FR2739623A1 - NEW P76 HELICOBACTER PYLORI MEMBRANE PROTEIN - Google Patents

Abstract

The invention relates to a protein of helicobacter pylori in a substantially purified form, susceptible of being obtained from a membrane fraction of helicobacter pylori and of which the molecular weight after electrophoresis on polyacrylamide gel 10 % in the presence of SDS is approximately of 76 kD.

Description

 The subject of the present invention is newly obtained proteins of Helicobacter pylori in substantially purified form, as well as pharmaceutical compositions containing them.

 H. pylori is a gram-negative bacterium found exclusively to date on the surface of the lining of the stomach in humans and more particularly around crater lesions of gastric and duodenal ulcers. This bacterium is currently recognized as the etiological agent of antral gastritis and appears as one of the co-factors required for the development of ulcers. Moreover, it appears that the development of gastric carcinomas may be associated with the presence of H. pylori.

 It therefore appears highly desirable to develop a vaccine for the purpose of preventing or treating H. pneumoniae infections. Such a vaccine would most likely be subunit in nature.

Different JI proteins. pylori have been characterized or isolated to date. It includes urease, composed of two subunits A and B of 30 and 67 kD respectively, the vacuolar cytotoxin of 87 kD (VacA), an immunodominant antigen of 128 kD associated with the cytotoxin, heat shock proteins HspA and
HspB 13 and 58 kD respectively. a 54 kD catalase, a 20 kD fibrillar hemagglutinin (HpaA), a 15 kD protein rich in histidine (Hpn); a 30 kD outer membrane protein, as well as a HopA, HopB, porin family
HopC and HopD, with molecular weights between 48 and 67 kD.

 Some of these proteins have already been proposed as potential vaccine antigens. However, the search for new antigens must be pursued, particularly since it is expected that, to obtain an optimized vaccine effect, several antigens will probably have to be incorporated in a vaccine.

This is why the subject of the invention is an H protein. pylori in substantially purified form, obtainable from a membrane fraction of H. pylori and whose molecular weight after electrophoresis on polyacrylamide gel 10% in the presence of SDS, appears to be of the order of 76 kD
By "substantially purified form" is meant a preparation in particular free of the cytoplasmic and periplasmic proteins of H. pylori.

Membrane protein whose apparent molecular weight is of the order of 76 kD is obtainable by a process in which
(i) H. pylori bacteria are extracted with n-octyl ss-D
glucopyranoside 1%, followed by centrifugation
(ii) recovering a supernatant which is dialyzed against 75 mM PBS pH
7.2, followed by centrifugation
(iii) recovering the membrane fraction consisting of the centrifugation pellet that
the aqueous medium is resuspended, advantageously in carbonate buffer pH 9.5
containing 5% zwittergents 3-14
(iv) the membrane fraction is subjected to anion exchange chromatography
on a Q-Sepharose column in 0.1 - 0.5 M NaCl gradient, advantageously in
carbonate buffer pF1 9.5 to 0.1 0% zwittergent 3- 14,
(v) the fraction eluted with 0.25 - 0.35 M NaCl is recovered and subjected to
cation exchange chromatography on a gradient S-Sepharose column
0 - 0.5 M NaCl, advantageously in pH 5 acetate buffer at 0.1% of
zwittergent 3-14 (advantageously, the 0.25 - 0.35 M NaCl fraction is
first dialyzed against acetate buffer pH 5 0.1% zwittergent 3-14); and
(vi) the fraction eluted with 0.15 M NaCl is recovered.

 The 76 kD membrane protein has the presumed biological function of being a porin, presumably located at the level of the outer membrane. Exner et al, Infect.

Immun. (Apr. 1995) 63 1567 characterizes a family of porins that each have SDS-Page electrophoresis, a different migration profile depending on whether they have been heated to 95 ° C (conventional process) or not. the case as regards the 76 kD protein according to the invention, in reflection of its gel migration, is not modified with a sample heated to 60 ° C. and not at 98 ° C.
The N-terminal sequence of the 76 kD protein is as follows (code one letter) EDDOFYTSVGYQIGEAAQMV.

 According to one particular embodiment, a protein according to the invention may in particular be obtained by purification from H. Or expressed recombinantly in a heterologous system. In the latter case, the protein may have post translational modifications that are not identical to those of the corresponding protein from the original strain.

 The subject of the invention is also the protein line t.ci of H. pylori or a polypeptide in substantially purified form, which is capable of being recognized by an antiserum or a monoclonal antibody established against a protein according to the invention. As regards the polypeptides, these include polypeptides derived from a protein according to the invention by mutation of one or more amino acids, e.g., by deletion.

Finally, the invention also relates to a pharmaceutical composition, in particular intended for the prevention or treatment of an H. pylori infection which comprises as active principle a protein or a polypeptide according to the invention, the use of a protein or a polypeptide according to the invention as a therapeutic or prophylactic agent, the use of a protein or a polypeptide according to the invention for the manufacture of a medicament for the prevention or treatment of an H. infection. and a method of preventing or treating an H. loll infection by administering to an individual a prophylactically or therapeutically effective amount of a protein or polypeptide of the invention.
A composition according to the invention can be manufactured in a conventional manner. In particular, a protein according to the invention is combined with an adjuvant, a diluent or a support that is acceptable from a pharmaceutical point of view. A composition according to the invention may be administered by any conventional route in use in the field of vaccines, in particular orally or parenterally. The administration may take place as a single or repeated dose one or more times after a certain interval interval. The appropriate dosage varies depending on various parameters, for example, the individual being treated or the mode of administration.
In more detail. By way of example, it is proposed to administer a protein or a polypeptide according to the invention orally. For this purpose, a protein or a polypeptide according to the invention may be encapsulated alone or in the presence of other H.sub.2 proteins. pylori in gelatin capsules to protect the antigen from degradation by gastric juice or well administered in the presence of sodium bicarbonate. Such formulations have already been used for pharmaceutical compositions (Black et al., Dev Biol Stand.

(1983) 53). The protein may also be encapsulated in microspheres of PLGA (glycolic acid and lactic acid copolymers) according to the protocol described elsewhere (Eldridge et al, Curr Top Microbiol Immuno (1989) 146, 59-66). may also be included in liposomes prepared according to conventional methods widely described ("Liposomes a practical approach, Ed RRC New, D. Rickwood & BD Hames, 1990,
Oxford University Press, ISBN 0-19-963077-1).

 Regardless of the formulation, the amount of protein administered to the human orally is of the order of I to 10 mg per dose, and it is recommended at least 3 taken at intervals of 4 weeks.

 Alternatively, a protein or a polypeptide according to the invention can be administered parenterally. To do this, a protein or a polypeptide according to the invention is adsorbed on alumina gel in a completely conventional manner. The protein in solution at 1 mg / ml in a buffer whose pH is close to 6.5 is brought into contact for 1 hour with aluminum hydroxide at 10 mg / ml measured at AL. The final composition of the preparation is the following protein with catalase activity 50 lig / ml, AL "250 Fug / ml, merthiolate 1/10 000, all in PBS.

 As in the case of oral administration, 3 injections are recommended each spaced 4 weeks from the previous one.

The invention is illustrated below with reference to the following figures
Figure 1, column 4 shows the analysis of the CS2d membrane fraction by 10% polyacrylamide gel electrophoresis and staining with Coomassie blue. The molecular weight markers appear in column 1.

 Figure 2, column 4, shows the 10% polyacrylamide gel electrophoresis and Coomassie blue staining analysis, of the 76 kD protein purified from a preparative gel. Molecular weight markers appear in columns I and 8.

FIG. 3 shows, after 10% polyacrylamide gel electrophoresis and staining with Coomassie blue, the electrophoretic profile of the fraction D resulting from the Q-Sepharose column chromatography of the Cs2d membrane fraction (column 3) and the fraction Resulting from S-Sepharose column chromatography of fraction D (column 4). Column 1 is molecular weight markers and Column 2 is CS2d membrane fraction
EXAMPLE 1 Preparation of Membrane Fractions
Culture
Strain H. pilori ATCC 43579 is cultured in a liquid medium in a
fermentor of 101.

A freeze of germs in glycerol is used for seeding a bottle of 75
cm2 containing so-called "biphasic" medium (a solid phase in Colombia agar
containing 6% of fi-ais sheep blood and a liquid phase of soy trypticase
containing 20% fetal calf senim) After 24 hours of culture in
microaerophilic conditions, at 37 C, the liquid phase of this culture is used to
sese several bottles of 75 cm2 in biphasic medium in the absence of blood of
sheep. After 24 hours of culture, the liquor phase makes it possible to seed a
biofermentor of 2 1 in tryptic soybean liquid medium containing beta
cyclodextrin at 10 g / l. This culture with OD 1.5-1.8 is inoculated in a fermenter of
10 1 in a liquid medium.After 24 hours of culture, the bacteria are harvested by
centrifugation at 4000 xg for 30 minutes at 4 ° C. A culture of 10 liters of H.

pylori ATCC 43579 fermenter under microaerophilic atmosphere provides
about 20 to 30 g (wet weight) of bacteria.

1B - Extraction with n octyl pD glucopyranoside (OG)
The seed pellet obtained above is washed with 500 ml of PBS
(Phosphate buffer saline, NaCl 7.650 g, disodium phosphate 0.724 g phosphate
monopotassic 0.210 g per liter, pH 7.2) per liter of culture. Then the germs
are again centrifuged under the same conditions.

The resulting bacterial pellet (C1) is resuspended in a solution of OG
(Sigma) at 1% (30 ml / liter of culture). The bacterial suspension is incubated during
1 hour at room temperature with magnetic stirring, then centrifuged at 17,600
xg for 30 minutes at 4 ° C
The supernatant (S2) obtained is dialyzed (MWCO = 10,000 Da, Spectra / por)
overnight at 40C with magnetic stirring against 2 times 1 liter of PBS diluted
1/2. The precipitate formed during the dialysis is recovered by centrifugation at 2600x
g for 30 minutes at 4 ° C The supernatant (S2d) is removed and the pellet (Cs2d) which
contains outer membrane proteins, is stored at -200C.

The resuspension of the pellet is carried out in 20 mM Tris-HCl buffer.
pH 7.5 and Pefabloc 1001lu (buffer A).

1C - Analysis of fractions membl an.lil es
The membrane fraction C52d is analyzed by gel electrophoresis
polyacrylamide in the presence of SDS according to the method of Laemmli (1970). The
Proteins are visualized after staining with Coomassie blue.

The protein profile of the membrane fraction shows the presence of 4 bands
major at 76, 67, 50 and 30 kDa (Figure 1 column 4).

EXAMPLE 2 Purification of the 76 kD Protein by Preparative SDS-PAGE
Electrophoresis is carried out on polyacrylamide gel according to the method of
Laemmli (1970) with a concentration gel of 5% and a separation gel of 10%. The membrane fraction is resuspended in buffer A, and then diluted half in the 2X sample buffer. The mixture is heated for 5 minutes at 95 ° C. About 19 mg of proteins are deposited on a gel of size 16 × 12 cm and 5 mm thick Premigration is carried out at 50 V for 2 hours, followed by migration. at 65 V overnight The staining of Coomassie blue R250 gel (0.05% in ultrafiltered water) allows good visualization of the bands.

 The major band at 76 μl was cut with a scalpel and ground with ultra-turrax in the presence of 10 or 20 ml of 25 mM Tris-HCl extraction buffer pH 8.8, 8 M urea, 10% SDS, phenyl methyl sulfonyl fluoride (PMSF) 100, uM and Pefabloc 100 M (buffer C). The crushed material is filtered on a Millipore AP20 prefilter (#filter = 4.7 cm, spore = 20 μm) using an extruder at a pressure of 7 bar at room temperature, then washed with 5 to 10 ml of buffer C and filtered as before. The two filtrates obtained from the two ground materials are collected.

The filtrate is precipitated with 3 volumes of a mixture of 50% methanol 75% and isopropanol 75%, then ultracentrifuged at 240,000 xg for 16 hours at 10 C on a rotor 70
TFT (J8-55, Beckman).

 The pellet is taken up in 2 ml of 10 mM NaPO 4 solubilization buffer pH 7.0, 0.1 M NaCl, 0.1% Sarkosyl, 100 μM PMSF, 100 μM Pefabloc, and 6 M urea (D buffer).

The solubilized sample is dialyzed successively against 100 ml of buffer D at 4 M urea and 0.1% Sarkosyl, against 100 ml of buffer D at 2 M urea and 0.5% Sarkosyl and cons twice 100 ml of buffer D without urea and 0.5% of Sarkosyl. The dialysis is carried out for 1 hour with magnetic stirring at room temperature. The final dialysate is incubated for 30 minutes in an ice bath and then centrifuged at low speed for 10 minutes at 4 ° C. (Biofuge A, Heraeus Sepatech). The supernatant is recovered, filtered on filter
Millipore at 0.45 m and stored at -20 ° C.

 SDS-PAGE analysis is performed (Figure 2, column 4). The analysis of the electrophoretic profile of the fraction shows that it is pure with a single band on gel.

EXAMPLE 3 Purification of the 76 kD Membrane Protein from the
CS2d membrane fraction
3A - Separation of the protons from the CS2d membrane fiction on the column
Q-Sepharose
One hundred and forty mg of protein from the CS2d membrane fraction
solubilized were deposited on a column Q Sepharose prepared as follows
A column of Q Sepharose is prepared according to the indications of the manufacturer
(Pharmacia) for a gel volume of 40 ml (= 2.5 cm, h = 8 cm) .The column is
washed and then equilibrated with 50 mM NaCO3 buffer pH 9.5, Pefabloc 100, μM and
Zwittergent 3-14 at 0.1%. The chromatography was followed by a UV detection at 280 nm at the exit of the column
After the column was washed with equilibrium buffer (50 mM NaC03 pH 9.5, Pefabloc 100 μM and Zwittergent 3-14 0.1%) until back to baseline, wash with NaCI 0. 1 M in equilibrium buffer was carried out until the return to the baseline (fraction A), followed by a NaCl gradient of 0.1 to 0.5 M in equilibrium buffer (fractions B, C, D and E), followed by washing with 1M NaCl in equilibrium buffer (fraction F). The results are shown in the table below.

<Tb>

fractions <SEP> elution <SEP> total proteins <SEP>
<tb><SEP> (mg)
<tb><SEP> A <SEP> wash <SEP> NaCl <SEP> 0.1M <SEP> 18
<tb><SEP> B <SEP> NaCl0.1-0.2M <SEP> 14
<tb><SEP> C <SEP> NaClO, 25M <SEP> 10
<tb><SEP> D <SEP> NaCl <SEP> 0.25-0 <SEP> 35M <SEP> 27
<tb><SEP> E <SEP> NaCl <SEP> 0.35-0.45M <SEP> 26
<tb><SEP> F <SEP> wash <SEP> NaCI <SEP> 1M <SEP> 20
<Tb>
The protein balance shows us that 55% of the proteins are eluted during the NaCl gradient 0, I-05N1, 15% are eluted during washing with 0.1M NaCl and 15% are eluted during washing with 1M NaCl.

 The SDS-PAGE profiles of each of the fractions obtained after passage of the CS2d membrane fraction on the Q Sepharose column are different. In addition, some proteins are enriched with respect to the initial CS2d membrane fraction. In particular, the protein of 76 kDa is enriched on the profile corresponding to fraction D, the purification of which is continued as follows.

3B - Purification on S-Sepharose column, of the 76 kDa protein from
fraction D obtained on colony Q Sepharose
A column of S Sepharose is prepared according to the manufacturer's indications (Pharmacia) for a gel volume of about 10 ml (= 1.5 cm, h = 5 cm) (maximum 10 ml).
mg protein / ml gel). The column is washed and then equilibrated with the acetate buffer
50 mM pH 5.0, Pefabloc 100 M and Zwittergent 3-14 at 0.1%.

The fraction D dialysed beforehand against the equilibrium buffer (acetate 50
mM pH 5.0, Pefabloc 100 Cthl and Zwittergent 3-14 0.1%) is deposited on the column
of Sepharose. The column is then washed with the same buffer until
the absorbance at 280 nm is stabilized. About 3 column volumes are needed
for the return to the baseline. The proteins are eluted by a gradient of 0 to 0.5
M NaCl in equilibrium buffer (10 times VT), followed by washing with buffer
equilibrium containing 0.5 and 112; 1 NaCl (4 times VT). The fractions collected are
analyzed, collected and preserved as before.

D fraction exiting during the 0-0.5M NaCl gradient, between 0.15 and 0.20 M
NaCl has been recovered. On this fraction, an analysis by SDS-PAGE and by western
blot is performed. The results are shown in Figure 3.

These results show that after two ion exchange columns, the protein
of 76 kDa is enriched but remains contaminated with a protein of 54 kDa and
a 67 kDa protein (column 4). The 54 kDa protein does not react with
anti-catalase antibody, therefore it does not correspond to catalase,
even the 67 kDa protein does not react with the anti-ureB antibody in western
blot, therefore it does not correspond to the subunit B of urease. Analysis by
western blot in the presence of an ante anti-76 kDa serum shows that weak bands
observed below the 76 kDa band correspond to a degradation of the
76 kDa protein, since they react weakly with this antiserum (column 7).

EXAMPLE 4 Preparation of a hyperinuclear serum against the protein
76kD memlvranail e.

A polyclonal serum specific for the major membrane protein of H. pylori is obtained by hyperimmunization of rabbits respectively with SDS purified antigen
Preparative PAGE The first OJ injection (multisite and intramuscular subcutaneous) is carried out with a preparation containing 50 L of membrane protein emulsified in Freund's complete adjuvant, then the recalls J21 and J42 are made by injection of 25 μg of membrane protein into incomplete adjuvant of Freund. The animals are sacrificed at J60.

The sera obtained are decomplemented for 30 minutes at 56 ° C. and sterilized by membrane filtration with a porosity of 0.22 m (Millipore).

 The antiserum reacts with the 76 kD protein as isolated according to Example 3.

Claims (6)

claims 1. Helicobacter pylori protein in substantially purified form,  likely to be obtained from a membrane fraction of H. pylori and  whose molecular weight after 10% polyacrylamide gel electrophoresis  in the presence of SDS, appears on the order of 76 kD. 2. A protein according to claim 1, the apparent molecular weight of which is  the order of 76 kD and which is likely to be obtained by a process in  which:  (i) H. pylori bacteria are extracted with n-octyl ss-D  glucopyranoside 1%, followed by centrifugation;  (ii) recovering a supernatant which is dialyzed against PBS 75  mM pH 7.2, followed by centrifugation;  (iii) recovering the membrane fraction consisting of the centrifugation pellet  that we resuspend in an aqueous medium;  (iv) the membrane fraction is subjected to an exchange chromatography  of anions on Q-Sepharose column in gradient of 0.1 - 0.5 M NaCl;  (v) recovering the fraction eluted with 0.25 - 0.35 M NaCl, which is subjected to  cation exchange chromatography on a column of S-Sepharose in  gradient of NaCl O - 0.5 M; and  (vi) the fraction eluted with 0.15-0.20 M NaCl is recovered. 3. A protein according to claim 1 or 2, which has a biological function  to be a porine. 4. A protein according to claim 1, 2 or 3, which has an N-terminal sequence  the amino acid sequence EDDGFYTSVGYQIGEAAQMV. 5. A protein or polypeptide derived by mutation of a protein according to one of  Claims 1 to 4, in substantially purified form, which is capable of  to be recognized by an antiserum established against a protein according to one of the  Claims I to 4. 6. A pharmaceutical composition for prevention or treatment  H. pjioii infection, which comprises as active ingredient a protein  or a polypeptide according to one of claims I to 5.