NEISSERIA MENINGITIDIS TRYPSIN-LIKE SERINE PROTEASE POLYPEPTIDES AND COMPOSITIONS THEREOF
20180125961 · 2018-05-10
Inventors
- Nadège ARNAUD-BARBE (Saint Sorlin, FR)
- Geneviève RENAULD-MONGENIE (Chaponost, FR)
- Bachra Rokbi (Lyon, FR)
Cpc classification
C07K2319/40
CHEMISTRY; METALLURGY
C12Y304/21072
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to novel polypeptides derived from Neisseria meningitidis proteins, in particular auto-transporters of the trypsin-like serine protease subclass, such as IgA1P, App and AusI, and their use in immunogenic compositions i.a., vaccine compositions for the prevention and/or treatment of meningococcal infections. In particular, it provides fragments of IgA1P, App and AusI and polypeptides comprising or consisting of these fragments and fusions thereof, which may be used in immunogenic compositions, for example vaccine compositions.
Claims
1. An isolated polypeptide comprising or consisting of: (I): (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 -peptide domain of a trypsin-like serine protease auto-transporter of N. meningitidis; or (iii) a protease domain, an -peptide domain and a part of a -domain 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 (II) a first fragment fused to a second fragment: (1) said first fragment consisting of: a protease domain or a protease sub-domain of a first trypsin-like serine protease auto-transporter of N. meningitidis, or a mutant of a protease domain or a protease sub-domain of a first trypsin-like serine protease auto-transporter of N. meningitidis 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, (2) said second fragment consisting of: an -peptide domain of a second trypsin-like serine protease auto-transporter of N. meningitidis; 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.
2. An isolated polypeptide according to claim 1, wherein said fragment consists of the protease domain of the trypsin-like serine protease auto-transporter of N. meningitidis which is IgA1P, App or AusI.
3. An isolated polypeptide according to claim 1, wherein said fragment consists of a protease domain and all or part of an -peptide domain of the trypsin-like serine protease auto-transporter of N. meningitidis which is IgA1P, App or AusI.
4. An isolated polypeptide according to claim 1, wherein said fragment consists of the protease domain, the -peptide domain and a part of a -domain, of the trypsin-like serine protease auto-transporter of N. meningitidis which is IgA1P, App or AusI.
5. An isolated polypeptide according to claim 1, comprising or consisting 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 -peptide domain of said second trypsin-like serine protease auto-transporter.
6. An isolated polypeptide according to claim 5, wherein said first trypsin-like serine protease auto-transporter is IgA1P and said second trypsin-like serine protease auto-transporter is App or AusI.
7. An isolated polypeptide according to claim 2, which has 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.
8. An isolated polypeptide according to claim 4, which has 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.
9. An isolated polypeptide according to claim 4, which has 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: 3 starting from position 40, 41, 42, 43, 44, 45 or 46 and ending at position 1220, 1220, 1221, 1223, 1224, 1225, 1226 or 1227.
10. An isolated polypeptide according to claim 4, which has 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.
11. An isolated polypeptide according to claim 1, comprising or consisting of a first fragment fused to a second fragment wherein said first fragment has 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 wherein said second fragment has at least 90% identity with an amino acid sequence of (i) App of N. meningitidis MC58 shown in SEQ ID NO: 3 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: 5 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.
12. An isolated polypeptide according to claim 7, wherein said first fragment has 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 wherein said second fragment has at least 90% identity with an amino acid sequence of (i) App of N. meningitidis MC58 shown in SEQ ID NO 3: 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 5: 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.
13. An isolated polypeptide according to claim 8, wherein said first fragment has 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 wherein said second fragment has at least 90% identity with an amino acid sequence of (i) App of N. meningitidis MC58 shown in SEQ ID NO 3: 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 3: 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 5: 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 5: 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.
14. An isolated polypeptide according to claim 1, wherein the serine protease activity is inactivated by amino acid substitution in the catalytic triad or in the serine protease motif.
15. An isolated polypeptide according to claim 14, wherein the amino acid substitution occurs at the serine residue of the catalytic triad.
16. A nucleic acid encoding a polypeptide according to claim 1.
17. A vector comprising a nucleic acid according to claim 16.
18. A host cell comprising a nucleic acid according to claim 16.
19. (canceled)
20. (canceled)
21. A vaccine composition comprising a polypeptide according to claim 1.
22. A method of production of a polypeptide according to claim 1, the method comprising expression of said polypeptide from a vector comprising a nucleic acid encoding the polypeptide of claim 1.
23. An isolated polypeptide according to claim 4, wherein said fragment consists of the protease domain, the -peptide domain and the two first -sheets of the -domain, of the trypsin-like serine protease auto-transporter of N. meningitidis which is IgA1P, App or AusI.
24. A host cell comprising a vector according to claim 17.
25. A method of treating N. meningitidis B infection in a subject, the method comprising administering to the subject an effective amount of a polypeptide according to claim 19.
Description
BRIEF DESCRIPTION OF THE FIGURES
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[0325]
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[0327]
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[0330]
[0331]
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BRIEF DESCRIPTION OF THE SEQUENCE LISTING
SEQ ID NO:
[0336] 1 Amino acid sequence of IgA1 protease from N. meningitidis B strain MC58
2 Coding nucleic acid sequence of IgA1 protease from N. meningitidis B strain MC58
3 Amino acid sequence of App from N. meningitidis B strain MC58
4 Coding nucleic acid sequence of App from N. meningitidis B strain MC58
5 Amino acid sequence of AusI from N. meningitidis B strain MC58
6 Coding nucleic acid sequence of AusI from N. meningitidis B strain MC58
EXPERIMENTAL
AMaterial & Methods
Constructs
[0337] The sequence information with respect to the iga, app and ausI genes of the N. meningitidis MC58 genome (respectively NMB0700, NMB1985 and NMB1998) was retrieved 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. This sequence information is particularly useful for designing the primers.
[0338] The genomic DNA of strain MC58 was purified using a purification kit (Roche). In order to generate constructs, ORFs (open reading frame) were amplified by PCR from the purified genomic DNA using appropriate primers. 5 or 3 primers were designed so that a His-tag may be introduced (and when appropriate a spacer between the His-tag and the N-ter amino acid of the protein) as well as restriction sites. Then the PCR products were first cloned in an intermediate cloning vector and then transferred into the expression plasmid pET-cer which is a pET-28 plasmid (Novagen) stabilized by insertion of a stabilizing element (cer fragment).
[0339] A large number of IgA1P constructs were produced: SP502, SP503, SP528, SP530, SP531, SP532, SP533, SP548, SP550. They are more particularly described as follows:
[0340] SP502 and SP503 both start with Alanine 28 and ends with Alanine 1584 (amino acid numbering is based on the complete IgA1P amino acid sequence NMB0700). 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). SP503 further comprises the Ser 267 Val mutation.
[0341] SP528 and SP548 both start with Alanine 28 and end with Alanine 1005 (amino acid numbering is based on the complete IgA1P amino acid sequence NMB0700) and comprise the Ser 267 Val mutation. In SP528, 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). In SP548, a His-tag is added at the C-ter end, without spacer.
[0342] SP530 and SP550 both consist from N-ter to C-ter in (i) the IgA1P sequence exhibiting the Ser 267 Val mutation, starting with Alanine 28 and 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). In SP530, 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). In SP550, a His-tag is added at the C-ter end, without spacer.
[0343] SP532 consist from N-ter to C-ter in (i) the IgA1P sequence exhibiting the Ser 267 Val mutation, starting with Alanine 28 and 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 Serine 1224 (amino acid numbering is based on the complete App amino acid sequence NMB1985). 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).
[0344] SP531 and SP533 consist from N-ter to C-ter in (i) the IgA1P sequence exhibiting the Ser 267 Val mutation, starting with Alanine 28 and ending with Glutamic acid 966 (amino acid numbering is based on the complete IgA1P amino acid sequence NMB0700) fused to (ii) the AusI sequence starting with Alanine 974 and ending with Alanine 1161 (SP531) or Serine 1198 (amino acid numbering is based on the complete AusI amino acid sequence NMB1998). 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).
[0345] App and AusI constructs were also produced: respectively (i) SP534, SP535, and (ii) SP536, SP537. They are described as follows:
[0346] SP534 and SP535 both start with Glycine 43 and end with Serine 1224 (amino acid numbering is based on the complete App amino acid sequence NMB1985). 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). SP535 further comprises the Ser 267 Val mutation.
[0347] SP536 and SP537 both start with Serine 26 and end with Serine 1198 (amino acid numbering is based on the complete AusI amino acid sequence NMB1998). A His-tag is added at the N-ter end, separated from Serine 26 by a spacer constituted with four glycines and one serine (N-ter to C-ter). SP537 further comprises the Ser 241 Val mutation.
[0348] As a matter of additional guidance, the construction of the expression plasmids for SP502 and SP503 is further described as follows:
[0349] First the ORF is amplified by PCR from the purified MC58 genomic DNA, using appropriate primers and the Platinum Pfx Polymerase (Invitrogen) according to the protocol of the supplier. Then the PCR product is cloned into the intermediate vector: PCR Blunt TOPO vector (PCR-TOPO-BluntII) according to the protocol of the supplier. The ligation product is transformed into competent cells TOP10 bacteria supplied with the kit (Invitrogen). The selection of recombinant clones is performed on LB+kanamycin. The plasmid is checked by enzymatic digestion and verification of the restriction profile. The sequencing of the insert validates the plasmid.
[0350] Then the ORF is extracted from the intermediate vector by appropriate enzymatic digestion (double digestion enzyme NcoI+BamHI restriction sites) using the originally inserted restriction sites for transfer into an expression plasmid. The extracted fragment of interest is isolated by agarose gel migration and cutting the corresponding bands and then purified by electro-elution. Plasmid pET-cer is prepared using the same protocol. The extracted fragment of interest is then assembled with the pET-cer using T4 DNA ligase (Invitrogen) according to the protocol of the supplier to give the expression plasmid pSP502. The ligation product is transformed into competent TOP10 bacteria. The selection of recombinant clones is performed on LB+kanamycin. The resulting plasmid pSP502 is checked by enzymatic digestion and verification of the restriction profile. The sequencing of the insert validates the plasmid in which the ORF is placed.
[0351] In order to produce the plasmid able to express the ORF encoding SP503 a further mutagenesis step to suppress the active catalytic site is achieved as follows:
[0352] Overlap extension PCR using the pSP502 is performed to amplify the ORF in two overlapping PCR fragments. The overlapping central primers were designed to introduce the mutation. A third reaction was then used to assemble the first two fragments into one. The reactions are performed with Platinum Taq DNA Polymerase High Fidelity (Invitrogen) according to the protocol of the supplier. The two overlapping central primers also insert an original restriction site to facilitate the selection of clones carrying the mutation. The mutated ORF is selected using the restriction site created during the mutagenesis; and substituted for the corresponding non-mutated ORF into pSP502 to give SP503.
[0353] The nucleotide sequences encoding the IgA1P constructs SP528 and SP548; the App constructs SP534 and SP535; the AusI constructs SP536 and SP537; as well as the IgA1P fusion constructs SP530, SP531, SP532, SP533 and SP550; were conceived and designed by bioinformatics using the software pack Vector NTI (Invitrogen) and accordingly, chemically synthetized de novo (Geneart). The synthetized sequences were cloned in an intermediate plasmid of pUC type; then transferred into the pET-cer plasmid to be placed under the control of the T7 promoter (from pET-28).
Transformation into the Expression Strain
[0354] The expression plasmids were transformed into the expression E. coli strain BL21 (DE3) (Novagen) according to the protocol of the supplier. The selection of recombinant clones was performed on LB+kanamycin.
Protein Expression and Purification
Cell Culture
[0355] BL21 (DE3) E. coli strains transformed by one of the plasmids pSP502, pSP503, pSP528, pSP530, pSP531, pSP532, pSP533, pSP548, pSP550, pSP534, pSP535, pSP536 and pSP537 were seeded at a ratio 1:500 in Luria Bertani broth (LB) medium supplemented with kanamycin 30 g/ml and 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 the induction is pursued at 37 C. for 3 hrs. Bacterial cells are harvested by centrifugation and pellets stored at 20 C.
Purification of Recombinant Proteins
[0356] Upon thawing, bacteria were washed in PBS and centrifuged. Pellet (P0) was resuspended in a buffer (PBS or Tris-HCl pH 8) containing lysozyme 100 g/ml, MgCl2 1 mM and Triton X-100 0.1%; and incubated 15 min at 4 C. The suspension was viscous, free of visible aggregates and slightly translucent. Benzonase (1 U/ml final) was then added and the mixture was sonicated. The viscosity of the suspension must have disappeared. The insoluble proteins were pelleted after centrifugation. The pellet (P1) was resuspended in a buffer (PBS or Tris-HCl, pH 8) containing Triton X-100 0.1% and Urea 2 M. After centrifugation, the pellet (P2) was resuspended in a buffer (Tris-HCl 50 mM, pH 9) containing NaCl 300 mM, Urea 8 M and, optionally Triton X-100 0.1%. Upon centrifugation, the supernatant containing the His-tag protein was recovered and diluted to Urea 4 M while adding Tris-HCl 50 mM pH 9, NaCl 300 mM and zwittergent 3.14 (1% final).
[0357] The diluted supernatant was further purified by nickel chelation chromatography on an IMAC column using an imidazole elution gradient (0 to 250 mM). Elution fractions containing the protein were pooled and extensively dialysed against a buffer (Tris-HCl 20 mM, NaCl 150 mM or PBS pH 8) containing Urea 4 M to remove imidazole. Refolding was achieved by further dialysis against a buffer (Tris-HCl 20 mM, NaCl 150 mM or PBS pH 8) containing arginine 0.5 M.
[0358] In what follows, the purification of MC58 IgA1 protease SP503, SP548 and SP550 is more particularly described as an additional matter of example.
Preparation of MC58 IgA1P, SP503, SP548 and SP550 Extracts for Purification on an IMAC Column.
[0359] The bacterial pellets corresponding to 500 ml of culture are gently washed in PBS and bacterial suspensions are centrifuged. Pellets (P0) are resuspended in PBS pH 8 (SP503, SP550) or Tris-HCl 50 mM pH 8 (SP548); each buffer being complemented with lysosyme 100 l/ml, MgCl2 1 mM, Triton X100 0.1%. Incubation is achieved at 4 C. 15 min under mild stirring.
[0360] Benzonase is added at about 1 unit/ml. Suspension are further incubated at 4 C. 15-30 min. For SP548, the suspensions are then gently sonicated 1 min in ice and stirred 20 min at 4 C.
[0361] Suspensions are centrifuged 20 min at 30 000 g, 4 C. Pellets (P1) are resuspended in PBS pH 8 (SP503, SP550) or Tris-HCl 50 mM pH 8 (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 SP503, SP548 and SP550 pellets (P2) are resuspended in Tris-HCl 50 mM, NaCl 300 mM, Urea 8 M, pH 9.0 (complemented with Triton X-100 0.1% for SP548 and SP550). 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 IgA1P SP503, SP548 and SP550
[0362] The supernatant is diluted to a final concentration of Tris-HCl 50 mM, NaCl 300 mM, Urea 4 M, pH 9.0. Zwittergent 3.14 is added to 1% final.
[0363] An IMAC column (Chelating Sepharose Fast Flow from GE HealthCare) is prepared with 50 ml of a chelating gel charged with nickel (NiSO4 10% in water). 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.
[0364] About 100-150 ml of the SP503, SP548 or SP550 diluted supernatant to be purified are applied onto the equilibrated column.
[0365] About 3 column volumes of Buffer A are added. Then 3 column volumes of a gradient is applied to: 100 to 80% buffer A+0% to 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.
[0366] 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.
[0367] 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.
[0368] 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).
[0369] 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).
Immunogenicity, Bactericidal Activity & Flow Cytometry Analysis
Bacterial Strains and Growth Conditions
[0370] A set of 26 wild-type serogroup B N. meningitidis isolates that were isolated from geographically distinct locations at different date of isolation and that represented diverse MLST clonal complexes were selected for this study. They are listed in Table 3. 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).
[0371] MenB strains were grown overnight at 37 C. with 10% CO.sub.2 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 M desferal which is a chelator of divalent cations. Cultures were analyzed after 2.5 hours that correspond to an early exponential growth phase.
Production of Mice Antisera
[0372] To obtain specific immune sera, outbred CD1 mice were immunized 3 times on days 0, 21 and 35, by subcutaneous route, with 10 g/mouse of the antigen of interest co-injected with adjuvant AF04 [oil-in-water emulsion as described in WO 07/006939, containing the Eisai product ER 804057 (also known as E6020, described in U.S. Pat. No. 7,683,200) as TLR4-agonist. AF04 is described in Examples 1 and 2 of WO 07/080308].
[0373] Blood samples were collected on day 42. 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 heat-inactivated for 30 min at 56 C. They were stored at 20 C. until the assays were performed.
Serum Bactericidal Activity Assay
[0374] N. meningitidis strains were grown overnight at 37 C. with 10% CO.sub.2 on BHI agar (Difco) plates. The bacteria were then harvested from the plates and inoculated into BHI broth (Difco) alone or supplemented with 30 M desferal which is a chelator of divalent cations. 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. (1997) 4 (5): 522). Briefly, 50 l of two-fold serial dilutions of serum were added to 96-well microtiter plates (Nunc) and incubated with 25 l of a meningococci suspension adjusted to 410.sup.3 CFU/ml and 25 l of baby rabbit complement. After 1 hr of incubation at 37 C., 50 l of the mixture from each well was plated onto MHA plates. The plates were incubated overnight at 37 C. in 10% CO.sub.2. The bactericidal titer of each serum was expressed as the inverse of the last dilution of serum at which 50% killing was observed compared to the complement control.
[0375] The SBA assay is commonly acknowledged as a surrogate of protection for vaccines against N. meningitidis. 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
[0376] 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 1PBS (Eurobio). The final pellet was resuspended in PBS with 1% of bovine albumin (BSA, Eurobio) at a density of 10.sup.8 CFU/ml. To 20 l of bacteria, 20 l of dilutions of pooled serum were added in 96 deep-well plate (Ritter). For each serum, 3 dilutions were tested on a range going from 1/5 to 1/1000. The plate was incubated for 1 h at 37 C. with shaking. The bacteria were centrifuged, washed once with PBS 1% BSA and resuspended with 100 l of goat anti-mouse IgG (H and L chains) conjugated to 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 analyzed on a Cytomics FC500 flow cytometer (Beckman Coulter). The fluorescent signal obtained for bacteria incubated with the polyclonal antisera specific for proteins injected with adjuvant was compared to the signal obtained for bacteria incubated with the antisera of mice injected with buffer+adjuvant.
[0377] 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.
BResults
[0378] All the constructs were administered to mice in the presence of adjuvant AF04. Polyclonal antisera thereof were individually assayed for serum bactericidal activity (SBA) against 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 viable bacterial cells using flow cytometry (FACS analysis).
[0379] The individual sera raised to the constructs were first assayed for bactericidal activity against the homologous strain MC58. Results are expressed in terms of (i) GMTs (geometric mean titers), (ii) number of responders exhibiting a bactericidal titer superior or equal to 16 and, (iii) seroconversion compared to the negative control (fold-increase). Results are shown in Table 4 below. As used in the tables and Figures herein, the terms seroconversion, seroconversion compared to the control, seroconversion compared to the corresponding buffer and fold-increase are to be considered equivalent.
[0380] The constructs were also assayed for bactericidal activity against a panel of heterologous strains (Cross-bactericidal activity). Results are expressed in terms of seroconversion compared to a negative control (fold-increase). It is considered that cross-bactericidal activity is encountered when the fold-increase is superior or equal to 8. Results are summarized in Table 5 and detailed in
TABLE-US-00005 TABLE 4 SBA against the homologous strain MC58 Homologous SBA data GMT (% of responders 16) His- Fold- Construct Control Control Experiment Constructs tag increase under test 1 2 A SP9 IgA1P (28- N-ter X 1.2 2.8 (0%) 2.3 (0%) 2.6 (0%) 972) Ser 267 SP502 IgA1P (28- N-ter X 180.8 415.9 (100%) 1584) Ser 267 SP503 IgA1P (28- N-ter X 238.6 548.7 (100%) 1584) Val 267 C SP503 IgA1P (28- N-ter X 79.0 955.4 (100%) 12.1 (30%) 4.9 (10%) 1584) Val 267 SP528 IgA1P (28- N-ter X 9.1 109.7 (100%) 1005) Val 267 SP530 IgA1P (28- N-ter X 4 48.5 (100%) 966) - App (1061-1187) Val 267 SP531 IgA1P (28- N-ter X 4.9 59.7 (90%) 966) - Ausl (974-1161) Val 267 SP532 IgA1P (28- N-ter X 1.72 21.1 (60%) 966) - App (1061-1224) Val 267 SP533 IgA1P (28- N-ter X 3.5 42.2 (80%) 966) - Ausl (974-1198) Val 267 D SP503 IgA1P (28- N-ter X 157 362 (100%) 2.3 (0%) 2.5 (0%) 1584) Val 267 SP548 IgA1P (28- C-ter X 111 256 (100%) 1005) Val 267 SP550 IgA1P (28- C-ter X 111 256 (100%) 966) - App (1061-1187) Val 267 E SP534 App (43-1224) N-ter X 16.2 42.2 (80%) 2.6 3.0 Ser 267 SP535 App (43-1224) N-ter X 32.5 84.4 (90%) Val 267 SP536 Ausl (26- N-ter X 3 8.0 (30%) 1198) Ser 241 SP537 Ausl (26- N-ter X 1.6 4.3 (20%) 1198) Val241
[0381] Results are discussed in more details below:
Mutated, Truncated and/or Hybrid IgA1P
SBA Against the Homologous Strain
[0382] A first set of truncated IgA1Ps, less hydrophobic than natural complete IgA1P, were produced and tested for SBA with the aim of determining the extent of truncation that would lead to positive SBA. Therefore, a short IgA1P was made the amino acid sequence of which corresponds to the protease domain with a deletion of about 30 amino acids at its C-terminal extremity (construct SP9). A much longer IgA1P was also made the amino acid sequence of which corresponds to the full-length IgA1P sequence deleted of all beta sheets except the 2 first ones (construct SP502). In addition to this, the catalytic site was inactivated by replacing Ser 267 with Alanine in SP502, leading to SP503.
[0383] Results are shown in Section A of Table 4. They showed that (i) the removal of all but the 2 first beta sheets was not detrimental to SBA and (ii) a truncated IgA1P should at least contain the entire protease domain or, if not, contain additional sequence corresponding to the alpha-peptide e.g., the alpha-peptide of another serine-protease.
[0384] These findings and hypotheses were further tested in second and third experiments for which additional truncated IgA1Ps were made, all of which bearing the mutation Ser 267 Val:
[0385] (i) Truncated IgA1P, the amino acid sequence of which corresponds exclusively to the entire protease domain (SP528, SP548).
[0386] (ii) Truncated IgA1P, the amino acid sequence of which corresponds to the protease domain with a deletion of about 35 amino acids at its C-terminal extremity, further fused to the MC58 App 1061-1187 sequence (SP530, SP550) or the MC58 AusI 974-1161 sequence (SP531) which both corresponds to the alpha-peptide domain.
[0387] (iii) Truncated IgA1P, the amino acid sequence of which corresponds to the protease domain with a deletion of about 35 amino acids at its C-terminal extremity, further fused to the MC58 App 1061-1224 sequence (SP532) or the MC58 AusI 974-1198 sequence (SP533) which both corresponds to the alpha-peptide domain followed by the first two beta-strands.
[0388] These constructs were assayed for SBA together with SP503 for which the best results were to be seen in the first experiment. Results are to be seen in Sections C and D of Table 4. They reveal that a truncated IgA1P corresponding to the entire protease domain is effective in raising SBA. In addition to this, the fusion of a partial IgA1P protease domain to an App or AusI structure equivalent to the IgA1P alpha-peptide domain results in hybrids exhibiting positive SBA. Interestingly, SP528 and SP530 exhibit a modest fold-increase although they induce a bactericidal titer >16 in 100% of mice. Moving the His-tag to the C-ter (SP548 & SP550 respectively) allows significant improvement.
Mutated, Truncated and/or Chimeric IgA1P
SBA Against Heterologous Strains
[0389] In a first set of two independent experiments A & B, the cross-bactericidal activity of mouse pooled sera raised against SP503 was assayed against a panel of 25 strains including the homologous strain MC58, most of them being spread over 5 major epidemiological clusters (ST32, ST11, ST41/44, ST8, ST269). Strains were cultured in BHI+desferal, 2 hr 30 except strain NGH41, cultured for 4 hrs in BHI agar. Results are shown in
[0390] In a second and similar experiment C, SP503 was assayed together with SP528 and SP530 for cross-SBA against a panel of 26 strains including the homologous strain MC58. Results are shown in
[0391] In a third and similar experiment D, SP503 was assayed together with SP548 and SP550 for cross-SBA against a panel of 20 strains including the homologous strain MC58. Results are shown in Table 5 above and in
Truncated and/or Mutated App & AusI
Homologous & Heterologous SBA
[0392] In Experiment E, truncated App constructs SP534 and SP535 as well as truncated AusI constructs SP536 and SP537 were tested for SBA against a panel of 20 strains. Results are shown in
[0393] Antisera raised against truncated App either mutated or not (SP534 & SP535) exhibit significant bactericidal activity against the homologous strain and high cross-SBA coverage. While antisera raised against truncated AusI, mutated or not (SP536 & SP537), do not show any bactericidal activity against the homologous strain, they are able to cross-react with strains of the ST269 complex and therefore are of potential interest.
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