590, e.g. in position 1584.
Another example of an IgA1P fragment may be a fragment of a variant of MC58 IgA1P, said fragment essentially consisting of the IgA1P protease domain, the α-peptide domain and part of the beta-domain e.g. comprising at least one and no more than eleven beta-sheets and being described by reference to the MC58 amino acid sequence reported in SEQ ID NO: 1, which comprises (has) an amino acid sequence
(i) starting with the amino acid corresponding to the amino acid in position 27 or 28 and ending with the amino acid corresponding to the amino acid in position 1584;
or (ii) starting with the amino acid corresponding to the amino acid in any one of positions 27 or 28 to 78, preferably 27 or 28 to 58, more preferably 27 or 28 to 38, and ending with the amino acid corresponding to the amino acid in any one of positions 1505 to 1600, preferably 1550 to 1590, e.g., in position 1584, in SEQ ID NO: 1.
According to a preferred embodiment, an isolated polypeptide in accordance with the invention may have an amino acid sequence having at least 90% identity with, and preferably may consist in, the amino acid sequence of the IgA1P of N. meningitidis MC58 shown in SEQ ID NO: 1 starting from position 26, 27, 28, 29 or 30, an preferably 28, and ending at position 1582, 1583, 1584, 1585 or 1586, and preferably 1584.
According to another preferred embodiment, an isolated polypeptide in accordance with the invention may have an amino acid sequence having at least 90% identity with, and preferably may consist in, the amino acid sequence of the IgA1P of N. meningitidis MC58 shown in SEQ ID NO: 1 starting from position 26, 27, 28, 29 or 30, an preferably 28, and ending at position 1003, 1004, 1005, 1006 or 1007, and preferably 1005.
The polypeptide in accordance with the invention, and in particular those preferred embodiments may further comprise a mutation in the catalytic site as described below to reduce or suppress the catalytic activity, preferably at the Serine in position 267. Preferably, the Serine may be change for a Valine.
According to one embodiment, an isolated polypeptide in accordance with the invention may have an amino acid sequence having at least 90% identity with the amino acid sequence of the App of N. meningitidis MC58 shown in SEQ ID NO: 2 starting from position 40, 41, 42, 43, 44, 45 or 46 and ending at a position selected from positions 1057 to position 1204.
According to one embodiment, an isolated polypeptide in accordance with the invention may have an amino acid sequence having at least 90% identity with the amino acid sequence of the App of N. meningitidis MC58 shown in SEQ ID NO: 2 starting from position 40, 41, 42, 43, 44, 45 or 46 and ending at position 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059 or 1060.
According to one embodiment, an isolated polypeptide in accordance with the invention may have an amino acid sequence having at least 90% identity with the amino acid sequence of the App of N. meningitidis MC58 shown in SEQ ID NO: 2 starting from position 40, 41, 42, 43, 44, 45 or 46 and ending at a position between 1170 and 1204 inclusive
According to one embodiment, an isolated polypeptide in accordance with the invention may have an amino acid sequence having at least 90% identity with the amino acid sequence of the App of N. meningitidis MC58 shown in SEQ ID NO: 2 starting from position 40, 41, 42, 43, 44, 45 or 46 and ending at position 1220, 1220, 1221, 1223, 1224, 1225, 1226 or 1227.
As a matter of example, an App fragment may essentially consist of the protease domain of:
(i) MC58 App comprising (having) the amino acid sequence shown in SEQ ID NO: 2 starting with the amino acid in position 40, 41, 42, 43, 44, 45 or 46 and ending with amino acid in position 1052, 1053, 1055, 1056, 1057, 1058, 1059 or 1060; or
(ii) a variant of MC58 App, said fragment being described by reference to the MC58 amino acid sequence reported in SEQ ID NO: 2 as comprising (having) an amino acid sequence (i) starting with an amino acid corresponding to the amino acid in position 40, 41, 42, 43, 44, 45 or 46 and ending with the amino acid corresponding to the amino acid in position 1052, 1053, 1055, 1056, 1057, 1058, 1059 or 1060, in SEQ ID NO: 2.
Still as a matter of example, an App fragment may also be the MC58 App fragment essentially consisting of the App protease domain and all or part of the α-peptide domain and e.g., comprising (having) the amino acid sequence shown in SEQ ID NO: 2
(i) starting with the amino acid in position 42 or 43 and ending with the amino acid in position 1175 or 1187; or
(ii) starting with the amino acid in any one of positions 42 or 43 to 92, preferably 42 or 43 to 72, more preferably 42 or 43 to 52, and ending with the amino acid in any one of positions 1060 or 1160 to 1210, preferably 1170 to 1200, e.g., in position 1175 or 1187.
Another example of an App fragment may be a fragment of a variant of MC58 App, said fragment essentially consisting of the App protease domain and all or part of the α-peptide domain and being described by reference to the MC58 amino acid sequence reported in SEQ ID NO: 2, which comprises (has) an amino acid sequence
(i) starting with the amino acid corresponding to the amino acid in position 42 or 43 and ending with the amino acid corresponding to the amino acid in position 1175 or 1187; or
(ii) starting with the amino acid corresponding to the amino acid in any one of the positions 42 or 43 to 92, preferably 42 or 43 to 72, more preferably 42 or 43 to 52, and ending with the amino acid corresponding to the amino acid in any one of positions 1060 or 1160 to 1210, preferably 1170 to 1200, e.g., in position 1175 or 1187, in SEQ ID NO: 2.
Still as a matter of example, an App fragment may also be the MC58 App fragment essentially consisting of the App protease domain, the α-peptide domain and part of the beta-domain and e.g., comprising (having) the amino acid sequence shown in SEQ ID NO: 2
(i) starting with the amino acid in position 42 or 43 and ending with the amino acid in position 1224; or
(ii) starting with the amino acid in any one of positions 42 or 43 to 92, preferably 42 or 43 to 72, more preferably 42 or 43 to 52, and ending with the amino acid in any one of positions 1175 to 1240, preferably 1210 to 1230, e.g., in position 1224.
Another example of an App may be a fragment of a variant of MC58 App, said fragment essentially consisting of the App protease domain, the α-peptide domain and part of the beta-domain and being described by reference to the MC58 amino acid sequence reported in SEQ ID NO: 2, which comprises (has) an amino acid sequence
(i) starting with the amino acid corresponding to the amino acid in position 42 or 43 and ending with the amino acid corresponding to the amino acid in position 1224; or
(ii) starting with the amino acid corresponding to the amino acid in any one of the positions 42 or 43 to 92, preferably 42 or 43 to 72, more preferably 42 or 43 to 52, and ending with the amino acid corresponding to the amino acid in any one of positions 1175 to 1240, preferably 1210 to 1230, e.g., in position 1224, in SEQ ID NO: 2.
According to a preferred embodiment, an isolated polypeptide in accordance with the invention may have an amino acid sequence having at least 90% identity with, and preferably may consist in, the amino acid sequence of the App of N. meningitidis MC58 shown in SEQ ID NO: 2 starting from position 41, 42, 43, 44 or 45, and preferably 43, and ending at position 1122, 1123, 1224, 1225 or 1126, and preferably 1224.
The polypeptide in accordance with the invention, and in particular those preferred embodiments may further comprise a mutation in the catalytic site as described below to reduce or suppress the catalytic activity, preferably at the Serine in position 267. Preferably, the Serine may be change for a Valine.
According to one embodiment, an isolated polypeptide in accordance with the invention may have an amino acid sequence having at least 90% identity with the amino acid sequence of the AusI of N. meningitidis MC58 shown in SEQ ID NO: 3 starting from position 26, 27, 28, 29, 30 or 31 and ending at a position selected from position 969 to position 1177.
According to one embodiment, an isolated polypeptide in accordance with the invention may have an amino acid sequence having at least 90% identity with the amino acid sequence of the AusI of N. meningitidis MC58 shown in SEQ ID NO: 3 starting from position 26, 27, 28, 29, 30 or 31 and ending at position 966, 967, 968, 969, 970, 971 or 972.
According to one embodiment, an isolated polypeptide in accordance with the invention may have an amino acid sequence having at least 90% identity with the amino acid sequence of the AusI of N. meningitidis MC58 shown in SEQ ID NO: 3 starting from position 26, 27, 28, 29, 30 or 31 and ending at a position between 1131 and 1177 inclusive.
According to one embodiment, an isolated polypeptide in accordance with the invention may have an amino acid sequence having at least 90% identity with the amino acid sequence of the AusI of N. meningitidis MC58 shown in SEQ ID NO: 3 starting from position 26, 27, 28, 29, 30 or 31 and ending at position 1194, 1195, 1196, 1197, 1198, 1199, 1200, 1201 or 1202.
As a matter of example, an AusI fragment may essentially consist of the protease domain of:
(i) MC58 AusI comprising (having) the amino acid sequence shown in SEQ ID NO: 3 starting with the amino acid in position 26, 27, 28, 29, 30 or 31 and ending with amino acid in position 965, 966, 967, 968, 969, 970, 971 or 972; or
(ii) a variant of MC58 AusI, said fragment being described by reference to the MC58 amino acid sequence reported in SEQ ID NO: 3 as comprising (having) an amino acid sequence starting with an amino acid corresponding to the amino acid in position 26, 27, 28, 29, 30 or 31 and ending with the amino acid corresponding to the amino acid in position 965, 966, 967, 968, 969, 970, 971 or 972.
Still as a matter of example, an AusI fragment may be the MC58 AusI fragment essentially consisting of the AusI protease domain and all or part of the α-peptide domain and e.g., comprising (having) the amino acid sequence shown in SEQ ID NO: 3
(i) starting with the amino acid in position 26 or 27 and ending with the amino acid in any one of the positions 1130 to 1180, e.g., in position 1161; or
(ii) starting with the amino acid in any one of positions 26 or 27 to 76, preferably 26 or 27 to 56, more preferably 26 or 27 to 36, and ending with the amino acid in any one of positions 972 or 1130 to 1180, e.g., in position 1161.
Another example of an AusI fragment may be a fragment of a variant of MC58 AusI, said fragment essentially consisting of the AusI protease domain and all or part of the α-peptide domain and being described by reference to the MC58 amino acid sequence reported in SEQ ID NO: 3, which comprises (has) an amino acid sequence
(i) starting with the amino acid corresponding to the amino acid in position 26 or 27 and ending with the amino acid corresponding to the amino acid in any one of the positions 1130 to 1180, e.g., in position 1161; or
(ii) starting with the amino acid corresponding to the amino acid in any one of the positions 26 or 27 to 76, preferably 26 or 27 to 56, more preferably 26 or 27 to 36, and ending with the amino acid corresponding to the amino acid in any one of positions 972 or 1130 to 1180, e.g., in position 1161, in SEQ ID NO: 3
Still as a matter of example, an AusI fragment may be the MC58 AusI fragment essentially consisting of the AusI protease domain, the α-peptide domain and part of the beta domain and e.g., comprising (having) the amino acid sequence shown in SEQ ID NO: 3
(i) starting with the amino acid in position 26 or 27 and ending with the amino acid in any one of the positions 1198; or
(ii) starting with the amino acid in any one of positions 26 or 27 to 76, preferably 26 or 27 to 56, more preferably 26 or 27 to 36, and ending with the amino acid in any one of positions 1130 or 1180 to 1210, preferably 1190 to 1200, e.g., in position 1198.
Another example of an AusI fragment may be a fragment of a variant of MC58 AusI, said fragment essentially consisting of the AusI protease domain, the α-peptide domain and part of the beta-domain and being described by reference to the MC58 amino acid sequence reported in SEQ ID NO: 3, which comprises (has) an amino acid sequence
(i) starting with the amino acid corresponding to the amino acid in position 26 or 27 and ending with the amino acid corresponding to the amino acid in any one of the positions 1130 to 1180; or
(ii) starting with the amino acid corresponding to the amino acid in any one of the positions 26 or 27 to 76, preferably 26 or 27 to 56, more preferably 26 or 27 to 36, and ending with the amino acid corresponding to the amino acid in any one positions 1130 or 1180 to 1210, preferably 1190 to 1200, e.g., in position 1198, in SEQ ID NO: 3.
According to a preferred embodiment, an isolated polypeptide in accordance with the invention may have an amino acid sequence having at least 90% identity with, and preferably may consist in, the amino acid sequence of the AusI of N. meningitidis MC58 shown in SEQ ID NO: 3 starting from position 24, 25, 26, 27 or 28, and preferably 26, and ending at position 1196, 1197, 1198, 1199 or 1200, and preferably 1198.
The polypeptide in accordance with the invention, and in particular those preferred embodiments may further comprise a mutation in the catalytic site as described below to reduce or suppress the catalytic activity, preferably at the Serine in position 241. Preferably, the Serine may be change for a Valine.
As already mentioned above, in some embodiments, the full-length mature trypsin-like serine protease auto-transporter i.a., IgA1P, App or AusI, or a fragment thereof i.a., or a construct of a first fragment fused to a second fragment, such as described above, may be mutated so that it lacks or has reduced trypsin-like serine protease activity and/or does not contain any cleavage site susceptible/able to be cleaved by a trypsin-like serine protease. As a result of the mutation, the auto-transporter remains in a precursor state, the N-terminal protease sub-domain not being cleaved from the rest of the molecule. For example, protease activity may be reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99 or 100% compared to the wild type sequence. Protease activity may be assayed by, for example, the methods described in Vitovski et al., (1999) FASEB J. 13: 331.
Preferably, the full-length mature trypsin-like serine protease auto-transporter or a fragment thereof i.a., such as described above lacks trypsin-like serine protease activity. As already mentioned above, the catalytic triad of the serine protease auto-transporters responsible for the protease activity includes a Serine residue. In order to reduce or abolish the serine protease activity, any of the amino acids present in the catalytic triad (located in the protease sub-domain) may be mutated, advantageously by amino acid substitution. In a particular embodiment, one way to achieve that goal may be to substitute the Serine residue in the catalytic triad by any other amino acid, advantageously by Glycine, Threonine, Alanine, Leucine, Isoleucine or Valine, this latter amino acid being preferred.
Examples of useful mutated full-length mature IgA1P, App or AusI proteins include in particular any one of the MC58 full-length mature IgA1P, App and AusI proteins or variants thereof as described above, each being mutated so that it lacks or has reduced trypsin-like serine protease activity and/or does not contain any cleavage site susceptible/able to be cleaved by a trypsin-like serine protease.
Examples of useful mutated IgA1P, App or AusI fragments include in particular any one of the MC58 IgA1P, App or AusI fragments or variants thereof as described above, each being mutated so that it lacks or has reduced trypsin-like serine protease activity and/or does not contain any cleavage site susceptible/able to be cleaved by a trypsin-like serine protease.
As already mentioned above, the catalytic triad of IgA1P from N. meningitidis strain MC58 of SEQ ID NO: 1 is generally considered to be 101H 150D 267S. The catalytic triad of App from N. meningitidis strain MC58 of SEQ ID NO: 2 is generally considered to be 115H 158D 267S. The catalytic triad of AusI from N. meningitidis strain MC58 of SEQ ID NO: 3 is generally considered to be 100H 135D 241S. The catalytic residues of proteins from other N. meningitidis strains may be determined by reference to the corresponding MC58 amino acid sequences as described herein, for example by reference to SEQ ID NO: 1 in the case of IgA1P.
The catalytic triad of MC58 IgA1P is composed of His101, Asp150 and Ser267 in SEQ ID NO: 1. Accordingly, the catalytic triad of MC58 IgA1P variants is composed of amino acids corresponding to His101, Asp150 and Ser267 in the amino acid sequence of SEQ ID NO: 1; and accordingly, the mutation occurs at the position corresponding to His101, Asp150 and Ser267 of the amino acid sequence of SEQ ID NO: 1. While any of the amino acids being or corresponding to His101, Asp150 and Ser267 of the amino acid sequence of SEQ ID NO: 1 may be mutated, e.g. by substitution, it is preferred to substitute the serine residue in the catalytic triad by any other amino acid, advantageously by e.g. Glycine, Threonine, Alanine, Leucine, Isoleucine and Valine, this latter amino acid being preferred.
A useful mutation as described above may be introduced in any of the IgAIP polypeptides (IgAIP or fragments) thereof i.a., as described above. In particular, the mutation that may be introduced in MC58 IgA1P polypeptide sequences is S267V. In a similar manner, the mutation that may be introduced in MC58 variant polypeptide sequences is the substitution of the Serine residue in the catalytic triad with Valine.
As a matter of non-limiting illustration, particular examples include:
In some embodiments, a fragment polypeptide comprises or consists of an IgAIP fragment which essentially consists of:
The catalytic triad of MC58 App is composed of His115, Asp158 and Ser267 in SEQ ID NO: 2. Accordingly, the catalytic triad of MC58 App variants is composed of amino acids corresponding to His15, Asp158 and Ser267 in the amino acid sequence of SEQ ID NO: 2; and accordingly, the mutation occurs at the position corresponding to His115, Asp158 and Ser267 of the amino acid sequence of SEQ ID NO: 2. While any of the amino acids being or corresponding to His115, Asp158 and Ser267 of the amino acid sequence of SEQ ID NO: 2 may be mutated e.g. by substitution, it is preferred to substitute the serine residue in the catalytic triad by any other amino acid, advantageously by e.g. Glycine, Threonine, Alanine, Leucine, Isoleucine and Valine, this latter amino acid being preferred.
A useful mutation as described above may be introduced in any of the App proteins or fragments thereof as described above. In particular, the mutation that may be introduced in MC58 App polypeptide sequences is S267V. In a similar manner, the mutation that may be introduced in MC58 variant App polypeptide sequences is the substitution of the Serine residue in the catalytic triad with Valine.
As a matter of non-limiting illustration, particular examples include:
The catalytic triad of MC58 AusI is composed of His100, Asp135 and Ser241 in SEQ ID NO: 3. Accordingly, the catalytic triad of MC58 AusI variants is composed of amino acids corresponding to His100, Asp135 and Ser241 in the amino acid sequence of SEQ ID NO: 3; and accordingly, the mutation occurs at the position corresponding to His100, Asp135 and Ser241 of the amino acid sequence of SEQ ID NO: 3. While any of the amino acids being or corresponding to His100, Asp135 and Ser241 of the amino acid sequence of SEQ ID NO: 3 may be mutated e.g. by substitution, it is preferred to substitute the serine residue in the catalytic triad by any other amino acid, advantageously by e.g. Glycine, Threonine, Alanine, Leucine, Isoleucine and Valine, this latter amino acid being preferred.
A useful mutation as described above may be introduced in any of the AusI proteins or fragments thereof as described above. In particular, the mutation that may be introduced in MC58 AusI polypeptide sequences is S241V. In a similar manner, the mutation that may be introduced in MC58 variant AusI polypeptide sequences is the substitution of the Serine residue in the catalytic triad with Valine.
As a matter of non-limiting illustration, particular examples include:
In other words, the IgA1P antigen may be an IgA1P polypeptide comprising or consisting of:
A full-length mature IgA1P protein (i.e., a full-length mature MC58 IgA1P protein or a variant thereof), being optionally mutated as described above, which comprises (has) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the MC58 amino acid sequence reported in SEQ ID NO: 1, starting with the amino acid in position 27, 28, 29, 30, 31, or 32 and ending with the amino acid in position 1815;
An IgA1P fragment (i.e., a MC58 IgA1P fragment or a variant thereof) being optionally mutated as described above, which comprises (has) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the MC58 amino acid sequence reported in SEQ ID NO: 1, starting with the amino acid in position 27, 28, 29, 30, 31, or 32 and ending with the amino acid in position 1002, 1003, 1004, 1005, 1006, 1007 or 1008;
An IgA1P fragment (i.e., a MC58 IgA1P fragment or a variant thereof) being optionally mutated as described above, which comprises (has) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the MC58 amino acid sequence reported in SEQ ID NO: 1, starting with the amino acid in position 27, 28, 29, 30, 31, or 32 and ending with the amino acid at a position selected from positions 1008 to 1505 inclusive;
An IgAIP fragment (i.e., a MC58 IgAIP fragment or a variant thereof) being optionally mutated as described above, which comprises (has) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the MC58 amino acid sequence reported in SEQ ID NO: 1, starting with the amino acid in position 27, 28, 29, 30, 31, or 32 and ending with the amino acid in position 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507, 1508, 1509 or 1510;
An IgA1P fragment (i.e., a MC58 IgA1P fragment or a variant thereof) being optionally mutated as described above, which comprises (has) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the MC58 amino acid sequence reported in SEQ ID NO: 1, starting with the amino acid in position 27, 28, 29, 30, 31, or 32 and ending with the amino acid in any one of positions 1506 to 1700, preferably 1506 to 1600, more preferably 1550 to 1600; OR
An IgA1P fragment (i.e., a MC58 IgA1P fragment or a variant thereof) being optionally mutated as described above, which comprises (has) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the MC58 amino acid sequence reported in SEQ ID NO: 1, starting with the amino acid in position 27, 28, 29, 30, 31, or 32 and ending with amino acid in position 1580, 1581, 1582, 1583, 1584, 1585, 1586, 1587, or 1588.
In other words, the App antigen may be an App polypeptide comprising or consisting of:
A full-length mature App protein (i.e., a full-length mature MC58 App protein or a variant thereof) being optionally mutated as described above, which comprises (has) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the MC58 amino acid sequence reported in SEQ ID NO: 2, starting with the amino acid in position 40, 41, 42, 43, 44, 45 or 46 and ending with the amino acid in position 1457;
An App fragment (i.e., a MC58 App fragment or a variant thereof) being optionally mutated as described above, which comprises (has) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the MC58 amino acid sequence reported in SEQ ID NO: 2, starting with the amino acid in position 40, 41, 42, 43, 44, 45 or 46 and ending with the amino acid in position 1052, 1053, 1054, 1055, 1056, 1057, 1058, 105 or 1060;
An App fragment (i.e., a MC58 App fragment or a variant thereof) being optionally mutated as described above, which comprises (has) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the MC58 amino acid sequence reported in SEQ ID NO: 2, starting with the amino acid in position 40, 41, 42, 43, 44, 45 or 46 and ending with the amino acid at a position selected from positions 1057 to 1204 inclusive;
An App fragment (i.e., a MC58 App fragment or a variant thereof) being optionally mutated as described above, which comprises (has) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the MC58 amino acid sequence reported in SEQ ID NO: 2, starting with the amino acid in position 40, 41, 42, 43, 44, 45 or 46 and ending with the amino acid at a position between 1170 and 1204 inclusive;
An App fragment (i.e., a MC58 App fragment or a variant thereof) being optionally mutated as described above, which comprises (has) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the MC58 amino acid sequence reported in SEQ ID NO: 2, starting with the amino acid in position 40, 41, 42, 43, 44, 45 or 46 and ending with the amino acid in any one of positions 1205 to 1400, preferably 1205 to 1300, more preferably 1220 to 1260; OR
An App fragment (i.e., a MC58 App fragment or a variant thereof) being optionally mutated as described above, which comprises (has) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the MC58 amino acid sequence reported in SEQ ID NO: 2, starting with the amino acid in position 40, 41, 42, 43, 44, 45 or 46 and ending with amino acid in position 1220, 1221, 1222, 1223, 1224, 1225, 1226, or 1227.
In other words, the AusI antigen may be an AusI polypeptide comprising or consisting of:
A full-length mature MC58 AusI protein (i.e., a full-length mature MC58 AusI protein or a variant thereof) being optionally mutated as described above, which comprises (has) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the MC58 amino acid sequence reported in SEQ ID NO: 3, starting with the amino acid in position 26, 27, 28, 29, 30 or 31 and ending with amino acid in position 1431;
An AusI fragment (i.e., a MC58 AusI fragment or a variant thereof) being optionally mutated as described above, which comprises (has) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the MC58 amino acid sequence reported in SEQ ID NO: 3, starting with the amino acid in position 26, 27, 28, 29, 30 or 31 and ending with amino acid in position 1052, 966, 967, 968, 969, 970, 971 or 972;
An AusI fragment (i.e., a MC58 AusI fragment or a variant thereof) being optionally mutated as described above, which comprises (has) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the MC58 amino acid sequence reported in SEQ ID NO: 3, starting with the amino acid in position 26, 27, 28, 29, 30 or 31 and ending with amino acid at a position selected from positions 969 to 1177;
An AusI fragment (i.e., a MC58 AusI fragment or a variant thereof) being optionally mutated as described above, which comprises (has) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the MC58 amino acid sequence reported in SEQ ID NO: 3, starting with the amino acid in position 26, 27, 28, 29, 30 or 31 and ending with amino acid at a position between 1131 and 1177 inclusive;
An AusI fragment (i.e., a MC58 AusI fragment or a variant thereof) being optionally mutated as described above, which comprises (has) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the MC58 amino acid sequence reported in SEQ ID NO: 3, starting with the amino acid in position 26, 27, 28, 29, 30 or 31 and ending with amino acid in any one of positions 1178 to 1300, preferably 1178 to 1250, more preferably 1180 to 1220; OR
An AusI fragment (i.e., a MC58 AusI fragment or a variant thereof) being optionally mutated as described above, which comprises (has) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the MC58 amino acid sequence reported in SEQ ID NO: 3, starting with the amino acid in position 26, 27, 28, 29, 30 or 31 and ending with amino acid in position 1220, 1195, 1196, 1197, 1198, 1199, 1200, or 1201.
For use in the composition of the present invention, the trypsin-like serine protease auto-transporter antigen may also be i.a., a fusion polypeptide comprising or consisting of:
a first fragment fused to a second fragment:
(1) said first fragment consisting of:
(2) said second fragment consisting of:
wherein the first and second trypsin-like serine protease auto-transporters are different; and
wherein the C-terminus of the first fragment is fused to the N-terminus of the second fragment,
wherein said polypeptide does not comprise the said full-length mature trypsin-like serine protease auto-transporter of N. meningitidis.
A trypsin-like serine protease auto-transporter of N. meningitidis suitable for the invention may be IgA1P, App or AusI.
Advantageously, in the fusion polypeptide, the first fragment may essentially consist of (be) the protease domain or protease sub-domain, and preferably is a protease sub-domain, of the first trypsin-like serine protease auto-transporter. In an advantageous and independent manner, the second fragment may essentially consist of (be) the α-peptide domain, and optionally part of the β-domain, of the second trypsin-like serine protease auto-transporter.
In some embodiments, in the fusion polypeptide, (i) the first fragment essentially consists of (is) the protease domain or protease sub-domain of the first trypsin-like serine protease auto-transporter and (ii) the second fragment essentially consists of the α-peptide domain and part of the β-domain of the second trypsin-like serine protease auto-transporter.
According to one embodiment, a polypeptide of the invention may comprise or consist of a first fragment fused to a second fragment wherein said first fragment consists of a protease sub-domain of said first trypsin-like serine protease auto-transporter and said second fragment consist of an α-peptide domain, optionally with a part of a 1-domain, of said second trypsin-like serine protease auto-transporter.
According to a preferred embodiment, a polypeptide of the invention may comprise or consist of a first fragment fused to a second fragment wherein said first fragment consists of a protease sub-domain of said first trypsin-like serine protease auto-transporter which is IgAIP, and said second fragment consist of an α-peptide domain, optionally with a part of a β-domain, of said second trypsin-like serine protease auto-transporter which is App or AusI.
In a particular embodiment, part of the β-domain of the second fragment useful in the fusion polypeptide comprises at least one and no more than eleven β-sheets; preferably from two to eight β-sheets, more preferably from two to four β-sheets, most preferably two β-sheets. In practice, the C-terminus of the α-peptide domain is fused to the N-terminus of the β-domain which comprises from N-ter to C-ter, at least the first beta-sheet; (ii) first and second beta-sheets; (iii) first, second and third beta-sheets; (iv) first, second, third and fourth beta-sheets; (v) first, second, third, fourth and fifth beta-sheets; (vi) first, second, third, fourth, fifth and sixth beta-sheets; (viii) first, second, third, fourth, fifth, sixth and seventh beta-sheets; or (viii) first, second, third, fourth, fifth, sixth, seventh, and eighth beta-sheets; option (ii) being preferred.
For use in the fusion polypeptide, the first fragment may be mutated in the catalytic triad as described above with respect to the full-length mature trypsin-like serine protease auto-transporter of N. meningitidis, such as IgA1P, App or AusI, or the described fragments thereof. Indeed, the catalytic triad is located in the protease sub-domain. It may also be not mutated, especially when this first fragment essentially consists of the protease sub-domain that is the protease domain lacking the C-terminus amino acids containing the auto-cleavage site.
The first and second fragments may independently be a trypsin-like serine protease fragment of an MC58 strain or a variant thereof.
According to one embodiment, an isolated peptide in accordance with the invention comprising or consisting of a first fragment fused to a second fragment may comprise or consist of a first fragment having at least 90% identity with an amino acid sequence of the IgA1P of N. meningitidis MC58 shown in SEQ ID NO: 1 starting from position 27, 28, 29, 30, 31 or 32 and ending at position 1002, 1003, 1004, 1005, 1006, 1007 or 1008;
and may comprise or consist of a second fragment having at least 90% identity with an amino acid sequence of
(i) App of N. meningitidis MC58 shown in SEQ ID NO: 2 starting from position 1057, 1058, 1059, 1060, 1061 or 1062 and ending at a position between 1170 and 1204 inclusive; or
(ii) AusI of N. meningitidis MC58 shown in SEQ ID NO: 3 starting from position 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979 or 980 and ending at a position between 1131-1177 inclusive.
According to another embodiment, an isolated peptide in accordance with the invention comprising or consisting of a first fragment fused to a second fragment may comprise or consist of a first fragment having at least 90% identity with an amino acid sequence of the IgA1P of N. meningitidis MC58 shown in SEQ ID NO: 1 starting from position 27, 28, 29, 30, 31 or 32 and ending at position 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 9701, 971, 972, 973, 974, 975, 976, 977, 978, 979 or 980;
and may comprise or consist of a second fragment having at least 90% identity with an amino acid sequence of
(i) App of N. meningitidis MC58 shown in SEQ ID NO: 2 starting from position 1057, 1058, 1059, 1060, 1061 or 1062 and ending at a position between 1170 and 1204 inclusive; or
(ii) AusI of N. meningitidis MC58 shown in SEQ ID NO: 3 starting from position 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979 or 980 and ending at a position between 1131 and 1177 inclusive.
According to another embodiment, an isolated peptide in accordance with the invention comprising or consisting of a first fragment fused to a second fragment may comprise or consist of a first fragment having at least 90% identity with an amino acid sequence of the IgA1P of N. meningitidis MC58 shown in SEQ ID NO: 1 starting from position 27, 28, 29, 30, 31 or 32 and ending at position 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 9701, 971, 972, 973, 974, 975, 976, 977, 978, 979 or 980;
and may comprise or consist of a second fragment having at least 90% identity with an amino acid sequence of
(i) App of N. meningitidis MC58 shown in SEQ ID NO: 2 starting from position 1057, 1058, 1059, 1060, 1061 or 1062 and ending at a position between 1170 and 1204 inclusive, and preferably at a position 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190 or 1191; or
(ii) App of N. meningitidis MC58 shown in SEQ ID NO: 2 starting from position 1057, 1058, 1059, 1060, 1061 or 1062 and ending at a 1220, 1221, 1222, 1223, 1224, 1125, 1226, 1227, or 1228; or
(iii) AusI of N. meningitidis MC58 shown in SEQ ID NO: 3 starting from position 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979 or 980 and ending at a position between 1131 and 1177 inclusive, and preferably at a position 1157, 1158, 1159, 1160, 1161, 1162, 1163, 1164, or 1165; or
(iv) AusI of N. meningitidis MC58 shown in SEQ ID NO: 3 starting from position 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979 or 980 and ending at a position between 1194, 1195, 1196, 1197, 1198, 1199, 1200, 1201 or 1202.
In a particular embodiment, the above constructs may further comprise a mutation in the catalytic site as previously described to reduce or suppress the catalytic activity. The mutation may in particular intervene at the Serine in position 267, which may, for instance, be replaced with a Valine.
As a matter of non-limiting illustration, the first fragment may be e.g. the MC58 IgAIP protease sub-domain and may be fused to the alpha-peptide domain of e.g. App of a variant of the MC58 strain.
In some embodiments, the first fragment in the fusion polypeptide essentially consists of the protease domain or the protease sub-domain of IgAIP, mutated or not as described above, and is fused to the second fragment which essentially consists of the α-peptide domain and optionally, part of the β-domain of App or AusI.
A particular example of these embodiments is an IgA1P-App fusion polypeptide which essentially consists of the IgA1P protease sub-domain in which the Ser residue of the catalytic triad is optionally mutated by substitution with e.g., Valine, fused to the App α-peptide domain.
As a matter of example, the fusion polypeptide comprises or consists of the protease sub-domain or protease domain of MC58 IgA1P, optionally bearing a mutation in the catalytic triad, fused to the alpha-peptide domain of MC58 App or AusI. Accordingly, such an MC58 fusion polypeptide may comprise or consist of:
Another example of a fusion polypeptide may be a fusion polypeptide which may be described by reference to the MC58 amino acid sequences reported in SEQ ID NO: 1, 2 and/or 3 as comprising or consisting of:
Specific non-limiting examples include i.a.:
In some other embodiments, the first fragment in the fusion polypeptide essentially consists of the protease domain or the protease sub-domain of App, mutated or not as described above, and is fused to the second fragment which essentially consists of the α-peptide domain and optionally of part of the 3-domain of IgA1P or AusI.
Still in some other embodiments, the first fragment in the fusion polypeptide essentially consists of the protease domain or the protease sub-domain of AusI, mutated or not as described above, and is fused to the second fragment which essentially consists of the α-peptide domain and optionally of part of the β-domain of App or IgA1P.
In some embodiments, the fusion polypeptide has first and second amino acid sequences, the C-terminus of the first sequence being fused to the N-terminus of the second sequence,
wherein the first sequence has at least 90% identity with the amino acid sequence of the IgA1P of N. meningitidis MC58 shown in SEQ ID NO: 1 starting from position 27, 28, 29, 30, 31 or 32 and ending at position 1002, 1003, 1004, 1005, 1006, 1007 or 1008; and
wherein the second sequence has at least 90% identity with the amino acid sequence of:
(i) App of N. meningitidis MC58 shown in SEQ ID NO: 2 starting from position 1057, 1058, 1059, 1060, 1061 or 1062 and ending at a position between 1170 and 1204 inclusive, preferably position 1187; or
(ii) AusI of N. meningitidis MC58 shown in SEQ ID NO: 3 starting from position 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979 or 980 and ending at a position between 1131-1177 inclusive, preferably position 1161; or
(iii) App of N. meningitidis MC58 shown in SEQ ID NO: 2 starting from position 1057, 1058, 1059, 1060, 1061 or 1062 and ending at a position 1220, 1221, 1222, 1223, 1224, 1225 and 1226, preferably position 1224; or
(iv) AusI of N. meningitidis MC58 shown in SEQ ID NO: 3 starting from position 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979 or 980 and ending at a position 1195, 1196, 1197, 1198, 1199, 1200, 1201, preferably position 1198.
According to another embodiment, an isolated peptide in accordance with the invention comprising or consisting of a first fragment fused to a second fragment may comprise or consist of a first fragment having at least 90% identity with an amino acid sequence of the App of N. meningitidis MC58 shown in SEQ ID NO: 2 starting from position 40, 41, 42, 43, 44, 45 or 46 and ending at position 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059 or 1060;
and may comprise or consist of a second fragment having at least 90% identity with an amino acid sequence of
(i) IgA1P of N. meningitidis MC58 shown in SEQ ID NO: 1 starting from position 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1110, 1111, 1112, 1113 or 1114 and ending at a position selected from position 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507, 1508, 1509 and 1510; or
(ii) AusI of N. meningitidis MC58 shown in SEQ ID NO: 3 starting from position 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979 or 980 and ending at a position between 1131-1177 inclusive.
In some embodiments, the fusion polypeptide has first and second amino acid sequences, the C-terminus of the first sequence being fused to the N-terminus of the second sequence,
wherein the first sequence has at least 90% identity with the amino acid sequence of the App of N. meningitidis MC58 shown in SEQ ID NO: 2 starting from position 40, 41, 43, 44, 45 or 46 and ending at position 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059 or 1060; and
wherein the second sequence has at least 90% identity with the amino acid sequence of
(i) IgA1P of N. meningitidis MC58 shown in SEQ ID NO: 1 starting from position 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1110, 1111, 1112, 1113 or 114 and ending at a position selected from position 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507, 1508, 1509 and 1510; or
(ii) AusI of N. meningitidis MC58 shown in SEQ ID NO: 3 starting from position 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979 or 980 and ending at a position between 1131-1177 inclusive, preferably position 1161.
(iii) IgA1P of N. meningitidis MC58 shown in SEQ ID NO: 1 starting from position 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1110, 1111, 1112, 1113 or 114 and ending at a position selected from position 1580, 1581, 1582, 1583, 1584, 1585, 1586, 1587, and 1588, preferably 1584; or
(iv) AusI of N. meningitidis MC58 shown in SEQ ID NO: 3 starting from position 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979 or 980 and ending at a position 1195, 1196, 1197, 1198, 1199, 1200, 1201, preferably position 1198.
According to another embodiment, an isolated peptide in accordance with the invention comprising or consisting of a first fragment fused to a second fragment may comprise or consist of a first fragment having at least 90% identity with an amino acid sequence of the AusI of N. meningitidis MC58 shown in SEQ ID NO: 3 starting from position 26, 27, 28, 29, 30 or 31 and ending at position 966, 967, 968, 969, 970, 971 or 972;
and may comprise or consist of a second fragment having at least 90% identity with an amino acid sequence of
(i) App of N. meningitidis MC58 shown in SEQ ID NO: 2 starting from position 1057, 1058, 1059, 1060, 1061 or 1062 and ending at a position between 1170 and 1204 inclusive; or
(ii) IgA1P of N. meningitidis MC58 shown in SEQ ID NO: 1 starting from position 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1110, 1111, 1112, 1113 or 1114 and ending at a position selected from position 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507, 1508, 1509 and 1510.
In some embodiments, the fusion polypeptide has first and second amino acid sequences, the C-terminus of the first sequence being fused to the N-terminus of the second sequence,
wherein the first sequence has at least 90% identity with the amino acid sequence of the AusI of N. meningitidis MC58 shown in SEQ ID NO: 3 starting from position 26, 27, 28, 29, 30 or 31 and ending at position 966, 967, 968, 969, 970, 971 or 972; and
wherein the second sequence has at least 90% identity with the amino acid sequence of
(i) App of N. meningitidis MC58 shown in SEQ ID NO: 2 starting from position 1057, 1058, 1059, 1060, 1061 or 1062 and ending at a position between 1170 and 1204 inclusive, preferably position 1187; or
(ii) IgA1P of N. meningitidis MC58 shown in SEQ ID NO: 1 starting from position 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1110, 1111, 1112, 1113 or 114 and ending at a position selected from position 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507, 1508, 1509 and 1510; or
(iii) App of N. meningitidis MC58 shown in SEQ ID NO: 2 starting from position 1057, 1058, 1059, 1060, 1061 or 1062 and ending at a position 1220, 1221, 1222, 1223, 1224, 1225 and 1226, preferably position 1224; or
(iv) IgA1P of N. meningitidis MC58 shown in SEQ ID NO: 1 starting from position 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1110, 1111, 1112, 1113 or 114 and ending at a position selected from position 1580, 1581, 1582, 1583, 1584, 1585, 1586, 1587, and 1588, preferably 1584.
In some embodiments, the fusion polypeptide has first and second amino acid sequences, the C-terminus of the first sequence being fused to the N-terminus of the second sequence,
wherein the first sequence has at least 90% identity with the amino acid sequence of the IgA1P of N. meningitidis MC58 shown in SEQ ID NO: 1 starting from position 27, 28, 29, 30, 31 or 32 and ending at position 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 9701, 971, 972, 973, 974, 975, 976, 977, 978, 979 or 980; and
wherein the second sequence has at least 90% identity with the amino acid sequence of
(i) App of N. meningitidis MC58 shown in SEQ ID NO: 2 starting from position 1057, 1058, 1059, 1060, 1061 or 1062 and ending at a position between 1170 and 1204 inclusive, preferably position 1187; or
(ii) AusI of N. meningitidis MC58 shown in SEQ ID NO: 3 starting from position 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979 or 980 and ending at a position between 1131 and 1177 inclusive, preferably position 1161; or
(iii) App of N. meningitidis MC58 shown in SEQ ID NO: 2 starting from position 1057, 1058, 1059, 1060, 1061 or 1062 and ending at a position 1220, 1221, 1222, 1223, 1224, 1225 and 1226, preferably position 1224; or
(iv) AusI of N. meningitidis MC58 shown in SEQ ID NO: 3 starting from position 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979 or 980 and ending at a position 1195, 1196, 1197, 1198, 1199, 1200, 1201, preferably position 1198.
In some embodiments, the fusion polypeptide has first and second amino acid sequences, the C-terminus of the first sequence being fused to the N-terminus of the second sequence,
wherein the first sequence has at least 90% identity with the amino acid sequence of the App of N. meningitidis MC58 shown in SEQ ID NO: 2 starting from position 40, 41, 43, 44, 45 or 46 and ending at position 950, 951, 952, 953, 954, 956, 957, 958, 959 or 960; and
wherein the second sequence has at least 90% identity with the amino acid sequence of
(i) IgA1P of N. meningitidis MC58 shown in SEQ ID NO: 1 starting from position 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1110, 1111, 1112, 1113 or 114 and ending at a position selected from position 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507, 1508, 1509 and 1510; or
(ii) AusI of N. meningitidis MC58 shown in SEQ ID NO: 3 starting from position 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979 or 980 and ending at a position between 1131 and 1177 inclusive, preferably position 1161.
(iii) IgA1P of N. meningitidis MC58 shown in SEQ ID NO: 1 starting from position 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1110, 1111, 1112, 1113 or 114 and ending at a position selected from position 1580, 1581, 1582, 1583, 1584, 1585, 1586, 1587, and 1588, preferably 1584; or
(iv) AusI of N. meningitidis MC58 shown in SEQ ID NO: 3 starting from position 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979 or 980 and ending at a position 1195, 1196, 1197, 1198, 1199, 1200, 1201, preferably position 1198.
In some embodiments, the fusion polypeptide has first and second amino acid sequences, the C-terminus of the first sequence being fused to the N-terminus of the second sequence,
wherein the first sequence has at least 90% identity with the amino acid sequence of the AusI of N. meningitidis MC58 shown in SEQ ID NO: 3 starting from position 26, 27, 28, 29, 30 or 31 and ending at position 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872 or 873; and
wherein the second sequence has at least 90% identity with the amino acid sequence of
(i) App of N. meningitidis MC58 shown in SEQ ID NO: 2 starting from position 1057, 1058, 1059, 1060, 1061 or 1062 and ending at position 1170-1204, preferably position 1187; or
(ii) IgA1P of N. meningitidis MC58 shown in SEQ ID NO: 1 starting from position 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1110, 1111, 1112, 1113 or 114 and ending at a position selected from position 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507, 1508, 1509 and 1510; or
(iii) App of N. meningitidis MC58 shown in SEQ ID NO: 2 starting from position 1057, 1058, 1059, 1060, 1061 or 1062 and ending at a position 1220, 1221, 1222, 1223, 1224, 1225 and 1226, preferably 1224; or
(iv) IgA1P of N. meningitidis MC58 shown in SEQ ID NO: 1 starting from position 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1110, 1111, 1112, 1113 or 114 and ending at a position selected from position 1580, 1581, 1582, 1583, 1584, 1585, 1586, 1587, and 1588, preferably 1584
‘At least 90% identity’ naturally encompasses at least 91, 92, 93, 94, 95, 96, 97, 98, 99 and/or 100% identity.
The “fusion polypeptides” in accordance with the invention, in particular as described above may further comprise a mutation in the catalytic site as previously described to reduce or suppress the catalytic activity. The mutation may in particular intervene at the Serine position, which may, for instance, be replaced with a Valine.
Subtilisin-Like Serine Protease Auto-Transporter Antigen of N. Meningitidis: NalP
WO 00/26375 relates to several virulence factors of N. meningitidis for use as a vaccinal agent. One of these factors is the outer membrane protein ORF047 of N. meningitidis strain ATCC13090 of serogroup B, also designated under the reference NMB1969 (Tettelin et al., Science (March 2000) 287: 1809). This protein has been further identified as an auto-transporter lipoprotein with subtilisin-like serine protease activity, by Turner et al., Infect. Immun. (2002) 70: 4447 and originally called AusP or AspA for “auto-transported serine protease A”. Later on, in van Ulsen et al., Mol. Microbiol. (2003) 50 (3): 1017, this protein was called NalP for “N. meningitidis auto-transporter lipoprotease”. The nalP gene has been shown to be subject to phase variation.
Although the terms “ORF047”, “NMB1969”, “AspA” and “NalP” may be used interchangeably, the term “NalP” has been selected for further use hereinafter.
Like all auto-transporters, NalP is produced as a precursor of about 112 kDa, comprising a cleavable signal peptide, a N-terminal passenger domain and a C-terminal, outer-membrane-based beta-domain, this latter comprising in sequence, a N-terminal translocator domain, an alpha-domain helix and a C-terminal beta-core composed of 12 beta-sheets. The precursor is transported to the outer membrane and the C-terminal domain remains surface-exposed while the N-ter passenger domain of about 70 kDa is processed and secreted (released into the bacterial environment) upon auto-cleavage due to the subtilisin-like serine protease activity of NalP. van Ulsen et al., Mol. Microbiol. (2003) 50 (3): 1017 has additionally shown that the lipidated form is an intermediate in the secretion process as the secreted 70 kDa form is not lipidated.
The domain structure of NalP is further described in Table 3 below by reference to NalP of strain MC58: SEQ ID NO: 4; numbering starts in amino acid position 1 with the Met initiation codon, followed by the peptide leader sequence; the mature protein being considered to start with the Cysteine residue in position 28. The N-terminal passenger domain of NalP is responsible for the subtilisin-like serine protease activity. The serine protease catalytic site is constituted by a triad [Asp/His/Ser respectively at positions 138, 157 and 426 in NalP of strain MC58]. The twelve beta-sheets in the form of a barrel constitute a hydrophilic pore filled by the alpha-domain in the form of an alpha-helix.
It shall be understood that the domain definitions by reference to amino acid positions may vary slightly as it is not always possible to precisely define a domain to the exact amino acid. For example, the domains may be defined slightly differently by different workers or they may be defined differently in different strains of N. meningitidis. Thus, said domains may be defined according to the locations given herein, or according to said locations +/−1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids N-terminal or C-terminal of said locations.
The same holds true for the location of the twelve beta-sheets of strain MC58 beta-core; however it is indicated that they are located approximately as follows:
1st β-sheet L817-E831; 2nd β-sheet E836-G853; 3rd 1-sheet T856-E871; 4th β-sheet A874-A891; 5th β-sheet G895-S913; 6th β-sheet H919-V937; 7th β-sheet D946-Q960; 8th β-sheet L977-P993; 9th β-sheet A998-D1009; 10th β-sheet T1039-G1052; 11th β-sheet W1055-S1066; 12th β-sheet Y1069-F1082.
The sequences of the nalP gene (ORF047) and NalP protein thereof were initially described in patent application WO 00/26375 (Sanofi Pasteur/INSERM). Full-length sequences were identified by analysis of the genome sequence of N. meningitidis serogroup B strain ATCC 13090, available in the database Pathoseq® of Incyte Pharmaceuticals.
nalP gene and NalP protein sequences of N. meningitidis strains MC58 (serogroup B), Z2491 (serogroup A) and FAM18 (serogroup C) are respectively disclosed in Tettelin et al., Science, March 2000, 287: 1809 or WO 00/66791; Parkhill et al., Nature (March 2000) 404: 502; and Bentley et al., PLoS Genet., 3, e23 (2007). MC58 NalP is also designated as NMB1969 and sequences thereof are available under accession number NP_003112 (version NP as submitted on Mar. 17, 2000). Z2491 NalP is also designated as NMA0478. FAM18 NalP is also designated as NMC1943.
For use in the present invention, the NalP antigen may be a NalP polypeptide comprising or essentially consisting of:
By “full-length mature NalP” is meant the NalP protein lacking the NalP signal peptide.
Accordingly, by “full-length mature NalP” is meant the full-length mature NalP comprising (having) (i) a naturally-occurring full-length mature amino acid sequence or (ii) a naturally-occurring full-length mature amino acid sequence lacking at most the first 50 N-terminus amino acids and/or at most the last 20 C-terminus amino acids or (iii) a naturally-occurring full-length mature amino acid sequence fused to the 1, 2 or 3 amino acids of the C-terminus of the signal sequence.
Advantageously, the full-length mature NalP is not lipidated. To that end, the full-length mature NalP may lack at least the cysteine residue in position 1 of the mature form (position 28, by reference to SEQ ID NO: 4).
An example of a useful full-length mature NalP protein is the full-length mature NalP protein of strain MC58. Accordingly, this MC58 NalP protein comprises (has) the amino acid sequence shown in SEQ ID NO: 4, starting with the amino acid in position 28, 29 or 30, preferably 29 or 30, and ending with the amino acid in position 1082. Other useful MC58 NalP proteins comprise (have) the amino acid sequence shown in SEQ ID NO: 4, starting with the amino acid in any one of the positions 28 to 78, preferably 28 to 58, more preferably 28 to 38, most preferably in position 28, 29 or 30, and ending with the amino acid in any one of the positions 1070 to 1082.
Other useful full-length mature NalP proteins include variants of MC58 NalP protein which may be described by reference to the MC58 amino acid sequence reported in SEQ ID NO: 4. These variants comprise (have) an amino acid sequence starting with an amino acid corresponding to the amino acid in position 28, 29 or 30, preferably 29 or 30, and ending with an amino acid corresponding to the amino acid in position 1082. Other useful variants of the MC58 NalP protein, still described by reference to the MC58 amino acid sequence reported in SEQ ID NO: 4, comprise (have) an amino acid sequence starting with an amino acid corresponding to the amino acid in any one of the positions 28 to 78, preferably 28 to 58, more preferably 28 to 38, and ending with an amino acid corresponding to the amino acid in any one of positions 1070 to 1082.
NalP is a protein with low solubility due to the presence of the beta-core and accordingly, recombinant expression and purification of full-length NalP may be difficult to achieve. This is the reason why, in a preferred embodiment, the NalP antigen may be produced as a truncated NalP, at least partially lacking the beta-core.
Accordingly, for use in the present invention, the NalP antigen may also be a NalP polypeptide comprising:
The term “fragment” of a reference sequence refers to a chain of contiguous nucleotides or amino acids that is shorter than the reference sequence.
Advantageously, the NalP polypeptide is the NalP fragment or the mutant of the NalP fragment, as described.
Although the mutated NalP for use in the composition of the invention and polypeptides of the present invention are more particularly exemplified herein by reference to the amino acid sequence of MC58 NalP protein (that naturally-occurs in N. meningitidis strain MC58), polypeptides of any naturally-occurring/allelic variant of N. meningitidis strain M982/B16B6/MC58 are also encompassed within the scope invention as well as any variant that may result from genetic engineering. By extension, the term variant is therefore applied to amino acid sequences, proteins or fragments thereof other than MC58 sequences, proteins or fragments thereof. Variations in amino acid sequence may be introduced by substitution, deletion or insertion of one or more codons into the nucleic acid sequence encoding the protein that results in a change in the amino acid sequence of the protein without substantially affecting the tri-dimensional structure and/or the biological and/or immunogenic properties. Typically, the variation may result for an amino acid substitution that may be conservative or non-conservative, preferably conservative. A conservative substitution is an amino acid substitution in which an amino acid is substituted for another amino acid with similar structural and/or chemical properties.
In what follows, variants and/or mutants are described by reference to the amino acid sequence of reference (SEQ ID NO: 4). Such a description by reference is based on the prerequisite of optimal sequence alignment in order to determine the amino acid in the variant sequence that corresponds to the amino acid defined as being in a specific position in the amino acid of reference.
The NalP passenger domain at the N-terminus of the NalP fragment may be the MC58 NalP passenger domain comprising (having) the amino acid sequence starting in position 28, 29 or 30 or in any one of position 28 to 78, preferably 28 to 58, more preferably 28 to 38, and ending in position 774 in SEQ ID NO: 4. It may also be a variant of the MC58 NalP passenger domain comprising (having) an amino acid sequence described by reference to the MC58 amino acid sequence reported in SEQ ID NO: 4, starting with an amino acid corresponding to the amino acid in position 28, 29 or 30 or in any one of the positions 28 to 78, preferably 28 to 58, more preferably 28 to 38, and ending with an amino acid corresponding to the amino acid in position 774. Preferably, an NalP passenger domain described above may start at position 29.
Advantageously, the NalP fragment comprises at the C-terminus at least one and no more than eight, six, four, or preferably two NalP beta sheets. In some embodiments, the NalP fragment comprises (i) the first beta-sheet; (ii) first and second beta-sheets; (iii) first, second and third beta-sheets; (iv) first, second, third and fourth beta-sheets; (v) first, second, third, fourth and fifth beta-sheets; (vi) first, second, third, fourth, fifth and sixth beta-sheets; (viii) first, second, third, fourth, fifth, sixth and seventh beta-sheets; or (viii) first, second, third, fourth, fifth, sixth, seventh, and eighth beta-sheets.
As a matter of example, a useful NalP fragment is the MC58 NalP fragment comprising at the C-terminus, two beta-sheets comprising (having) the amino acid sequence starting in any one of positions 815 to 820, e.g. in position 817 and ending in any one of positions 850 to 855, e.g. in position 853. Still as a matter of example, a useful NalP fragment is a variant of the MC58 NalP fragment comprising two beta-sheets comprising (having) an amino acid sequence described by reference to the MC58 amino acid sequence reported in SEQ ID NO: 4, starting with an amino acid corresponding to the amino acid in any one of the positions 815 to 820, e.g. in position 817, and ending with an amino acid corresponding to the amino acid in any one of positions 850 to 855, e.g. in position 853.
For use in the present invention, the NalP fragment advantageously comprises a C-ter truncated NalP C-terminal domain including the translocator domain, the alpha-peptide and as described above, at least one and no more than eleven NalP beta-sheets.
An example of a useful NalP fragment is a fragment of the full-length mature NalP protein of strain MC58. Accordingly, this MC58 NalP fragment comprises (has) the amino acid sequence shown in SEQ ID NO: 4, starting with the amino acid in position 28, 29 or 30 and ending with the amino acid in position 853. Other useful MC58 NalP fragments comprise (have) the amino acid sequence shown in SEQ ID NO: 4, starting with the amino acid in position 28, 29 or 30 or in any one of the positions 28 to 78, preferably 28 to 58, more preferably 28 to 38, and ending with the amino acid in any one of positions 840 to 860, preferably 850 to 855.
Other useful NalP fragments include variants of MC58 NalP fragments which may be described by reference to the MC58 amino acid sequence reported in SEQ ID NO: 4. These variants comprise (have) an amino acid sequence starting with an amino acid corresponding to the amino acid in position 28, 29 or 30 and ending with an amino acid corresponding to the amino acid in position 853. Other useful variants of the MC58 NalP fragment, still described by reference to the MC58 amino acid sequence reported in SEQ ID NO: 4, comprise (have) an amino acid sequence starting with an amino acid corresponding to the amino acid in position 28, 29 or 30 or in any one of the positions 28 to 78, preferably 28 to 58, more preferably 28 to 38, and ending with an amino acid corresponding to the amino acid in any one of positions 840 to 860, preferably 850 to 855.
As already mentioned above, in some embodiments, the full-length mature NalP protein or the NalP fragment is mutated so that it lacks or has reduced subtilisin-like serine protease activity and/or does not contain any cleavage site susceptible/able to be cleaved by a subtilisin-like serine protease i.a., NalP. As a result of the mutation, NalP remains in a precursor state, the N-terminal passenger domain not being cleaved from the C-terminal domain. For example, protease activity may be reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99 or 100% compared to the wild type sequence. Protease activity may be evaluated by assaying the ability to cleave auto-transporter proteins, for example by Western Blot as described in for example Roussel-Jazédé et al., Infect Immun. (2010) 78 (7): 3083; van Ulsen P, et al., Mol Microbiol. (November 2003) (3): 1017 and Serruto et al., PNAS February 2010 107 (8): 3770.
Preferably, the full-length mature NalP protein or the NalP fragment lacks subtilisin-like serine protease activity. In order to reduce or abolish the serine protease activity, any of the amino acids present in the catalytic triad may be mutated, advantageously by amino acid substitution. In a particular embodiment, one way to achieve that goal may be to substitute the Serine residue in the catalytic triad by any other amino acid, advantageously by Glycine, Threonine, Valine, Leucine, Isoleucine or Alanine, preferably by Valine or Alanine, and more preferably by Alanine.
The catalytic triad of MC58 NalP is composed of Asp138, His157 and Ser426 in SEQ ID NO: 4. Accordingly, the catalytic triad of variant of MC58 NalP is composed of amino acids corresponding to Asp138, His157 and Ser426 in the amino acid sequence of SEQ ID NO: 4; and accordingly, the mutation occurs at the position corresponding to Asp138, His157 or Ser426 of the amino acid sequence of SEQ ID NO: 4.
Examples of useful mutated full-length mature NalP protein include in particular any one of the MC58 NalP full-length mature NalP protein or variants thereof as described above, each being mutated so that it lacks or has reduced subtilisin-like serine protease activity and/or does not contain any cleavage site susceptible/able to be cleaved by a subtilisin-like serine protease.
Examples of useful mutated NalP fragment include in particular any one of the MC58 NalP fragments or variants thereof as described above, each being mutated so that it lacks or has reduced subtilisin-like serine protease activity and/or does not contain any cleavage site susceptible/able to be cleaved by a subtilisin-like serine protease.
According to a preferred embodiment, an isolated polypeptide according to the invention may be a polypeptide lacking protease activity, and wherein the NalP passenger domain portion of said polypeptide has an amino acid substitution at the position corresponding to Asp138, His157 or Ser426 of the amino acid sequence of SEQ ID NO:1.
According to a preferred embodiment, an isolated polypeptide according to the invention may be a polypeptide wherein the residue corresponding to Ser426 of SEQ ID NO: 1 is substituted by Ala.
According to a preferred embodiment, an isolated polypeptide according to the invention may be a NalP fragment comprising no more than two NalP beta sheets.
As a matter of non-limiting illustration, particular examples include:
In some embodiments, a NalP antigen may be a polypeptide comprising or consisting of a NalP fragment which essentially consists of the passenger domain, the translocator domain, the alpha-peptide and the first and second beta-sheets; wherein the Ser residue of the catalytic triad in the NalP passenger domain is optionally mutated by substitution.
According to a preferred embodiment, an isolated polypeptide according to the invention may be a fragment of full-length mature NalP protein of N. meningitidis MC58 or of a NalP variant having at least 95% identity with the NalP protein of N. meningitidis MC58.
In an embodiment, said NalP fragment comprises an amino acid sequence which has at least 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 7.
According to a preferred embodiment, an isolated polypeptide according to the invention may be NalP fragment comprising the amino acid sequence of SEQ ID NO: 7.
In other words, a NalP antigen may be a polypeptide comprising or being:
A full-length mature MC58 NalP protein or a variant thereof, each of the protein or the variant thereof being optionally mutated as described above, which comprises (has) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the MC58 amino acid sequence reported in SEQ ID NO: 4, starting with the amino acid in position 28 and ending with amino acid in position 1082.
A MC58 NalP fragment or a variant thereof, each of the fragment or the variant thereof being optionally mutated as described above, which comprises (has) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the MC58 amino acid sequence reported in SEQ ID NO: 4, starting with the amino acid in position 28, 29 or 30 and ending with amino acid in position 853.
TbpB of N. meningitidis
The human transferrin receptor of N. meningitidis is an outer-membrane complex composed of two subunits: the human transferrin-binding protein A (TbpA, also called Tbp1) and the human transferrin-binding protein B (TbpB also called Tbp2).
Two major human transferrin-binding protein B (TbpB) families have to date been documented in N. meningitidis, defining two isotypes within the species: strains of TbpB of isotype I are characterized by a tbpB gene of 1.8 kb, and those of isotype II are characterized by a tbpB gene of 2.1 kb. Herein after the term “isotype” is indifferently applied to the TbpB antigen and to the strain expressing the specific TbpB antigen. Strains of isotype I are essentially found within the ST-11 clonal complex, and those of isotype II, are spread over i.a. the ST-8, ST-18, ST-32 and ST-41/44 clonal complexes (Harrison et al., BMC Microbiol. 2008, 8: 66). The B16B6 (serogroup B) and FAM18 (serogroup C) strains are representatives of isotype I; the MC58, M982, BZ83 and 8680 serogroup B strains are representatives of isotype II.
A TbpB antigen of N. meningitidis is defined as being an antigen able to be recognized in an immunoassay (e.g., Western Blot) by an antiserum raised in a mammal against a purified full-length TbpB of N. meningitidis of either isotypes. For use in the present invention, it may be i.a., a full-length TbpB or an immunogenic fragment thereof; a full-length TbpB or a fragment thereof comprising the site for binding to human transferrin (hTf), which bears a mutation in the binding site so that it is unable to bind to hTf (Renauld-Mongenie et al., J. Bacteriol. (2004) 186 (3): 850); or TbpB chimeras such as the fusion product between (i) a first TbpB of a first strain or an immunogenic fragment thereof and (ii) a second TbpB of a second strain or an immunogenic fragment thereof; or a fusion product between a TbpB or an immunogenic fragment thereof and another protein of N. meningitidis.
Although for the purposes of the invention, a TbpB antigen of any isotype can be used without distinction, it is preferred to use at least a TbpB antigen of isotype II.
Still preferably, the composition of the invention may comprise both a TbpB antigen of isotype II and a TbpB antigen of isotype I.
The TbpB of N. meningitidis, as naturally produced by N. meningitidis, is a lipoprotein. For use in the composition of the invention, the TbpB antigen can be lipidated or not. Accordingly, the immunogenic or vaccinal composition/combination may comprise:
(i) a non-lipidated TbpB isotype II antigen;
(ii) a non-lipidated TbpB isotype II antigen and a lipidated TbpB isotype I antigen;
(iii) a lipidated TbpB isotype II antigen and a lipidated TbpB isotype I antigen;
(iv) a non-lipidated TbpB isotype II antigen and a non-lipidated TbpB isotype I antigen;
(v) a lipidated TbpB isotype II antigen and a non-lipidated TbpB isotype I antigen;
(vi) a non-lipidated TbpB isotype I antigen;
(vii) a lipidated TbpB isotype II antigen; or
(viii) a lipidated TbpB isotype I antigen.
According to an advantageous embodiment, when the immunogenic or vaccinal composition/combination comprises at least one TbpB antigen, this TbpB antigen is of isotype II antigen.
The open reading frame (ORF) or tbpB gene encoding the TbpB antigen of several strains of N. meningitidis, and the amino acid sequence of the corresponding protein, are already known. For instance, the tbpB and TbpB precursor sequences of the N. meningitidis strains M982 (isotype II) and B16B6 (isotype I), were disclosed in Legrain et al., Gene (1993) 130 (1): 73. The amino acid sequence of TbpB of strain M982 is shown in SEQ ID NO: 5. The amino acid sequence of TbpB of strain B16B6 is shown in SEQ ID NO: 6 [as well as in patent EP 586 266, respectively designated under the names “Tbp2-2169” (Seq Id No 7-8 in EP 586 266) and “Tbp2-2394” (Seq Id No 1-2 in EP 586 266)]. M982 and B16B6 TbpB sequences (amino acid and nucleotides sequences) may be retrieved from the NCBI website, under the respective Genbank accession numbers Z15130.1 and Z15129.1. Those web pages refer to Tbp2, the previous term for TbpB. Both M982 and B16B6 TbpB sequences include a signal peptide from position 1 to 20, the mature form starting in position 21 with a Cysteine residue which is the residue onto which the lipid chain is attached, upon lipidation; which Cysteine residue is omitted when TbpB is recombinantly produced in a non-lipidated form.
Within the N. meningitidis species and even strain isotypes, the amino acid sequence of TbpB proteins may display a certain degree of variability from one strain to another, without this affecting the biological function. This is then referred to as an “allelic variant” to a protein of a specific (reference) strain, with an identical function.
Thus, it will be easily understood that the present invention is not limited to the use of a TbpB defined by a particular amino acid sequence. Although any reference to an amino acid sequence is made by way of non-limiting illustration, it is indicated that TbpB protein of isotype II or I may exhibit an amino acid sequence which shows at least 80%, 85%, 90%, 95% 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of respectively:
In other words, it is indicated that for use in the present invention a TbpB antigen may be a polypeptide comprising or consisting of:
A full-length mature TbpB protein or a fragment thereof, which comprises (has) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the M982 amino acid sequence reported in SEQ ID NO: 5, starting with the amino acid in position 21 and ending with amino acid in position 711, the percent identity being calculated upon global or local alignment.
A full-length mature TbpB protein or a fragment thereof, which comprises (has) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the B16B6 amino acid sequence reported in SEQ ID NO: 6, starting with the amino acid in position 21 and ending with amino acid in position 599, the percent identity being calculated upon global or local alignment.
Preferably, the N. meningitidis TbpB isotype II antigen is a TbpB antigen of strain M982. Preferably also, the N. meningitidis TbpB isotype I antigen is a TbpB antigen of strain B16B6.
It is also indicated that annotated genome sequences of the meningococcal strains MC58 (serogroup B), Z2491 (serogroup A) and FAM18 (serogroup C) are respectively disclosed in Tettelin et al., Science, March 2000, 287: 1809 or WO 00/66791; Parkhill et al., Nature (March 2000) 404: 502; and Bentley et al., PLoS Genet., 3, e23 (2007). The tbpB/TbpB sequences of the MC58 strain disclosed in Tettelin et al. (above) or WO 00/66791 are designated under the reference number NMB0460; they are also available under accession number NP_273507 (version NP_273507.1 as submitted on Mar. 17, 2000).
The TbpB antigen, either lipidated or non-lipidated, may be recombinantly produced as a full-length protein. When the TbpB antigen is produced as a non-lipidated full-length protein, the cysteine residue in position 21 (position 1 at the N-terminal end of the mature form) may be absent.
When the TbpB antigen is produced as a lipidated full-length protein, for use in the present invention, it may conveniently be incorporated into liposomes, preferably cationic liposomes, optionally together with an N. meningitidis lipooligosaccharide (LOS) as described in WO 10/130896. A preferred LOS is described in WO 10/130898.
The TbpB antigen may be also produced as a truncated protein. Indeed, It has been already shown that the N-ter TbpB fragment and the C-ter TbpB fragment are each able to induce serum bactericidal activity (Rokbi et al., Infect. Immun. (2000) 68 (9): 4938). For use in the present invention, an example of an N-ter fragment of M982 TbpB has an amino acid sequence starting with the cysteine residue in position 21 or the leucine residue in position 22 and ends with the glutamic acid residue in position 371. An example of a C-ter fragment of M982 TbpB has an amino acid sequence starting with the asparagine residue in position 372 and ends with the glutamine residue in position 711. A truncated TbpB protein used in the present invention as the TbpB antigen is preferably a C-ter TbpB fragment.
Unless otherwise indicated, all the antigens/polypeptides/fragments I constructs/amino acid sequences are described throughout the specification from the N-terminus end to the C-terminus end. As a matter of example, a fragment described as consisting of the protease domain, the α-peptide domain and part of the beta-domain of the trypsin-like serine protease auto-transporter of N. meningitidis shall be understood as a fragment consisting of, from N-ter to C-ter, the protease domain, the α-peptide domain and part of the beta-domain, the C-ter of the protease domain being fused to the N-ter of the α-peptide domain, the C-ter of which being fused to the N-ter of ‘part of the beta-domain’. Fusion is conveniently achieved by covalent peptidic bound (amide linkage CO—NH).
Any of the antigens for use in the composition according to the invention may be synthetized by any method well-known from the skilled person. Such methods include biological production methods by recombinant technology and means. In particular, nucleotide sequences encoding the N. meningitidis proteins and corresponding amino acid sequences thereof may be retrieved from a number of bioinformatics websites such as the site of the European Bioinformatics Institute or the National Center for Biotechnology Information (US). Any desired encoding sequences may be conceived and designed by bioinformatics according to methods and software known in the art, such as the software pack Vector NTI of Invitrogen; chemically synthetized de novo; and finally cloned into expression vectors available in the art. Methods of purification that can be used are also well-known from the skilled person.
In the context of the invention the term “about” as used herein when referring to a measurable value, such as an amount, duration or a number, such as the number of amino acids in an amino acid sequence, is meant to encompass variations of ±5%.
In the context of the invention the term “a” or “an” entity refers to one or more of that entity. For example “a polynucleotide”, “an isolated peptide”, “a fusion peptide”, “an isolated polynucleotide” is understood to represent respectively at least one or more “polynucleotide”, at least one or more “isolated peptide”, at least one or more “fusion peptide”, at least one or more “isolated polynucleotide”.
In the context of the invention and throughout the specification, the terms such as “comprising”, “having”, “including” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. All reference to “comprising” or “having” also includes the embodiments “essentially consisting of”, “consisting of” and “being”. Terms such as the embodiments “essentially consisting of”, “consisting of” and “being” have the meaning ascribed to them in most patent jurisdictions, preferably in the jurisdiction in question; e.g., they imply the exclusion of all, most or all but a negligible amount of other elements, or they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel property.
Compositions
In a first embodiment, a composition of the invention may comprise at least two Neisseria meningitidis antigens selected from the group consisting of a trypsin-like serine protease auto-transporter antigen, a NalP antigen and a TbpB antigen, said antigens being as-above described.
According to one embodiment, a composition in accordance with the invention may comprise (i) a trypsin-like serine protease auto-transporter antigen and (ii) an NalP antigen and/or TbpB antigen.
Preferably, a TbpB antigen may be of isotype II.
More preferably, a composition comprising a TbpB antigen of isotype II may further comprise a TbpB antigen of isotype I.
According to one embodiment, a composition in accordance with the invention may comprise an additional N. meningitidis protein antigen.
According to one embodiment, a composition in accordance with the invention may be a bivalent composition comprising a trypsin-like serine protease auto-transporter antigen and an NalP antigen.
According to one embodiment, a composition in accordance with the invention may be a bivalent composition comprising a trypsin-like serine protease auto-transporter antigen and a TbpB antigen.
According to one embodiment, a composition in accordance with the invention may be a trivalent composition comprising (i) a trypsin-like serine protease auto-transporter antigen, (ii) an NalP antigen and (iii) a TbpB antigen.
According to one embodiment, a composition in accordance with the invention may be a trivalent composition comprising (i) a trypsin-like serine protease auto-transporter antigen, (ii) a TbpB antigen of isotype II and (iii) a TbpB antigen of isotype I.
According to one embodiment, a composition in accordance with the invention may be a quadrivalent composition comprising (i) an IgA1P antigen, (ii) an NalP antigen, (iii) a TbpB antigen of isotype II and (iv) a TbpB antigen of isotype I.
Preferably, the TbpB antigen of isotype II is the TbpB of strain M982.
Also preferably, the TbpB antigen of isotype I is the TbpB of strain B16B6.
In some embodiments the TbpB of isotype I is lipidated.
Preferably, an NalP antigen is of strain MC58. More preferably, an NalP antigen comprises the amino acid sequence SEQ ID NO:7.
According to one embodiment, a trypsin-like serine protease auto-transporter antigen of the composition of the invention may be the IgA1P antigen.
Preferably, an IgA1P antigen may be of strain MC58. More preferably, an IgAIP antigen may comprise an amino acid sequence selected from SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.
In some embodiments, a composition in accordance with the invention may further comprise a N. meningitidis LOS.
Preferably, the LOS is detoxified in liposomes.
In some embodiments of the invention, an immunogenic composition may comprise at least two antigens independently selected from:
(I) An IgA1P antigen comprising or consisting of:
According to particular embodiments, an immunogenic composition of the invention can be bivalent, trivalent, quadrivalent or pentavalent. In bivalent, trivalent, quadrivalent and pentavalent compositions of the invention, the number of N. meningitidis protein antigens is respectively limited to two, three, four and five.
By «bivalent composition» is meant a composition in which there are only two protein antigens of N. meningitidis. One of these two proteins antigens is a trypsin-like serine protease auto-transporter (trypsine-like SPAT) and the second one can be selected from the group consisting of a NalP antigen and a TbpB antigen. Accordingly, in a bivalent composition of the invention, the N. meningitidis proteins consist of (i) the trypsin-like SPAT and the NalP antigen; or (ii) the trypsin-like SPAT and the TbpB antigen. In this latter alternative, the TbpB antigen is preferably of isotype II.
Examples of bivalent compositions include the following bivalent compositions:
In a particular embodiment, a bivalent composition according to the invention comprises:
(I) An IgA1P antigen which comprises or consists of:
In another particular embodiment, a bivalent composition according to the invention comprises:
(I) A TbpB antigen, preferably non-lipidated, preferably of isotype II; and
(II) A NalP antigen which comprises a NalP fragment which essentially consists of the NalP passenger domain, the translocator domain, the alpha-peptide and the first and second beta-sheets; wherein the Ser residue of the catalytic triad in the NalP passenger domain is optionally mutated by substitution with e.g., Alanine.
According to a preferred embodiment, a bivalent composition in accordance with the invention may comprise:
According to another preferred embodiment, a bivalent composition in accordance with the invention may comprise:
According to another preferred embodiment, a bivalent composition in accordance with the invention may comprise:
By «trivalent composition» is meant a composition in which there are only three protein antigens of N. meningitidis. One of these three proteins antigens is a trypsine-like serine protease auto-transporter (trypsine-like SPAT); the second and the third proteins antigens can be independently selected from the group consisting of a NalP antigen, a TbpB antigen of isotype I and a TbpB antigen of isotype II.
Accordingly, in a trivalent composition of the invention, the N. meningitidis proteins consist of the trypsine-like SPAT, the NalP antigen and the TbpB antigen, this latter being preferably of isotype II. Alternatively, in a trivalent composition of the invention, the N. meningitidis proteins consist of the trypsine-like SPAT, the TbpB antigen of isotype II and the TbpB antigen of isotype serotype I. In a further embodiment, in a trivalent composition of the invention, one of the three protein antigens is the trypsine-like SPAT; the second protein antigen is a NalP antigen or a TbpB antigen, this latter being preferably of isotype II; and the third protein antigen is a N. meningitidis protein antigen which is an additional protein antigen i.e., other than a trypsine-like SPAT, NalP and TbpB antigen.
In a specific embodiment, this additional protein antigen may be the factor H binding protein (fHBP) antigen of N. meningitidis. A fHBP antigen of N. meningitidis is defined as being an antigen able to be recognized in an immunoassay (e.g., Western Blot) by an antiserum raised in a mammal against a purified full-length fHBP of N. meningitidis. For use in the present invention, it may be i.a., a full-length fHBP, lipidated or not, mutated in the fHBP binding site or not, or an immunogenic fragment thereof; or fHBP chimeras such as the fusion product between (i) a first fHBP of a first strain or an immunogenic fragment thereof and (ii) a second fHBP of a second strain or an immunogenic fragment thereof; or a fusion product between a fHBP or an immunogenic fragment thereof and another protein of N. meningitidis.
The fHBP of strain MC58 is designated under the reference NMB1870. Sequences may be retrieved from the National Center for the Biotechnology Information (NCBI) web site at http://www.ncbi.nlm.nih.gov with the accession number NC_003112.2. NMB1870 is also known as GNA1870 or LP2086. Its vaccinal use is reported in i.a., Giuliani et al., PNAS, (July 2006) 103 (29): 10834; Giuliani et al., Infect. Immun. (February 2005) 73 (2): 1151; Fletcher et al., Infect. Immun. (2004) 72: 2088; and Masignani et al., J. Exp. Med. (March 2003) 197 (6): 789).
The fHBP antigen may thus comprise NMB1870, an allelic variant or a fragment thereof or any chimeric or fusion derivatives as mentioned above.
Examples of trivalent compositions include the following trivalent compositions:
In a particular embodiment, a trivalent composition according to the invention comprises:
(I) An IgA1P antigen which comprises or consists of:
According to a preferred embodiment, a trivalent composition in accordance with the invention may comprise:
According to another preferred embodiment, a trivalent composition in accordance with the invention may comprise:
According to another preferred embodiment, a trivalent composition in accordance with the invention may comprise:
By «quadrivalent composition» is meant a composition in which there are only four protein antigens of N. meningitidis. One of these four protein antigens is a trypsine-like serine protease auto-transporter (trypsine-like SPAT); the second and the third proteins antigens are a NalP antigen, a TbpB antigen, preferably of isotype II. The fourth protein antigen can be independently selected from a TbpB antigen of isotype I and any other additional N. meningitidis protein antigen which is not a trypsine-like SPAT, NalP and TbpB antigen. In a specific embodiment, this additional protein antigen may be the fHBP antigen of N. meningitidis.
Accordingly, in a quadrivalent composition of the invention, the N. meningitidis proteins consist of the trypsine-like SPAT, the NalP antigen, the TbpB antigen of isotype II and the TbpB of isotype I. Alternatively, in a quadrivalent composition of the invention, the N. meningitidis proteins consist of the trypsine-like SPAT, the TbpB antigen of isotype II and the TbpB antigen of isotype serotype I and any other additional N. meningitidis protein antigen which is not a trypsine-like SPAT, NalP and TbpB antigen. In a specific embodiment, this additional protein antigen may be the fHBP antigen of N. meningitidis.
Examples of quadrivalent compositions include the following quadrivalent compositions:
In a particular embodiment, a quadrivalent composition according to the invention comprises:
(I) An IgA1P antigen which comprises or consists of:
According to a preferred embodiment, a quadrivalent composition in accordance with the invention may comprise:
According to a preferred embodiment, a quadrivalent composition in accordance with the invention may comprise:
By «pentavalent composition» is meant a composition in which there are only five protein antigens of N. meningitidis. One of these five proteins antigens is a trypsine-like serine protease auto-transporter (trypsine-like SPAT); the second, the third and fourth protein antigens are a NalP antigen, a TbpB antigen of isotype II and a TbpB antigen of isotype I, and the fifth protein antigen is any other additional N. meningitidis protein antigen which is not a trypsine-like SPAT, NalP and TbpB antigen. In a specific embodiment, this additional protein antigen may be the fHBP antigen of N. meningitidis.
Examples of pentavalent compositions include the following pentavalent compositions:
The compositions according to the invention, such as the bivalent, trivalent, quadrivalent and pentavalent compositions described above, can comprise other components that are no protein antigen of N. meningitidis. As a matter of example, these compositions can comprise a lipooligosaccharide (LOS) of N. meningitidis, an adjuvant and/or an excipient.
LOS
A lipooligosaccharide (LOS) antigen of N. meningitidis may also be included in the immunogenic or vaccinal composition/combination of the invention. This is in particular advantageous when said composition/combination comprises a lipidated TbpB antigen, as lipidated TbpB antigen has been reported to have an adjuvant effect of the immune response induced by LOS (patent application WO2010130896).
LOS is a major constituent of the bacterial outer membrane and is anchored into the lipid outer layer of the membrane by its Lipid A moiety, the polysaccharide chains being exposed to the surface. The LOS structure varies depending on the N. meningitidis strain. Strains have been classified into several immunotypes (IT L1 to IT L13) based on their specific LOS structure, as a function of their reactivity with a series of antibodies that recognize various LOS epitopes (Achtman et al., J. Infect. Dis. (1992) 165: 53). As a direct consequence of this, the LOS from these N. meningitidis strains may by extension, also be referred to LOS of immunotype L1 to L13. The differences between immunotypes originate from variations in the composition and in the conformation of the oligosaccharide chains.
The conformation of the oligosaccharide chain is represented by formula I:
wherein KDO is 3-deoxy-D-manno-2-octulosonic acid, Glc is glucose, Hep is heptose, GlcNAc is N-acetyl glucosamine,
and wherein R1 and R2 are as shown in the table below:
The structure of LOS of immunotypes 1 to 11 is also reported in particular in WO 10/130898.
For use in the composition of the invention, the LOS may be of any immunotype e.g. L2 or L3, optionally modified by genetic engineering as described in U.S. Pat. No. 5,705,161 or WO 04/014417 or WO 01/022994, so that the LOS does not bear the Galβ1-4GlcNAcβ1-3 Galβ1-4Glcβ1-4 carbohydrate unit or lacto-N-neotetraose unit which constitutes an epitope which can potentially cross-react with human erythrocytes. Accordingly, it is possible to envisage using a LOS from a strain of immunotype L2 or L3 in which a gene involved in the biosynthesis of the alpha chain has been inactivated by mutation, so as to obtain an incomplete LNnT structure. Such mutations are proposed in patent application WO 04/014417. This involves extinguishing, by mutation, the IgtB, IgtE (or IgtH), IgtA or IgtA and IgtC genes. Thus, it appears to be possible and advantageous to use an LOS originating from an N. meningitidis strain of immunotype L2 or L3 mutated as described in WO 04/014417.
Alternatively, it may be also advisable to use an LOS which does not possess this lacto-N-neotetraose unit. It may therefore be particularly advantageous to use a LOS of immunotype L6 or L8, preferably L6, or an L6 LOS as described in WO 10/130898, modified by genetic engineering i.a., to bear an additional phospho-ethanolamine group.
LOS is a toxic compound that may be first detoxified before use in mammals. Various detoxification methods are reported in the art, including chemical, enzymatic, genetic methods e.g., as respectively described in U.S. Pat. No. 6,531,131, U.S. Pat. No. 6,482,807 and U.S. Pat. No. 6,887,483. Detoxification may also be achieved by association of LOS with a peptide analog of polymixin B as described in WO 06/108586 or by incorporation of LOS into a delivery structure (e.g., liposomes) as described in U.S. Pat. No. 5,888,519. Advantageously, the LOS may be incorporated into liposomes which may be anionic or cationic, preferably cationic e.g. involving a cationic lipid such as ethyl-PC as described in e.g. U.S. Pat. No. 5,698,721, U.S. Pat. No. 5,902,802 or U.S. Pat. No. 5,651,981. A detoxification method including the production of LOS incorporated into cationic liposomes is described in WO 10/130896.
LOS of N. meningitidis such as those described above, may act as a vaccinal antigen and may also have an adjuvant effect on the immune response induced by the protein antigens. Accordingly, when LOS is used in a composition of the invention, there may be no need to add an additional adjuvant such as those cited below. In a particular embodiment, LOS in liposomes is added to any of the bivalent, trivalent, quadrivalent or pentavalent compositions described above, i.a., when one of the protein antigens is a lipidated TbpB antigen.
Accordingly, in a particular embodiment, a composition of the invention comprising a N. meningitidis LOS (e.g., formulated in LOS-liposomes) as described hereinabove, advantageously comprises at least one first lipidated TbpB antigen of either isotype I or II, preferably isotype I. When such a composition comprises a second Tbp, this TbpB may be lipidated or not, and is of the isotype differing from that of the first lipidated B. For the purposes of the present invention, the LOS may be obtained by conventional means. In particular, it can be extracted from a bacterial culture, and then purified according to conventional methods. Many methods of production are described in the literature. By way of example, mention is made, i.e., of Westphal & Jann, (1965) Meth. Carbohydr. Chem. 5: 83; Gu & Tsai, 1993, Infect. Immun. 61 (5): 1873; Wu et al., 1987, Anal. Biochem. 160: 281 and U.S. Pat. No. 6,531,131. A LOS preparation can be quantified according to well-known procedures. Assaying of KDO by high performance anion exchange chromatography (HPAEC-PAD) is a method which is most particularly suitable.
Adjuvants
In a particular embodiment of the invention, the immunogenic composition further comprises one or several adjuvant(s).
The term “adjuvant” as used herein denotes a product which, added to the content of an immunogenic composition, in particular to a vaccine, increases the intensity of the immune reaction induced in the mammalian host to which said composition is administered. An adjuvant may in particular increase the quantity/quality of specific antibodies e.g. bactericidal antibodies, which said host is capable of producing after administration of said composition and thus increases the efficiency of the immune response.
The adjuvant (s) that can be used in the context of the invention include adjuvants promoting a Th1 and/or Th2 immune response. Accordingly, for use in the composition of the invention, an adjuvant may be a Th1, Th2 or Th1/Th2 adjuvant. The meaning given to “Th1, Th2 or Th1/Th2 adjuvant” shall be the meaning commonly acknowledged by the scientific community. A Th1 adjuvant promotes an immune response characterized by the predominant production of IFN-γ and/or IL-2 cytokines. A Th2 adjuvant promotes an immune response characterized by the predominant production of e.g., IL-4, IL-5, IL-6 and/or IL-10 cytokines. A Th1/Th2 adjuvant favours a balanced cytokine production (balanced immune response).
Examples of adjuvants promoting a Th1-type immune response include but are not limited to agonists of Toll-like receptors (TLRs), in particular to agonists of TLR4, which may be formulated or not. Typical formulation of a TLR agonist such as a TLR4 agonist, include oil-in-water emulsions. LPS derivatives like 3-De-O-acylated Monophosphoryl Lipid A (3D-MPL) described in WO 94/00153 or a 3D-MPL derivative named RC-529 described in U.S. Pat. No. 6,113,918 are well known TLR4 agonists; Other TLR4 agonists which share structural similarity with monophosphoryl lipid A, referred to as aminoalkyl glucosaminide phosphates (AGPs), are described in U.S. Pat. No. 6,113,918, U.S. Pat. No. 6,303,347, and WO 98/50399. Other synthetic TLR4 agonists are described in US 2003/0153532. Among these synthetic agonists, reference is made of a chemical compound named as E6020 and referenced in the Chemical Abstract Services (CAS) registry as CAS Number 287180-63-6 as particularly suitable Th1-adjuvant in the context of the invention. The chemical formula of the disodic salt is C83H63N4O19P2, 2Na and the developed chemical formula is as follows:
The R configuration (R,R,R,R) of the four asymetric carbons is preferred. The synthesis process is described in WO2007/005583. E6020 is preferably formulated in an oil-in-water emulsion and more particularly formulated in an oil-in-water emulsion (such as the one described in WO 07/006939), according to the process as described in the patent application WO 2007/080308.
Examples of adjuvants promoting a Th2-type immune response include but are not limited to aluminium salts and especially aluminium oxy hydroxide (also called for sake of brevity aluminium hydroxide) or aluminum hydroxy phosphate (also called for sake of brevity aluminum phosphate). When an aluminium salt is used, the protein antigens may advantageously be adsorbed onto the aluminium salt.
Excipients
In a composition of the invention, the active ingredients i.a., the protein antigens, may be formulated together with a pharmaceutically-acceptable excipient such as a pharmaceutically acceptable diluent or carrier. In a particular embodiment, the composition of the invention may comprise a buffer and/or an isotonic agent such as sodium chloride or sugars e.g. sucrose; and/or a stabilizing agent such as histidine.
An immunogenic composition according to the invention is useful for inducing an immune response in a mammal, in particular humans, against N. meningitidis of any serogroup, in particular against serogroup B. This immune response includes in particular, a bactericidal immune response wherein bactericidal antibodies are induced against N. meningitidis. By “bactericidal antibody” is meant antibodies able to kill the bacteria in the presence of complement (which is a component of the humoral immune system of mammals). The antibodies produced as part of the immune response upon administration of the immunogenic composition may be identified as “bactericidal antibodies” in a serum bactericidal assay using an appropriate source of complement, according to methods known in the art.
An immunogenic composition according to the invention is particularly useful for inducing an immune response i.a., a bactericidal immune response, against N. meningitidis strains of (i) the clonal complexes of the hyper-invasive lineage (invasive clonal complexes); (ii) the clonal complexes wherein strains of serogroup B are prevalent (highly represented), those complexes being or not prevalent worldwide, advantageously prevalent worldwide; and/or (iii) clonal complexes ST8, ST11, ST18, ST32, ST41/44, ST162 and/or ST269. The immunogenic composition is more particularly useful against N. meningitidis strains of serogroup B belonging to clonal complexes, such as the ST8, ST11, ST18, ST32, ST41/44, ST162 and/or ST269 complex(es). The immunogenic composition may be characterized by strain coverage of at least 70%. In other words, it may induce a bactericidal immune response against at least 70%, 75%, 80%, 85%, 90%, 95% or 100% of N. meningitidis strains of the clonal complexes specified above, in particular the ST8, ST11, ST18, ST32, ST41/44, ST162 and/or ST269 complex(es).
Strain coverage may be determined as described in the experimental part of the specification, involving in particular (i) the selection of a collection of strains representative of the most important clonal complexes e.g. including ST8, ST11, ST18, ST32, ST41/44, ST162 and/or ST269 complex(es) and (i) the achievement of an SBA assay against each of the strains of the collection, such as described in the experimental part. Briefly, the whole test consist in administering the composition to a mammal, one or several times at appropriate interval; collecting the sera that may optionally be pooled (within a group of mammals submitted to identical administration); culturing the strains of the collection; and testing the individual sera or pooled serum and/or dilutions thereof against each strain in an SBA assay, such as the one described in the experimental part. The percentage of coverage is determined on the basis of the number of strains responding positively—that is, against which the bactericidal titer of e.g., the pooled serum, meets (e.g., equals or is superior to) the threshold value considered as indicative of a positive surrogate of protection—over the total number of strains tested. Alternatively, the bactericidal titer of individual sera within a group of mammals as defined above, may be determined and the geometric mean titer (GMT) established. In that case, the strain is considered to respond positively when the GMT meets (e.g., equals or is superior to) the threshold value considered as indicative of a positive surrogate of protection.
According to an embodiment, the invention relates to a composition comprising a combination of antigens as above-described together with a pharmaceutically acceptable diluent or carrier.
According to another embodiment, the invention relates to a combination of antigens as above-described or a composition as above-described, for use as a vaccine, and more preferably for use for the prevention and/or treatment of N. meningitidis B infection.
According to another embodiment, the invention relates to a vaccine composition comprising a combination of antigens as above-described, or a composition as above-described.
According to an embodiment, the invention relates to an immunogenic composition comprising a combination of antigens as above-described.
An immunogenic composition according to the invention may be used as a pharmaceutical composition, in a prophylactic or therapeutic manner. Typically, it may be used as a vaccine composition for protecting against N. meningitidis infections e.g., for treating or preventing N. meningitidis infections. N. meningitidis induces a large range of infections from asymptomatic carriage to invasive diseases e.g., meningitidis and/or septicemia. Typically, the immunogenic or pharmaceutical composition of the invention comprises a therapeutically or prophylactically effective amount of each of the protein antigens. A therapeutically and/or prophylactically effective amount of a protein antigen may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the protein antigen, to elicit a desired therapeutic and/or prophylactic result.
The composition according to the invention may be administered as a dose wherein the amount of each of the protein antigens depends on various conditions including e.g., the weight, the age and the immune status of the recipient. As a matter of guidance, it is indicated that a dose of the composition of the invention may comprise a therapeutically and/or prophylactically effective amount of each of the protein antigen, which may be from 10 μg to 1 mg, e.g. about 50 μg per protein antigen included in the composition of the invention.
‘Prevention’ refers to prophylactic treatment, wherein a composition of the invention is administered to an individual with no symptoms of meningitis and/or septicaemia and/or no detectable N. meningitidis infection. Said prophylactic treatment is preferably administered with the aim of preventing or reducing future N. meningitidis infection.
Within the meaning of the invention, the terms “for preventing or for prevention” intend to mean, with reference to an N. meningitidis infection, a reduction of risk of occurrence of said infection and/or symptoms associated with said infection.
‘Treatment’ includes both therapeutic treatment and prophylactic or preventative treatment, wherein the object is to prevent or slow down the infection or symptoms of disease. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. The terms ‘therapy’, ‘therapeutic’, ‘treatment’ or ‘treating’ include reducing, alleviating or inhibiting or eliminating the symptoms or progress of a disease, as well as treatment intended to reduce, alleviate, inhibit or eliminate said symptoms or progress.
A further object of the invention is to provide a method of inducing an immune response, in particular a bactericidal immune response, against N. meningitidis, in particular against N. meningitidis of serogroup B, which comprises administering to an individual in need an immunogenic composition according to the invention. Still within the scope of the invention, it is provided a method of treating or preventing a N. meningitidis infection, in particular against N. meningitidis of serogroup B, which comprises administering to a patient in need a composition according to the invention.
In order to achieve the desirable effect, the composition of the invention may be administered as a primary dose, in a primary immunisation schedule, one or several times, e.g., two or three times, at appropriate intervals defined in terms of week or advantageously, month. In a particular embodiment, the interval between the primary doses may be not less than one or two months, depending on the conditions of the subject receiving the doses. If needed, the primary doses may possibly be followed by a booster dose of the composition of the invention, which may be administered e.g., from at least 6 months, preferably at least one year to two-five years, after the last primary dose.
The composition according to the invention may be administered by any conventional routes in use in the vaccine field e.g. by parenteral route such as the subcutaneous or intramuscular route. In a particular embodiment, the composition is suitable for injection and formulated accordingly. It may be in a liquid form or in a solid form that, before administration, may be extemporaneously suspended in a pharmaceutically-acceptable diluent.
Also provided, is a combination of antigens of the invention in the manufacture of a medicament for the preventive or therapeutic treatment of a N. meningitidis infection, e.g. an infection of N. meningitidis of serogroup B, such as meningitis.
Also provided, is a combination of antigens or composition of the invention for use in a method of inducing an immune response to N. meningitidis, in particular N. meningitidis of serogroup B. Also provided, is a combination of antigens or composition of the invention for use in a method of preventing or treating a N. meningitidis infection, e.g. an infection of N. meningitidis of serogroup B, such as meningitis. In some embodiments, said method comprises administering said combination of antigens or composition to a subject, in particular a subject in need thereof. According to one embodiment, a method of the invention may comprise the step of observing a preventing and/or a treating effect with regard to a N. meningitidis infection.
Also provided is a method of inducing an immune response to N. meningitidis, in particular N. meningitidis of serogroup B, which comprises administering a combination of antigens or composition of the invention to an individual in need thereof. Also provided is a method of preventing or treating of meningitis, in particular N. meningitidis infection, e.g. an infection of N. meningitidis of serogroup B, such as meningitis, which comprises administering a combination of antigens or composition of the invention to an individual in need thereof.
The invention will be further illustrated by the following figures, sequences and experimental part.
SEQ ID No:
1 IgA1P from N meningitidis strain MC58 amino acid sequence
2 App from N meningitidis strain MC58 amino acid sequence
3 AusI from N meningitidis strain MC58 amino acid sequence
4 NalP from N meningitidis strain MC58 amino acid sequence
5 TbpB from N meningitidis strain M982 amino acid sequence
6 TbpB from N meningitidis strain B16B6 amino acid sequence
7 NalP construct SP509 amino acid sequence, without the His-tag
8 IgA1P construct SP503 amino acid sequence, without the His-tag
9 IgA1P construct SP548 amino acid sequence, without the His-tag
10 MC58 IgA1P-App fusion construct SP550 amino acid sequence, without the
His-tag
A—Materials & Methods
Non-His-tag Proteins
M982 TbpB
Preparation of E. coli BL21 (D3) pSP314
The expression strain is the E. coli BL21 (DE3) strain containing the pSP314 plasmid. This plasmid derives from plasmid pET28 (Novagen). The sequence encoding TbpB from strain M982 without the peptide signal sequence and the first cysteine residue of the mature protein (but with an initiation codon), is placed under the control of the T7 promoter (from pET28). This plasmid contains also a kanamycin resistant gene and a plasmid stability element.
Culture E. coli BL21 (D3) pSP314
Preculture at a 3 L Scale
A working seed vial of E. coli BL21 (D3) pSP314 was thawed and used to inoculate shake flasks containing a classical E. coli culture medium comprising salts and yeast extract at an inoculation rate of 0.5 ml inoculum/500 ml medium in 2 L flasks. Incubation was pursued overnight at 37° C.
Culture in 30 L Fermentor
The preculture is transferred to a 30 L fermentor containing a classical E. coli culture medium comprising salts and yeast extract at a 5% inoculation rate to a final volume of 20 L. The culture is pursued overnight (16 hrs; pH 7.0; 37° C.: pO2: 30%; speed: 200 rpm to max). At O.D. 600 nm 25-35, IPTG (isopropyl β-D-1-thiogalactopyranoside) is added for 3 hrs to a final concentration of 1 mM. Then the temperature is decreased to 10° C. and the bacteria are harvested by centrifugation (20 min; 7 000 g). Cell pellets are frozen at −20° C. and then stored at −35° C.
Preparation of a Cell Extract Before Purification
Frozen cell pellets are thawed at +5° C. during 16-96 hrs and resuspended in a lysis buffer Tris 50 mM pH 9 (ratio 150 g of wet cell weight per liter of buffer) by mechanical mixing with an ultraturrax at 13 000-16 000 rpm. MgCl2 at a final concentration of 1 mM and benzonase (endonuclease) at 30 UI/g of wet cells are added to the suspension before cell disruption.
Cell disruption is performed at 5° C. by high pressure homogenization (PANDA 2K, Niro Soavi) under 1300 bars. The bacterial lysate is clarified by centrifugation at 13 000 g during 2 hrs. The supernatant is finally filtrated on a 0.45 μm Durapore membrane (Millipore).
Purification
A small scale lot was produced using three purification steps including (i) a first capture step by anion exchange chromatography (AEX), (ii) an intermediate step by hydrophobic chromatography (HIC) and (iii) a final step by ultrafiltration on a 30 kDa membrane using an Ultracel cassette from Millipore. The purity of TbpB small scale lot was about 87%.
A pilot scale lot was produced using four main purification steps including (i) a first capture step by anion exchange chromatography (AEX), (ii) an intermediate step by hydrophobic chromatography (HIC); (iii) a concentration step on a 10 kDa membrane using Ultracel cassette from Millipore and (iv) a size exclusion chromatography (SEC). The purity of TbpB pilot scale lot was superior to 90%.
Purification of the Small Scale Lot
First Capture Chromatography: Anion Exchange Chromatography
The clarified lysate in Tris 50 mM, pH 9 obtained from cell disruption was applied onto a Capto Q gel (GE HealthCare) packed in a 1.8 L BPG column from GE Healthcare (3.8 mg of total protein/L of gel) previously equilibrated with Tris 50 mM, pH 9. The chromatography was performed at a flow rate of 300 cm/hr. The column was washed with Tris 50 mM, pH 9 and the protein was eluted by step with 4 CV of Tris 50 mM, NaCl 150 mM, pH 9.
Recovered fractions were analysed by SDS-PAGE (M982 TbpB as produced≈72 kDa) and those containing M982 TbpB were pooled and stored at 5° C. before further purification.
Intermediate Chromatography: Hydrophobic Chromatography
The 0.22 μm filtrate was adjusted to 1 M ammonium sulfate, and to pH 7.5 by HCl addition and then filtered on 0.45 μm filter.
The 0.45 μm filtrate was divided in two fractions and processed in two runs onto a 80 ml Phenyl Sepharose High Sub 6 Fast flow gel (GE HealthCare) packed into an XK16 column from GE Healthcare and previously equilibrated with phosphate 50 mM, ammonium sulfate 1 M, pH 7.5. The chromatography was performed at a flow rate of 300 cm/hr. The collected flow-through (TbpB is not retained on the column) of the two runs were pooled and stored at 5° C. before further purification.
Concentration/Diafiltration Step:
The HIC Flow-through was submitted to ultrafiltration on a 30 kDa Ultracel membrane using a Pellicon 3 cassette (Millipore) for buffer exchange against PBS and product concentration. For further use, the preparation was concentrated at 1.3 mg/ml M982 TbpB in PBS.
Purification of the Pilot Scale Lot
First Capture Chromatography: Anion Exchange Chromatography
The clarified lysate in Tris 50 mM, pH 9 obtained from cell disruption was applied onto a Capto Q gel (GE HealthCare) packed into a 3.8 L axichrom column from GE Healthcare (18 mg of total protein/ml of gel) and previously equilibrated with Tris 50 mM, pH 9. The chromatography was performed at a flow rate of 300 cm/hr. The column was washed with Tris 50 mM, pH 9 and the protein was eluted by steps with 4 CV of Tris 50 mM, NaCl 150 mM, pH 9.
Recovered fractions were analysed by SDS-PAGE (M982 TbpB=72 kDa) and those containing M982 TbpB were pooled, filtered onto a 0.22 μm 1900 cm2 Optical XL4 filter (Durapore, Millipore) and stored at 5° C. before further purification.
Intermediate Chromatography: Hydrophobic Chromatography
The 0.22 μm filtrate was adjusted at 1 M Ammonium sulfate, and at pH 7.5 by HCl addition and then filtered on 0.22 μm filter.
The 0.22 μm Q eluate was loaded onto a Phenyl Sepharose High Sub 6 Fast flow gel (GE HealthCare) gel (Axichrom from GE Healthcare) packed into a 0.6 L BPG column, previously equilibrated with Tris 50 mM, ammonium Sulfate 1 M, pH 7.5. The chromatography was performed at a flow rate of 300 cm/hr. The collected flow-through (TbpB is not retained on the column) was filtrated onto a 0.22 μm 1900 cm2 Optical XL4 filter (Durapore, Millipore) before further purification.
Polishing Step for the Pilot Scale Lot: Size Exclusion Chromatography (SEC)
The 0.22 μm filtrate was submitted to a concentration/diafiltration step by tangential flow filtration on 10 kDa Ultracel membrane using three 0.1 m2 Pellicon 3 cassettes (Millipore) for buffer exchange and product concentration before the final SEC.
The cassettes were first conditioned in buffer Tris 50 mM, NaCl 100 mM, pH 7.5. A polarization step with the HIC flow-through was then performed during 10 min. The product was then pre-concentrated and diafiltered against 10 diavolumes of Tris 50 mM, NaCl 100 mM, pH 7.5. Then, the product was concentrated to the minimum working volume (0.4 L) and the membranes were rinsed with the diafiltration buffer. The final retentate collected was <350 ml (<7% of the SEC column volume).
After concentration, the product was applied on a SEC column using a Superdex 75 sorbent. The product was loaded on the column (<7% of the column volume on a 4.9 L BPG column) at a flow rate of 30 cm/h in a Tris 50 mM, NaCl 100 mM, pH 7.5 buffer. During elution, fractions were collected and each fractions analyzed by SDS PAGE. Fractions containing poor level of 20 kDa impurities were pooled.
The pooled fractions were diluted in Tris 50 mM, NaCl 100 mM, pH 7.5 to a concentration of 2 mg/ml and stabilized in 10% sucrose and stored at −70° C. For use, the thawed solution is diluted to a final concentration of 1 mg/ml in Tris 50 mM, NaCl 100 mM, pH 7.5, sucrose 5%.
B16B6 TbpB
The expression strain is the E. coli BL21 strain containing the pTG9216 plasmid. This plasmid contains in particular a kanamycin-selectable marker and the polynucleotide encoding the mature TbpB from N. meningitidis B16B6 fused to the E. coli RlpB (real lipoprotein B) signal sequence and placed under the control of the arabinose promoter (araB). As a result of this, the expressed TbpB is lipidated.
The expression strain is cultured as described in WO 10/130896 for the preparation of M982 TbpB. LPS is extracted and cell lysis is performed as described in WO 10/130896. After lysis, membranes are washed in Tris-HCl 50 mM, Urea 1 M; then in Tris-HCl 50 mM, EDTA 25 mM; and finally in Tris-HCl 50 mM. Membranes are solubilized to extract B16B6 TbpB as described in WO 10/130896.
B16B6 TbpB is purified by two successive anion exchange chromatographies followed by a cation exchange chromatography as described in WO 10/130896. Eluted fractions recovered from each chromatography are analysed by SDS-PAGE (B16B6 TbpB=68 kDa) and those containing B16B6 TbpB are pooled.
Dialysis and concentration of the pooled fractions recovered from the last chromatography are achieved as described in WO 10/130896. Purified B16B6 TbpB is finally treated by adsorption on Bio-Beads™ SM-2 in order to remove the excess Elugent™ detergent (surfactant constituted of alkyl glucosides which could destabilize the LPS L8 liposomes) as described in WO 10/130896.
B16B6 TbpB is stored in PBS merthiolate 0.001% at −80° C.
Preparation and Purification of His-Tag Protein Constructs
M982 His-TbpB
The sequence encoding the mature TbpB protein from strain M982 was produced from the tbpB sequence available from the database Entrez Gene du NCBI (National Center for the Biotechnology Information) at http://www.ncbi.nlm.nih.gov under the accession number Z15130 corresponding to the M982 tbp locus. The sequence encoding the mature protein [without the peptide signal sequence and the first amino acid (Cys); starting with Leu in position 2] was placed in a pET28 plasmid (Novagen), in frame with and immediately downstream a His-tag encoding sequence, under the control of the T7 promoter. The M982 His-TbpB expression plasmid contains a kanamycin resistant gene and a plasmid stability element.
E. coli BL21 (DE3) transformed with this plasmid was cultured at 37° C. in Luria-Bertani medium complemented with 50 μg/ml kanamycine. The expression of M982 His-TbpB was induced by adding 1 mM IPTG. The expression was pursued for 3 hrs.
Bacteria were harvested by centrifugation and pellets are washed with Tris 20 mM, NaCl 300 mM, pH 8.0. The bacterial suspension was centrifuged (6 000 g 15 min) and pellets were resuspended in Tris 20 mM, NaCl 300 mM, pH 8.0, complemented with lysozyme (100 pg/ml) MgCl2 1 mM and Triton X100 0.1%. Incubation was pursued 15 min at 4° C. under mild stirring.
One Unit/ml of benzonase was added. Incubation was pursued 15 min at 4° C. under mild stirring. The suspension was then centrifuged at 30 000 g for 20 min at 4° C. The supernatant was recovered.
The supernatant was loaded at a flow rate of 2 ml/min on a His-trap column FF previously equilibrated with buffer A (Tris 20 mM, NaCl 300 mM pH 8.0). The column was then submitted to a linear imidazole gradient from 0 to 250 mM. M982 His-TbpB was eluted in fractions at about 250 mM imidazole. Fractions were pooled and dialyzed against PBS for 48 hrs and dialyzed against Tris 20 mM, NaCl 300 mM, pH 8.0. The dialysate is diluted 5-fold with a solution of Tris 20 mM, NaCl 300 mM and imidazole 20 mM. The diluted dialysate was then submitted to second purification step identical to the previous one. Fractions eluted at 250 mM imidazole are recovered, pooled and extensively dialysed against PBS. The dialysate was diluted with PBS 10% glycerol to a protein concentration of about 1 mg/ml.
B16B6 His-TbpB (also Called SP544)
The sequence encoding the mature TbpB protein from strain B16B6 was produced from the tbpB sequence available from the database Entrez Gene du NCBI (National Center for the Biotechnology Information) at http://www.ncbi.nlm.nih.gov under the accession number Z15129 corresponding to the B16B6 tbp locus. The sequence encoding the mature protein [without the peptide signal sequence and the first amino acid (Cys); starting with Leu in position 2] was placed in a pET28b plasmid (Novagen), in frame and immediately downstream a His-tag encoding sequence, under the control of the T7 promoter to give plasmid pSP544 (which also contains a kanamycin resistant gene and a plasmid stability element).
BL21 (DE3) E. coli strain transformed with plasmid pSP544 is cultured at 37° C. during 20 hrs in Auto Induction Medium (Formedium™) supplemented with kanamycin 30 μg/ml.
Cells are harvested by centrifugation at 5 000 g, 15 min, 4° C. Pellets corresponding to 6 L of cultures are resuspended in 240 mL of lysis buffer Tris 50 mM pH7.5, NaCl 300 mM, β-mercaptoethanol 5 mM, MgCl2 2 mM, leupeptine 1 mM, pepstatine 1 mM, CHAPS 0.1%, urea 0.5 M. The suspensions are incubated 1 hr at room temperature under shaking. Cells are disrupted by 4 cycles at Pin 1100 bars and Pout 110 bars. Benzonase is added at 1 mM and incubation is pursued 1 hour at 4° C. under shaking. Soluble and insoluble proteins are separated by centrifugation at 46,000 g, 30 min at 4° C.
Purification is performed onto affinity resin Ni-Sepharose 6 Fast Flow 6 ml (Amersham GE Healthcare) in column XK16 (Amersham GE Healthcare). Soluble fraction from lysis is loaded on Ni-Sepharose equilibrated in Buffer A (Tris 50 mM, pH 7.5, NaCl 300 mM, CHAPS 0.1%, urea 0.5 M, β-mercaptoethanol 1 mM). Steps of 30 column volumes (CV) were performed at 2% of buffer B (Tris 50 mM pH 7.5, NaCl 300 mM, CHAPS 0.1%, urea 0.5 M, β-mercaptoethanol 1 mM, imidazole 1 M, followed by a linear gradient up to 100% of buffer B (30 CV).
Fractions eluted from the column are analysed by SDS-PAGE and those containing SP544 (MW≈64.3 kDa) are pooled and submitted to size-exclusion chromatography.
Ni-Sepharose pool is loaded on an S-200 preparative gel filtration column and eluted in Tris-HCl 50 mM, pH 7.5, NaCl 150 mM, β-mercaptoethanol 1 mM. Fractions eluted from the column are analysed by SDS-PAGE and those containing SP544 (MW≈64.3 kDa) are pooled and dialyzed against Tris-HCl 50 mM, pH 7.5, NaCl 10 mM, 1-mercaptoethanol 1 mM. The dialysate is then submitted to ion-exchange chromatography.
The dialysate is loaded on 4 mL of an anion exchange (ANX) resin previously equilibrated in buffer Tris-HCl 50 mM pH 7.5, NaCl 10 mM, β-mercaptoethanol 1 mM. Elution with 30 column volumes is performed at 0 to 50% of buffer B (Tris-HCl 50 mM pH 7.5, NaCl 1 M, β-mercaptoethanol 1 mM).
Fractions eluted from the column are analysed by SDS-PAGE and those containing SP544 (MW=64.3 kDa) are pooled and dialysed against Tris-HCl 50 mM, pH 7.5, NaCl 10 mM, β-mercaptoethanol 1 mM, glycerol 10% (v/v). The dialysate is concentrated 4-fold on Amicon 3 kDa MWCO (Molecular Weight Cut Off) device to give an SP544 solution at 0.52 mg/mL that is stored at −80° C.
Recombinant Expression of MC58 NalP SP509 Construct
The expression strain is the E. coli BL21 (DE3) strain containing the pSP509 plasmid. This plasmid derives from vector pET28 (Novagen). The sequence encoding a truncated and mutated NalP from strain MC58 (NalP TR mut; also called SP509) is placed under the control of the T7 promoter (from pET28). This plasmid contains also a kanamycin resistant gene and a plasmid stability element.
The sequence encoding the NalP TR mut (SP509) was produced from the nalP gene (NMB1969) of the N. meningitidis MC58 genome available from the Entrez Gene database of the NCBI (National Center for the Biotechnology Information) at http://www.ncbi.nlm.nih.gov under the accession number NC_003112. The NalP TR mut (SP509) starts with Glycine 30 and ends with Glycine 853 (amino acid numbering is based on the complete amino acid sequence of NMB1969) and with insertion of the Ser 426 Ala mutation generated by overlap PCR extension. A His-tag is added at the N-ter end, without spacer. The amino acid sequence of SP509 without the his-tag is shown in SEQ ID NO: 7.
E. coli BL21 (DE3) strain (Novagen) was transformed with pSP509 according to the supplier recommendations.
BL21 (DE3) E. coli strain transformed by plasmid pSP509 is seeded at a ratio 1:500 in Luria Bertani broth (LB) medium supplemented with kanamycin 30 pg/ml and cultured at 37° C. under stirring (220 rpm) up to a O.D.600 nm of from 0.6 to 0.8. The IPTG is added at 1 mM final and induction is pursued 3 hrs at 37° C. Bacterial cells are harvested by centrifugation and pellets stored at −20° C.
Recombinant Expression of MC58 IgA1 Protease SP503, SP548 and SP550 Constructs
The sequences encoding IgA1P SP503, SP548 and SP550 were produced from the iga gene (NMB0700) and the app gene (NMB1985) of the N. meningitidis MC58 genome available from the Entrez Gene data-base of the NCBI (National Center for the Biotechnology Information) at http://www.ncbi.nlm.nih.gjov under the accession number NC_003112.
The sequences and the expression plasmids containing them (respectively pSP503, pSP548 and pSP550) were conceived and designed by bioinformatics using the software pack Vector NTI (Invitrogen).
Each of pSP503, pSP548 and pSP550 derives from vector pET28 (Novagen). The respective encoding sequence is placed under the control of the T7 promoter (from pET28). This vector also contains a kanamycin resistant gene and a plasmid stability element.
SP503 starts with Alanine 28 and ends with Alanine 1584 (amino acid numbering is based on the complete IgA1P amino acid sequence NMB0700) and comprises the Ser 267 Val mutation. A His-tag is added at the N-ter end, separated from Ala 28 by a spacer constituted with four glycines and one serine (N-ter to C-ter). The amino acid sequence of SP503 without the his-tag is shown in SEQ ID NO: 8.
SP548 starts with Alanine 28 and ends with Alanine 1005 (amino acid numbering is based on the complete IgA1P amino acid sequence NMB0700) and comprises the Ser 267 Val mutation. A His-tag is added at the C-ter end, without spacer. The amino acid sequence of SP548 without the his-tag is shown in SEQ ID NO: 9
SP550 consists from N-ter to C-ter in (i) the IgA1P sequence exhibiting the Ser 267 Val mutation, starting with Alanine 28, ending with Glutamic acid 966 (amino acid numbering is based on the complete IgA1P amino acid sequence NMB0700) fused to (ii) the App sequence starting with Glutamine 1061 and ending with Alanine 1187 (amino acid numbering is based on the complete App amino acid sequence NMB 1985). A His-tag is added at the C-ter end, without spacer. The amino acid sequence of SP550 without the his-tag is shown in SEQ ID NO: 10
E. coli BL21 (DE3) strain (Novagen) was transformed with each of pSP503, pSP548 and pSP550 according to the supplier recommendations.
Transformed BL21 (DE3) E. coli strains are seeded at a ratio 1:500 in LB medium supplemented with kanamycin 30 pg/ml and cultured about at 37° C. under stirring (220 rpm) up to a O.D.600 nm of from 0.6 to 0.8. The IPTG is added at 1 mM final and induction is pursued 3 hrs at 37° C. Bacterial cells are harvested by centrifugation and pellets stored at −20° C.
Preparation of MC58 NalP SP509 and IgA1P, SP503, SP548 and SP550 Extracts for Purification on an IMAC Column.
The bacterial pellets corresponding to 500 ml of culture are gently washed in PBS and bacterial suspensions are centrifuged. Pellets are resuspended in PBS (SP509, SP503, SP550) or Tris HCl 50 mM (SP548); each buffer being complemented with lysosyme 100 μl/ml, MgCl2 1 mM, Triton X100 0.1%. Incubation is achieved at 4° C. 15 min under mild stirring.
Benzonase is added at about 1 unit/ml. Suspension are further incubated at 4° C. 15-30 min. For SP509 and SP548, the suspensions are then gently sonicated 1 min in ice and stirred 20 min at 4° C.
Suspensions are centrifuged 20 min at 30 000 g, 4° C. Pellets are resuspended in PBS (SP509, SP503, SP550) or Tris HCl 50 mM (SP548); each buffer being complemented with Triton X100 0.1% and urea 2 M. The suspensions are incubated for 1 hr at 4° C. under mild stirring and centrifuged 20 min at 30 000 g 4° C.
The SP509 pellets are resuspended in Tris-HCl 20 mM, NaCl 300 mM, Guanidine 6 M and DTT (dithiothreitol) 5 mM, pH 8.0. The suspensions are incubated for 1 hr and centrifuged 20 min at 30 000 g 4° C. Supernatants are recovered.
The SP503, SP548 and SP550 pellets are resuspended in Tris-HCl 50 mM, NaCl 300 mM, Urea 8 M, pH 9.0. The suspensions are incubated at 4° C. overnight under mild stirring and then centrifuged 20-30 min at 30 000 g 4° C. Supernatants are recovered.
Purification of MC58 NalP SP509
The supernatant is recovered and applied onto an IMAC column (HPLC Biorad Biologic) previously (i) washed with 6 column volumes of water and (ii) equilibrated with 3 column volumes of Buffer A (Tris-HCl 20 mM, NaCl 300 mM, Guanidine 6 M, DTT 1 mM, pH 8.0). 6 column volumes of Buffer A are added. Purification program once the supernatant is applied on the column, is as follows: 6 CV 100% buffer A; 3 CV gradient 100% buffer A→92%+0% buffer B (buffer A+250 mM imidazole)→8%; 6 CV 92% buffer A+8% buffer B; 6 CV gradient 92% buffer A→0%+8% buffer B→100%; 3 CV 100% buffer B.
Fractions containing SP509 eluted at about 20 mM imidazole are pooled and dialysed overnight against PBS urea 4 M to remove guanidine. SP509 solution is kept at −80° C.
Before use, SP509 in PBS urea 4 M is extemporaneously dialysed against PBS arginine 0.5 M for renaturation.
Purification of MC58 IgA1P SP503, SP548 and SP550
The supernatant is diluted vol./vol. to a final concentration of Tris 50 mM, NaCl 300 mM Urea 4 M, Zwittergent 3.14 1%, pH 9.0.
An IMAC column is prepared with 50 ml of a chelating gel charged with nickel. The column is equilibrated with buffer A (Tris-HCl 50 mM, NaCl 300 mM, Urea 4 M, pH 9.0) at a flow rate of 2 ml/min. This flow rate is applied to the following purification steps.
About 100-150 ml of the SP503, 548 or 550 solution to be purified are applied onto the equilibrated column.
About 3 column volumes of Buffer A are added. Then 3 column volumes of a gradient is applied to: 100 to 80% buffer A +20% buffer B (Buffer A +250 mM Imidazole). This is followed by (i) 3 column volumes of 80% buffer A +20% buffer B; and then (ii) 4 column volumes of buffer B.
The SP503 fractions eluted at 50 mM imidazole are pooled and dialysed overnight against PBS urea 4 M and stored at −80° C. After dialysis against 4 M urea, about 19 mg of SP503 are recovered (about 0.3 mg/ml). Before use, SP503 is renatured by extensive dialysis against Tris-HCl 20 mM, NaCl 150 mM, Arginine 0.5 M, pH 8.0. The final SP503 concentration is about 0.40 mg/ml.
The SP548 and SP550 fractions each elute at 250 mM imidazole. Fractions are pooled and dialysed overnight against Tris HCl 20 mM, NaCl 150 mM, urea 4 M, pH 8.0.
After dialysis against 4 M urea, about 50 mg of SP548 are recovered (about 3.40 mg/ml). The concentration is decreased to about 0.4 mg/mL. SP548 is renatured by extensive dialysis against Tris-HCl 20 mM, NaCl 150 mM, Arginine 0.5 M, pH 8.0 and stored at −80° C. (0.50 mg/ml).
After dialysis against 4 M urea, about 50 mg of SP550 are recovered (about 3.15 mg/ml). The concentration is decreased to about 0.7 mg/ml. SP550 is renatured by extensive dialysis against Tris-HCl 20 mM, NaCl 150 mM, Arginine 0.5 M, pH 8 and stored at −80° C. (0.75 mg/ml).
AF04 Adjuvant
AF04 is constituted with (i) an oil-in-water emulsion as described in WO 07/006939 and (ii) the Eisai product ER 804057 (also known as E6020, described in U.S. Pat. No. 7,683,200) which is a TLR4-agonist. AF04 in PBS is more particularly described in Examples 1 and 2 of WO 07/080308.
Liposomes
Liposomes LPS L8
LPS L8 and liposomes LPS L8 are produced as described in WO 10/130896. The final liposome LPS L8 preparation for use in the third experiment (see below) contains 108 μg/ml LPS L8, 4.7 mg/ml EDOPC and 2.20 mg/ml DOPE in Tris 10 mM, NaCl 150 mM, merthiolate 0.001%, pH 7.2.
Empty Liposomes (Liposomes without LPS L8)
Empty liposomes are prepared as liposomes LPS L8 with the proviso that the volume of LPS L8 at 1 mg/ml in Tris 10 mM, octyl β-D-glucopyranoside (OG) 100 mM (added to the EDOPC:DOPE suspension) is replaced by an identical volume of Tris 10 mM, OG 100 mM. The final empty liposome preparation for use in the third experiment (see below) contains 4.7 mg/ml EDOPC and 2.51 mg/ml DOPE in Tris 10 mM, NaCl 150 mM, merthiolate 0.001%, pH 7.2.
Immunogenicity. Bactericidal Activity & Flow Cytometry Analysis
Bacterial Strains and Growth Conditions
A set of 36 wild-type serogroup B N. meningitidis strains isolated from geographically distinct locations at different dates of isolation and representing diverse MLST clonal complexes were selected for this study. They are listed in Table 5. The majority of the strains were kindly provided by Drs D. A Caugant (NIPH, Norway), D. Martin (EZR, New-Zealand), M. K Taha (IP, Paris), M. A. Diggle (SHLMPRL, Scotland), L. Saarinen (NPHI, Finland).
MenB strains were grown overnight at 37° C. with 10% CO2 on Brain Heart Infusion (BHI) agar (Difco) plates. Then, the bacteria were harvested from plates and inoculated into BHI broth (Difco) alone or supplemented with or without 30 μM desferal which is a chelator of divalent cations. Cultures were analysed after 2.5 hrs that correspond to an early exponential growth phase.
C: Dr D. A. Caugeant, NIPH, Norway; D: Dr M. A. Diggle, SHLMPRL, Scotland; M: Dr D. Martin, ESR New-Zealand; S: Dr L. Saarinen, NPHI, Finland; T: Dr M. K. Taha, I P, Paris: S Pasteur: Sanofi Pasteur.
The following Table 6 describes more precisely the strains listed, in particular with regard to the IgA1P, TbpB and NalP expression.
Production of Mouse or Rabbit Antisera
To obtain specific immune sera, outbred CD1 mice or rabbits were immunized 3 times on days 0, 21 and 35 (mouse) or 42 (rabbit), by subcutaneous (mouse) or intramuscular (rabbit) route, with 10 pg/mouse or rabbit of the antigen of interest under a volume of 0.2 ml, in the presence of various adjuvants.
When more than one antigen were concomitantly administered, the two or three antigens were respectively administered as bivalent or trivalent preparation.
Blood samples were collected on day 42 (mouse) or 56 (rabbit). Blood samples were collected in Vacutainer vials containing a coagulation activator and a serum separator gel (BD, Meylan France). Tubes were centrifuged for 20 min at 2600 g in order to separate serum from cells. Sera were transferred into Nunc tubes and de-complemented by heat-inactivation for 30 min at 56° C. They were stored at −20° C. until the assays were performed.
IgG Purification from Rabbit Antisera
IgGs were purified on an Hi Trap rProtein A FF column (GE Healthcare) using the AKTAdesign™ system chromatography, according to the supplier recommendations.
Serum Bactericidal Activity Assay
N. meningitidis strains were grown overnight at 37° C. with 10% CO2 on BHI agar (Difco) plates. The bacteria were then harvested from the plates and inoculated into BHI broth (Difco) alone or supplemented with 30 pM desferal which is a chelator of divalent cations so that TbpB be expressed. The cultures were analyzed after 2.5 hours, which corresponds to early exponential growth phase. The bactericidal activity of specific mouse sera was evaluated using as complement source pooled baby rabbit serum as described earlier with slight modifications (Rokbi et al., Clin. Diagnostic Lab. Immunol. (September 1997) 4 (5): 522). Briefly, 50 μl of two-fold serial dilutions of IgG solutions or serum were added to 96-well microtiter plates (Nunc) and incubated with 25 μl of a meningococci suspension adjusted to 4×103 CFU/ml and 25 μl of baby rabbit complement. After 1 hr of incubation at 37° C., 50 μl of the mixture from each well was plated onto MHA plates. The plates were incubated overnight at 37° C. in 10% CO2. The bactericidal titer of each serum or IgG preparation was expressed as the inverse of the last dilution of serum at which ≥50% killing was observed compared to the complement control.
The SBA assay is commonly acknowledged as a surrogate of protection for N. meningitidis vaccines. When the SBA titer is superior or equal to 16 in homologous SBA assay, or superior or equal to 8 in heterologous SBA, protection is considered to be met.
Flow Cytometry Analysis
The ability of polyclonal antisera elicited by the recombinant proteins to bind to the surface of live MenB strains was determined using a flow cytometric detection of indirect fluorescence assay. A culture sample was centrifuged and washed once with 1×PBS (Eurobio). The final pellet was resuspended in PBS with 1% of bovine serum albumin (BSA, Eurobio) at a density of 108 CFU/ml. To 20 μl of bacteria, 20 μl of dilutions of pooled serum were added in 96 deep-well plate (Ritter). For each pool of serum, several dilutions were tested on a range going from 1/5 to over 1/2000. The plate was incubated for 1h at 37° C. with shaking. The bacteria were centrifuged, washed once with PBS 1% BSA and resuspended with 100 μl of goat anti-mouse IgG (H and L chains) conjugated to fluorescein isothiocyanate (FITC) (Southern Biotech) diluted 100-fold. The plate was incubated for 30 minutes at 37° C. with shaking in the dark. The bacteria were washed twice with PBS 1% BSA and fixed with 0.3% formaldehyde in PBS buffer overnight at +4° C. in the dark. The bacteria were centrifuged, the formaldehyde solution was discarded and the bacteria were finally washed once and dissolved in PBS 1% BSA. The fluorescent staining of bacteria was analysed on a Cytomics FC500 flow cytometer (Beckman Coulter). The fluorescent signal obtained for bacteria incubated with the polyclonal antisera or purified IgGs thereof specific for proteins injected with adjuvant was compared to the signal obtained for bacteria incubated with the antisera of mice injected with buffer+adjuvant.
B—Results
Mouse antisera raised against one or several antigens were individually assayed for serum bactericidal activity (SBA) against the homologous strain or as a pool against a panel of heterologous strains. In addition, pools of sera were assessed for their ability to recognize the targeted protein at the surface of live bacterial cells using flow cytometry (FACS analysis).
In a first experiment, three monovalent preparations comprising each the M982 His-TbpB, the MC58 NalP SP509 and the MC58 IgA1P SP503 administered as a single product were compared to:
(A) three bivalent preparations comprising:
(B) a trivalent preparation comprising M982 His-TbpB, MC58 NalP SP509 and MC58 IgAIP SP503.
All of these proteins were produced as described above. Monovalent, bivalent and trivalent compositions (10 pg per protein in 200 μl per injection) were prepared in PBS pH 7.0, 2.5% squalene, 4 μg TLA4E (AF04).
Two negative controls were added: PBS with or without the adjuvant AF04 (2.5% squalene, 4 μg TLA4E).
SBA Against Strain MC58
Groups of 10-15 mice were immunized as described above with one of the monovalent, bivalent or trivalent compositions. Sera were collected as described above and individually assayed in the SBA test against strain MC58 grown 2.5 hrs in the presence of Desferal. Results were as shown in the following table 7, expressed in terms of (i) geometric mean titers (GMTs); (ii) percentage of responders exhibiting an antiserum with an SBA titer superior or equal to 16; and (iii) fold increase of the geometrical mean titers compared to the corresponding buffer.
The monovalent M982 His-TbpB induced a strong SBA activity against the homologous strain (not shown in Table 7). The monovalent NalP SP509 and IgA1P SP503 both induced high SBA titers against the homologous strain MC58.
SBA titers against MC58 (GMTs) induced by bivalent compositions were similar to those obtained with the respective monovalent compositions, indicating that no negative interference occurred when the antigens were mixed. Additionally, the percentage of outbred CD1 murine responders was >90%; which is considered as a very positive result.
The highest GMT was obtained with the trivalent composition.
Heterologous SBA and Flow Cytometry Analysis
The pools of sera raised to the constructs were assessed for bactericidal activity against a panel of 26 strains including the homologous strains MC58 and M982, and representative of the major epidemiological clusters (ST32, ST11, ST41/44, ST8 and ST269). SBA results were expressed in term of fold-increase (FI) of the geometric mean titers compared to the negative control including the AF04 adjuvant. It is considered that cross-bactericidal activity is met when the fold-increase is superior or equal to 8. Surface Exposure (SE) is expressed in terms of detection level ranging from [—] to [++++] depending on the highest dilution of the pooled antisera at which surface exposure is detected: [—] at a dilution <1/20e; [+] at a 1/20e dilution: [++] at a 1/200e dilution; [+++] at a 1/2000e dilution; and [++++] at a dilution >1/2000e.
Results are shown in
The compositions of the invention give satisfactory immune responses. A coverage superior or equal to 85% is considered as being very satisfactory.
In a second experiment, three monovalent preparations comprising each a different MC58 IgA1P construct (SP503, SP548 or SP550) administered as a single product were compared with trivalent preparations comprising (i) MC58 IgA1P SP503 or SP548, or MC58 IgA1P-App fusion construct SP550; (ii) MC58 NalP SP509; and (iii) M982 TbpB.
Two negative controls were added: PBS with or without the adjuvant AF04 (2.5% squalene, 4 μg TLA4E).
M982 TbpB was recombinantly expressed from E. coli BL21 (DE3) transformed with pSP314, purified from the small scale lot and formulated in PBS as described above. Monovalent composition for injection is prepared at 10 μg/dose (200 μl) in PBS, 2.5% squalene, 4 pg TLA4E (AF04).
SP509, SP503, SP548 and SP550 were produced as described above. Monovalent SP509, SP503, SP548 and SP550 compositions as well as trivalent compositions (10 pg per protein in 200 μl per injection) were prepared in Tris 20 mM, NaCl 150 mM, Arginine 0.5 M, pH 8.0, 2.5% squalene, 4 pg TLA4E (AF04).
Homologous SBA
Groups of 10-15 mice were immunized as described above with one of the monovalent or trivalent compositions. Sera were collected as described above and individually assayed in the SBA assay against the homologous strain MC58 or strain M982 (strain MC58 is M982-like) grown 2.5 hrs in the presence of Desferal. Results were as shown in the following table 9, expressed in terms of (i) geometrical means titers (GMTs); (ii) percentage of responders exhibiting an antiserum with an SBA titer superior or equal to 16; and (iii) fold increase of the geometrical mean titers compared to the corresponding buffer.
Results shown in Table 9 reveal that:
Heterologous SBA and Flow cytometry (FACS) Analysis
The pools of sera raised to the constructs were assayed for bactericidal activity against a panel of 20 strains including the homologous strains MC58 and M982. SBA results were expressed in term of fold-increase of the geometrical mean titers compared to the negative control including the AF04 adjuvant. It is considered that cross-bactericidal activity is met when the fold-increase is superior or equal to 8. The ability of the pools of sera to bind to the bacterial surface was also examined. Surface Exposure (SE) is expressed in terms of detection level ranging from [—] to [++++] depending on the highest dilution of the specific pooled antisera at which surface exposure is detected: [—] at a dilution <1/20e; [+] at a 1/20e dilution: [++] at a 1/200e dilution; [+++] at a 1/2000e dilution; and [++++] at a dilution >1/2000e.
Results are shown in
In a third experiment, three quadrivalent compositions were assayed for cross-SBA. They were as follows:
M982 TbpB+MC58 NalP SP509+MC58 IgA1P-App fusion construct SP550+B16B6 TbpB lipidated+liposomes LPS L8 [negative control: liposomes without LPS (empty liposomes)]
M982 TbpB+MC58 NalP SP509+MC58 IgA1P-App fusion construct SP550+B16B6 His-TbpB+AF04 [negative control: AF04 in PBS]
M982 TbpB+MC58 NalP SP509+MC58 IgA1P-App fusion construct SP550+B16B6 His-TbpB+empty liposomes [negative control: liposomes without LPS (empty liposomes)].
The primary objective of the experiment was to evaluate the adjuvant effect of L8 LPS formulated in liposomes, co-injected with the lipidated B16B6 TbpB produced as described above, on the antibody responses raised against M982 TbpB, MC58 NalP SP509 and MC58 IgA1P-App fusion construct SP550. This adjuvant effect of LPS liposomes was also compared to the adjuvant effect of the adjuvant formulation AF04.
The second objective was to compare the vaccine coverage measured by cross-SBA generated by compositions A, B and C.
M982 TbpB was recombinantly expressed from E. coli BL21 (DE3) transformed with pSP314, purified from the pilot scale lot and formulated in Tris 50 mM, NaCl 100 mM, sucrose 5%, pH 7.5, as described above. MC58 IgA1P-App fusion construct SP550, MC58 NalP SP509, B16B6 TbpB and B16B6 His-TbpB were produced as described above.
Compositions contained 10 pg of each protein and (i) 40 pg of liposomes under a volume of 500 μl or AF04 (2.5% squalene, 10 μg TLA4E/dose). The dilution buffer for liposomes was Tris 10 mM, NaCl 150 mM, merthiolate 0.001%, pH 7.2.
Rabbits were immunized intramuscularly (2 sites; 2×250 μl) at DO, D21 and D42. Sera are collected at D56 and IgGs purified as described above. Purified IgGs are used in the SBA test described above against a panel of 30 strains. SBA results were expressed in term of fold-increase (FI) of the geometric mean titers compared to that the appropriate negative control. It is considered that cross-bactericidal activity is met when the fold-increase is superior or equal to 8. The ability of the IgGs purified from the pooled antisera to bind to the bacterial surface was also examined. Surface Exposure (SE) is expressed in terms of detection level ranging from [—] to [++++] depending on the highest dilution of the specific purified IgGs at which surface exposure is detected: [—] at a dilution <1/20e; [+] at a 1/20e dilution: [++] at a 1/200e dilution; [+++] at a 1/2000e dilution; and [++++] at a dilution >1/2000e.
Results are shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 14306978 | Dec 2014 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2015/079021 | 12/8/2015 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2016/091902 | 6/16/2016 | WO | A |
| Number | Date | Country |
|---|---|---|
| 2207302 | Jun 1997 | CA |
| 2267066 | Apr 1999 | CA |
| WO 9713860 | Apr 1997 | WO |
| WO 9907741 | Feb 1999 | WO |
| WO 0071725 | Nov 2000 | WO |
| WO 2004014418 | Feb 2004 | WO |
| WO 2005032583 | Apr 2005 | WO |
| WO 2011051893 | May 2011 | WO |
| WO 2016091890 | Jun 2016 | WO |
| WO 2016091912 | Jun 2016 | WO |
| Entry |
|---|
| Van Ulsen et al (Molecular Microbiology vol. 50 (3), pp. 1017-1030) (Year: 2003). |
| Harrison et al (BMC Microbiology vol. 8 (66) pp. 1-10) (Year: 2008). |
| Sequence Listing from PCT published application No. WO 2011/051893 published May 5, 2011. |
| Sequence Listing from PCT published application No. WO 99/07741 published Feb. 18, 1999. |
| International Search Report dated Apr. 28, 2016 of PCT/EP2015/079021 filed Dec. 8, 2015, 9 pages. |
| European Search Report dated May 6, 2015 of European Application No. 14306978 filed Dec. 9, 2014, 7 pages. |
| Number | Date | Country | |
|---|---|---|---|
| 20180125959 A1 | May 2018 | US |
