WO2002090383A2 - M. catarrhalis antigens - Google Patents

M. catarrhalis antigens Download PDF

Info

Publication number
WO2002090383A2
WO2002090383A2 PCT/GB2002/002205 GB0202205W WO02090383A2 WO 2002090383 A2 WO2002090383 A2 WO 2002090383A2 GB 0202205 W GB0202205 W GB 0202205W WO 02090383 A2 WO02090383 A2 WO 02090383A2
Authority
WO
WIPO (PCT)
Prior art keywords
protein
catarrhalis
sequence
composition
fragment
Prior art date
Application number
PCT/GB2002/002205
Other languages
French (fr)
Other versions
WO2002090383A3 (en
Inventor
Keith Peek
Mark Wilkinson
Suzanne Thomson
Original Assignee
Provalis Uk Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB0111492A external-priority patent/GB0111492D0/en
Priority claimed from GB0112448A external-priority patent/GB0112448D0/en
Priority claimed from GB0119479A external-priority patent/GB0119479D0/en
Application filed by Provalis Uk Limited filed Critical Provalis Uk Limited
Priority to AU2002307943A priority Critical patent/AU2002307943A1/en
Publication of WO2002090383A2 publication Critical patent/WO2002090383A2/en
Publication of WO2002090383A3 publication Critical patent/WO2002090383A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • C07K14/212—Moraxellaceae, e.g. Acinetobacter, Moraxella, Oligella, Psychrobacter
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to novel proteins derived from Moraxella catarrhalis, and the use of the proteins as antigens and/or imr ⁇ unogens in medicine, particularly in the preparation of vaccines and in diagnosis, as well as screening the proteins as potential antimicrobial targets.
  • Moraxella catarrhalis is a Gram negative diplococcus that is an obligate parasite of the mucous membranes of humans, especially the nasopharynx. Generally it is of low virulence but can cause meningitis, bacteremia, empyema, pericarditis, endocarditis and pneumonia in certain susceptible groups including the elderly, chronic obstructive pulmonary disease (COPD) patients, the immunocomprimised and patients on ventilators.
  • COPD chronic obstructive pulmonary disease
  • Otitis media can have serious consequences for children, resulting in permanent damage to the eardrum and impaired hearing. If untreated or unrecognised in children it can impair learning capacity and delay speech development (J. O. Klein. 2001. Vaccine 19: S2-S8.).
  • a vaccine for preventing M. catarrhalis induced otitis media would have a number of advantages for use in children. It would act as a prophylactic approach preventing infection and associated complications. This would significantly reduce the prescription of antibiotics, resulting in reduced health care cost and a reduction in the selective pressure on bacteria to develop resistant strains.
  • Vaccination of rabbits with a formalin inactivated whole-cell preparation of Moraxella catarrhalis has allowed the identification of a number of antigenic proteins that might represent potential vaccine candidates.
  • Candidate antigens were selected from whole-cell protein extracts by identifying those proteins that bind IgG antibodies in sera prepared from rabbits vaccinated sub-cutaneously with inactivated whole cells of Moraxella catarrhalis. By using this technique we have isolated and identified a number of novel antigens for use as vaccines or in the diagnosis of disease.
  • a protein from M. catarrhalis which has the NH 2 - terminal sequence:
  • bracketed amino acids represent an alternative to the preceding amino acid and X represents any amino acid.
  • the proteins of the present invention are isolatable from M. catarrhalis and may be provided in substantially pure form. For example, it may be provided in a form that is substantially free of other proteins.
  • the proteins of the invention are useful as antigenic material.
  • Such material can be "antigenic” and/or “immunogenic”.
  • antigenic is taken to mean that the protein is capable of being used to raise antibodies or indeed is capable of inducing an antibody response in a subject.
  • immunogenic is taken to mean that the protein is capable of eliciting a protective immune response in a subject.
  • the protein may be capable of not only generating an antibody response but, in addition, a non-antibody based immune responses.
  • proteins which include one or more additions, deletions, substitutions or the like are encompassed by the present invention.
  • the determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art.
  • An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-226%, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
  • the NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410 have incorporated such an algorithm.
  • Gapped BLAST can be utilised as described in Altschul et al.
  • PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.).
  • BLAST Altschul et al.
  • Gapped BLAST Altschul et al.
  • PSI-Blast programs the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
  • homologues or derivatives the degree of identity with a protein as described herein is less important than that the homologue or derivative should retain its antigenicity and/or immunogenicity to M. catarrhalis.
  • homologues or derivatives having at least 60% similarity (as discussed above) with the proteins or polypeptides described herein are provided.
  • homologues or derivatives having at least 70% similarity, more preferably at least 80% similarity are provided.
  • homologues or derivatives having at least 90%, 95%, 96, 97, 98, 99 or even 99.8% or greater similarity are provided.
  • the homologues or derivatives could be fusion proteins, inco ⁇ orating moieties which render purification easier, for example by effectively tagging the desired protein or polypeptide. It may be necessary to remove the "tag” or it may be the case that the fusion protein itself retains sufficient antigenicity to be useful.
  • the invention therefore provides, in a second aspect, a protein, which is a homologue or derivative of the proteins of the first aspect of the invention. It is well known that is possible to screen an antigenic or immunogenic protein or polypeptide to identify epitopic regions, i.e. those regions that are responsible for the protein or polypeptide' s antigenicity or i munogenicity. Thus, in a third aspect of the invention, there is provided one or more antigenic or immunogenic fragments
  • antigenic fragments of the proteins of the invention, or of homologues or derivatives thereof.
  • the fragment may comprise the N-terminal sequence as described above.
  • fragments and or homologues and/or derivatives can be tested to determine whether serum raised against M. catarrhalis reacts against the fragment and/or homologue and/or derivative in question.
  • the fragments of the present invention should include one or more such epitopic regions or be sufficiently similar to such regions to retain their antigenic/immunogenic properties.
  • the degree of identity is perhaps irrelevant, since they may be 100% identical to a particular part of a protein or polypeptide, homologue or derivative as described herein.
  • the key issue, once again, is that the fragment retains the antigenic/immunogenic properties of the protein from which it is derived.
  • homologues, derivatives and fragments possess at least a degree of the antigenicity/immunogenicity of the protein or polypeptide from which they are derived.
  • the proteins of the present invention, or antigenic fragments thereof can be provided alone, as a purified or isolated preparation. They may be provided as part of a mixture with one or more other M. catarrhalis proteins of the invention, or antigenic fragments thereof, or one or more other catarrhalis antigenic proteins or fragments thereof.
  • the invention provides an antigen composition comprising one or more proteins of the invention and/or one or more antigenic fragments thereof. Such a composition can be used for the detection and/or diagnosis of M. catarrhalis.
  • the composition comprises one or more additional M. catarrhalis antigens/immunogens.
  • the present invention provides a method of detecting and/or diagnosing M. catarrhalis which comprises:
  • the protein, antigenic fragment thereof or antigen composition of the present invention can be used to detect IgA, IgM or IgG antibodies.
  • the sample to be tested will be a biological sample, e.g. a sample of blood or saliva.
  • the invention provides the use of an antigenic protein, antigenic fragment thereof or an antigenic composition of the present invention in detecting and/or diagnosing M. catarrhalis in vitro.
  • the antigenic proteins, antigenic fragments thereof or antigenic composition of the present invention can be provided as a kit for use in the in vitro detection and/or diagnosis of M. catarrhalis.
  • the present invention provides a kit for use in the detection and/or diagnosis of M. catarrhalis, which kit comprises one or more antigenic protein(s) or antigenic fragment(s) thereof or an antigenic composition of the present invention.
  • the antigenic protein, antigenic fragment thereof or antigen composition of the invention can be used to induce an immune response against M. catarrhalis.
  • the invention provides the use of an antigenic protein of the invention, an antigenic fragment thereof or an antigen composition of the invention in medicine.
  • the present invention provides a composition capable of eliciting an immune response in a subject, which composition comprises a protem, an antigenic fragment thereof, or an antigen composition of the invention.
  • the composition will be a vaccine composition, optionally comprising one or more suitable adjuvants.
  • a vaccine composition may be either a prophylactic or therapeutic vaccine composition.
  • the vaccine compositions of the invention can include one or more adjuvants.
  • adjuvants examples well-known in the art include inorganic gels, such as aluminium hydroxide, and water-in-oil emulsions, such as incomplete Freund's adjuvant.
  • inorganic gels such as aluminium hydroxide
  • water-in-oil emulsions such as incomplete Freund's adjuvant.
  • Other useful adjuvants will be well known to the skilled person.
  • the present invention provides:
  • Cells were harvested from cultures by centrifugation at 4000 x g for 2.5 hours and washed with one volume of 25 mM sodium phosphate buffer pH 7.0. The cells were resuspended in 20 ml 25 mM sodium phosphate buffer pH 7.0 containing 1 mM l-(2- Ammoethyl)benzenesulfonylfluoride-HCl (AEBSF protease inhibitor) and disrupted by sonication using a Sanyo Soniprep 150 (19 mm probe) at an amplitude of 6 ⁇ m for
  • Chromatography fractions were run on 4-12% Novex Bis-Tris gels (Invitrogen) according to manufacturer's instructions. Briefly, 65 ⁇ l of sample was mixed with 25 ⁇ l 4 x LDS NuPAGE sample buffer and 10 ⁇ l of NuPAGE 10 x reducing agent. samples were heated at 70°C for 10 minutes and then centrifuged at 16,000 x g for 5 minutes. 10 ⁇ l of sample was loaded per lane and 5 ⁇ l of Novex SeeBlue molecular weight markers were loaded in one lane of the gel. Proteins were separated with 1 x NuPAGE MOPS running buffer containing 500 ⁇ l of NuPAGE running buffer antioxidant per 200ml of running buffer. Gels were run at 200 volts until the dye front reached the bottom of the gel (approximately 45 minutes). Gels were removed from the casting plates and either coomassie stained, silver stained or Western blotted.
  • nitrocellulose membrane was then washed twice in TTBS and incubated for 2 hours with swine anti-rabbit IgG horse radish peroxidase (HRP) conjugate (1:500 dilution in TTBS containing 1 % (w/v) BSA). A 1:500 dilution of avidin peroxidase was added to the conjugate, which binds to the biotinylated molecular weight markers. Following incubation of the conjugate, the membrane was washed twice in TTBS and twice in TBS for 5 minutes each.
  • HRP horse radish peroxidase
  • the blot was then developed by incubating with the HRP-substrate, 4-chloronapthol (4-CN ; 30 mg 4- CN in 10 ml methanol plus 50 ml TBS and 30 ⁇ l of 30 % (v/v) H 2 O 2 ). After a sufficient time, to visualise the protein antigens, the reaction was stopped by immersing the nitrocellulose membrane in ddH 2 O for 10 minutes.
  • the antigens contained in column fractions were separated by SDS-PAGE using NuPAGE 4-12 % Bis/Tris gels and then transfened onto polyvinylidene difluoride membrane using BioRad mini Trans-blot apparatus as described above. After transfer, the membrane was stained with 0.25 % (w/v) Coomassie Brilliant Blue R-250 dissolved in 40 % (v/v) methanol for 5 minutes, followed by destaining in 50 % (v/v) methanol. The membrane was dried in air between filter paper at room temperature and stained proteins were cut out and placed in the upper cartridge of the 471 A NH 2 - terminal sequencer (Applied Biosystems). The NH 2 -amino acid sequence was determined by Edman degradation. Results
  • Moraxella catarrhalis proteins were separated by Mono Q anion exchange chromatography by using a 0-1 M NaCl gradient. The chromatography profile can be seen in Figure 1. Fractions 36, 37, 51, 52, 53, 56, 57, 59, 60, 61, 63, 65, 66, 67 were analysed by SDS-PAGE using 4-12% T Bis/Tris NuPAGE gels and were silver stained or Western blotted. Western blots were immunostained with 30 day Moraxella catarrhalis immune serum. The silver-stained gel can be seen in Figure 2. The gel shows numerous proteins that could be of interest, especially in fractions 60-67.
  • ID-1 has an NH 2 - terminal amino acid sequence of AFTLPELGYSYDALEPGFDK(N)EA(T)XM(G)L.
  • Bracketed amino acids represent an alternative for the preceding residue and X represents an unidentified amino acid.
  • a BLAST (blastp) search of the first 20 amino acids was performed at the NCBI internet site (www.ncbi.nlm.nih.gov), using the BLOSUM62 matrix with the default settings. Fifty-five hits were returned showing homology to the enzyme superoxide dismutase (SOD) from various bacterial species.
  • SOD superoxide dismutase
  • ID-2 the NH 2 -terminal amino acid sequence was MKQPV(T)RVAXT.
  • a BLAST search (ignoring the bracketed amino acid) using the PAM30 matrix reveals the first 4 hits showing homology to the enzyme malate dehydrogenase. The best alignment was 90 % over the 10 amino acids to malate dehydrogenase isolated from Deinococcus radiodurans.
  • a translated DNA (TBLASTN) search again gave the best match to the malate dehydrogenase from Deinococcus radiodurans.
  • Cell pellets from 5 ml of culture were mixed with 1.25 ml of 5 x SDS sample buffer (10 % (v/v) glycerol, 10 % (w/v) SDS, 5 % (v/v) 2-mercaptoethanol (added fresh) in 315 mM Tris-Cl pH 6.8 and coloured with bromophenol blue).
  • the sample was boiled for 10 minutes and then centrifuged at 16,000 x g for 10 minutes. The resulting supernatant was used for preparative electrophoresis.
  • a BioRad Mini Prep Cell was used to separate proteins by their size from whole cell M. catarrhalis.
  • a 9 % acrylamide gel (3.0 ml of acrylamide/bis-acrylamide (30% T30 2.7% C), 2.5 ml 3 M Tris-Cl pH 8.85 and 4.5 ml of ddH 2 O), was poured into the casting tube according to manufacturers instructions.
  • Electrophoresis buffer (25 mM Tris, 192 mM Glycine and 0.1% (w/v) SDS) was placed into the upper and lower chamber.
  • Elution buffer 50 mM Tris-Cl, 0.1 % (w/v) SDS pH 8.5
  • was placed into the elution chamber which was mixed with the proteins as they were eluted from the bottom of the gel.
  • the antigens were separated by SDS-PAGE using 10 % T acrylamide gel and then transferred onto polyvinylidene dif ⁇ uoride membrane and protein sequenced as described in example 2.
  • FIG. 4a and 4b show SDS-PAGE gels of fractions collected from a preparative electrophoresis experiment. Silver staining of these fractions after SDS- PAGE shows proteins ranging from 6 kDa to 300 kDa in size.
  • Figure 5 shows the same fractions Western blotted and immunostamed. Many of these proteins are very antigenic.
  • Figure 6 shows a repeated Western blot and immunostain of fractions selected for further investigation in order to identify proteins for NH -terminal amino acid sequencing. Two of these proteins are indicated on the gel by arrows (Fig. 6), these are designated ID-3 and ID-4. NH 2 -terminal amino acid sequencing results for these proteins showed that ID-3 is superoxide dismutase with the same amino acid sequence as ID-1 isolated by anion exchange chromatography and described in example 2
  • ID-4 had an NH 2 -terminal amino acid sequence of TTQNNQQNGKN.
  • This protein showed no significant matches after a blastp search using the PAM30 matrix. Modify PAM30 by adjusting the expected value from 10 to 100 matched the sequence to the internal portion of a number of transferrin binding proteins (best match 72 % ID over the 11 amino acids). Further refinement using the PSI-BLAST iteration program gave hits to a number of other proteins, the best of which was a 63 % identity to the ⁇ H 2 - terminal amino acid sequence of a vacuolar protein. Overall, the BLAST searches do not reveal convincing data on the homology or identification of this protein. It can therefore be acknowledged that this is a novel protem of unknown function.
  • a Moraxella catarrhalis cell pellet (5ml (0.87g)) was extracted with 1.25 ml of 5 x SDS sample buffer and boiled for 10 minutes as described for preparative electrophoresis. The sample was centrifuged at 16,000 x g for 10 minutes and the supernatant removed. 900 ⁇ l of this supernatant was mixed with 100 ⁇ l of 100% (w/v) trichloroacetic acid (TCA) and stored in the freezer for 10 minutes. The sample was then centrifuged for 5 minutes at 4,000 x g. The resulting protein pellet was washed once in 1 ml of ice cold acetone and centrifuged at 4,000 x g for 5 minutes. The pellet was then solubilised in 1 ml of 2-dimensional rehydration buffer (8 M Urea, 2 % (w/v) CHAPS and bromophenol blue).
  • the strip was removed from the machine and placed in 10 ml of SDS equilibration buffer containing 100 mg of DTT (50 mM Tris, 6 M Urea, 30 % (v/v) glycerol, 2 % (w/v) SDS and a trace of bromophenol blue).
  • DTT 50 mM Tris, 6 M Urea, 30 % (v/v) glycerol, 2 % (w/v) SDS and a trace of bromophenol blue.
  • the strip was then loaded onto a 9 % T SDS polyacrylamide gel in a SE600 Hoefer electrophoresis unit. The gel was run at 100 volts, 45 mA for 30 minutes and then 200 volts, 90 mA until the dye front reached the bottom of the gel.
  • the antigens were separated by 2-dimensional electrophoresis and then transferred onto polyvinylidene difluoride membrane using BioRad semi-dry Transblot apparatus as described above.
  • the proteins were then sequenced as described in example 2
  • FIG. 7 shows a Western blot of a 2-D electrophoresis gel that has been immunostamed with M. catarrhalis immune serum. The blot shows a large number of strongly antigenic proteins, all of which are labelled with their unique identifier, from I2D-5-26.
  • Figure 8 shows a coomassie stained Western blot of the same preparation used for NH 2 - terminal amino acid sequencing and labelled with the corresponding antigenic proteins.
  • I2D-17 had the same NH 2 -terminal sequence as ID-4 from preparative electrophoreisis and as I2D-19, which suggest that it may exist as several isoforms.
  • I2D 18 had the Table 1. NH -terminal Sequences of Moraxella catarrhalis proteins separated by 2-D Electrophoresis. Bracketed residue indicate an alternative to the preceding amino acid. X indicates any other amino acids.
  • This buffer was added to the slices in 10-20 ⁇ l aliquots, allowing each gel slice to take up the buffer before adding the next aliquot. When completely re-swollen the slices were incubated at 37 ° C overnight. At the end of the incubation period excess RHB was removed to a second eppendorf tube and peptides were extracted from the gel slices with 2 lots of 60% v/v acetonitrile containing 0.1% v/v TFA. These washes were pooled with the excess buffer, concentrated by centrifugal evaporation and applied to a Brownlee Aquapore C4 RP-HPLC column (220 x 2.1 mm) equilibrated in 0.08% v/v TFA.
  • sample 40 ⁇ l of sample was added to 10 ⁇ l of 5X SDS sample buffer (10% (v/v) glycerol, 10% (w/v) SDS, 5% (v/v) 2-mercaptoethanol (freshly added) in 315 mM Tris HCl pH 6.8 coloured with bromophenol blue). Samples were heated for five minutes at 95 °C and then centrifuged at 16,000 x g for five minutes. 7 ⁇ l of sample or 5 ⁇ l of molecular weight markers (Sigma Dalton NII-LTM, except for Western blots, where BioRad prestained broad range markers were used) were loaded onto each lane, using a 25 ⁇ l Hamilton syringe.
  • 5X SDS sample buffer 10% (v/v) glycerol, 10% (w/v) SDS, 5% (v/v) 2-mercaptoethanol (freshly added) in 315 mM Tris HCl pH 6.8 coloured with bromophenol blue).
  • the blot was then incubated, overnight at 4°C with primary antibodies (1:800 dilution of 120 day Moraxella catarrhalis immune serum (supplied by Provalis PLC) in TTBS containing 1% (w/v) fat free dried milk powder).
  • primary antibodies (1:800 dilution of 120 day Moraxella catarrhalis immune serum (supplied by Provalis PLC) in TTBS containing 1% (w/v) fat free dried milk powder).
  • the nitrocellulose membrane was then washed three times in TTBS prior to incubation, for four hours, with secondary antibodies - swine anti-rabbit IgG horseradish peroxidase
  • Moraxella catarrhalis cells were harvested from cultures by centrifugation at 5,850 x g for 5 minutes at 4°C and washed with 1 volume of Tris buffer (TB; 50 mM Tris HCl pH 8.0). The cells were then resuspended in 20 ml TB and disrupted by sonication using a MSE 100 watt Ultrasonic Disintegrator at % power for 10 minutes while on ice. Cell debris and unbroken cells were removed by centrifugation at 25,100 x g for 30 minutes at 4°C. The resulting supernatant was then subjected to purification by affinity chromatography.
  • Proteins from the sonicate supernatant of Moraxella catarrhalis were separated using affinity chromatography. Briefly, a 10 ml affinity gel was poured (Cibacron Blue 3GA dye (Sigma C-1285, lot no. 31K7002)) into an Amersham Pharmacia C 10/10 column and equilibrated with three bed volumes of Tris buffer (TB; 10 mM Tris HCl pH 8.0) at a flow rate of 0.5 ml/min. The sonicate supernatant was loaded onto the column at a flow rate of 0.5 ml/min and washed with TB, to remove non-adsorbed proteins, until the absorbance at 280nm returned to zero.
  • Tris buffer TB; 10 mM Tris HCl pH 8.0
  • the mobile phase was then changed for TB containing 200 mM NaCl and washed at a flow rate of 0.5 ml/min until the absorbance at 280 nm returned to zero. At this point the mobile phase was changed once more for TB, this time containing 500 mM NaCl and washed at a flow rate of 0.5 ml min until the absorbance at 280 nm returned to zero. Eluant from both stages was collected as separate fractions and tested for enzyme activity protein, content and analysed by SDS-PAGE, prior to overnight dialysis, against 1.5 litres of 50 mM Tris HCl pH 8.0 at 4°C.
  • Dialysed proteins were separated by ion exchange chromatography using a 1 ml Mono Q (Amersham Pharmacia) column. Briefly, the entire dialysed sample was loaded onto a 1 ml Mono Q column, pre-equilibrated with 25 mM Tris HCl pH 8.0, using a Pharmacia FPLC at 0.5 ml/min. Unbound material was eluted by washing the column with 25 mM Tris HCl pH 8.0, until the absorbance, at 280nm, returned to zero.
  • Protein was eluted using a 0 - 0.1 M NaCl gradient in 25 mM Tris HCl pH 8.0, over 5 column volumes, followed by a 0.1 - 0.3 M NaCl gradient in 25 mM Tris HCl pH 8.0 over 25 column volumes and finally a 0.3 - 1.0 M NaCl gradient in 25 mM Tris HCl pH 8.0 over 2.5 column volumes.
  • the eluate was continuously monitored at 280 nm and fractions collected conesponding to the major peaks, were tested for protein content, enzyme activity and analysed by SDS-PAGE and Western blotting.
  • Unbound material was eluted by washing the column with 25 mM Tris HCl pH 8.0 containing 1.5 M ammonium sulphate, until the absorbance, at 280nm, returned to zero.
  • Malate dehydrogenase was eluted using a 1.5 - 0 M ammonium sulphate gradient in 25 mM Tris HCl pH 8.0, over 10 column volumes.
  • the eluate was continuously monitored at 280 nm and the fraction collected conesponding to the major peak, was tested for protein content, and analysed by SDS- PAGE (coomassie and silver stained) and Western blotting. Purified protein was subjected to overnight dialysis against 1.5 litres 10 mM ammonium bicarbonate, after which the sample was freeze dried using a Freezemobile freeze drier.
  • malate dehydrogenase was further confirmed by NH 2 amino acid terminal sequencing of the first 9 residues of the protein band, shown in figure 10. This revealed two sequences. The predominant sequence being NEIVVYSAR and the lesser MKQPVRVAV. The latter is an exact match to that of malate dehydrogenase from Moraxella catarrhalis. A BLAST search of the other sequence gave a best match to D-lactate dehydrogenase from Agrobacterium tumefaciens.
  • BHI Brain heart infusion
  • the washed bacterial pellet was resuspended in 20 ml of 1 M sodium acetate and 0.01 M ⁇ - mercaptoethanol which was then stined at room temperature for 45 min before 80 ml of 5% (w/v) Zwittergent 3-14 in 0.5 M calcium chloride was added and stined for a further 90 min at room temperature.
  • Ethanol was added to a final concentration of 20% (v/v) and the suspension left overnight at 4 C before centrifugation at 17,000 x g for 10 min at 4 C. The supernatant was collected and the ethanol concentration adjusted to 80% (v/v). This suspension was left overnight at 4 C before centrifugation at 17,000 x g for 20 min at 4°C.
  • the protein pellet was resuspended in a buffer containing 0.05% (w/v) Zwittergent 3-14, 0.05 M Tris, and 0.01 M EDTA, pH 8, and stined at room temperature for 1 h before centrifuging at 12,000 x g for 10 min at 4 C.
  • the supernatant was dialysed overnight against distilled water at 4 C, frozen to -70 C and then lyophilized.
  • the protein extract was resuspended to a concentration of approximately 18mg/ml in Buffer A (25mM Tris-HCl, pH 8.1) before loading onto a BioRad Q5 anion-exchange column. 1ml aliquots were loaded on each run (seven runs in total). The column was washed with Buffer A for 5mins at 1 ml/min. The proteins were eluted using a combination of continuous and step gradients from 100% Buffer A to 100% Buffer B (25mM Tris-HCl + 0.5M NaCl, pH 8.1). The gradient was followed by a 4 min wash with 100% Buffer B followed by a 1 min wash with 100% Buffer A. Fractions 9-10 (see Fig. 16 peaks 1 and 2) from each run were pooled and freeze-dried.
  • Buffer A 25mM Tris-HCl, pH 8.1
  • the freeze dried protein was redissolved in 1.5 ml distilled H 2 O and 1.5 ml reducing buffer (62.5 mM Tris, [pH 6.8], 10% [vol/vol] glycerol, 2% [wt/vol] SDS, 5%
  • FIG 16 shows the chromatogram from which superoxide dismutase (SOD) was purified. Peaks 1 and 2 were pooled and freeze-dried prior to preparative electrophoresis. SOD was found to elute from the gel in fractions 49-54.
  • Figure 17 shows, by analytical SDS-PAGE, that these fractions contain apparently purified SOD. These fractions were pooled and following solubilisation and dialysis into PBS used for immunisation studies.
  • the animals are sedated with ketamine/xylazine in phosphate-buffered saline (PBS) delivered intraperitoneally.
  • PBS phosphate-buffered saline
  • the small intestine was exposed through a midline abdominal incision, and the antigen was injected subserosally into each Peyer's patch with a 27-gauge needle.
  • the protein was prepared by emulsification of purified protein in a 1 :1 mixture of incomplete Freund's adjuvant (IF A) (Difco Laboratories, Detroit, Mich.) and PBS, and a total inoculum of lO ⁇ l, was administered to each experimental animal in the immunisation group.
  • IF A incomplete Freund's adjuvant
  • Blood was collected by heart puncture, fransfened to a sterile container, clotted, centrifuged at 2000 ⁇ m and aliquots of serum stored at -20°C for antibody analysis.
  • the lungs, trachea and heart were excised intact, the heart and connective tissues were removed and the lungs were placed in 2ml of sterile PBS and homogenised with a Heidolph Diax 600 homogeniser set at 9,500 ⁇ m with no load.
  • the lung homogenate was assessed for the presence of bacteria by plating serial dilutions (20 ⁇ l in a 10-fold series) onto chocolate blood agar for CFU determination.
  • Immunoblot Assay For immunoblot assay proteins were separated by SDS-PAGE and elecfrophoretically fransfened to nitrocellulose (0.2- ⁇ m pore size, BioRad Laboratories, Nth Ryde,
  • TTBS-5% skim milk for 90 min with gentle agitation.
  • the membrane was Washed twice for 5 min in TTBS followed by a third 5 min wash in TBS, and the blot developed in a solution of TBS containing 0.05% (wt/vol) 4-chloro-l-naphthol (BioRad Laboratories), 16.7% (vol/vol) methanol and 0.015% (v/v) H2O2.
  • the reaction was stopped by washing in distilled water.
  • Figure 18 and Table 2 show results of challenge experiments after immunization with M. catarrhalis antigens. Mice immunized IPP and boosted IT with purified superoxide dismutase showed statistically enhanced clearance of bacteria from the lungs compared to sham immunised animals protein
  • the mixture was incubated for 2 hrs at 37°C or until the suspension was clear and 8ml of phenol (saturated with lOmM Tris-HCl and ImM EDTA) added followed by gentle mixing for 30sec at 37°C.
  • the solution was centrifuged at 8,000 ⁇ m for 15 mins at 4°C and the DNA containing phase removed and extracted in 5ml phenol/chloroform/isoamyl alcohol (25:24:1). This was gently mixed on ice for 30 sees and centrifuged at 8,000 ⁇ m for 15 mins at 4°C.
  • Bacteria containing the plasmid were cultured in LB broth with the relevant antibiotic and grown in a shaker incubator at 37°C. The cells were pelleted and resuspended in 50mM Tris-Cl, pH 8.0, lOmM EDTA, lOO ⁇ g/ml RNase A before adding 200mM NaOH, 1% SDS and incubating at RT for 5 min. Prechilled 3.0M potassium acetate, pH 5.5 was added and incubated on ice for 15-20 min prior to centrifugation at
  • DNA was precipitated with isopropanol, centrifuged at 15,000xg for 30 min at 4°C and the pellet washed with 70% ethanol by centrifugation at 15,000xg for 10 min at RT. The supernatant was removed and the pellet air dried before resuspending in ImM EDTA,
  • insert purified DNA and plasmid were mixed with the digestion enzymes in the buffer recommended by the supplier for the enzymes. The mixture was incubated at 37°C for 2 hours. Generally a ratio of 3 : 1 is used for insert: vector.
  • DNA and plasmid vectors were then purified by agarose electrophoresis and eluted from the gel using a Qiagen min elute kit. Purified DNA and vector were then mixed with lOxLigation buffer (MBI Fermentas, Progen) and nanopure water (all kept on ice). Ligation enzyme (MBI Fermentas, Progen) was added and incubated at 16°C overnight. Prior to transformation of E. coli the mixture was heat inactivated.
  • the cells were washed by suspending in ice cold 0.1M calcium chloride solution, centrifuged at 6,000rpm for 20 minutes at 4°C and the cell pellet resuspended to a final volume of 1.5mls in (ice cold) 0.1M calcium chloride/ glycerol solution (85:15). Aliquot of the cells (lOO ⁇ l volumes) were placed into sterile 1.5ml microfuge tubes and stored at -80°C.
  • Bacteria were grown in LB broth containing the relevant antibiotic until they reached an optical density of between 0.7-0.9 at 600nm.
  • the protein expression was induced by addition of IPTG to a concentration of 1 mM (or as otherwise determined) and the cells incubated for between 1 and 5 hours (as determined).
  • the bacteria were harvested by centrifugation at 4000xg for 20 min, the pellet resuspended in sonication buffer (50 mM sodium phosphate, 300 mM NaCl, ⁇ H8, 10 mM imidazole) and frozen at -70°C for between 0.5 h and overnight.
  • sonication buffer 50 mM sodium phosphate, 300 mM NaCl, ⁇ H8, 10 mM imidazole
  • the cell suspension was sonicated 4 times, centrifuged at 11,000 rpm for 20 min and the supernatant retained for extraction of the recombinant protein using the Ni-NTA resin.
  • the Qiagen protocol was followed and the protein is extracted into phosphate buffer.
  • the recombinant protein was extracted into a buffer containing 8 M Urea (Qiagen).
  • I2D-21 gave a best match (40% identity) to disulphide bond isomerase A (DSBA) protein from Pseudomonas aeruginosa.
  • DSBA protein catalyses the formation of disulphide bonds.
  • the enzyme can be membrane bound and is present in the periplasm where it ensures the correct folding of membrane and secreted proteins.
  • I2D- 22 gave a good match of 45% over part of the sequence (residues 82-261) to a probable adhesin component from Neisseria meningitidis (group A strain Z2491). These proteins generally function to facilitate adhesion of bacteria to surfaces or other cells. These can be important virulence factors involved in the colonisation and maintenance of infections. I2D-23 showed the best match of 69% identity to a histone-like (HU) protein from Pseudomonas putida.
  • HU histone-like
  • FIG. 20 shows the induction and expression of ID-2 and I2D-14.
  • Figures 21 and 22 show the purified proteins after Ni chromatography that were subsequently used for immunisation studies.
  • Figure 23 shows the induction, expression and purification of superoxide dismutase (I2D-18). I2D-20 and I2D-21 were also prepared in the same way, Figures 24 and 25 show that the proteins were purified to apparent homogeniety and these were used for immunisation studies.
  • I2D-18 superoxide dismutase
  • Table 5 and Fig 26 show the recovery of bacteria from the lungs of mice challenged with M. catarrhalis. Mice immunised with recombinant I2D-18 did not appear to afford any clearance of bacteria from the lungs. This was in contrast to the purified native protein (see Example 6). However, different immunisation regimes were used that may account for this difference. Nevertheless, for recombinant I2D-20 significant clearance of bacteria was observed using the s.c. route of vaccination for both BAL and LH recovered bacteria demonstrating it is also a good vaccine candidate. Using sera collected from immunised mice western blotting was performed using whole-cell extracts from a number of M. catarrhalis strains. Figure 27 shows the results for antisera raised against recombinant I2D-18. Although antibodies recognized the purified recombinant protein no reaction was observed to whole-cell extracts. The reasons for this are as yet unclear.
  • Rats were immunised 3 times on days 0, 14 and 21.
  • the first immunisation was intrapeyer's patch (IPP) in Freund's incomplete adjuvant, followed by two intratracheal boosts (IT) in PBS. Serum was collected on day 28.
  • IPP intrapeyer's patch
  • IT intratracheal boosts
  • Serum was collected on day 28.
  • 5x10 cells was used to vaccinate animals.
  • OMP vaccine 20 ⁇ g of OMP protein was used to vaccinate animals.
  • FIG. 30 shows the results of probing with antisera raised to whole-cell M. catarrhalis. Bands are clearly visible for I2D-22, 12D-16, 12D19 and I2D-14, demonstrating the immunogenicity and presence of these proteins in a killed whole-cell M. catarrhalis vaccine.
  • Figure 31 show the results using antisera to OMP proteins. The blot is not developed for I2D-22, 12D-16 and I2D-19, which may be due to poor transfer indicated by the non-staining areas in these lanes. However, I2D- 14 and I2D-23 were detected and this also demonstrates the presence of these antigens in OMP preparations and their immunogenicity. Both these vaccine preparations have been shown to be protective in animal models of M. catarrhalis infection.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Proteins derivered from Moraxella catarrhalis are described. These proteins can be used as antigens and or immunogens in medicine, in particular in the preparations of vaccines. They can also be used in diagnosis, and for screening as potential antimicrobial targets.

Description

Antigens
The present invention relates to novel proteins derived from Moraxella catarrhalis, and the use of the proteins as antigens and/or imrήunogens in medicine, particularly in the preparation of vaccines and in diagnosis, as well as screening the proteins as potential antimicrobial targets.
Moraxella catarrhalis is a Gram negative diplococcus that is an obligate parasite of the mucous membranes of humans, especially the nasopharynx. Generally it is of low virulence but can cause meningitis, bacteremia, empyema, pericarditis, endocarditis and pneumonia in certain susceptible groups including the elderly, chronic obstructive pulmonary disease (COPD) patients, the immunocomprimised and patients on ventilators.
More commonly it is associated with middle ear infections (otitis media) in children and sinusitis in adults. It is the third most common cause of otitis media in children;
Haemophilus influenzae and Streptococcus pneumoniae accounting for the remainder of cases. M. catarrhalis colonizes the throat of about 5% of healthy children and approximately 12% of all children will have at least one episode of middle ear infection caused by this organism. Total expenditure for otitis media in the United States is estimated to be in excess of $5 billion (Gates, G.A., in The sixth International Symposium on recent advances in otitis media, Hamilton, Ontario: Decker 1996, 1-4.)
Otitis media can have serious consequences for children, resulting in permanent damage to the eardrum and impaired hearing. If untreated or unrecognised in children it can impair learning capacity and delay speech development (J. O. Klein. 2001. Vaccine 19: S2-S8.).
First line treatment involves the use of antibiotics, but most strains of M. catarrhalis carry a β-lactamase on their outer membrane and are thus resistant to penicillins. Fortunately most strains are still susceptible to co-amoxiclav, cefuroxime, tetracyclines and erythromycin. In severe chronic cases, the use of antibiotics is often combined with surgery in which a tympanostomy tube is inserted into the eardrum to aid the drainage of fluid trapped in the middle ear, thus relieving earache and improving hearing. Otitis media still remains the most common cause of antibiotic prescriptions in children. Increased antibiotic resistance is an ever-present threat, which could undermine the current therapeutic treatment of otitis media.
A vaccine for preventing M. catarrhalis induced otitis media would have a number of advantages for use in children. It would act as a prophylactic approach preventing infection and associated complications. This would significantly reduce the prescription of antibiotics, resulting in reduced health care cost and a reduction in the selective pressure on bacteria to develop resistant strains. Several reports have suggested that vaccination could work in preventing otitis media and that protection from infection by M. catarrhalis would come primarily from bactericidal IgG3 antibodies that promote complement dependent killing of the bacteria.
A number of potential vaccine candidates have been identified on the surface of the bacterium and are currently undergoing pre-clinical evaluation (for a review see McMichael J.C. 2000. Microbes and Infection 2, 561-568).
Because there is no effective human vaccine against Moraxella catarrhalis there is a continuing need to search for candidates in order to be able to select the best antigen or combination of antigens that will protect against infection.
Vaccination of rabbits with a formalin inactivated whole-cell preparation of Moraxella catarrhalis has allowed the identification of a number of antigenic proteins that might represent potential vaccine candidates. Candidate antigens were selected from whole-cell protein extracts by identifying those proteins that bind IgG antibodies in sera prepared from rabbits vaccinated sub-cutaneously with inactivated whole cells of Moraxella catarrhalis. By using this technique we have isolated and identified a number of novel antigens for use as vaccines or in the diagnosis of disease.
Thus in a first aspect, there is provided a protein from M. catarrhalis which has the NH2- terminal sequence:
I. MAFTLPELGYSYDALEPGFDK(J )EA(T)XM(G)L; π. MKQPV(T)RVAXT; m. TTQN QQNGKNAINTS(X)AAG(X)LS(X)NAIA(S)T(S)RL;
IV. GVSFAKDIGDKLFHR(S)N(P)K(A)KQ(E)D(P)T(A)AQE(P)I(T)AN(A)LL;
V. ADFNKTLDAGNVDDQ(G)I;
VI. MIQDIFTDLE; vπ. MQNEΠ QAGG; VIII N(E orK)FVEDQD(X)YQ(X)VLP;
IX A(Q)AIINQTffEFXTQAYVNG(X)E(X);
X MNKSELVDG(T)IAQXAGLT; Or contains the sequence:
XI KLGNITSPSGDSA; and/or XII FXPFNLN
Where bracketed amino acids represent an alternative to the preceding amino acid and X represents any amino acid.
The proteins of the present invention are isolatable from M. catarrhalis and may be provided in substantially pure form. For example, it may be provided in a form that is substantially free of other proteins.
As discussed herein, the proteins of the invention are useful as antigenic material. Such material can be "antigenic" and/or "immunogenic". Generally, "antigenic" is taken to mean that the protein is capable of being used to raise antibodies or indeed is capable of inducing an antibody response in a subject. "Immunogenic" is taken to mean that the protein is capable of eliciting a protective immune response in a subject. Thus, in the latter case, the protein may be capable of not only generating an antibody response but, in addition, a non-antibody based immune responses.
The skilled person will appreciate that homologues or derivatives of the proteins of the invention will also find use in the context of the present invention, i.e. as antigenic/immunogenic material. Thus, for instance proteins which include one or more additions, deletions, substitutions or the like are encompassed by the present invention. In addition, it may be possible to replace one arnino acid with another of similar "type". For instance, replacing one hydrophobic amino acid with another. One can use a program such as the CLUSTAL program to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or similarity
(identity plus conservation of amino acid type) for an optimal alignment. The percent identity of two amino acid sequences or of two nucleic acid sequences is determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the first sequence for best alignment with the sequence) and comparing the amino acid residues or nucleotides at corresponding positions. The "best alignment" is an alignment of two sequences which results in the highest percent identity. The percent identity is determined by the number of identical amino acid residues or nucleotides in the sequences being compared (i.e., % identity = number of identical positions/total number of positions x 100).
The determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-226%, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. The NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410 have incorporated such an algorithm. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilised as described in Altschul et al.
(1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilising BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
Both types of analysis are contemplated in the present mvention.
In the case of homologues and derivatives, the degree of identity with a protein as described herein is less important than that the homologue or derivative should retain its antigenicity and/or immunogenicity to M. catarrhalis. However, suitably, homologues or derivatives having at least 60% similarity (as discussed above) with the proteins or polypeptides described herein are provided. Preferably, homologues or derivatives having at least 70% similarity, more preferably at least 80% similarity are provided. Most preferably, homologues or derivatives having at least 90%, 95%, 96, 97, 98, 99 or even 99.8% or greater similarity are provided.
In an alternative approach, the homologues or derivatives could be fusion proteins, incoφorating moieties which render purification easier, for example by effectively tagging the desired protein or polypeptide. It may be necessary to remove the "tag" or it may be the case that the fusion protein itself retains sufficient antigenicity to be useful.
The invention therefore provides, in a second aspect, a protein, which is a homologue or derivative of the proteins of the first aspect of the invention. It is well known that is possible to screen an antigenic or immunogenic protein or polypeptide to identify epitopic regions, i.e. those regions that are responsible for the protein or polypeptide' s antigenicity or i munogenicity. Thus, in a third aspect of the invention, there is provided one or more antigenic or immunogenic fragments
(hereinafter referred to as "antigenic fragments" for convenience) of the proteins of the invention, or of homologues or derivatives thereof. In particular, the fragment may comprise the N-terminal sequence as described above.
Methods well known to the skilled person can be used to test fragments and or homologues and/or derivatives for antigenicity/immunogenicity. For example, the fragments and/or homologues and/or derivatives can be tested to determine whether serum raised against M. catarrhalis reacts against the fragment and/or homologue and/or derivative in question. Thus, the fragments of the present invention should include one or more such epitopic regions or be sufficiently similar to such regions to retain their antigenic/immunogenic properties. Thus, for fragments according to the present invention the degree of identity is perhaps irrelevant, since they may be 100% identical to a particular part of a protein or polypeptide, homologue or derivative as described herein. The key issue, once again, is that the fragment retains the antigenic/immunogenic properties of the protein from which it is derived.
What is important for homologues, derivatives and fragments is that they possess at least a degree of the antigenicity/immunogenicity of the protein or polypeptide from which they are derived.
The proteins of the present invention, or antigenic fragments thereof, can be provided alone, as a purified or isolated preparation. They may be provided as part of a mixture with one or more other M. catarrhalis proteins of the invention, or antigenic fragments thereof, or one or more other catarrhalis antigenic proteins or fragments thereof. In a fourth aspect, therefore, the invention provides an antigen composition comprising one or more proteins of the invention and/or one or more antigenic fragments thereof. Such a composition can be used for the detection and/or diagnosis of M. catarrhalis. In one embodiment, the composition comprises one or more additional M. catarrhalis antigens/immunogens.
In a fifth aspect, the present invention provides a method of detecting and/or diagnosing M. catarrhalis which comprises:
(a) bringing into contact with a sample to be tested an antigenic protein, or an antigenic fragment thereof, or an antigen composition of the invention; and
(b) detecting the presence of antibodies to M. catarrhalis.
In particular, the protein, antigenic fragment thereof or antigen composition of the present invention can be used to detect IgA, IgM or IgG antibodies. Suitably, the sample to be tested will be a biological sample, e.g. a sample of blood or saliva.
In a sixth aspect, the invention provides the use of an antigenic protein, antigenic fragment thereof or an antigenic composition of the present invention in detecting and/or diagnosing M. catarrhalis in vitro.
The antigenic proteins, antigenic fragments thereof or antigenic composition of the present invention can be provided as a kit for use in the in vitro detection and/or diagnosis of M. catarrhalis. Thus, in a seventh aspect, the present invention provides a kit for use in the detection and/or diagnosis of M. catarrhalis, which kit comprises one or more antigenic protein(s) or antigenic fragment(s) thereof or an antigenic composition of the present invention.
In addition, the antigenic protein, antigenic fragment thereof or antigen composition of the invention can be used to induce an immune response against M. catarrhalis. Thus, in an eighth aspect, the invention provides the use of an antigenic protein of the invention, an antigenic fragment thereof or an antigen composition of the invention in medicine.
In a ninth aspect, the present invention provides a composition capable of eliciting an immune response in a subject, which composition comprises a protem, an antigenic fragment thereof, or an antigen composition of the invention. Suitably, the composition will be a vaccine composition, optionally comprising one or more suitable adjuvants. Such a vaccine composition may be either a prophylactic or therapeutic vaccine composition.
The vaccine compositions of the invention can include one or more adjuvants. Examples well-known in the art include inorganic gels, such as aluminium hydroxide, and water-in-oil emulsions, such as incomplete Freund's adjuvant. Other useful adjuvants will be well known to the skilled person.
In yet further aspects, the present invention provides:
(a) the use of a protein, an antigenic fragment thereof, or an antigen composition of the invention in the preparation of an immunogenic composition, preferably a vaccine;
(b) the use of such an immunogenic composition in inducing an immune response in a subject; and
(c) a method for the treatment or prophylaxis of M. catarrhalis infection in a subject, or of vaccinating a subject against M. catarrhalis which comprises the step of administering to the subject an effective amount of a protein, at least one antigenic fragment thereof or an antigen composition of the mvention, preferably as a vaccine. Amongst other uses the vaccine may be used for the treatment or prevention of respiratory infections and otitis media. In an alternative approach, the proteins described herein, or fragments thereof, can be used to raise antibodies, which in turn can be used to detect the antigens, and hence M. catarrhalis. Such antibodies form another aspect of the invention. Antibodies within the scope of the present invention may be monoclonal or polyclonal.
Polyclonal antibodies can be raised by stimulating their production in a suitable animal host (e.g. a mouse, rat, guinea pig, rabbit, sheep, goat or monkey) when a protein as described herein, or a homologue, derivative or fragment thereof, is injected into the animal. If desired, an adjuvant may be administered together with the protein. Well- known adjuvants include Freund's adjuvant (complete and incomplete) and aluminium hydroxide. The antibodies can then be purified by virtue of their binding to a protein as described herein.
Monoclonal antibodies can be produced from hybridomas. These can be formed by fusing myeloma cells and spleen cells which produce the desired antibody and form an immortal cell line. Thus, the well-known Kohler & Milstein technique (Nature 256 (1975)) or subsequent variations upon this technique can be used.
Techniques for producing monoclonal and polyclonal antibodies that bind to a particular polypeptide/protein are now well developed in the art. They are discussed in standard immunology textbooks, for example in Roitt et al, Immunology second edition (1989), Churchill Livingstone, London.
In addition to whole antibodies, the present invention includes derivatives thereof which are capable of binding to proteins etc as described herein. Thus the present invention includes antibody fragments and synthetic constructs, and the term "antibody" as used herein is intended to include these. Examples of antibody fragments and synthetic constructs are given by Dougall et al in Tibtech 12372-379 (September 1994). Antibody fragments include, for example, Fab, F(ab')2 and Fv fragments. Fab fragments (These are discussed in Roitt et al [supra] ). Fv fragments can be modified to produce a synthetic construct known as a single chain Fv (scFv) molecule. This includes a peptide linker covalently joining Nh and Nj regions, which contributes to the stability of the molecule. Other synthetic constructs that can be used include CDR peptides. These are synthetic peptides comprising antigen-binding determinants. Peptide mimetics may also be used. These molecules are usually conformationally restricted organic rings that mimic the structure of a CDR loop and that include antigen-interactive side chains.
Synthetic constructs include chimaeric molecules. Thus, for example, humanised (or primatised) antibodies or derivatives thereof are within the scope of the present invention. An example of a humanised antibody is an antibody having human framework regions, but rodent hypervariable regions. Ways of producing chimaeric antibodies are discussed for example by Morrison et al in PΝAS, 81, 6851-6855 (1984) and by Takeda et al in
Nature. 314, 452-454 (1985).
Synthetic constructs also include molecules comprising an additional moiety that provides the molecule with some desirable property in addition to antigen binding. For example the moiety may be a label (e.g. a fluorescent or radioactive label, or latex or an equivalent solid physical label such as an erythrocyte). Alternatively, it may be a pharmaceutically active agent.
Antibodies, or derivatives thereof, find use in detection diagnosis of M. catarrhalis. Thus, in another aspect, the present invention provides a method for the detection/diagnosis of M. catarrhalis which comprises the step of bringing into contact a sample to be tested and antibodies capable of binding to one or more proteins of the invention, or to fragments thereof. In addition, so-called "Affibodies" may be utilised. These are binding proteins selected from combinatorial libraries of an alpha-helical bacterial receptor domain (Nord et al,). Thus, small protein domains, capable of specific binding to different target proteins can be selected using combinatorial approaches.
Gene cloning techniques may be used to provide a protein of the invention in substantially pure form. These techniques are disclosed, for example, in J. Sambrook et al Molecular Cloning 2nd Edition, Cold Spring Harbor Laboratory Press (1989). Thus, in a further aspect, the present invention provides a nucleic acid molecule comprising or consisting of a sequence which is:
(i) a DNA sequence coding for a protein or polypeptide as described herein or their RNA equivalents;
(ii) a sequence which is complementary to any of the sequences of (i);
(iiϊ) a sequence which has substantial identity with any of those of (i) and (ii);
(iv) a sequence which codes for a homologue, derivative or fragment of a protein as defined herein.
The nucleic acid molecules of the invention may include a plurality of such sequences, and/or fragments. The skilled person will appreciate that the present invention can include novel variants of those particular novel nucleic acid molecules which are exemplified herein. Such variants are encompassed by the present invention. These may occur in nature, for example because of strain variation. For example, additions, substitutions and/or deletions are included. In addition and particularly when utilising microbial expression systems, one may wish to engineer the nucleic acid sequence by making use of known preferred codon usage in the particular organism being used for expression. Thus, synthetic or non-naturally occurring variants are also included wάthin the scope of the invention.
The term "RNA equivalent" when used above indicates that a given RNA molecule has a sequence which is complementary to that of a given DNA molecule (allowing for the fact that in RNA "U" replaces "T" in the genetic code).
The determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. The NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410 have incoφorated such an algorithm. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention.
Another example of a mathematical algorithm utilised for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0) which is part of the CGC sequence alignment software package has incoφorated such an algorithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput. Appl. Biosci., 10 :3-5; and FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 55:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search.
When comparing nucleic acid sequences for the puφoses of determining the degree of homology or identity one can use programs such as BESTFIT and GAP (both from the
Wisconsin Genetics Computer Group (GCG) software package) BESTFIT, for example, compares two sequences and produces an optimal alignment of the most similar segments. GAP enables sequences to be aligned along their whole length and finds the optimal alignment by inserting spaces in either sequence as appropriate. Suitably, in the context of the present invention compare when discussing identity of nucleic acid sequences, the comparison is made by alignment of the sequences along their whole length.
Preferably, sequences which have substantial identity have at least 50% sequence identity, desirably at least 75% sequence identity and more desirably at least 90 or at least 95% sequence identity with said sequences, hi some cases, the sequence identity may be 99% or above.
Desirably, the term "substantial identity" indicates that said sequence has a greater degree of identity with any of the sequences described herein than with prior art nucleic acid sequences.
It should however be noted that, where a nucleic acid sequence of the present invention codes for at least part of a novel gene product, the present invention includes within its scope all possible sequence coding for the gene product or for a novel part thereof.
The nucleic acid molecule may be in isolated or recombinant form. It may be incoφorated into a vector and the vector may be incoφorated into a host. Such vectors and suitable hosts form yet further aspects of the present invention.
Therefore, for example, by using probes based upon the nucleic acid or amino acid sequences provided herein, genes in M. catarrhalis can be identified. They can then be excised using restriction enzymes and cloned into a vector. The vector can be introduced into a suitable host for expression.
Nucleic acid molecules of the present invention may be obtained from M. catarrhalis by the use of appropriate probes complementary to part of the sequences of the nucleic acid molecules. Restriction enzymes or sonication techniques can be used to obtain appropriately sized fragments for probing.
Alternatively, PCR techniques may be used to amplify a desired nucleic acid sequence. Thus the sequence data provided herein can be used to design two primers for use in PCR so that a desired sequence, including whole genes or fragments thereof, can be targeted and then amplified to a high degree. One primer will normally show a high degree of specificity for a first sequence located on one strand of a DNA molecule, and the other primer will normally show a high degree of specificity for a second sequence located on the complementary strand of the DNA sequence and being spaced from the complementary sequence to the first sequence. Typically primers will be at least 15-25 nucleotides long.
As a further alternative chemical synthesis may be used. This may be automated. Relatively short sequences may be chemically synthesised and ligated together to provide a longer sequence.
It is also possible to utilise the nucleic acid molecules of the present invention in the preparation of so-called DNA vaccines. Thus, the invention also provides a vaccine composition comprising one or more nucleic acid sequences as defined herein. The use of such DNA vaccines is described in the art. See for instance, Donnelly et al, Ann. Rev. Immunol, 15:617-648 (1997).
It will also be clear that the nucleic acid sequences described herein may be used to detect/diagnose M. catarrhalis. Thus, in yet a further aspect, the present invention provides a method for the detection/diagnosis of M. catarrhalis which comprises the step of bringing into contact a sample to be tested with at least one nucleic acid sequence as described herein. Suitably, the sample is a biological sample, such as a tissue sample or a sample of blood or saliva obtained from a subject to be tested. Such samples may be pre-treated before being used in the methods of the invention. Thus, for example, a sample may be treated to extract DNA. Then, DNA probes based on the nucleic acid sequences described herein (i.e. usually fragments of such sequences) may be used to detect nucleic acid from M. catarrhalis.
The present invention also provides a method of vaccinating a subject against M. catarrhalis which comprises the step of administering to a subject a nucleic acid molecule as defined herein; a method for the prophylaxis or treatment of M. catarrhalis infection which comprises the step of administering to a subject a nucleic acid molecule as defined herein; and a kit for use in detecting/diagnosing M. catarrhalis infection comprising one or more nucleic acid molecules as defined herein.
The proteins of the present invention are potential targets for anti-microbial therapy. It is necessary, however, to determine whether each individual protein is essential for the organism's viability. Thus, the present invention also provides a method of determining whether a protein or polypeptide as described herein represents a potential anti-microbial target which comprises inactivating said protein and determining whether M. catarrhalis is still viable, in vitro or in vivo.
A suitable method for inactivating the protein is to effect selected gene knockouts, i.e. prevent expression of the protein and determine whether this results in a lethal change. Suitable methods for carrying out such gene knockouts are described in Li et al, P.N.A.S., 94:13251-13256 (1997).
In a final aspect, the present invention provides the use of an agent capable of antagonising, inhibiting or otherwise interfering with the function or expression of a protein or polypeptide of the invention in the manufacture of a medicament for use in the treatment or prophylaxis of M. catarrhalis infection. Preferred features of each aspect of the invention as for each other aspect, mutatis mutandis.
The invention will now be described with reference to the following examples which should not be construed as limiting the invention in any way.
The examples refer to the accompanying drawings in which:
Figure 1. Anion-exchange chromatography elution profile of M. catarrhalis sonicate. 3ml of M. catarrhalis sonicate (0.56mg/ml) was diluted with 12 ml of 20 mM Tris pH
8.0. The 15 ml sample was loaded onto a 1 ml Mono Q column pre-equilibrated with 20 mM Tris pH 8.0 at 1.0 ml/min. Proteins were eluted with a 0-0.5 M NaCl gradient in 20 mM Tris pH 8.0 over 15 column volumes at 1 ml/min, followed by a 0.5-1 M NaCl gradient in 20 mM Tris pH 8.0 over 2 column volumes. 1 ml fractions were collected and are denoted by numbered lines. The absorbance of the eluate was continuously monitored at 280 nm;
Figure 2. SDS-PAGE Bis/Tris NuPAGE analysis of M. catarrhalis proteins separated by Mono Q anion exchange chromatography. Proteins were separated on a 4-12% T Bis/Tris polyacrylamide gel and silver stained. Lane 1 shows molecular weight markers with sizes given in kDa. Lanes 2-14 represent fractions 36, 51, 52, 53, 56, 57, 59, 60, 61, 63, 65, 66, 67 respectively that were eluted from the Mono Q column using a 0-1M NaCl gradient;
Figure 3. Western Blot of Mono Q separated M. catarrhalis proteins, immunostained with Rabbit anti- catarrhalis 90-day serum. Lane 1 shows NuPAGE SeeBlue markers with molecular weight given in kDa. Lanes 2-14 are fractions 36, 51, 52, 53, 56, 57, 59, 60, 61, 63, 65, 66, and 67 respectively that were eluted from the Mono Q column using a 0-1M NaCl gradient; Figure 4. SDS-PAGE analysis of M. catarrhalis proteins following preparative electrophoresis using 10 % T polyacrylamide gel. Lanes 1 and 13 NuPAGE SeeBlue molecular weight standards. Lane 2, M. catarrhalis SDS-extracted whole cells, lane 3- 10 fractions 1, 7, 14, 21, 28, 35, 42 and 49 respectively and lanes 11, 12, 14-19 are fractions 56, 63, 70, 77, 84, 91, 98 and 105 respectively;
Figure 5. Western Blot and Immunostain of fractions from Preparative Electrophoresis of M. catarrhalis SDS-extracted whole cell proteins. Lane 1 and lane 12 are NuPAGE SeeBlue molecular weight standards with molecular weight given in kDa. Lane 2, SDS-extracted whole cells M. catarrhalis proteins, lanes 3-9, fractions 1, 7, 14, 21, 28,
35 and 42 respectively and lanes 10, 11, 13-19 are fractions 49, 56, 63, 70, 77, 84, 91, 98 and 105 respectively;
Figure 6. Western Blot and Immunostain of Fractions from Preparative Electrophoresis of M. catarrhalis SDS-extracted whole cell proteins. Lanes 1 and 14 are BioRaD biotinylated broad range molecular weight standards with molecular weights given in kDa. Lanes 2-10 are fractions 14, 17, 18, 19, 25, 26, 33, 40, 41 respectively and lanes 11-13 and 15-18 are fractions 46, 53, 55, 64, 71, 72, and 73 respectively;
Figure 7. Western Blot and immunostain of a 2-Dimensional Electrophoresis gel of M. catarrhalis SDS-extracted whole cell proteins.
Figure 8. Coomassie stained Western blot of a 2-Dimensional electrophoresis gel of M. catarrhalis SDS-extracted whole cell proteins.
1
Figure 9: Cibachrom blue affinity chromatography chromatogram of whole-cell sonicate extract of M. catarrhalis. Figure 10: Coomassie stained 12.5% SDS-PAGE gel of affinity chromatography fractions of whole-cell sonicate extract of M. catarrhalis. Lane 4 was transferred onto a polyvinylidene diflouride membrane and the protein band indicated by the arrow subjected to NH2 sequencing.
Figure 11 : Ion exchange chromatography chromatogram
Figure 12: Immunoblot (lane 2) and silver stained (lane 3) of ion exchange peak 2.
Figure 13: Hydrophobic interaction chromatography chromatogram
Figure 14: Silver stained (lane 1) and coomassie stained (lane 2) 12.5% SDS- PAGE gel of hydrophobic interaction purified malate dehydrogenase (ID-2)
Figure 15: Immunoblot of purified malate dehydrogenase (ID-2) (lanes 3, 6 & 7) lane 1 contains a positive control (whole cell extract)
Figure 16. The profile of absorbance (y-axis) verses fraction (x-axis) following anion exchange chromatography of the M. catarrhalis extract.
Figure 17. Native I2D-18 (Superoxide dismutase) fractions from 9% prep cell
Figure 18. Clearance of M. catarrhalis from the lungs after challenge in naϊve mice and those immunised with native manganese superoxide dismutase (I2D- Figure 19. Predicted DNA and Protein Sequences of M. catarrhalis Antigens
Figure 20. SDS-PAGE gel showing induction and Expression of M. catarrhalis ID-2 and I2D-14
Figure 21. SDS-PAGE gel showing the purification of recombinant M. catarrhalis ID-2 protein
Figure 22. SDS-PAGE gel showing purified recombinant M. catarrhalis I2D- 14 protein
Figure 23. SDS-PAGE of purified recombinant M. catarrhalis I2D-18 (manganese superoxide dismutase)
Fig. 24. SDS-PAGE and Coomassie Blue staining of recombinant I2D20
Fig. 25. SDS-PAGE and Coomassie Blue staining of recombinant I2D21.
Figure 26. Clearance of Moraxella catarrhalis from the lungs after challenge in naive mice and those immunised with recombinant manganese superoxide dismutase (I2D-18) and I2D20.
Figure 27. I2D-18 IgG immunoblot using 1/20 sera dilution of pooled sera from mice immunised with I2D-18.Shows recognition of I2D-18, but no recognition of a band in the M.catarrhalis cell lysates Figure 28. I2D-20 IgG immunoblot using 1/20 sera dilution of pooled sera from mice immunised with I2D-20. Shows recognition of the recombinant protein and a corresponding protein expressed in six strains of M. catarrhalis
Figure 29. Coomassie stained SDS-PAGE gel of proteins and cell lysates used for Western Blots
Figure 30. Western Blot using Non-Immune Sera
Figure 31. Western Blot: Anti- Whole Killed Cell
Figure 32. Western Blot using Outer Membrane Protein antisera (20ig)
Example 1- Preparation of Immune Sera
Culture of Moraxella catarrhalis
Moraxella catarrhalis strain 25238 was obtained from ATCC. Stocks of M. catarrhalis were stored at -80°C in tryptone soya broth containing 20 % (v/v) glycerol. In the lab, starter cultures were grown by streaking a loopful of stock onto chocolate agar plates and growing in an atmosphere containing 5% C02 overnight at 37°C. Single colonies from agar plates were inoculated into 4 x 250 ml of brain heart infusion broth in 500 ml shake flasks. Cultures were grown for 16-20 hours at 37°C in a shaker incubator at 250 φm.
Preparation of Whole-Cell Vaccine
Cultures were grown as above and then formalin added to a final concentration of 1 % formaldehyde. The formaldehyde containing culture was then incubated for a further 16 h at 37 °C. Cells were harvested by centrifugation at 8000 x g for 5 min. The supernatant was discarded and the cell pellets resuspended in sterile phosphate buffered saline (PBS). Cells were recovered by centrifugation and washed a further two times in sterile PBS. Cell pellets were frozen at -80 °C and then lyophilised for 16 h at room temperature in a Birchover freeze drier. Lyophilised cells were stored at - 80 °C until ready for use.
Immunisations
Two Rabbits were immunised by subcutaneous administration with 1 ml of 1 mg/ml of formalin killed whole cell M. catarrhalis lyophilisate in Freund's complete adjuvant. Booster immunisations were given on a 30-day basis for 6 months with 1 ml of 1 mg/ml lyophilisate in incomplete Freund's adjuvant. 10 mis of rabbit serum were taken 24 hours before each immunisation on days 0, 30, 60, 90, 120 and 150. After 6 months the rabbits were killed. Sera was prepared by- storing whole blood samples in the freezer for 16-20 hours. Blood clots were removed and the serum was centrifuged at 4000 x g for 10 minutes. Supernatants were removed and stored at -20°C.
Example 2-Extraction and Purification Moraxella catarrhalis Antigens
Subcellular fractionation
Cells were harvested from cultures by centrifugation at 4000 x g for 2.5 hours and washed with one volume of 25 mM sodium phosphate buffer pH 7.0. The cells were resuspended in 20 ml 25 mM sodium phosphate buffer pH 7.0 containing 1 mM l-(2- Ammoethyl)benzenesulfonylfluoride-HCl (AEBSF protease inhibitor) and disrupted by sonication using a Sanyo Soniprep 150 (19 mm probe) at an amplitude of 6 μm for
25 cycles of 30 s on and 60 s off. Cell debris and unbroken cells were removed by centrifugation at 11,000 x g for 30 min. The resulting supernatant was then used for locating the antigens.
Anion Exchange Chromatography
Proteins from the sonicate supernatant of M. catarrhalis were separated by Anion exchange chromatography using a 1 ml Mono Q (Amersham Pharmacia) column. Briefly, 3 ml of M. catarrhalis sonicate (0.56 mg/ml) was diluted with 12 ml of 20 mM Tris pH 8.0. The 15 ml sample was loaded onto a 1 ml Mono Q column pre- equilibrated with 20 mM Tris pH 8.0, using a BioCad Vision at 1.0 ml/min. Unbound material was washed from the column with 25 ml of 20 mM Tris pH 8.0. Protein was eluted by using a 0-0.5 M NaCl gradient in 20 mM Tris pH 8.0, over 15 column volumes at 1 ml/min, followed by a 0.5-1M NaCl gradient in 20 mM Tris pH 8.0 over 2 column volumes. The eluate was continuously monitored at 280 and 214 nm and 1 ml fractions were collected. Each fraction was tested for protein content and analysed by SDS-PAGE and Western blotting.
SDS-PAGE using Novex 4-12% T Bis/Tris Acrylamide Gels
Chromatography fractions were run on 4-12% Novex Bis-Tris gels (Invitrogen) according to manufacturer's instructions. Briefly, 65 μl of sample was mixed with 25 μl 4 x LDS NuPAGE sample buffer and 10 μl of NuPAGE 10 x reducing agent. samples were heated at 70°C for 10 minutes and then centrifuged at 16,000 x g for 5 minutes. 10 μl of sample was loaded per lane and 5μl of Novex SeeBlue molecular weight markers were loaded in one lane of the gel. Proteins were separated with 1 x NuPAGE MOPS running buffer containing 500 μl of NuPAGE running buffer antioxidant per 200ml of running buffer. Gels were run at 200 volts until the dye front reached the bottom of the gel (approximately 45 minutes). Gels were removed from the casting plates and either coomassie stained, silver stained or Western blotted.
Silver Staining
Silver staining was performed as described by Heukeshoven and Dernick (1985;
Electrophoresis 6, 103-112)
Western Blotting and Immunostaining
SDS-PAGE using NuPAGE 4-12% Bis/Tris gels was performed on column fractions as described above, except BioRad biotinylated Broad range molecular weight markers were used. Following electrophoresis, proteins were transfened onto nitrocellulose membrane for 1 hr at 65 volts using BioRad mini Trans-blot wet transfer equipment in accordance with manufacturer's instructions. Following Western blotting, the nitrocellulose membrane was blocked in 1 % (w/v) BSA in Tris buffered saline (TBS 20 mM Tris-HCl, 150 mM NaCl pH 7.5) for 30 minutes and then washed twice for 5 minutes each with Tween-Tris buffered saline (TTBS ; TBS containing 0.05 % (v/v) Tween 20). The blot was then incubated overnight with primary antibody (1:200 dilution of M. catarrhalis day 30 or day 90 immune serum in TTBS containing 1 % (w/v) BSA). The nitrocellulose membrane was then washed twice in TTBS and incubated for 2 hours with swine anti-rabbit IgG horse radish peroxidase (HRP) conjugate (1:500 dilution in TTBS containing 1 % (w/v) BSA). A 1:500 dilution of avidin peroxidase was added to the conjugate, which binds to the biotinylated molecular weight markers. Following incubation of the conjugate, the membrane was washed twice in TTBS and twice in TBS for 5 minutes each. The blot was then developed by incubating with the HRP-substrate, 4-chloronapthol (4-CN ; 30 mg 4- CN in 10 ml methanol plus 50 ml TBS and 30μl of 30 % (v/v) H2O2). After a sufficient time, to visualise the protein antigens, the reaction was stopped by immersing the nitrocellulose membrane in ddH2O for 10 minutes.
NH2-TermmaI Amino Acid Analysis
The antigens contained in column fractions were separated by SDS-PAGE using NuPAGE 4-12 % Bis/Tris gels and then transfened onto polyvinylidene difluoride membrane using BioRad mini Trans-blot apparatus as described above. After transfer, the membrane was stained with 0.25 % (w/v) Coomassie Brilliant Blue R-250 dissolved in 40 % (v/v) methanol for 5 minutes, followed by destaining in 50 % (v/v) methanol. The membrane was dried in air between filter paper at room temperature and stained proteins were cut out and placed in the upper cartridge of the 471 A NH2- terminal sequencer (Applied Biosystems). The NH2-amino acid sequence was determined by Edman degradation. Results
Purification of Moraxella catarrhalis Protein Antigens by Anion Exchange Chromatography
Moraxella catarrhalis proteins were separated by Mono Q anion exchange chromatography by using a 0-1 M NaCl gradient. The chromatography profile can be seen in Figure 1. Fractions 36, 37, 51, 52, 53, 56, 57, 59, 60, 61, 63, 65, 66, 67 were analysed by SDS-PAGE using 4-12% T Bis/Tris NuPAGE gels and were silver stained or Western blotted. Western blots were immunostained with 30 day Moraxella catarrhalis immune serum. The silver-stained gel can be seen in Figure 2. The gel shows numerous proteins that could be of interest, especially in fractions 60-67. The Western blot and immunostain of these fractions (Figure 3) shows that only a few of these proteins are immunogenic to the 30-day immune serum. Proteins ID-1 and ID-2 were two of the most antigenic proteins and were therefore selected for NH2-terminal amino acid sequencing.
NH2-Terminal Amino Acid Sequencing of Proteins
NH2-terminal amino acid sequencing of ID-1 and ID-2 showed that ID-1 has an NH2- terminal amino acid sequence of AFTLPELGYSYDALEPGFDK(N)EA(T)XM(G)L. Bracketed amino acids represent an alternative for the preceding residue and X represents an unidentified amino acid. A BLAST (blastp) search of the first 20 amino acids was performed at the NCBI internet site (www.ncbi.nlm.nih.gov), using the BLOSUM62 matrix with the default settings. Fifty-five hits were returned showing homology to the enzyme superoxide dismutase (SOD) from various bacterial species. The best alignment was with Mannheimia haemolytica serotype 1 that had an identity of 85 % over the first 20 amino acids of the above sequence. A translated DNA search (TBLASTN) returned 40 hits the best match giving an 85 % identity to a superoxide dismutase from Pasteurella haemolytica.
For ID-2 the NH2-terminal amino acid sequence was MKQPV(T)RVAXT. A BLAST search (ignoring the bracketed amino acid) using the PAM30 matrix reveals the first 4 hits showing homology to the enzyme malate dehydrogenase. The best alignment was 90 % over the 10 amino acids to malate dehydrogenase isolated from Deinococcus radiodurans. A translated DNA (TBLASTN) search again gave the best match to the malate dehydrogenase from Deinococcus radiodurans.
Example 3- Extraction and Purification of M. catarrhalis Protein Antigens by Preparative Electrophoresis
Sodium Dodecyl Sulphate (SDS) Extraction of Whole-Cell M. catarrhalis Protein
Antigens
Cell pellets from 5 ml of culture were mixed with 1.25 ml of 5 x SDS sample buffer (10 % (v/v) glycerol, 10 % (w/v) SDS, 5 % (v/v) 2-mercaptoethanol (added fresh) in 315 mM Tris-Cl pH 6.8 and coloured with bromophenol blue). The sample was boiled for 10 minutes and then centrifuged at 16,000 x g for 10 minutes. The resulting supernatant was used for preparative electrophoresis.
Preparative Electrophoresis
A BioRad Mini Prep Cell was used to separate proteins by their size from whole cell M. catarrhalis. A 9 % acrylamide gel (3.0 ml of acrylamide/bis-acrylamide (30% T30 2.7% C), 2.5 ml 3 M Tris-Cl pH 8.85 and 4.5 ml of ddH2O), was poured into the casting tube according to manufacturers instructions. Electrophoresis buffer (25 mM Tris, 192 mM Glycine and 0.1% (w/v) SDS) was placed into the upper and lower chamber. Elution buffer (50 mM Tris-Cl, 0.1 % (w/v) SDS pH 8.5) was placed into the elution chamber, which was mixed with the proteins as they were eluted from the bottom of the gel.
0.5 ml of the prepared sample was loaded onto the gel and run at 200 volts, 6 mA with a pump speed of 150 μl min. 500 μl fractions were collected and each was analysed by SDS-PAGE on a 10 % T acrylamide gel.
SDS-PAGE using 10 % T Acrylamide Gels
Fractions obtained from Preparative Electrophoresis were analysed by SDS-PAGE. 10 % T acrylamide gels were cast ( 3.3 ml of Acrylamide/Bis acrylamide stock (30 % T 2.7 % C ), 2.5 ml 3 M Tris-Cl pH 8.85, 4.2 ml ddH20, 100 μl 10 % (w/v) SDS, 10 μl TEMED and 100 μl of 50 mg/ml Ammonium persulphate) leaving enough space for a 4 % stacking gel (1.3 ml acrylamide/Bis-acrylamide stock (30 % T 2.7 % C), 5 ml
0.25 M Tris-Cl pH 6.8, 3.7 ml ddH20, 100 μl 10 % (w/v) SDS, 20 μl TEMED and 100 μl of 50 mg/ml Ammonium Persulphate). 40 μl of samples were added to 10 μl of 5 X SDS sample buffer (10 % (v/v) glycerol, 10 % (w/v) SDS, 5 % v/v 2-mercaptoethanol (added fresh) in 315 mM Tris-Cl pH 6.8 coloured with bromophenol blue). Samples were boiled for 5 minutes and then centrifuged at 16,000 x g for 5 minutes. BioRad
Broad range molecular weight markers were diluted 1 :20 with 1 x SDS sample buffer and also boiled for 5 minutes. 10 μl of each sample and molecular weight markers were loaded per lane using a Hamilton syringe. Gels were run at 200 volts until the dye front reached the bottom of the gel (approx. 45 minutes). Gels were removed from the glass plates and the stacking gel was discarded. The gels were either silver stained or Western blotted. Silver Staining
Silver staining was performed as described by Heukeshoven and Demick (1985; Electrophoresis 6, 103-112)
Western Blotting and Immunostaining
SDS-PAGE 10 % T acrylamide gels was performed as described above, except BioRad biotinylated Broad range molecular weight markers were used. Western blotting and immunostaining were performed as described in example 2.
NH -Terminal Amino Acid Analysis
The antigens were separated by SDS-PAGE using 10 % T acrylamide gel and then transferred onto polyvinylidene difϊuoride membrane and protein sequenced as described in example 2.
Results
Preparative electrophoresis was performed on M. catarrhalis SDS-extracted whole cells to separate proteins on the basis of molecular size in order to identify more antigenic proteins. Figure 4a and 4b show SDS-PAGE gels of fractions collected from a preparative electrophoresis experiment. Silver staining of these fractions after SDS- PAGE shows proteins ranging from 6 kDa to 300 kDa in size. Figure 5 shows the same fractions Western blotted and immunostamed. Many of these proteins are very antigenic. Figure 6 shows a repeated Western blot and immunostain of fractions selected for further investigation in order to identify proteins for NH -terminal amino acid sequencing. Two of these proteins are indicated on the gel by arrows (Fig. 6), these are designated ID-3 and ID-4. NH2-terminal amino acid sequencing results for these proteins showed that ID-3 is superoxide dismutase with the same amino acid sequence as ID-1 isolated by anion exchange chromatography and described in example 2
ID-4 had an NH2-terminal amino acid sequence of TTQNNQQNGKN. This protein showed no significant matches after a blastp search using the PAM30 matrix. Modify PAM30 by adjusting the expected value from 10 to 100 matched the sequence to the internal portion of a number of transferrin binding proteins (best match 72 % ID over the 11 amino acids). Further refinement using the PSI-BLAST iteration program gave hits to a number of other proteins, the best of which was a 63 % identity to the ΝH2- terminal amino acid sequence of a vacuolar protein. Overall, the BLAST searches do not reveal convincing data on the homology or identification of this protein. It can therefore be acknowledged that this is a novel protem of unknown function.
Due to the large number of antigens identified and the complexity of bands using the
1-D SDS-PAGE it was decided, that in order to obtain better resolution of the antigenic proteins for sequencing, 2-dimensional electrophoresis would be performed.
Example 4- Extraction and Purification of M. catarrhalis Protein Antigens by 2- Dimensional Gel Electrophoresis
Sodium Dodecyl Sulphate (SDS) Extraction and Preparation of Whole-Cell M. catarrhalis Protein Antigens for Electrophoresis
A Moraxella catarrhalis cell pellet (5ml (0.87g)) was extracted with 1.25 ml of 5 x SDS sample buffer and boiled for 10 minutes as described for preparative electrophoresis. The sample was centrifuged at 16,000 x g for 10 minutes and the supernatant removed. 900 μl of this supernatant was mixed with 100 μl of 100% (w/v) trichloroacetic acid (TCA) and stored in the freezer for 10 minutes. The sample was then centrifuged for 5 minutes at 4,000 x g. The resulting protein pellet was washed once in 1 ml of ice cold acetone and centrifuged at 4,000 x g for 5 minutes. The pellet was then solubilised in 1 ml of 2-dimensional rehydration buffer (8 M Urea, 2 % (w/v) CHAPS and bromophenol blue).
2-Dimensional Electrophoresis
50 μl of M. catarrhalis protein sample prepared as above was added to 250 μl of rehydration buffer containing 0.7 mg of DTT and 1.25 μl of IPG 3-10 buffer (Amersham Pharmacia UK) and 250 μl loaded onto the 13 cm pH 3-10 immobiline strip (Amersham Pharmacia UK) and was rehydrated overnight on a IPGphor electrophoreisis system as per manufacturers instructions (Amersham Pharmaica UK) at 30 volts for 195 volt hours followed by 60 volts for 360 volt hours. The proteins were separated according to their pi by running at 500 volts for 500 volt hours, 1000 volts for 1000 volt hours then 8000 volts for 20000 volt hours. After electrophoresis, the strip was removed from the machine and placed in 10 ml of SDS equilibration buffer containing 100 mg of DTT (50 mM Tris, 6 M Urea, 30 % (v/v) glycerol, 2 % (w/v) SDS and a trace of bromophenol blue). The strip was then loaded onto a 9 % T SDS polyacrylamide gel in a SE600 Hoefer electrophoresis unit. The gel was run at 100 volts, 45 mA for 30 minutes and then 200 volts, 90 mA until the dye front reached the bottom of the gel.
Western Blotting and Immunostaining
For Western blotting of 2-dimensional gels, a semi-dry system was used. Following electrophoresis, proteins were transfened onto PVDF membrane for 2.5 hr at 15 volts using BioRad semi-dry Transblot equipment in accordance with manufacturer's instructions. Following Western blotting, immunostaining was performed as described in Example 2. NH2-Terminal Amino Acid Analysis
The antigens were separated by 2-dimensional electrophoresis and then transferred onto polyvinylidene difluoride membrane using BioRad semi-dry Transblot apparatus as described above. The proteins were then sequenced as described in example 2
Results
2-Dimensional electrophoresis of M. catarrhalis proteins
In order to improve the separation and resolution of the numerous antigenic proteins identified by 1-D electrophoresis (described in examples 2 and 3), 2-D electrophoresis was performed on the SDS-extracts from M. catarrhalis whole cells. Figure 7 shows a Western blot of a 2-D electrophoresis gel that has been immunostamed with M. catarrhalis immune serum. The blot shows a large number of strongly antigenic proteins, all of which are labelled with their unique identifier, from I2D-5-26. Figure 8 shows a coomassie stained Western blot of the same preparation used for NH2- terminal amino acid sequencing and labelled with the corresponding antigenic proteins.
NH2-Terminal Amino Acid Analysis
The results of NH -terminal amino acid sequencing of antigens identified by 2-D electrophoresis and Western blotting are shown in Table 1. I2D-7 gave an identical sequence match to OMPCD protein of M. catarrhalis, which has previously been described as a promising vaccine candiate. It is an integral outer membrane protein with homology to the Opr F porin from Pseudomonas aeruginosa.
I2D-17 had the same NH2-terminal sequence as ID-4 from preparative electrophoreisis and as I2D-19, which suggest that it may exist as several isoforms. I2D 18 had the Table 1. NH -terminal Sequences of Moraxella catarrhalis proteins separated by 2-D Electrophoresis. Bracketed residue indicate an alternative to the preceding amino acid. X indicates any other amino acids.
Figure imgf000033_0001
same sequence as ID-1 isolated by anion exchange chromatography and ID-3 isolated by preparative electrophoresis.
I2D-14, 12D15 (i and ii), I2D-16 and I2D-20, ID21 and ID23 represent additional new sequences. A BLAST (blastp and tblastn) search was made on the sequences, omitting the bracketed amino acids, using the PAM30, Blosum 62 and Blosum 45 matrices. No significant matches were returned.
Example 5 In-Gel Digest of Antigens
For antigens where it was difficult to obtain NH2-terminal sequence of the intact protein proteolytic digestion was performed to obtain internal peptide sequences. Gel slices from 2-D SDS-PAGE containing the antigen of interest were cut in half, placed in an eppendorf tube and washed (2 x 30 min) with 50% (v/v) acetonitrile, 0.2M ammonium bicarbonate pH8.9 and then freeze-dried for 1 hour. The slices were re- swollen in RHB (0.2 M ammonium bicarbonate pH7.8, 0.02% tween 20) containing a quantity of trypsin equivalent to approximately 10 % (w/w) of target protein. This buffer was added to the slices in 10-20 μl aliquots, allowing each gel slice to take up the buffer before adding the next aliquot. When completely re-swollen the slices were incubated at 37°C overnight. At the end of the incubation period excess RHB was removed to a second eppendorf tube and peptides were extracted from the gel slices with 2 lots of 60% v/v acetonitrile containing 0.1% v/v TFA. These washes were pooled with the excess buffer, concentrated by centrifugal evaporation and applied to a Brownlee Aquapore C4 RP-HPLC column (220 x 2.1 mm) equilibrated in 0.08% v/v TFA. Peptides were separated with a 95 min. gradient of 0-64% v/v acetonitrile in 0.08% v/v TFA over 95 min and elution was monitored at 214 nm. Suitable peptides were subject to NH2-terminal sequencing by Edman degradation using an Applied Biosystems model 471A Protein Sequenator. Results
The in gel digestion of I2D-22 generated two peptides. The NH2-terminal sequences of these peptides were determined to be KLGNITSPSGDSA and FXPFNLN. A BLAST search on the NCBI web site did not return any significant homologies to these peptides.
Example 6. Purification of Native Moraxella catarrhalis Antigens for Immunization studies
A) Malate Dehydrogenase
Materials and Methods
Bacterial Strain
Moraxella catarrhalis strain 11020 was obtained from the Department of Microbiology, School of Biological Sciences, University of Liveφool.
Growth of Bacteria
1 litre of sterile brain heart infusion broth, contained in a 2 litre shake flask, was inoculated with 10 ml of prepared Moraxella catarrhalis cells taken from a cell bank. The culture was grown for 16 - 20 hours at 37°C in a shaker incubator at 180 φm.
SDS-PAGE
Chromatography fractions were analysed by SDS-PAGE. 12.5% acrylamide gels were cast (4.2 ml Acrylamide Bis acrylamide stock (30% T, 2.7% C), 2.5 ml 3 M Tris HCl pH 8.85, 3.3 ml ddH2O, 100 μl 10% (w/v) SDS, lOμl TEMED and 100 μl of 50 mgml"1 Ammonium persulphate) leaving enough space for a 4% stacking gel (1.3 ml Acrylamide/Bis acrylamide stock (30% T, 2.7% C), 1.0 ml 1.25 M Tris HCl pH 6.8, 6.7 ml ddH2O, 100 μl 10% (w/v) SDS, 20μl TEMED and 100 μl of 50 mgml"1 Ammonium persulphate). 40 μl of sample was added to 10 μl of 5X SDS sample buffer (10% (v/v) glycerol, 10% (w/v) SDS, 5% (v/v) 2-mercaptoethanol (freshly added) in 315 mM Tris HCl pH 6.8 coloured with bromophenol blue). Samples were heated for five minutes at 95 °C and then centrifuged at 16,000 x g for five minutes. 7 μl of sample or 5 μl of molecular weight markers (Sigma Dalton NII-L™, except for Western blots, where BioRad prestained broad range markers were used) were loaded onto each lane, using a 25 μl Hamilton syringe. Gels were run at 200 volts, 30 mA (per gel) until the dye front reached the bottom of the gel (approximately 60 minutes). Gels were removed from the glass plates and the stacking gel was discarded. The gels were either coomassie stained, silver stained or Western blotted.
Silver Staining
Silver staining was performed as described by Heukenshoven and Demick (1985; Electrophoresis 6, 103-112)
Western Blotting and Immunostaining
Western blots of 1-dimentional gels run were performed using a wet system. Following electrophoresis, proteins were fransfened onto nitrocellulose membrane for 1 hour at 76 volts, 400 mA using BioRad mini Trans-blot equipment in accordance with the manufacture's instructions. Following transfer, the nitrocellulose membrane was blocked in 1% (w/v) fat free dried milk powder in Tris buffered saline (TBS; 20 mM Tris-HCl, 150 mM ΝaCl pH 7.5) for 15 minutes and then washed three times for five minutes each with Tween-Tris buffered saline (TTBS; TBS containing 0.1% (v/v) freshly added Tween-20). The blot was then incubated, overnight at 4°C with primary antibodies (1:800 dilution of 120 day Moraxella catarrhalis immune serum (supplied by Provalis PLC) in TTBS containing 1% (w/v) fat free dried milk powder). The nitrocellulose membrane was then washed three times in TTBS prior to incubation, for four hours, with secondary antibodies - swine anti-rabbit IgG horseradish peroxidase
(HRP) conjugate (1:2000 dilution in TTBS containing 1% (w/v) fat free dried milk powder). Following incubation in secondary antibodies the membrane was washed in TTBS, three times for five minutes. The blot was then developed by incubating in 20ml TBS containing 0.5 ml diaminobenzidine (DAB; tetrahydrocholride form, made 20 mgml"1 in TBS) and lOμl H2O2. Once bands were sufficiently visible, incubation was terminated by washing the membrane in 20 ml dcffl^O three times for five minutes each.
NH2 Terminal Amino Analysis
Proteins were separated by SDS-PAGE using a 12.5% acrylamide gel and then fransfened onto a polyvinylidene difluoride (PNDF) membrane using BioRad mini Trans-blot apparatus as described above. After transfer, the membrane was stained with 0.25% (w/v) Coomassie Brilliant Blue R-250 dissolved in 40% (v/v) methanol. Followed by destaining in 50% (v/v) methanol. The membrane was briefly dried in air, before being placed between two filter papers to dry completely. The dried membrane was stored in a sealed bag at -20°C. Stained proteins were cut out and placed into the upper cartridge of the 471 A ΝH2-terminal sequencer (Applied Biosystems). The NH2-amino acid sequence was determined by the Edman degradation. Protein Assay
1 volume of BioRad protein assay concentrate was diluted with 4 volumes of ddH2O and filtered through a Whatman No. 1 filter paper. The prepared reagent was stored at 4°C in a brown bottle. 20 μl of protein sample was added to 1 ml of BioRad reagent and mixed by gently inverting the tube, several times. The reaction was left for five minutes, after which the absorbance was read at 595 nm against a reagent blank, on a LKB Biochrom ultraspec 4050 spectrophotometer. Protein concentrations were determined by comparing their A5 5 with a calibration curve, constructed earlier, for known concentrations of bovine gamma globulin.
Purification of Malate Dehydrogenase
Subcellular Fractionation
Moraxella catarrhalis cells were harvested from cultures by centrifugation at 5,850 x g for 5 minutes at 4°C and washed with 1 volume of Tris buffer (TB; 50 mM Tris HCl pH 8.0). The cells were then resuspended in 20 ml TB and disrupted by sonication using a MSE 100 watt Ultrasonic Disintegrator at % power for 10 minutes while on ice. Cell debris and unbroken cells were removed by centrifugation at 25,100 x g for 30 minutes at 4°C. The resulting supernatant was then subjected to purification by affinity chromatography.
Affinity chromatography
Proteins from the sonicate supernatant of Moraxella catarrhalis were separated using affinity chromatography. Briefly, a 10 ml affinity gel was poured (Cibacron Blue 3GA dye (Sigma C-1285, lot no. 31K7002)) into an Amersham Pharmacia C 10/10 column and equilibrated with three bed volumes of Tris buffer (TB; 10 mM Tris HCl pH 8.0) at a flow rate of 0.5 ml/min. The sonicate supernatant was loaded onto the column at a flow rate of 0.5 ml/min and washed with TB, to remove non-adsorbed proteins, until the absorbance at 280nm returned to zero. The mobile phase was then changed for TB containing 200 mM NaCl and washed at a flow rate of 0.5 ml/min until the absorbance at 280 nm returned to zero. At this point the mobile phase was changed once more for TB, this time containing 500 mM NaCl and washed at a flow rate of 0.5 ml min until the absorbance at 280 nm returned to zero. Eluant from both stages was collected as separate fractions and tested for enzyme activity protein, content and analysed by SDS-PAGE, prior to overnight dialysis, against 1.5 litres of 50 mM Tris HCl pH 8.0 at 4°C.
Ion Exchange Chromatography
Dialysed proteins were separated by ion exchange chromatography using a 1 ml Mono Q (Amersham Pharmacia) column. Briefly, the entire dialysed sample was loaded onto a 1 ml Mono Q column, pre-equilibrated with 25 mM Tris HCl pH 8.0, using a Pharmacia FPLC at 0.5 ml/min. Unbound material was eluted by washing the column with 25 mM Tris HCl pH 8.0, until the absorbance, at 280nm, returned to zero. Protein was eluted using a 0 - 0.1 M NaCl gradient in 25 mM Tris HCl pH 8.0, over 5 column volumes, followed by a 0.1 - 0.3 M NaCl gradient in 25 mM Tris HCl pH 8.0 over 25 column volumes and finally a 0.3 - 1.0 M NaCl gradient in 25 mM Tris HCl pH 8.0 over 2.5 column volumes. The eluate was continuously monitored at 280 nm and fractions collected conesponding to the major peaks, were tested for protein content, enzyme activity and analysed by SDS-PAGE and Western blotting.
Hydrophobic Interaction Chromatography
Malate dehydrogenase containing samples, identified by the enzyme activity assays were further purified by hydrophobic interaction chromatography using a 1 ml Phenyl Superose (Amersham Pharmacia) column. Briefly, ammonium sulphate was slowly added to the sample to bring the concentration, with respect to ammonium sulphate, to 1.5 M. The sample was then subjected to centrifugation at 13,000 x g for 5 minutes. The supernatant was loaded onto a 1 ml Phenyl Superose column, pre-equilibrated with 25 mM Tris HCl pH 8.0 containing 1.5 M ammonium sulphate, using a Pharmacia FPLC at 0.5 ml/min. Unbound material was eluted by washing the column with 25 mM Tris HCl pH 8.0 containing 1.5 M ammonium sulphate, until the absorbance, at 280nm, returned to zero. Malate dehydrogenase was eluted using a 1.5 - 0 M ammonium sulphate gradient in 25 mM Tris HCl pH 8.0, over 10 column volumes. The eluate was continuously monitored at 280 nm and the fraction collected conesponding to the major peak, was tested for protein content, and analysed by SDS- PAGE (coomassie and silver stained) and Western blotting. Purified protein was subjected to overnight dialysis against 1.5 litres 10 mM ammonium bicarbonate, after which the sample was freeze dried using a Freezemobile freeze drier.
Results
A process of centrifugation - sonication - centrifugation was used to isolated proteins from 1 litre of overnight Moraxella catarrhalis culture. Affinity chromatography was performed on this whole cell extract to separate proteins based on their ability to reversibly bind to Cibacron Blue 3GA dye. Figure 9 shows a chromatogram of this step. Enzyme activity assays revealed that the majority of the malate dehydrogenase was contained in the 16 ml (1.4 mgml"1) sample collected during elution with 500 mM NaCl. Figure 10 shows SDS-PAGE gels of the fractions collected during affinity chromatography. This sample was, after overnight dialysis, further purified by ion exchange chromatography
The presence of malate dehydrogenase was further confirmed by NH2 amino acid terminal sequencing of the first 9 residues of the protein band, shown in figure 10. This revealed two sequences. The predominant sequence being NEIVVYSAR and the lesser MKQPVRVAV. The latter is an exact match to that of malate dehydrogenase from Moraxella catarrhalis. A BLAST search of the other sequence gave a best match to D-lactate dehydrogenase from Agrobacterium tumefaciens.
Further separation of Moraxella catarrhalis proteins eluted with 500 mM NaCl was performed by Mono Q ion exchange chromatography, using a 0 - 1 M NaCl gradient. The chromatogram profile can be seen in figure 11. Two fractions were collected conesponding to the two main peaks shown in figure 11, both were tested for enzyme activity. Peak 1 gave no increase in absorbance at 340 nm, while the absorbance of peak 2 increased steadily over a period of 5 minutes. Figure 12 shows silver stained and immunoblotted SDS-PAGE gels of peak 2. 1.6 ml of solution eluted under peak 2 and had a concentration 1.0 mgml"1.
Affinity chromatography and ion exchange chromatography were repeated on two further 1 litre overnight Moraxella catarrhalis cultures. Ion exchange purified malate dehydrogenase from all three runs was pooled and brought to an ammonium sulphate concentration of 1.5 M by adding 1.65g of ammonium sulphate prior to separation, performed on a Phenyl Superose hydrophobic interaction chromatography column. The resultant chromatogram is shown in figure 13. Peak 1 contained 1.6 ml of elutant at a protein concentration of 0.63 mgml"1. Figure 14 shows a coomassie and silver stained SDS-PAGE gel of peak 1 and figure 15 shows an immunoblot of peak 1. After overnight dialysis, 0.85 mg of malate dehydrogenase remained in solution. This was subsequently freeze dried for storage and prior to testing.
B) Purification of Superoxide Dismutase
Methods
20ml of Brain heart infusion (BHI) broth was inoculated with 4-5 colonies of M. catarrhalis K65 strain and incubated overnight at 37°C in a shaker incubator. 2mls of the culture was added to each of 2 x 500ml flasks of BHI broth and incubated overnight at 37°C in a shaker incubator. The bacteria were pelleted at 10,000 φm for 15 mins at 4°C in a Beckman JA-2 centrifuge using a JA-14 rotor and then washed three times by resuspension in PBS followed by centrifugation. The proteins were extracted using a Zwittergent extraction and ethanol precipitation method. The washed bacterial pellet was resuspended in 20 ml of 1 M sodium acetate and 0.01 M β- mercaptoethanol which was then stined at room temperature for 45 min before 80 ml of 5% (w/v) Zwittergent 3-14 in 0.5 M calcium chloride was added and stined for a further 90 min at room temperature. Ethanol was added to a final concentration of 20% (v/v) and the suspension left overnight at 4 C before centrifugation at 17,000 x g for 10 min at 4 C. The supernatant was collected and the ethanol concentration adjusted to 80% (v/v). This suspension was left overnight at 4 C before centrifugation at 17,000 x g for 20 min at 4°C. The protein pellet was resuspended in a buffer containing 0.05% (w/v) Zwittergent 3-14, 0.05 M Tris, and 0.01 M EDTA, pH 8, and stined at room temperature for 1 h before centrifuging at 12,000 x g for 10 min at 4 C.
The supernatant was dialysed overnight against distilled water at 4 C, frozen to -70 C and then lyophilized.
The protein extract was resuspended to a concentration of approximately 18mg/ml in Buffer A (25mM Tris-HCl, pH 8.1) before loading onto a BioRad Q5 anion-exchange column. 1ml aliquots were loaded on each run (seven runs in total). The column was washed with Buffer A for 5mins at 1 ml/min. The proteins were eluted using a combination of continuous and step gradients from 100% Buffer A to 100% Buffer B (25mM Tris-HCl + 0.5M NaCl, pH 8.1). The gradient was followed by a 4 min wash with 100% Buffer B followed by a 1 min wash with 100% Buffer A. Fractions 9-10 (see Fig. 16 peaks 1 and 2) from each run were pooled and freeze-dried.
The freeze dried protein was redissolved in 1.5 ml distilled H2O and 1.5 ml reducing buffer (62.5 mM Tris, [pH 6.8], 10% [vol/vol] glycerol, 2% [wt/vol] SDS, 5%
[vol/vol] β-mercaptoethanol, 1.2 x 10~3% [wt/vol] bromophenol blue) and incubated at 37°C for 30 mins. Preparative SDS-PAGE to purify proteins was performed using the Bio-Rad Model 491 Prep Cell using a 40ml 9% T-l.42% C acrylamide/BIS (N,N'- methylene-bis acrylamide) separating gel with a 10-ml 4% T-0.36% C acrylamide/BIS stacking gel polymerized in a 37-mm (internal diameter [i.d.]) column. Fractions eluted from the column with 0.025M Tris-HCl, were concentrated by lyophilization and analysed for protein content by analytical SDS-PAGE. The superoxide dismutase was in fractions 49-54. These were pooled, freeze-dried and reconstituted in distilled H2O. The Tris buffer was removed by dialysis against PBS. The protein precipitated during this step and was reconstituted in 8 M Urea, 0.1 M Tris-HCl, 0.1 M NaH2PO4, pH 4.5. Buffer exchange into PBS was achieved using Centicon 10000 tubes.
Flow Diagram
Purification of Superoxide Dismutase
I M. catarrhalis K65 cultured in BHI Broth i Zwittergent-based extractraction
I
Anion exchange ,
Fractions 9-10 pooled
I
Preparative Electrophoresis - 9% resolving gel
Fractions 49-54
I
Dialysis against PBS
,
Resolubilisation in 8 M Urea
I
Buffer exchange into PBS
I
Immunisation studies Results
Figure 16 shows the chromatogram from which superoxide dismutase (SOD) was purified. Peaks 1 and 2 were pooled and freeze-dried prior to preparative electrophoresis. SOD was found to elute from the gel in fractions 49-54. Figure 17 shows, by analytical SDS-PAGE, that these fractions contain apparently purified SOD. These fractions were pooled and following solubilisation and dialysis into PBS used for immunisation studies.
Example 7 Immunisation and Challenge Studies in Mice using Native Moraxella catarrhalis Antigens.
Materials and Methods
Immunisation Regime
Protocol:
Day O IPP or subcutaneous immunisation with protein emulsified in
Incomplete Freund's adjuvant (IF A).
Day .14 IT or subcutaneously delivered boost with protein in PBS or TFA, respectively.
Day 21 Live pulmonary challenge with Moraxella catarrhalis strain K65 and clearance assessed. Animals
Balb/c mice were 6-8 weeks old, obtained from the Animal Resources Centre, Perth, WA, Australia and were maintained under SPF conditions until the start of the experiment. They were removed from behind the SPF barrier for immunisations and the final live bacterial challenge. At all other times, the animals remained in SPF cages and under SPF conditions.
Immunisation
Intra-Peyer's Patch Immunisation
The animals are sedated with ketamine/xylazine in phosphate-buffered saline (PBS) delivered intraperitoneally. The small intestine was exposed through a midline abdominal incision, and the antigen was injected subserosally into each Peyer's patch with a 27-gauge needle. The protein was prepared by emulsification of purified protein in a 1 :1 mixture of incomplete Freund's adjuvant (IF A) (Difco Laboratories, Detroit, Mich.) and PBS, and a total inoculum of lOμl, was administered to each experimental animal in the immunisation group.
Intratracheal boost immunisation
Animals were sedated with saffan anaesthesia via the tail vein and the protein in 20 μl PBS was introduced into the lungs of the animals via an IT cannula and dispersed with two 0.3ml-volumes of air. The non-immune animals received 20μl of PBS.
Bacterial challenge
M. catarrhalis was grown overnight on plates of brain heart infusion (BHI) agar supplemented with 50ml per litre of defibrinated horse blood (Amadeus International, Brooklyn, Nic, Australia). Plates were incubated overnight at 37°C in 5% CO2, the bacteria harvested and washed three times in PBS. The animals were sedated with saffan and a 20μl bolus of 5x10 CFU per ml of live M. catarrhalis K65 in PBS was introduced into the lungs via an IT cannula and dispersed by two 0.3 ml volumes of air. Animals were killed by an overdose of Nembutal administered by intraperitoneal injection 4 hours after lung inoculation.
Collection of Tissues
Serum collection
Blood was collected by heart puncture, fransfened to a sterile container, clotted, centrifuged at 2000φm and aliquots of serum stored at -20°C for antibody analysis.
Bronchoalveolar lavage collection
Lungs were lavaged with one 0.5ml-volume of PBS via the trachea, which had been exposed through an incision in the neck, and the bronchoalveolar lavage (BAL) fluid was assessed for bacterial numbers by plating onto chocolate blood agar, serial dilutions (20μl in a 10-fold series) of the washings for CFU determination. A lOOμl aliquot of BAL fluid was spun for lOmins at 4.5xg onto a microscope slide with a
Cytospin apparatus (Shanddon Inc., Pittsburgh Pa). The slides were fixed and stained in Diff Quick (Veterinary Medical Surgical Supply, Pty. Ltd., Maryville, NSW, Australia), and percentages were determined from three differential cell counts on each slide. BAL fluid was centrifuged at l,000φm for lOmin with a Jouan BR3.11 bench top centrifuge. The supernatant was removed, and aliquots were stored at -20°C. The pellet was resuspended in a known volume of PBS, and the total number of viable cells present in the BAL fluid was determined using a Neubauer haemocytometer and staining with methylene blue in 1.5% (vol/vol) acetic acid. Lung homogenate
The lungs, trachea and heart were excised intact, the heart and connective tissues were removed and the lungs were placed in 2ml of sterile PBS and homogenised with a Heidolph Diax 600 homogeniser set at 9,500φm with no load. The lung homogenate was assessed for the presence of bacteria by plating serial dilutions (20μl in a 10-fold series) onto chocolate blood agar for CFU determination.
Immunoblot Assay. For immunoblot assay proteins were separated by SDS-PAGE and elecfrophoretically fransfened to nitrocellulose (0.2-μm pore size, BioRad Laboratories, Nth Ryde,
N.S.W., Australia ) at a constant cunent of 0.8 mA/cm^ for 55 min in buffer containing 25 mM Tris, 192 mM glycine, pH 8.8. The nitrocellulose was then soaked in TBS (20 mM Tris, 500 mM NaCl, pH 7.5) for 10 min. Immunoblot preparations were blocked with TBS containing 5% (wt/vol) skim milk and using gentle agitation for 30 min. The membrane was washed twice by 5 min gentle agitation in TTBS (20 mM Tris, 500 mM NaCl, 0.05%Tween, pH 7.5). Mouse serum was diluted 20-fold in TTBS-5% (wt/vol) skim milk powder and the membrane exposed to this for 90 min with gentle agitation. After washing twice for 5 min with TTBS, the membrane was exposed to a 500-fold dilution of horseradish peroxidase conjugated anti-mouse IgG in
TTBS-5% skim milk for 90 min with gentle agitation. The membrane was Washed twice for 5 min in TTBS followed by a third 5 min wash in TBS, and the blot developed in a solution of TBS containing 0.05% (wt/vol) 4-chloro-l-naphthol (BioRad Laboratories), 16.7% (vol/vol) methanol and 0.015% (v/v) H2O2. The reaction was stopped by washing in distilled water.
Results
Figure 18 and Table 2 show results of challenge experiments after immunization with M. catarrhalis antigens. Mice immunized IPP and boosted IT with purified superoxide dismutase showed statistically enhanced clearance of bacteria from the lungs compared to sham immunised animals protein
Table 2. Recovery of bacteria from the lungs of non-immune and native SOD immunised mice 4h after challenge with 9 x 106 CFU of M. catarrhalis K65.
Figure imgf000049_0001
Example 8. Cloning, Expression and Purification of Moraxella catarrhalis Antigens
Materials and Methods
Standard Molecular Biology Techniques
Standard molecular biology techniques were carried out according to the texts, Molecular Cloning. A Laboratory Manual (Sambrook et al. 1989. Cold Spring Harbor Laboratory Press) and Cunent Protocols in Molecular Biology (Ausubel et al. 1999.
John Wiley & Sons).
Extraction of Genomic DNA
Bacteria (Moraxella catarrhalis K65; Kyd, Cripps, Muφhy. 1998. J. Med. Microbiol.
47: 159-168) were washed three times in PBS and the pellet resuspended in 10ml of 50mM Tris-HCl, pH8.0. 0.4ml of 0.4M EDTA was added and the mix incubated in a 37°C water bath for 20 mins before adding 0.4ml of 20mg/ml lysozyme. The cells were incubated for 10 mins at 37°C and 540μg (0.0005 ) Proteinase K added (final concentration of Proteinase K in 10.8ml of suspension is 50μg/ml) together with 0.4ml of 10% w/v SDS and lOμl of lOmg/ml Ribonuclease A. The mixture was incubated for 2 hrs at 37°C or until the suspension was clear and 8ml of phenol (saturated with lOmM Tris-HCl and ImM EDTA) added followed by gentle mixing for 30sec at 37°C. The solution was centrifuged at 8,000φm for 15 mins at 4°C and the DNA containing phase removed and extracted in 5ml phenol/chloroform/isoamyl alcohol (25:24:1). This was gently mixed on ice for 30 sees and centrifuged at 8,000φm for 15 mins at 4°C. The DNA phase was removed and fransfered to a new centrifuge tube containing 5ml chloroform/isoamyl alcohol (24:1), centrifuged at 8,000φm for 15 mins at 4°C. This step was repeated before the DNA was removed and 2 volumes of cold (-20°C) absolute ethanol used to precipitate the DNA. The DNA was extracted with a glass pipette, dipped in 70% ethanol and resuspended in l-2ml of TE (ImM EDTA, 10 mM
Figure imgf000051_0001
Extraction and Purification of Plasmid DNA
Bacteria containing the plasmid were cultured in LB broth with the relevant antibiotic and grown in a shaker incubator at 37°C. The cells were pelleted and resuspended in 50mM Tris-Cl, pH 8.0, lOmM EDTA, lOOμg/ml RNase A before adding 200mM NaOH, 1% SDS and incubating at RT for 5 min. Prechilled 3.0M potassium acetate, pH 5.5 was added and incubated on ice for 15-20 min prior to centrifugation at
20,000xg for 30 min at 4°C. The supernatant containing the plasmid was removed and applied to the spin column φreviously equilibrated with QBT - Equilibration buffer :750mM NaCl, 50mM MOPS pH 7.0, 15% isopropanol, 0.15% Triton-XlOO ). The column was washed twice with 1.0M NaCl, 50mM MOPS pH 7.0, 15% isopropanol and the DNA eluted with 1.25M NaCl, 50mM Tris-Cl pH 8.5, 15% isopropanol. DNA was precipitated with isopropanol, centrifuged at 15,000xg for 30 min at 4°C and the pellet washed with 70% ethanol by centrifugation at 15,000xg for 10 min at RT. The supernatant was removed and the pellet air dried before resuspending in ImM EDTA,
Figure imgf000051_0002
Polymerase Chain Reaction
All PCR experiments are individually optimised and the specific conditions are described for each gene. In general a reaction mix consisted of 1 μl (lOOng) primer 1 and 1 μl (1 OOng) primer 2, 0.4μl 1 OmM dNTPmix, 1 Oxcloned pfu DNA polymerase buffer (Stratagene #600250), 1.0 μl (2.5 U) pfu turbo and 40.6 μl nanopure water. Add lμl of template DNA (approx lOOng/μl) and the Sealed capillary or tube placed into the thermocycler and the program started. Cloning of Moraxella catarrhalis Antigen Genes
Searching of the NCBI databases (www.ncbi.nlm.nih.gov) failed to find any proteins with significant homology to any of the NH2-terminal sequences listed in Table 1. However, searching a Moraxella catarrhalis genome sequence allowed the identification of 9 putative open reading frames. The only proteins we were unable to identify in this genome were I2D-15 (i) and I2D-15 (ii). The identified open reading frame sequences were then used to design PCR primers for the cloning of individual genes. Primers were designed for amplification by PCR of the 5' and 3' ends of each antigen gene. Primer pairs for each of the antigen genes are shown in Table 3 along with the conditions for amplification. The predicted full length nucleotide sequences and derived protein sequences are shown in Fig. 19
CO c
CD O
m
CO
I m m
c m r
Figure imgf000053_0001
Table 3. PCR, Cloning and expression methods for Moraxella catarrhalis antigens
DNA Digestion and Ligation
In general, insert purified DNA and plasmid were mixed with the digestion enzymes in the buffer recommended by the supplier for the enzymes. The mixture was incubated at 37°C for 2 hours. Generally a ratio of 3 : 1 is used for insert: vector. DNA and plasmid vectors were then purified by agarose electrophoresis and eluted from the gel using a Qiagen min elute kit. Purified DNA and vector were then mixed with lOxLigation buffer (MBI Fermentas, Progen) and nanopure water (all kept on ice). Ligation enzyme (MBI Fermentas, Progen) was added and incubated at 16°C overnight. Prior to transformation of E. coli the mixture was heat inactivated.
Preparation of Competent Cells
Rubidium Chloride Method 2-5ml LB was inoculated with 1 colony of an E. coli strain (ie. XLl-Blue, JM109 etc) from an overnight culture, incubated in shaker at 37°C overnight and 1ml transferred to 100ml of LB. Bacteria were incubated in a shaker at 37°C for 3-4 hrs, until OD600 = 0.4. The bacteria were chilled on ice for 5 min, centrifuged for 10 min, 4°C and 1200g and the supernatant discarded. The pellet was resuspended in 40mls ice cold buffer l(30mM Kac, lOOmM RbCl, lOmM CaCl2 , 50mM MnCl2, 15% v/v glycerol) and incubated on ice for 5 min. The mix was centrifuged, the supernatant discarded and the pellet resuspend in 4mls ice cold buffer 2 (lOmM MOPS, 75mM CaCl2, lOmM RbCl, 15% v/v glycerol). The bacteria were chilled on ice prior to aliquoting in lOOμl vols and stored at -70°C.
Calcium Chloride Method
50mls of LB broth was inoculated with a single bacterial colony and incubate the solution overnight at 37°C with vigorous shaking lOmls of the overnight culture was used to inoculate 500mls of LB media. The flask was incubated at 37°C with vigorous shaking until the optical density reached 0.7-0.9 units (mid-log phase). The cell suspension was then centrifuged at 6,000rpm for 20 minutes at 4°C. The cells were washed by suspending in ice cold 0.1M calcium chloride solution, centrifuged at 6,000rpm for 20 minutes at 4°C and the cell pellet resuspended to a final volume of 1.5mls in (ice cold) 0.1M calcium chloride/ glycerol solution (85:15). Aliquot of the cells (lOOμl volumes) were placed into sterile 1.5ml microfuge tubes and stored at -80°C.
Heat Shock Transformation
1 vial of competent cells per transformation was thawed and the required amount of DNA (25-50ng) in a maximum volume of 15μl was added. The mixture was incubated on ice for 10 min prior to incubating for 45 seconds at 42°C. The cells were then placed on ice for 2 mins, 900μl SOC added and incubated with shaking at 37°C for 45-
60 mins. Cells were plated onto LB containing the required antibody selection and incubated overnight at 37 C.
Production and Purification of Recombinant Moraxella catarrhalis Antigens Induction and Extraction
Bacteria were grown in LB broth containing the relevant antibiotic until they reached an optical density of between 0.7-0.9 at 600nm. The protein expression was induced by addition of IPTG to a concentration of 1 mM (or as otherwise determined) and the cells incubated for between 1 and 5 hours (as determined). The bacteria were harvested by centrifugation at 4000xg for 20 min, the pellet resuspended in sonication buffer (50 mM sodium phosphate, 300 mM NaCl, ρH8, 10 mM imidazole) and frozen at -70°C for between 0.5 h and overnight. The cell suspension was sonicated 4 times, centrifuged at 11,000 rpm for 20 min and the supernatant retained for extraction of the recombinant protein using the Ni-NTA resin. For native purification, the Qiagen protocol was followed and the protein is extracted into phosphate buffer. For denaturing extraction, the recombinant protein was extracted into a buffer containing 8 M Urea (Qiagen).
Results
Cloning and Expression of Moraxella catarrhalis Antigen Genes
The predicted full length gene sequences were used to search the Genbank databases for homology to the derived protein sequences. Table 4 shows the results of these searches and describes the proteins with the best matches. ID-2 shows 75% identity to a malate dehydrogenase from Aquaspirillum arcticum. All other hits returned malate dehydrogenases from a variety of organisms. Although normally cytoplasmic, some MDH's have been found on the cell surface of certain bacteria. The precise location of Moraxella catarrhalis MDH is not known although homology indicates it is likely to be found in the cytosol. I2D-14 showed the best homology at 75% identity to an inorganic pyrophosphatase from Neisseria meningitidis (group B strains MD58, MC58 and group A strain Z2491). The protein also showed a good match (73% identity) to a pyrophosphatase from Haemophilus influenzae strain RdKW20. Pyrophosphatases can be induced under conditions of environmental stress. I2D-16 showed a best match to alkyl hydroperoxide reductase of several bacteria. The highest match was to alkyl hydroperoxide reductase from Bacteroides fragilis that gave 66% identitiy. Good matches were also obtained with Salmonella typhimurium, Bacillus subtilus, E.coli and Caulobacter crescentus. This enzyme reduces organic hydroperoxides and can protect against reactive nitrogen intermediates. As such it is thought to be an important factor in protection against host defenses such as phagocytosis and reactive oxygen intermediates produced during the respiratory burst. I2D-18 was also identified as a protein involved in protection against oxidative stress. The sequence gave a match to numerous manganese superoxide dismutases (Mn CO c
CD CO
m
CO
I m m
c m r
Figure imgf000057_0001
-4
Figure imgf000058_0001
CO c
CD CO
m
CO
I m m
73 c m r
Figure imgf000059_0001
CO c
CD CO
m
CO VO
I m m
73 c m r
Figure imgf000060_0001
* Internal sequence from IGD
SOD). The highest homology of 69% identity being with Mn SODs from Haemophilus ducreyi, Yersinia enterocolitica and Salmonella typhimurium all well known intracellular pathogens capable of surviving within macrophages and resisting their oxidative defense mechanisms. I2D- 19 showed highest similarity of 40% identity to acetoin reductase from
Staphylococcus aureus an enzymes involved in the production of 2,3 butanediol from acetoin during fermentation. For I2D-20 we were unable to identify any proteins with ascribed functions. The best match was 45% identity to a hypothetical protein from Pseudomonas aeruginosa. A portion of the protein showed a high homology to a putative peptidoglycan binding domain which may suggest the protein is bound to the cell wall or involved in cell wall biogenesis.
I2D-21 gave a best match (40% identity) to disulphide bond isomerase A (DSBA) protein from Pseudomonas aeruginosa. DSBA protein catalyses the formation of disulphide bonds. The enzyme can be membrane bound and is present in the periplasm where it ensures the correct folding of membrane and secreted proteins.
They have been identified as important virulence factors and mutant bacteria lacking a functional DSBA have altered membrane protein profiles and reduced virulence. I2D- 22 gave a good match of 45% over part of the sequence (residues 82-261) to a probable adhesin component from Neisseria meningitidis (group A strain Z2491). These proteins generally function to facilitate adhesion of bacteria to surfaces or other cells. These can be important virulence factors involved in the colonisation and maintenance of infections. I2D-23 showed the best match of 69% identity to a histone-like (HU) protein from Pseudomonas putida. In streptococci and Actinobacillus pleuropneumoniae they have been shown to be important virulence factors. They are found either in the periplasm or are membrane associated. Purification of Recombinant Moraxella catarrhalis Antigens
All recombinant protein were His-tagged and production was induced in culture with ΣPTG. After induction and harvesting the recombinant proteins were purified using Ni affinity chromatography. Figure 20 shows the induction and expression of ID-2 and I2D-14. Figures 21 and 22 show the purified proteins after Ni chromatography that were subsequently used for immunisation studies. Figure 23 shows the induction, expression and purification of superoxide dismutase (I2D-18). I2D-20 and I2D-21 were also prepared in the same way, Figures 24 and 25 show that the proteins were purified to apparent homogeniety and these were used for immunisation studies.
Example 9 Immunisation and Challenge Studies in Mice using Recombinant Moraxella catarrhalis Antigens.
Immunisation and challenge studies were performed as in example 7 using a subcutaneous regime.
Results
Table 5 and Fig 26 show the recovery of bacteria from the lungs of mice challenged with M. catarrhalis. Mice immunised with recombinant I2D-18 did not appear to afford any clearance of bacteria from the lungs. This was in contrast to the purified native protein (see Example 6). However, different immunisation regimes were used that may account for this difference. Nevertheless, for recombinant I2D-20 significant clearance of bacteria was observed using the s.c. route of vaccination for both BAL and LH recovered bacteria demonstrating it is also a good vaccine candidate. Using sera collected from immunised mice western blotting was performed using whole-cell extracts from a number of M. catarrhalis strains. Figure 27 shows the results for antisera raised against recombinant I2D-18. Although antibodies recognized the purified recombinant protein no reaction was observed to whole-cell extracts. The reasons for this are as yet unclear.
In contrast for I2D-20 antisera recognised one protein in all strains for which whole- cell extracts were tested, indicating that the protein is widespread and conserved between strains.
Table 5. Recovery of bacteria from the lungs of non-immune, recombinant SOD and I2D-20 immunised mice 4h after challenge with 9 x 106 CFU of M. catarrhalis K65.
Figure imgf000063_0001
Example 10: Immuno Western blotting of Antigens using sera prepared from rats immunised with killed whole-cell Moraxella catarrhalis and rats immunised with an outer membrane protein extract of Moraxella catarrhalis
Methods
Preparation of Moraxella catarrhalis antisera
Bacteria were grown as described in example 6. For the preparation of the whole cell vaccine the culture was treated with 1% formalin for 2 hours and cells collected by centrifugation followed by washing with PBS. For the outer membrane protein (OMP) vaccine cells were treated and proteins extracted using the detergent zwittergent as described in example 6.
Rats were immunised 3 times on days 0, 14 and 21. The first immunisation was intrapeyer's patch (IPP) in Freund's incomplete adjuvant, followed by two intratracheal boosts (IT) in PBS. Serum was collected on day 28. For the whole-cell vaccine, 5x10 cells was used to vaccinate animals. For the OMP vaccine 20 μg of OMP protein was used to vaccinate animals.
Immuno- Western Blotting
Inununoblottiing was performed as described in example 7 except a 1:5 dilution of sera from immunised rats was used to probe the blot and an anti-rat horse radish peroxidase conjugate was used to detect antibody bound to antigen. Results
Figure 29 shows a coomassie-stained SDS-PAGE gel of whole-cell lysates from E.coli clones expressing recombinant antigens I2D-22, 12D-16, 12D-19 and purified I2D-14 and I2D23. E.coli containing the vector but not expressing protein and a M. catarrhalis whole-cell lysate were used as controls. These same samples were also run and blotted onto nitrocellulose membranes, probed with antisera and developed. Figure 30 shows the results of probing with non- immune serum. Although some E.coli antigens bound antibodies in the antisera none of these corresponded to the recombinant proteins and little reactivity of antibodies was observed to the M. catarrhalis lysate. In contrast, Figure 30 shows the results of probing with antisera raised to whole-cell M. catarrhalis. Bands are clearly visible for I2D-22, 12D-16, 12D19 and I2D-14, demonstrating the immunogenicity and presence of these proteins in a killed whole-cell M. catarrhalis vaccine. Figure 31 show the results using antisera to OMP proteins. The blot is not developed for I2D-22, 12D-16 and I2D-19, which may be due to poor transfer indicated by the non-staining areas in these lanes. However, I2D- 14 and I2D-23 were detected and this also demonstrates the presence of these antigens in OMP preparations and their immunogenicity. Both these vaccine preparations have been shown to be protective in animal models of M. catarrhalis infection.

Claims

1. A protein from M. catarrhalis which has the NH2-terminal sequence:
I. MAFTLPELGYSYDALEPGFDK(N)EA(T)XM(G)L; π. MKQPV(T)RVAXT; m. TTQNNQQNGKNAINTS(X)AAG(X)LS(X)NAIA(S)T(S)RL;
IV. GVSFAKDIGDKLFHR(S)N(P)K(A)KQ(E)D(P)T(A)AQE(P)I(T)AN(A)LL
V. ADFN ILDAGNNDDQ(G)I VI. MIQDIFTDLE; vπ. MQΝEΓKQAGG;
VIII Ν(E orK)FVEDQD(X)YQ(X)VLP;
IX A(Q)AIINQTIPEFXTQAYVNG(X)E(X);
X MNKSELVDG(T)IAQXAGLT; Or contains the sequence:
XI KLGNITSPSGDSA; and/or Xπ FXPFNLN
2. A protein which is a homologue or derivative of a protein as defined in claim 1.
3. A protein as claimed in any preceding claim provided in substantially pure form.
4. An antigenic and or immunogenic fragment of a protein as claimed in any preceding claim.
5. A fragment as claimed in claim 4, comprising the sequence: I. MAFTLPELGYSYDALEPGFDK(N)EA(T)XM(G)L; π. MKQPV(T)RVAXT; m. TTQNNQQNGKVAIVTS(X)AAG(X)LS(X)NAIA(S)T(S)RL;
IV. GVSFAKDIGDKLFHR(S)N(P)K(A)KQ(E)D(P)T(A)AQE(P)I(T)AN(A)LL V. ADFN ILDAGNVDDQ(G)I
VI. MIQDIFTDLE; vπ. MQNEΓKQAGG;
VIII N(E or K)FVEDQD(X)YQ(X)VLP; rx A(Q)AIΓNQTΓPEFXTQAYVNG(X)E(X); X MNKSELVDG(T)IAQXAGLT; XI KLGNITSPSGDSA; or Xπ FXPFNLN
6. An antigen composition comprising one or more proteins as claimed in claims 1-3, and/or one or more fragments as claimed in claim 4 or claim 5.
7. An antigen composition as claimed in claim 4, which further comprises one or more other catarrhalis antigens and/or immunogens.
8. A protein as claimed in claim 1, a fragment as claimed in claim 4 or claim 5, or an antigen composition as claimed in claim 6 or claim 7 for use in the detection of M. catarrhalis.
A method of detecting and or diagnosing M. catarrhalis which comprises:
(a) bringing into contact with a sample to be tested a protein as claimed in any one of claim 1 to 3, a fragment as claimed in claim 4 or claim 5, or an antigen composition as claimed in claim 6 or claim 7; and
(b) detecting the presence of antibodies to M. catarrhalis.
10. An antibody capable of binding to a protein as defined in any one of claims 1 to 3.
11. A method for the detection/diagnosis of M. catarrhalis which comprises the step of bringing into contact a sample to be tested and an antibody as defined in claim 10.
12. A method as claimed in claim 9 or claim 11, wherein the sample is a sample of mucous, saliva or blood.
13. The use of a protein as claimed in any one of claims 1 to 3, a fragment as claimed in claim 4 or claim 5, an antigenic composition as claimed in claim 6 or claim 7, or an antibody as claimed in claim 11 in detecting and or diagnosing M. catarrhalis.
14. A method as claimed in any one of claims 9, 10 or 12 or the use as claimed in claim 13, wherein the detecting and/or diagnosing is carried out in vitro.
15. A kit for use in the detection and/or diagnosis of M. catarrhalis, which kit comprises a protein as claimed in any one of claims 1 to 3, a fragment as claimed in claim 4 or claim 5, an antigen composition as claimed in claim 6 or claim 7, or an antibody as claimed in claim 10.
16. The use of a protein as claimed in any one of claims 1 to 3, a fragment as claimed in claim 4 or claim 5, an antigenic composition as claimed in claim 6 or claim 7, or an antibody as claimed in claim 10 in medicine.
17. A composition capable of eliciting an immune response in a subject, which composition comprises a protein as claimed in any one of claims 1 to 3, a fragment as claimed in claim 4 or claim 5, or an antigen composition as claimed in claim 6 or claim 7.
18. A composition as claimed in claim 17 which is a vaccine composition, optionally further comprising one or more adjuvants.
19. The use of a protein as claimed in any one of claims 1 to 3, a fragment as claimed in claim 4 or claim 5, or an antigen composition as. claimed in claim 6 or claim 7 in the preparation of an immunogenic composition, preferably a vaccine.
20. The use of an immunogenic composition as claimed in claim 19 in inducing an immune response in a subject.
21. A method for the treatment or prophylaxis of M. catarrhalis infection in a subject, which comprises the step of administering to the subject an effective amount of a protein as claimed in any one of claims 1 to 3, a fragment as claimed in claim 4 or claim 5, or an antigen composition as claimed in claim 6 or claim 7.
22. A method as claimed in claim 21, wherein the subject is suffering from otitis media and /or a respiratory infection.
23. A method as claimed in claim 21 or claim 22, wherein the protein, fragment, or antigen composition is administered in the form of a vaccine.
24. A nucleic acid molecule comprising or consisting of a sequence which is:
(i) the equivalent DNA sequence of the proteins set out in claims 1 to 3, fragments as in claims 4 or claim 5 or antigen composition as claimed in claim 6 and claim 7 or their RNA equivalents; (ii) a sequence which is complementary to the sequence of (i); (iii) a sequence which codes for the same protein or polypeptide, as the sequence of (i) or (ii); (iv) a sequence which is has substantial identity with any of those of (i), (ii) and (iii); (v) a sequence which codes for a homologue, derivative or fragment of a protein as defined in claims 1 to 3 and fragments as in claims 4 and claim
5
25. A vector comprising a nucleic acid molecule as defined in claim 24.
26. A host cell transformed with a vector as defined in claim 25.
27. A vaccine composition comprising one or more nucleic acid molecules as defined in claim 24.
28. A method for the detection/diagnosis of M. catarrhalis which comprises the step of bringing into contact a sample to be tested and at least one nucleic acid as defined in claim 27.
29. A method of determining whether a protein as defined in any one of claims 1 to 3 represents a potential antimicrobial target, which method comprises inactivating said protein and determining whether M. catarrhalisis still viable in vitro or in vivo.
30. The use of an agent capable of antagonsing, inhibiting or otherwise interfering with the function or expression of a protein as defined in any one of claims 1 to 3 in the manufacture of a medicament for the treatment or prophylaxis of M. catarrhalis infection.
PCT/GB2002/002205 2001-05-10 2002-05-10 M. catarrhalis antigens WO2002090383A2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002307943A AU2002307943A1 (en) 2001-05-10 2002-05-10 M. catarrhalis antigens

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
GB0111492A GB0111492D0 (en) 2001-05-10 2001-05-10 Antigens
GB0111492.5 2001-05-10
GB0112448.6 2001-05-22
GB0112448A GB0112448D0 (en) 2001-05-22 2001-05-22 Antigens
GB0119479A GB0119479D0 (en) 2001-08-09 2001-08-09 Antigens
GB0119479.4 2001-08-09

Publications (2)

Publication Number Publication Date
WO2002090383A2 true WO2002090383A2 (en) 2002-11-14
WO2002090383A3 WO2002090383A3 (en) 2003-07-03

Family

ID=27256167

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2002/002205 WO2002090383A2 (en) 2001-05-10 2002-05-10 M. catarrhalis antigens

Country Status (2)

Country Link
AU (1) AU2002307943A1 (en)
WO (1) WO2002090383A2 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110904021A (en) * 2019-12-20 2020-03-24 北京市农林科学院 Preparation and transformation method of porcine pleuropneumonia actinobacillus competent cells

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001009331A2 (en) * 1999-07-30 2001-02-08 Smithkline Beecham Biologicals S.A. Moraxella catarrhalis antigen basb125

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001009331A2 (en) * 1999-07-30 2001-02-08 Smithkline Beecham Biologicals S.A. Moraxella catarrhalis antigen basb125

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE SWALL [Online] EBI; 1 March 2002 (2002-03-01) "Moraxella catarrhalis superoxide dismutase SODA" Database accession no. Q8VS37 XP002239116 *
DATABASE SWALL [Online] EBI; 1 May 2000 (2000-05-01) "B. fragilis alkyl hydroperoxide reductase subunit C" Database accession no. Q9RQ72 XP002239118 *
DATABASE SWALL [Online] EBI; 21 July 1986 (1986-07-21) "Rhizobium leguminosarum DNA binding protein HRL53" Database accession no. P02348 XP002239117 *
MCMICHAEL J C: "Vaccines for Moraxella catarrhalis" VACCINE, BUTTERWORTH SCIENTIFIC. GUILDFORD, GB, vol. 19, 8 December 2000 (2000-12-08), pages S101-S107, XP004227957 ISSN: 0264-410X *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110904021A (en) * 2019-12-20 2020-03-24 北京市农林科学院 Preparation and transformation method of porcine pleuropneumonia actinobacillus competent cells

Also Published As

Publication number Publication date
AU2002307943A1 (en) 2002-11-18
WO2002090383A3 (en) 2003-07-03

Similar Documents

Publication Publication Date Title
US10744192B2 (en) Vaccine
Billington et al. The Arcanobacterium (Actinomyces) pyogenes hemolysin, pyolysin, is a novel member of the thiol-activated cytolysin family
AU701340B2 (en) Vaccine for Branhamella catarrhalis
JPH11500624A (en) Proteinase K-resistant surface protein of Neisseria Meningitidis
JP4785014B2 (en) protein
Pannekoek et al. Identification and molecular analysis of a 63-kilodalton stress protein from Neisseria gonorrhoeae
JP4368944B2 (en) Helicobacter antigen for defense
Blanco et al. Recombinant Treponema pallidum rare outer membrane protein 1 (Tromp1) expressed in Escherichia coli has porin activity and surface antigenic exposure
US20090246228A1 (en) Moraxella catarrhalis Proteins
Harkness et al. Identification of two iron-repressed periplasmic proteins in Haemophilus influenzae
Yu et al. Analysis of the immunological responses to transferrin and lactoferrin receptor proteins from Moraxella catarrhalis
Webb et al. Immunization with recombinant transferrin binding protein B enhances clearance of nontypeable Haemophilus influenzae from the rat lung
US20040071713A1 (en) Psedomonas aeruginosa antigens
WO2002090383A2 (en) M. catarrhalis antigens
Gase et al. The lppC gene of Streptococcus equisimilis encodes a lipoprotein that is homologous to the e (P4) outer membrane protein from Haemophilus influenzae
Anvari et al. Biological activity of recombinant accessory cholerae enterotoxin (ace) on rabbit ileal loops and antibacterial assay
IL111102A (en) Proteins, peptides and oligopeptides of the outer membrane protein "cd" of branhamella catarrhalis, nucleic acids encoding them, method for their preparation and use thereof as vaccine formulations
KR100829380B1 (en) Fowl cholera vaccine composition comprising recombinant outer membrane protein h of pasteurella multocida
JP4151844B2 (en) Plasmid vector for expressing protective recombinant HAEMOPHIILS INFLUENZAE (H. influenzae) high molecular weight protein
Caetano et al. In Silico Prediction and Design of Uropathogenic Escherichia coli Alpha-Hemolysin Generate a Soluble and Hemolytic Recombinant Toxin. Microorganisms 2022, 10, 172
Tavakoli et al. Prokaryotic high-level expression system in producing adhesin recombinant protein E of nontypeable Haemophilus influenzae
WO2008127102A1 (en) S. pnuemoniae transcytosis protein
NZ541257A (en) 14 kDa Moraxella Catarrhalis protein
WO2000000614A9 (en) NOVEL ANTIGENS OF $i(HELICOBACTER PYLORI)

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP