Polypeptides derived from <i>Enterococcus </i>and their use for vaccination and the generation of therapeutic antibodies
11278609 · 2022-03-22
Assignee
Inventors
Cpc classification
C07K16/1228
CHEMISTRY; METALLURGY
A61K39/09
HUMAN NECESSITIES
International classification
A61K39/09
HUMAN NECESSITIES
C07K14/24
CHEMISTRY; METALLURGY
Abstract
Medicament for the treatment or the prevention of a bacterial infection, characterized in that it contains at least one polypeptide selected from the group of SEQ ID NO: 1 to SEQ ID NO: 9, and contiguous fragments thereof, wherein said at least one polypeptide or contiguous fragment thereof induces opsonic antibodies in a patient in need thereof. The polypeptides or the contiguous fragments thereof according to the present invention can be used for the preparation of a vaccine against an infection against Enterococcus.
Claims
1. A method for inducing in a subject opsonic antibodies against at least one polypeptide selected from the group consisting of SEQ ID NOs: 1 and 3-9, for treatment of an Enterococcus infection that can be treated by such opsonic antibodies, wherein the method comprises administering to the subject in need of such treatment, a composition comprising an effective amount of at least one polypeptide selected from the group consisting of SEQ ID NOs: 1 and 3-9 to induce opsonic antibodies in the subject; and confirming opsonic antibodies against at least one polypeptide selected from the group consisting of SEQ ID NOs: 1 and 3-9 in the subject through testing a serum sample for opsonic killing activity.
2. The method according to claim 1 wherein said polypeptide is covalently bound to a protein, a carbohydrate, and/or a glycoconjugate.
3. The method according to claim 1, wherein the composition comprises at least one pharmaceutically acceptable adjuvant.
4. The method according to claim 1, wherein the composition is a vaccine.
5. A polypeptide selected from the group consisting of SEQ ID NOs: 1 and 3-9, wherein said polypeptide is covalently bound to an immunocarrier.
6. The method, according to claim 1, wherein the infection is caused by E. faecium or E. faecalis.
7. The method, according to claim 4, wherein said vaccine is against an infection caused by Enterococcus faecium or E. faecalis.
8. The polypeptide, according to claim 5, wherein said immunocarrier is a protein, a carbohydrate, and/or a glycoconjugate.
9. The method according to claim 1, wherein the Enterococcus infection is caused by an Enterococcus bacterium expressing a polypeptide comprising any one of SEQ ID NOs: 1 and 3-9.
10. The method according to claim 1, wherein the subject is a human.
11. The method according to claim 1, wherein the composition comprises an effective amount of at least one polypeptide selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 9.
12. The polypeptide according to claim 5, which has an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 9.
Description
DESCRIPTION OF THE FIGURES
(1)
(2)
(3)
(4) SEQ ID NOs: 1 to 9 show the amino acid sequence of the polypeptides according to the invention derived from strain Enterococcus faecium E155, and Enterococcus faecalis. SEQ ID NOs: 10 to 167 show the amino acid sequence of preferred peptide fragments (epitopes) as active fragments derived from the polypeptides according to the invention.
EXAMPLES
(5) The bacterial strain used for all experiments was the Vancomycin-resistant E. faecium E155 strain, a clinical isolate that belongs to a genetic subpopulation of hospital-associated E. faecium responsible for worldwide emergence due to its multidrug-resistance and especially high level resistance to quinolone and ampicillin.
Example 1
Identification and Extraction of the Polypeptides According to the Present Invention
1. Surface Protein Extraction Using Trypsin Shaving Method
(6) Extractions were performed as described by Tjalsma et al. (2008). Briefly, two aliquots of 50 ml of bacterial cultures of the E155 strain were harvested at OD.sub.600=0.4 by centrifugation (10,000 rpm., 2 min) and washed twice with 4 ml Bicam (triethylammonium bicarbonate buffer 100 mM pH 8). The first aliquot was then mixed with a solution of trypsin in Bicam at final concentration of 10 μg/mL. The other aliquot was resuspended in Bicam without any trypsin. All the samples were incubated for 1 h at 37° C. under gentle shaking. After centrifugation (7,500 rpm., 5 min), the cell pellets were removed, and the supernatants were treated with 1 mM DTT for 30 min, followed by 1 mM iodoacetamide (IAA), also for 30 min at room temperature. Finally, fresh trypsin (0.5 mg) was added to all samples and tryptic cleavage was continued for 18 h at 37° C. In this way two samples were obtained for each experiment. These “shaved” proteins were identified by LC-MS/MS. For this protocol, the inventors analyzed 25 different conditions using different amounts of trypsin, different times of incubation with the enzyme and additional combined treatments with lysozyme and/or mutanolysin.
(7) After analysis of the 25 samples as obtained using the different conditions by nanoLC-MS/MS, a total of 401 proteins was identified using the Mascot software databases. Overall, 34 proteins were identified as surface proteins, 29 as present in both intra- and extracellular location, 315 as cytoplasmic proteins and 23 of unknown location. The results of this method demonstrate that this procedure is not an appropriate strategy to obtain mostly surface proteins, since only around 16% of the identified proteins belong to this category. Furthermore, this procedure is not an appropriate strategy as the sole strategy to obtain target polypeptides.
2. Extraction of Surface Proteins Under Strong Alkaline Conditions
(8) Surface-exposed proteins were extracted by exposure of cells to high pH using a protocol described by Hempel et al. (2011). Briefly, a cell pellet from a 50 ml culture (OD.sub.600=0.5) was washed with a PBS sucrose solution [NaCl 100 mM, sucrose 60 mM, sodium phosphate 55 mM (pH 7.2)] and then shaken gently for 1 h at room temperature in 2 ml NaOH glycine sucrose [glycine 50 mM, sucrose 60 mM (pH 12.4)]. After centrifugation (30 min, 10,000 g), 108 ml 1 M HCl and 100 ml 1 M Tris/HCl (pH 7.0) were added to 1 ml supernatant. Proteins were precipitated at 4° C. by addition of 8 ml cold acetone. The protein pellet obtained after centrifugation (10 min, 10,000 g) was resuspended in 20 ml Tris/HCl (pH 7.5).
(9) This protocol was used to extract peripheral proteins that are loosely attached to the membrane or cell wall. These proteins can be detached using treatment with a polar reagent like an alkaline pH solution since they are non-covalently attached to either the lipid layer or to integral membrane proteins by hydrophobic, electrostatic, or other interactions. Two different media where tested for this procedure, TSB and GM17, which are both rich laboratory mediums. The samples obtained under the different conditions were analyzed by nanoLC-MS/MS. A total of 329 proteins were identified using the mascot software databases. Overall, 47 proteins were identified as surface proteins, 16 as present in both intra- and extracellular location, 246 as cytoplasmic proteins and 20 of unknown location. As this method let to the isolation of intact proteins, the inventors were able to test their immunogenicity by immunodotblot and western blot. However, the results demonstrate that this method is also not the best strategy to obtain samples enriched in surface proteins because just around 19% of the predicted proteins belong to this category. Thus, this procedure is also not an appropriate strategy as the sole strategy to obtain target polypeptides.
3. Surface Proteins Extraction Using Biotinylation
(10) Surface-exposed proteins were labeled and extracted by exposure of cells to Sulfo-NHS-SS-Biotin using a protocol described by Hempel et al. (2011). Briefly, 100 mL of bacterial cultures at OD.sub.600=0.5 were harvested at 8,000×g for 5 min at 4° C. 0.2 g of cells (wet cell weight) were resuspended in 5 mL ice-cold PBS (pH 8.0) with 1 mM PMSF on ice. The biotinylation reaction was performed by adding 100 μL fresh Sulfo-NHS-SS-Biotin solution to 1 mL of intact cells, to give a final concentration of 1.5 mM Sulfo-NHS-SS-Biotin. A 1% (w/v) solution of Sulfo-NHS-SS-Biotin was prepared by adding 5 mg to 500 μL PBS (pH 8.0) immediately before use. Cells were incubated by gentle shaking for 1 h on ice. To stop the reaction and to remove nonreacted biotinylation reagent, cells were centrifuged at 8500×g for 1 min at 4° C. and washed three times with ice-cold PBS (pH 8.0)/500 mM glycine. A pellet of 1 mL reaction volume was resuspended in 500 μL PBS (pH 8.0) with 1 mM PMSF on ice and transferred to a 1.5 mL tube containing glass beads. The disruption of cells was performed mechanically in a FastPrep cell disruptor at 6 m/s.sup.2 twice for 30 s. The cell debris was recovered from the glass beads with a total of 3 mL of PBS (pH 8.0). The lysate was centrifuged (100,000×g for 1 h at 4° C.). The supernatant was then discarded. The cell debris were resuspended in a total of 400 μL of [PBS (pH 8.0), IAA (5%)] and homogenized in a FastPrep cell disrupter at 6 m/s.sup.2 twice for 30 s with 0.25 ml of glass beads. The proteins were the solubilized by addition of 100 μL of PBS (pH 8.0) with 1 mM PMSF, 4% CHAPS and 2% ASB-14. A second homogenization step was performed after detergent addition under the same conditions as mention above. Cell debris was removed by centrifugation (14,000 rpm, 15 min) after 1 h of incubation with the detergent.
(11) The biotinylated proteins were isolated and purified by NeutrAvidin agarose affinity-purification. For a reaction volume of 500 μL protein mixture 150 μL of NeutrAvidin agarose resin were washed twice with PBS (pH 8.0)/1% NP-40 and centrifuged at 1,000 rpm for 1 min at 4° C. The resin was mixed with the cell lysate for 1 h by gently shaking on ice. The supernatant was removed and the resin-bound complex washed 6 times with PBS (pH 8.0)/1% NP-40. Biotinylated proteins were eluted twice by incubation with 1 mL of elution buffer (5% mercaptoethanol in H.sub.2O) for 1 h with gentle shaking; supernatant was recovered after centrifugation at 1,000 rpm for 1 min and poured to 8 mL of cold acetone (−20° C., overnight). The precipitated proteins were harvested by centrifugation (8,500 rpm, 30 min, 4° C.) and washed twice with 1 mL of cold 98% ethanol (4° C.). The pellets were dried in a SpeedVac for 2 min and dissolved in 15 μl 6M urea/2M thiourea for 2 min at 80° C. The samples were loaded on a SDS-PAGE gel and the corresponding bands were excised from the gel and dehydrated with acetonitrile. Afterwards, samples were reduced and alkylated in two successive steps of 20 min with 0.5% Dithiothreitol and 5% Iodoacetamide. Samples were washed twice with 30% acetonitrile, 200 mM ammonium bicarbonate and subsequently digested overnight with 0.2 μg of trypsin (Promega). Peptides were obtained by covering the gel bands with water and incubating them in an ultrasonic bath for 15 min. Finally peptides samples were analyzed by nanoLC-MS/MS, sequence data were compared to the NCBI and MASCOT databases.
(12) Three different media where tested for this approach, the rich laboratory medium TSB, ccM17 MOPS, a carbon depleted laboratory medium and BHI (brain hearth infusion) medium supplemented with 30% of horse serum to mimic in vivo conditions. The samples obtained under the different conditions were analyzed by nanoLC-MS/MS. A total of 45 proteins were identified using the Mascot software databases. Overall, 27 proteins were identified as surface proteins, 6 as present in both intra and extra cellular location, and 12 as cytoplasmic proteins. The combined results demonstrated that this procedure is the best strategy to target preferentially surface proteins since 73% of the predicted proteins belonged to this category. Nevertheless, for the purposes of the present invention, all three approaches were combined.
4. Additional Surface Protein Identification from Transcriptional Analysis
(13) Real-time PCR experiments performed with cDNA synthesized from RNA extracted from an in vivo endocarditis model in E. faecalis closely related species revealed that over 300 genes were up-regulated under these conditions. Among these, 19 genes were identified to encode surface related proteins. These proteins were analyzed by online BLAST in the J. Craig Venter Institute database comparing the sequences in E. faecium completely sequenced strains. The adhesion lipoprotein (Adlip) and a protein showing homology to a periplasmic solute binding protein (PSB) were identified as the closest surface proteins related between the two strains and selected as targets for the overexpression experiments (see below).
6. MS Analyses
(14) MS analyses were performed after the overnight tryptic cleavage of protein samples obtained by shaving extraction. Trypsin-cleaved samples were desalted and concentrated on a tipmicroC18 Omix (Varian) before nano-liquid chromatography (LC)-MS-MS analysis. The chromatography step was performed using a Prominence nano-LC system (Shimadzu).
7. Summary of all the Extraction Methods
(15) A comparison between the proteins identified with the three methods allowed the inventors to establish that 22 extracellular proteins were detected by two of the three methods as used, and seven by all of them. Sag A was used as a control. Finally, nine proteins were selected for overexpression (see table 2). Seven of them were identified by the three extraction methods and the remaining two identified to be induced in vivo in the closely related species E. faecalis.
(16) TABLE-US-00003 TABLE 2 Comparison of the proteins identified by the different extraction methods Protein Abbreviation Locus Tag Method low affinity penicillin- PBP5 EFAU004_00870 Biotin, High pH, binding protein 5 (PBP5) Trypsin Basic membrane BML EFAU004_00080 Biotin, High pH, lipoprotein Trypsin peptidoglycan-binding LysM EFAU004_01059 Biotin, High pH, protein LysM Trypsin D-alanyl-D-alanine Dala EFAU004_01127 Biotin, High pH, carboxypeptidase Trypsin PpiC-type peptidyl-prolyl PpiC EFAU004_02526 Biotin, High pH, cis-trans isomerase Trypsin Enolase Enol EFAU004_02073 Biotin, High pH, Trypsin SCP-like extracellular SCP WP_002353118.1 Biotin, High pH, protein (serine protease) Trypsin Adhesion lipoprotein Adlip EFUG_02345 Biotin, High pH, Transcriptomic data Periplasmic solute binding PSB EFAU004_00598 Biotin, High pH, family Transcriptomic data
Example 2
Overexpression of the Polypeptides and Production of Polyclonal Antibodies
1. Cloning of the Genes Encoding the Protein Candidates
(17) The genes encoding the selected proteins were identified by in silico analysis using the E. faecium E155 genome sequence. Overexpression of the proteins was performed by cloning the corresponding genes into the expression vectors pQE30 or pET28a+. For the PBP5, BML, LysM, Dala, PpiC, Enol, PSB and Adlip the vector pQE30 was used, and the pET28a+ for the SCP. In addition, one more protein was overexpressed and purified, the Sag A protein supposed to be a promising vaccine target in E. faecium (Kropec et. al, 2011) was included as a positive control.
(18) Overexpression and purification of the H6-proteins. A QIAexpress system was used for the expression of a six-His-tagged recombinant proteins as follows. First, the genes were amplified by PCR using primers designed at the beginning of each gene (excluding the signal peptide base pairs) and one at the end of it. The PCR product was digested using the endonucleases BamHI and PstI for the genes encoding SagA, LysM, Dala, PpiC, Enol, Adlip and PSB; and BamHI and SacI for SCP, PBP5, BML and PpiC; and cloned into the corresponding restriction sites of the respective plasmid: pQE30 or pET28a+. The resulting plasmid, were then introduced in E. coli M15(pREP4) cells for the pQE30 and in E. coli BL21 for the pET28a+. Colonies were screened by PCR and the integrity of the constructions was controlled by sequencing.
2. Overexpression and Purification of the Protein
(19) The overexpression of all the proteins was carried out by inoculating an overnight culture in fresh LB media supplemented with the corresponding antibiotic. Bacteria were grown during 2 hours at 37° C. and shaking at 160 rpm before induction of protein expression by 0.5 mM IPTG, then, the culture was incubated for two additional hours under the same conditions. Cells were harvested by centrifugation and later disrupted using lysozyme and the FastPrep cell disrupter. Proteins were purified under denaturing conditions using Protino® Ni-NTA Agarose following the instructions of the manufacturer (Macherey-Nagel).
(20) Purified proteins were subject of SDS-PAGE with 10% acrylamide/bisacrylamide resolving gels (NuPAGE, Invitrogen) and stained with Coomassie brilliant blue (SimplyBlue SafeStain, Invitrogen) for protein detection and molecular size confirmation (see
Example 3
1. Production of Polyclonal Antibodies
(21) In order to produce anti-protein hyperimmune serum, eight New Zealand White rabbits (2.5 to 3.5 kg; Charles River Laboratories) were vaccinated with each protein (Enol, PpiC, Dala, LysM, BML, PBP5, Adlip and PSB) according to the immunization schedule (see Table 3). Preimmune serum was collected from the rabbits on days 0 and 7 prior to the first vaccination to be used as a negative control.
(22) TABLE-US-00004 TABLE 3 Immunization schedule for purified polypeptides Schedule for the immunization with the polypeptide Day No. Procedure 0 Pre Bleed 10-15 mL 7 Pre Bleed 10-15 mL 14 Immunization 1 s.c. (1FIA) 10 ug 28 Immunization 2 s.c. (1FIA) 10 ug 35 Boost 1 i.v. 5 ug (without FIA) 37 Boost 2 i.v. 5 ug (without FIA) 39 Boost 3 i.v. 5 ug (without FIA) 53 Test Bleed 2-10 mL 60 Boost 4 i.v. 5 ug (without FIA) 67 Boost 5 i.v. 5 ug (without FIA) 74 Terminal Bleed
(23) The sera were heat inactivated at 56° C. for 30 min and were then absorbed 1 h with heat killed cells of E. faecium E1162 treated with proteinase K.
2. Opsonophagocytic Assays
(24) An in vitro opsonophagocytic assay (OPA) was performed as described elsewhere (Huebner J, 1999) using baby rabbit serum as complement source and rabbit serum raised against purified proteins. Polymorphonuclear neutrophils (PMN's) were freshly prepared from human blood collected from healthy adult volunteers. Bacterial strains were grown to mid-log phase in TSB. For the assay, the following components were mixed: 100 μl of PMNs; 100 μl of 1:10 and 1:50 serum dilution, 100 μl of absorbed baby rabbit complement 1:30 dilution, and 100 μl of 1:150 dilution of bacteria E. faecium E155. The mixture was incubated on a rotor rack at 37° C. for 90 min, and samples were plated in duplicate at time 0 and after 90 min. Percent killing was calculated by comparing the colony counts of a control without PMN's to the colony counts after a 90-minute incubation at 37° C. (T90). For inhibition studies, rabbit serum was diluted 1:50 or 1:100 and incubated for 60 min at 4° C. with an equal volume of a solution containing 100 μg of the corresponding protein. Subsequently, the antiserum was used in the OPA as described above. Inhibition assays were performed at serum dilutions yielding 50-60% killing of the inoculum without the addition of the inhibitor. The percentage of inhibition of opsonophagocytic killing was compared to controls without inhibitor.
(25) The results are shown in
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