AU778957B2 - Bacterial membrane fractions with adjuvant effect - Google Patents

Abstract

The invention concerns the use of a membrane fraction of gram-negative bacteria, in particular Klebsiella pneumoniae combined with an antigen or hapten for preparing a pharmaceutical composition designed to direct the immune response towards a Th1 and/or a mixed Th1/Th2 type response directed against said antigen or hapten. The invention further concerns methods for preparing said membrane fractions and pharmaceutical compositions containing them and their uses for preventing and treating infectious diseases, in particular those involving paramyxovirus such as VRS, and cancers, in particular those with tumour-associated antigens.

Description

WO 00/54789 PCT/FR00/00622 BACTERIAL MEMBRANE FRACTIONS WITH ADJUVANT EFFECT The present invention relates to the use of a membrane fraction of gram-negative bacteria, in particular of Klebsiella pneumoniae, combined with an antigen or hapten, for the preparation of a pharmaceutical composition intended for orienting the immune response toward a Thl type and/or mixed Thl/Th2 type response directed against said antigen or hapten. This invention comprises, in addition, methods for the preparation of said membrane fractions and the pharmaceutical compositions containing them and their applications to the prevention and treatment of infectious diseases, in particular infections caused by paramyxoviruses such as RSV, and cancers, in particular those whose tumors are associated with tumor antigens.

Vaccination is an effective means for preventing or reducing in particular infections. The success of vaccination campaigns in this field has made it possible to extend the concept of vaccines to the fields of autoimmune diseases, cancer and fertility. On the other hand, vaccinating antigens alone are not always capable of inducing a rapid and sustained antibody response, which requires the presence of adjuvants, that is to say compounds which help (from the latin adjuvare: to help) them to induce such responses.

Adjuvants constitute a group of varied compounds with respect to their structure and their origin. There are thus, inter alia, in this category water-in-oil (incomplete Freund's adjuvant) or oil-in-water emulsions, compounds of bacterial origin such as lipopolysaccharide derivatives from Gram-negative bacteria and aluminum salts. Currently, only aluminum salts are used in humans as adjuvant for vaccine preparations.

2 The development of an antibody response directed against an antigen requires a series of complex events.

It involves cells presenting the antigen, regulatory T lymphocytes (Th for T "helper"), and antibody-producing B lymphocytes. Two types of Th lymphocytes may be distinguished according to the profile of cytokines produced: type 1 Th lymphocytes producing IFN-y and IL-2 and promoting the formation of IgG2a in mice, and type 2 Th lymphocytes producing IL-4, IL-5 and with formation of IgG1 in mice (Mosmann, T.R. and Sad S. Immunol. Today 1996, 17:138). Moreover, it has been shown that, for the same given antigen, it is the adjuvant which orients toward the predominant isotype during the antibody response (Toellner et al. J.

Exp. Med. 1998, 187:1193). Thus, it is known that aluminum salts, such as Alhydrogel, induce, in mice, an essentially Th2 type response and promote the formation of IgGl or even of IgE (Allison A.C. In Vaccine design The role of cytokine networks Vol. 293, 1-9 Plenum Press 1997), which can pose problems in subjects with an allergic predisposition. Furthermore, according to the therapeutic target envisaged, a Thl or mixed (Thl/Th2) type response may be desired.

Thus, there is currently a need to have available novel adjuvants capable of inducing an immune response of the Thl or mixed (Thl/Th2) type, preferably a mixed Thl/Th2 response for which the Thl response is close to or greater than the Th2 response.

Surprisingly, the authors of the present invention have demonstrated particular properties of the membrane fraction of a gram-negative bacterium Klebsiella pneumoniae (called FMKp), in particular membrane fractions obtained by methods as described below in the examples. The authors have indeed discovered that said membrane fraction FMKp, combined with an antigen, not only had the capacity to increase the antibody response directed against said antigen but also had the capacity 3 to reorient the cytokine response toward a Thl/Th2 profile, thus corresponding to the particular adjuvant activity sought, this being regardless of the mode of administration of said membrane fractions.

Thus, the subject of the present invention is the use of a membrane fraction of gram-negative bacteria, in particular of Klebsiella pneumoniae, combined with an antigen or hapten for orienting the immune response toward a Thl type and/or mixed Thl/Th2 type response directed against said antigen or hapten, or for the preparation of a pharmaceutical composition intended for orienting the immune response toward a Thl type and/or mixed Thl/Th2 type response directed against said antigen or hapten.

By orientation of the immune response toward a Thl and/or mixed Thl/Th2 type response, there is preferred in particular orientation of the immune response which promotes the induction of a Thl response relative to the Thl/Th2 response obtained with the alum adjuvant.

By orientation of the immune response toward a Thl and/or mixed Thl/Th2 type response, there is more particularly preferred an orientation of the immune response which increases the titer of IgG2a antibodies directed against the associated antigen by a factor of at least 10, preferably of at least 25, 50 and 100 relative to the IgG2a titer obtained with the alum adjuvant.

In a particularly preferred manner, the immune response is oriented toward a Thl and/or mixed Thl/Th2 type response in which the Thl response is close to or greater than the Th2 response. The expression "close to" will be understood to mean a response which, when expressed as titer of IgG2a antibodies directed against the associated antigen, is at least equal to times, preferably at least equal to 0.75 times, the 4 titer of IgGl antibody directed against said antigen, with a titer of IgG antibody directed against the associated antigen close to or greater than the titer of IgG antibody directed against the associated antigen obtained with the alum or Freund's adjuvant.

The invention also relates to the use according to the invention, characterized in that the membrane fraction comprises at least membrane fractions of two different strains of bacteria.

The expression membrane fraction of a bacterium is understood to mean, in the present invention, any purified or partially purified membrane fraction or extract obtained from a culture of said bacterium and whose method of preparation comprises at least a step of lysing the bacteria obtained after culture and a step of separating the fraction containing the membranes of said bacteria from the total lysate obtained after the lysis step, in particular by centrifugation or filtration.

The expression membrane fraction of the bacterium when said bacterium is Klebsiella pneumoniae is also understood to mean, in the present invention, protein an active fraction of the membrane fraction of Klebsiella pneumoniae, having an amino acid sequence SEQ ID No. 2, or one of its fragments.

According to the invention, the membrane fractions may be prepared according to methods known to a person skilled in the art, such as for example the method described by Haeuw J.F. et al. (Eur. J. Biochem, 255, 446-454, 1998) According to a particular embodiment, the invention relates to a use according to the invention, characterized in that the membrane fraction is prepared by a method comprising the following steps: 5 a) culture of said bacteria in a culture medium allowing their growth followed by centrifugation of said culture; b) where appropriate, deactivation of the lytic enzymes of the bacterial pellet obtained in step followed by centrifugation of the suspension obtained; c) extraction and removal of nonmembrane proteins and of nucleic acids from the pellet obtained in step a) or b) by at least one cycle of washing the pellet in an extraction solution; d) digestion of the membrane pellet obtained in step c) in the presence of proteolytic enzymes, followed by centrifugation; e) at least one cycle of washing of the pellet obtained in step d) in physiological saline and/or in distilled water; and f) ultrasonication of the pellet obtained in step e).

Step b) of deactivation of the lytic enzymes of the bacterial pellet obtained in step a) may be carried out by any known methods of deactivation of enzymes, such as in particular by heating the resuspended bacterial pellet at a temperature preferably of close to 100 0 C or by adding an inhibitor of the activity of these enzymes.

Step c) of extraction and removal of the nonmembrane proteins and of the nucleic acids from the pellet obtained in step a) or b) may be carried out, for example, by at least one cycle of washing of the pellet in an extraction solution corresponding to the addition of a hypertonic solution (extraction solution), preferably a saline solution having a molarity of close to 1 M, followed, after a contact period which is sufficient for the desired effect, by centrifugation of the suspension obtained and removal of the supernatant obtained after said centrifugation, it being possible for this washing cycle to be repeated several times.

6 Step d) of digestion of the membrane pellet obtained in step c) may be carried out in the presence of a solution of proteolytic enzymes such as, for example, trypsin, chymotrypsin, or any known enzyme with proteolytic activity, the reaction conditions, pH of the solution, temperature and duration of the reaction, being preferably adjusted to the optimum conditions for the activity of the chosen enzyme(s), followed by centrifugation, it being possible for this digestion cycle to be repeated several times with the same enzyme, the same combination of enzymes or with a different enzyme for each digestion cycle performed.

Step e) of washing the pellet obtained in step d) is carried out by taking up the pellet in physiological saline or in distilled water followed, after a sufficient period of contact, by centrifugation, it being possible for this washing cycle to be repeated several times.

Finally step f) of ultrasonication of the pellet is intended in particular to disintegrate and homogenize the membrane fraction obtained at the end of step e).

The ultrasonication conditions (duration and intensity) will be determined by persons skilled in the art, for example, according to the quantity of membrane fraction to be treated.

According to another particular embodiment, the invention relates to a use according to the invention, characterized in that the membrane fraction is prepared by a method comprising the following steps: a) culture of said bacteria in a culture medium allowing their growth, followed, where appropriate, by centrifugation; b) freezing of the culture medium or of the pellet obtained in step a) followed by thawing and drying of the cells; 7 c) removal, by means of a DNase, of the nucleic acids from the dry cells obtained in step b) which have been resuspended; d) grinding of the cells obtained in step c) and clarification of the suspension obtained; e) precipitation, in an acid medium, of the suspension obtained in step d) and removal of the pellet; f) neutralization of the supernatant obtained in step e) containing the membrane suspension, followed by dialysis and concentration of the membrane suspension; and g) sterilization of the concentrated membrane suspension obtained in step f) The thawing conditions in step b) of the method below will of course be determined by persons skilled in the art according to the initial quantity of pellet to be treated, preferably carried out at 4 0 C for at least 48 hours for the equivalent of 1 kg of dry cells.

In step the removal of the nucleic acids is carried out, for example, by the addition of a DNase, at a final concentration of 5 mg/ml of a cell suspension at a concentration equivalent to 5% of dry cells.

The grinding of the cells obtained in step c) may be carried out by means of any system or apparatus known to a person skilled in the art for grinding cells, such as presses or preferably such as grinding in a Manton Gaulinet loop for 30 minutes.

The clarification of the suspension obtained after grinding may be carried out by means of any system or apparatus known to a person skilled in the art for the clarification of ground products of bacterial cells such as the Sharpless system.

8 Step e) of precipitation in acid medium of the suspension obtained in step d) may be carried out, for example, with acetic acid. The precipitation is followed by the removal of the pellet by means of a Sharpless-type system and by recovering of the supernatant.

Step f) consists in a step in which the supernatant, obtained after precipitation in acid medium, is neutralized, diluted, dialyzed and then concentrated.

Finally, the last step consists in a step of sterilizing the membrane fraction concentrate obtained in the preceding step such as, for example, by heating at 1210C for about 35 minutes.

The invention particularly relates to the use according to the invention, characterized in that the membrane fraction is the Klebsiella pneumoniae protein having the sequence SEQ ID No. 2, or one of these fragments.

The expression protein P40 fragment is understood to mean in particular any fragment having an amino acid sequence contained in the amino acid sequence of protein P40 capable of increasing a nonspecific immune response and/or capable of inducing an antitumor immune response, and comprising at least 5 amino acids, preferably at least 10 amino acids and more preferably at least 15 amino acids.

Of course, said protein P40, or its fragments, may be obtained by chemical synthesis or in the form of recombinant peptides.

The invention particularly relates to the use according to the invention, characterized in that said antigen or hapten is chosen from the antigens or haptens specific to an infectious agent, such as a virus, a bacterium, a 9 fungus or a parasite, or from the antigens associated with tumor cells.

According to the invention, said antigens or haptens are preferably chosen from peptides, lipopeptides, polysaccharides, oligosaccharides, nucleic acids, lipids or any compound capable of specifically directing the immune response toward a Thl type and/or mixed Thl/Th2 type response against an antigen or hapten specific to an infectious agent or an antigen associated with a tumor cell.

Of course, said antigen or hapten, when it is of a peptide nature, may be obtained by chemical or recombinant synthesis.

The methods for preparing recombinant peptides are nowadays well known to persons skilled in the art and will not be developed in the present description. Among the cells which may be used for the production of these recombinant peptides, there may of course be mentioned bacterial cells (Olins P.O. and Lee 1993, Recent advances in heterologous gene expression in E. coli Curr. Op. Biotechnology 4:520-525), but also yeast cells (Buckholz 1993, Yeast Systems for the Expression of Heterologous Gene Products. Curr. Op.

Biotechnology 4:538-542), as well as animal cells, in particular mammalian cell cultures (Edwards C.P. and Aruffo 1993, Current applications of COS cell based transient expression systems. Curr. Op. Biotechnology 4, 558-563) but also insect cells in which methods may be used involving for example baculoviruses (Luckow 1993, Baculovirus systems for the expression of human gene products. Curr. Op. Biotechnology 4, 564-572).

The invention comprises, in addition, the use according to the invention, characterized in that said antigen or hapten is coupled or mixed with said membrane fraction, 10 in particular covalently coupled with at least one of the compounds contained in the membrane fraction.

In a preferred embodiment, the invention comprises the use according to the invention, characterized in that said antigen or hapten is covalently coupled with a supporting peptide to form a complex capable of binding specifically to mammalian serum albumin, preferably said supporting peptide is a peptide fragment derived from streptococcal G protein, in particular the C-terminal fragment called BB.

Of course, said complex may be prepared by genetic recombination.

The chimeric or hybrid complex may be produced by recombinant DNA techniques by insertion or addition of a sequence encoding said antigen or hapten of a protein nature to a DNA sequence encoding said peptide fragment of the streptococcal G protein.

The methods for the synthesis of hybrid molecules include the methods used in genetic engineering for constructing hybrid polynucleotides encoding the desired polypeptide sequences. Reference may be advantageously made, for example, to the technique for producing genes encoding fusion proteins which is described by D.V. Goeddel (Gene expression technology, Methods in Enzymology, Vol. 185, 3-187, 1990) According to the present invention, the covalent coupling may be carried out by chemical synthesis. In a particular embodiment of the invention, it will be possible for one or more linking elements to be introduced into at least one of the compounds contained in the membrane fraction and/or in said antigen or hapten to facilitate the chemical coupling.

11 Preferably, said linking element introduced is an amino acid.

According to the invention, it is possible to introduce one or more linking elements, in particular amino acids, to facilitate the coupling reactions between a compound of the membrane fraction, and said antigen or hapten. The covalent coupling between said compound of the membrane fraction and said antigen or hapten according to the invention may be achieved at the N- or C-terminal end of said compound of the membrane fraction or of said antigen or hapten, if the latter are for example of a peptide nature. The bifunctional reagents allowing the coupling will be determined according to the end which is chosen for the coupling and the nature of said antigen or hapten to be coupled.

The invention also comprises the use according to the invention, characterized in that the coupling between said antigen, hapten or complex and at least one of the compounds contained in the membrane fraction is carried out by genetic recombination when said antigen, hapten or complex and said membrane compound are of a peptide nature.

The coupling between said antigen, hapten or complex, and at least one of the compounds contained in the membrane fraction may indeed be carried out by genetic recombination. It will be possible, for example, before extracting its membrane fraction, to transform said gram-negative bacterium beforehand with a vector containing a nucleic construct encoding an antigen of interest or said complex, such that the bacterium thus transformed expresses the antigen of interest or said complex attached to the membrane or anchored in the membrane of said bacterium. Such methods for expressing recombinant proteins attached to the membrane are well known and require, for example, the presence of a 12 specific regulatory sequence, such as a signal peptidetype sequence.

The subject of the invention is also the use according to the invention, characterized in that the pharmaceutical composition comprises, in addition, an agent which makes it possible to carry said membrane fraction associated with said antigen, hapten or complex in a form which makes it possible to enhance its stability and/or its immunogenicity, such as in the form of an oil-in-water or water-in-oil type emulsion, or in the form of a particle of the liposome, microsphere or nanosphere type or any type of structure allowing the encapsation and the presentation, in particulate form, of said membrane fraction associated with said antigen, hapten or complex.

The invention also relates to the use according to the invention, characterized in that said agent is chosen from aluminum salts, calcium salts, compounds of plant origin such as Quil A or saponin, or compounds of bacterial origin such as the derivatives of cholera, pertussis or tetanus toxoid or of the E. coli thermolabile toxin.

Also included in the present invention is the use according to the invention, characterized in that the pharmaceutical composition comprises, in addition, an agent which makes it possible to regulate the immune response induced by said membrane fraction combined with said antigen, hapten or complex.

Among said regulatory agents, cytokines, growth factors, hormones or cellular components such as nucleic acids, a protein of the family of heat shock proteins or ribosomes are in particular preferred.

The subject of the invention is also the use according to the invention, for the preparation of a 13 pharmaceutical composition intended for the prevention or treatment of infectious diseases of viral, bacterial, fungal or parasitic origin, or for the prevention or treatment of cancers, in particular cancers in which the tumors are associated with tumor antigens.

Among said infectious diseases of viral origin, the infectious diseases caused by paramyxoviruses, in particular by the parainfluenzae virus and more preferably by the respiratory syncytial virus (RSV) are particularly preferred.

In a particular embodiment, the use according to the invention is characterized in that said antigen associated with the membrane fraction comprises the peptide G2Na, a fragment of the G protein of the virus having an amino acid sequence SEQ ID No. 4, a peptide homologous to G2Na whose sequence exhibits at least 80%, preferably 90%, 95% and 99% identity, after alignment with the sequence SEQ ID No. 4, or the peptide G2Na or one of its homologs, covalently coupled with a C-terminal fragment (BB) of the streptococcal G protein to form a complex capable of binding to mammalian serum albumin, peptide BB as described in the documents Power et al., 1997 (Virology, 230, 155-166) and WO 96/14416.

The expression "percentage, degree or level of identity" between two nucleic acid or amino acid sequences for the purposes of the present invention is understood to mean a percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being randomly distributed over their full length. The sequence comparisons between two nucleic acid or amino acid sequences are traditionally carried out by 14 comparing these sequences after having aligned them in an optimum manner, said comparison being carried out by segment or by "comparison window" to identify and compare the local regions of sequence similarity. The optimum alignment of the sequences for the comparison may be carried out either manually or by means of the local homology algorithm by Smith and Waterman (1981) [Ad. App. Math. 2:482], by means of the local homology algorithm by Neddleman and Wunsch (1970) Mol. Biol.

48:443], by means of the method of search for similarity by Pearson and Lipman (1988) [Proc. Natl.

Acad. Sci. USA 85:2444], by means of computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or by the comparison software packages BLAST N or BLAST P).

The percentage identity between two nucleic acid or amino acid sequences is determined by counting these two sequences optimally aligned by the comparison window in which the region of the nucleic acid or amino acid sequence to be compared may comprise additions or deletions relative to the reference sequence for an optimum alignment between these two sequences. The percentage identity is calculated by determining the number of identical positions for which the nucleotide or the amino acid residue is identical between the two sequences, by dividing this number of identical positions by the total number of positions in the comparison window and by multiplying the result obtained by 100 in order to obtain the percentage identity between these two sequences.

For example, it will be possible to use the BLAST program, "BLAST 2 sequences", available on the site http://www.ncbi.nlm.nih.gov/gorf/bl2.html, the parameters used being those given by default (in particular for the parameters "open gap penaltie": and "extension gap penaltie": 2; the template chosen 15 being for example the template "BLOSUM 62" proposed by the program), the percentage identity between the two sequences to be compared being calculated directly by the program.

In another aspect, the invention relates to a method for preparing a membrane fraction of gram-negative bacteria, in particular Klebsiella pneumoniae, characterized in that it comprises the following steps: a) culture of said bacteria in a culture medium allowing their growth followed by centrifugation of said culture; b) where appropriate, deactivation of the lytic enzymes of the bacterial pellet obtained in step followed by centrifugation of the suspension obtained; c) extraction and removal of nonmembrane proteins and of nucleic acids from the pellet obtained in step a) or b) by at least one cycle of washing the pellet in an extraction solution; d) digestion of the membrane pellet obtained in step c) in the presence of protease enzymes, followed by centrifugation; e) at least one cycle of washing of the pellet obtained in step d) in physiological saline and/or in distilled water; and f) ultrasonication of the pellet obtained in step e).

The invention also comprises the method for preparing a membrane fraction of gram-negative bacteria, in particular Klebsiella pneumoniae, characterized in that it comprises the following steps: a) culture of said bacteria in a culture medium allowing their growth, followed, where appropriate, by centrifugation; 16 b) freezing of the culture medium or of the pellet obtained in step a) followed by thawing and drying of the cells; c) removal, by means of a DNase, of the nucleic acids from the dry cells obtained in step b) which have been resuspended; d) grinding of the cells obtained in step c) and clarification of the suspension obtained; e) precipitation, in an acid medium, of the suspension obtained in step d) and removal of the pellet; f) neutralization of the supernatant obtained in step e) containing the membrane suspension, followed by dialysis and concentration of the membrane suspension; and g) sterilization of the concentrated membrane suspension obtained in step f) The membrane fractions capable of being obtained by said methods indeed form part of the invention.

The content of proteoglycan of the membrane fractions capable of being obtained by said methods, an active ingredient of FMKp, represented by the sum of the contents of galactose and of protein, is preferably between: for galactose: between 1.2 g/l and 3.4 g/l; for the proteins: between 7.5 g/l and 14.9 g/l.

More preferably, this content will be: for galactose: between 1.6 g/l and 2.6 g/l; for the proteins: between 9.3 g/l and 11.7 g/l.

The invention relates, in addition, to the pharmaceutical compositions comprising a membrane fraction capable of being obtained by the methods according to the invention, preferably, said 17 pharmaceutical compositions comprise, in addition, an antigen, a hapten or a complex, as defined above, associated with said membrane fraction, such as in particular viral antigens or complexes specific to paramyxoviruses, or the antigens associated with tumor cells.

Of course, said pharmaceutical compositions according to the invention may comprise, in addition, the agents such as the vehicles and the regulatory agents defined above.

In a preferred embodiment, the pharmaceutical composition according to the invention is characterized in that said antigen associated with the membrane fraction comprises the peptide G2Na having the sequence SEQ ID No. 4 of the respiratory syncytial virus, one of its homologs as defined above, said peptide G2Na, or one of its homologs, covalently coupled with a C-terminal fragment (BB) of the streptococcal G protein to form a complex capable of binding to mammalian serum albumin.

The legend to the figures and examples which follow are intended to illustrate the invention without in any way limiting the scope thereof.

Legend to the figures: Figure 1: BBG2Na adjuvanted with FMKp dose-response study (serum anti-G2Na IgG titers).

p 0.05 (compared with the PBS group).

Figure 2: BBG2Na adjuvanted with FMKp anti-G2Na IgG1 and IgG2a titers.

Figure 3: BBG2Na adjuvanted with FMKp protection study.

Figure 4: Adjuvant effect of FMKp toward Immugrip (influenza vaccine).

18 p 0.05 compared with the nonadjuvanted group on the same day of sample collection.

Example 1: Production of the membrane fraction of K. pneumoniae (FMKp) Method No. 1 The extraction of the K. pneumoniae 1145 membranes from the centrifugation pellet of the step is preferably preceded by a step of destroying the lytic enzymes of the cellular components obtained in the pellet, for example by heating the latter at 100 0 C, optionally after redissolving in solution.

The actual extraction of the membranes from the centrifugation pellet is preferably carried out by treating the cellular components of the pellet, after optional destruction of the lytic enzymes, with a saline solution, for example 1 M sodium chloride, once or several times, followed by centrifugation, preferably at 20,000 g, of the suspension obtained, the supernatant from this centrifugation, which is eliminated, contains nonmembrane impurities such as proteins and nucleic acids, while the pellet contains the membranes.

After separation of the saline solution containing the impurities, the membranes are digested in the presence of proteolytic enzymes, preferably trypsin and chymotrypsin, in solution at pH 8 at 370C for 4 hours.

After digestion, the solution is homogenized by ultrasonication. The product thus obtained constitutes the membrane fraction called FMKp.

The supernatant obtained is again centrifuged under the same conditions, preferably at 140,000 g.

19 Preparation of the membrane glycopeptides This fraction is prepared from the pellet obtained by centrifugation at 40,000 g for 20 minutes. Said pellet is resuspended in physiological saline and then this suspension is heated for 10 minutes at 100 0 C on a boiling water bath to inactivate the lytic enzymes.

After cooling, the medium is centrifuged for 30 min at 20,000 g. The pellet obtained is extracted twice with 1M NaCl in order to eliminate the proteins and the nucleic acids. The membranes are recovered by centrifugation for 30 minutes at 20,000 g.

They are then subjected to digestion with trypsin at pH 8 and at 370C for 4 hours and then with chymotrypsin under the same conditions.

The membranes are then recovered by centrifugation at 2000 g for 30 minutes, washed with physiological saline and then with distilled water and are subjected to disintegration by ultrasound for 15 minutes.

Method No. 2 After thawing at +4 0 C for 48 h minimum, 1 kg of dry K. pneumoniae cells is resuspended in solution at dry cells. The DNase is added at 5 mg/l. Grinding in a Manton Gaulin loop is then carried out for 30 min, followed by clarification on SHARPLES at 50 l/h, followed by precipitation with acetic acid at pH 4.2 0.1 for 30 min. The pellet is removed (SHARPLES at 25 l/h) and the supernatant is neutralized, diluted to twice the initial volume with osmosed water. Constant-volume dialysis is then carried out on PUF 100 up to 800 Qcm, followed by concentration of the membrane suspension (MS) thus obtained, to 11 1/kg of dry cells. The MS is then autoclaved at +121 0 C for 35 min and preserved at +4 0 C for 6 weeks.

20 Characteristics of the FMKp By definition, the content of proteoglycan, an active ingredient of FMKp, is equal to the sum of the contents of galactose and of proteins.

Galactose: on average 2.2 g/l Proteins: on average 10.5 g/l.

Example 2: Adjuvant effect of FMKp on a recombinant protein, BBG2Na BBG2Na is a recombinant protein produced in E. coli. It consists of the peptide G2Na having the sequence SEQ ID No. 4, the fragment of the G protein of the respiratory syncytial virus (RSV) type A extending from residue 130 to residue 230, fused with BB, a fragment of the streptococcal G protein, having the capacity to bind to serum albumin. BBG2Na is an anti-RSV vaccine (Power U.

Virology 1997, 230:155-166).

BALB/c mice receive 2 subcutaneous injections of 20 tg of BBG2Na and various quantities of FMKp. Blood samples are collected on D28 and the serum antibody titers are determined by ELISA with G2Na in solid phase. The results obtained are illustrated by figure 1.

Surprisingly, they show that FMKp significantly increases the anti-G2Na IgG response; the anti-G2Na IgG titer reached is similar to those induced by alum or Freund's adjuvant. The effect is dose-dependant: it is observed from 5 jig of FMKp, is maximum from 50 gg of FMKp and remains stable with 100 ig of FMKp. FMKp is therefore a potential adjuvant for BBG2Na.

To know the effect of FMKp on the orientation of the immune response, in terms of Thl/Th2 response, the anti-G2Na IgG1 and IgG2a titers were determined on sera obtained as specified above. The results (figure 2) show that, surprisingly, FMKp is capable of modifying 21 the anti-G2Na IgGl/IgG2a ratio, in contrast to that which is observed with alum, for which the predominant isotype is IgG1. This profile is close to that induced by Freund's adjuvant. This indicates that FMKp may be used as immunity adjuvant to induce a mixed (Thl/Th2) type response.

The animals immunized as described above received a viral challenge by the nasal route with 105 TCID 50 of RSV-A. This was carried out 3 weeks after the last immunization. Five days after the viral challenge, the animals were sacrificed and the lungs removed in order to determine the RSV-A titers. The results (figure 3) show that the animals which received BBG2Na adjuvanted with FMKp are protected against an RSV-A challenge.

In conclusion, FMKp makes it possible to reorient the antibody response without affecting the capacity to protect mice against an RSV-A challenge.

Example 3: Adjuvant effect of FMKp on an inactivated virus (influenza vaccine) BALB/c mice receive a single injection of 0.01 jg of Immugrip TM (influenza vaccine marketed by Laboratoires INAVA), and various quantities of FMKp. The products are coadministered. The injection is performed subcutaneously at DO. Blood samples are collected at D7, D14 and D21. The anti-Immugrip serum IgG antibody titer is determined by ELISA with Immugrip at 2 gg/ml in solid phase. The results presented (figure 4) show that FMKp significantly increases the anti-Immugrip antibody titer, this being from the lowest dose of FMKp, namely 0.1 jg. The adjuvant effect is dosedependant. It is observed, interestingly, that the presence of FMKp induces the generation of an earlier antibody response, obtained from D7, compared with the nonadjuvanted Immugrip control. This effect is not UU4btYiBaV4 22 obtained with the reference adjuvant, complete Freund's adjuvant (CFA).

As used herein, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude other additives, components, integers or steps.

Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment, or any form of suggestion, that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as relevant by a person skilled in the art.

*o ooo o* EDITORIAL NOTE APPLICATION NUMBER 32980/00 The following Sequence Listing pages 1 to 4 are part of the description. The claims pages follow on pages "23" to Wo 00/54789 1 WO 005478 -1-PCT/FROO/ 00622 SEQUENCE LISTING <110> PIERRE FABRE MEDICAMENT <120> USE OF BACTERIAL MEMBRANE FRACTIONS WITH ADJUVANT EFFECT, THEIR METHODS OF PREPARATION AND PHARMACEUTICAL COMPOSITION CONTAINING THEM <130> D17975 <140> FR 99 03513 <141> 1999-03-15 <160> <170> <210> <211> <212> <213> <220> <221> <222> <220> <221> <222> <220> <221> <222> 4 Patentln Vers. 1 1035

DNA

Kiebsielia pneumoniae exon (1032) intron (1033) (1035)

CDS

(1032) <400> 1 atg aaa Met Lys 1 gca att ttc Ala Ile Phe 5 ggt ggt aaa Gly Gly Lys gta ctg aat gcg Val Leu Asn Ala gc t Ala 10 cag Gin ccg aaa gat aac Pro Lys Asp Asn acc tgg Thr Trp tat Tyr gca Ala ctg ggt tgg Leu Gly Trp tat cac gac Tyr His Asp tac ggt aac Tyr Gly Asn ctt ggt gct Leu Gly Ala ggt Gly ttc cag aac Phe Gin Asn aac Asn 40 ggt Gly ggt ccg acc Gly Pro Thr cgt Arg ccg Pro acc ggt ttc Thr Gly Phe aac gat cag Asn Asp Gin tac ctc ggt Tyr Leu Gly ggt gcg ttc Gly Ala Phe tac cag gtt Tyr Gin Val ttc gaa Phe Giu atg ggt tat Met Gly Tyr gtt Val1 gac Asp 70 ttc Phe ctg ggc cgt Leu Giy Arg atg Met 75 gtt Val1 tat aaa ggc Tyr Lys Gly agc Ser gac aac ggt Asp Asn Gly gc t Ala atc Ile aaa gct cag Lys Ala Gin ggc Gly 90 gac Asp cag ctg acc Gin Leu Thr gct aaa Ala Lys ctg ggt tac Leu Gly Tyr ggc atg gtt Gly Met Val 115 gtt tcc cgt Val Ser Arg ccg Pro 100 tgg Trp act gac gat Thr Asp Asp atc tac acc Ile Tyr Thr cgc gct gac Arg Ala Asp tcc Ser 120 act Thr ggc aac tac Gly Asn Tyr gct Ala 125 gta Val1 cgt ctg ggc Arg Leu Gly 110 tct acc ggc Ser Thr Gly ttt gct ggc Phe Ala Gly agc gaa cac Ser Giu His 130 ggc gta Glv Val gac Asp 135 act Thr ggc gtt tcc cca Gly Val Ser Pro gag tgg gct Giu Trp Ala 145 cag Gin gtt Val 150 atc Ile cgt gac atc Arg Asp Ile gc t Ala 155 act Thr cgt ctg gaa Arg Leu Glu tac Tyr 160 tgg gtt aac Trp Val Asn aac Asn 165 ctg ggc gac gcg Gly Asp Ala ggc Gly 170 tcc gtg ggt acc Val Gly Thr cgt cct Arg Pro 175 cag gaa gat aac ggc atg agc ctg ggc gtt tac cgc ttc ggt 2 Asp Asn Gly Met Leu Ser Leu Gly Val Ser Tyr Arg Phe Gly Gin Glu 180 gat Asp gct Ala aaa Lys 225 act Thr ggc Gly gag Glu ccg Pro act Thr 305 tgc Cys gaa Glu gct Ala acc Thr 210 gct Ala cag Gin tac Tyr aaa Lys gct Ala 290 ggc Gly ctg Leu gtt Val gca Ala 195 aag Lys acc Thr ctg Leu acc Thr cgt Arg 275 ggc Gly aac Asn gct Ala gta Val ccg Pro cac His ctg Leu agc Ser gac Asp 260 get Ala aaa Lys acc Thr ccg Pro act Thr 340 gtt Val ttc Phe aaa Lys aac Asn 245 cgc Arg cag Gin ate Ile tgt Cys gat Asp 325 cag Gin gtt Val acc Thr ccg Pro 230 atg Met ate Ile tcc Ser tcc Ser gac Asp 310 cgt Arg ccg Pro get Ala ctg Leu 215 gaa Glu gat Asp ggt Gly gtt Val gct Ala 295 aac Asn cgt Arg gcg Ala 185 ccg get Pro Ala 200 aag tct Lys Ser ggt cag Gly Gin ccg aaa Pro Lys tec gaa Ser Glu 265 gtt gac Val Asp 280 cgc ggc Arg Gly gtg aaa Val Lys gta gag Val Glu ggt taa Gly Asn Ala Trp Ser 25 Asn Asn 40 Gly Tyr Leu Gly Ala Gin Asp Leu 105 Ser Lys 120 Thr Gly Arg Asp ccg Pro gac Asp cag Gin gac Asp 250 get Ala tac Tyr atg Met get Ala ate Ile 330 Ala 10 Gin Gly Gin Arg Gly 90 Asp Gly Val Ile 190 gct Ala gtt Val gct Ala 235 ggt Gly tac Tyr ctg Leu ggt Gly cgc Arg 315 gaa Glu Pro Tyr Pro Val Met 75 Val Ile Asn Ser Ala 155 ccg Pro ctg Leu 220 ctg Leu tcc Ser aac Asn gtt Val gaa Glu 300 get Ala gtt Val Lys His Thr Asn Ala Gin Tyr Tyr Pro 140 Thr get ccg Ala Pro 205 ttc aac Phe Asn gat cag Asp Gin get gtt Ala Val cag cag Gin Gin 270 get aaa Ala Lys 285 tec aac Ser Asn gcc ctg Ala Leu aaa ggc Lys Gly Asp Asn Asp Thr Arg Asn Pro Tyr Tyr Lys Leu Thr Thr Arg 110 Ala Ser 125 Val Phe Arg Leu gaa Glu ttc Phe ctg Leu gtt Val 255 ctg Leu ggc Gly ccg Pro ate Ile tac Tyr 335 Thr Gly Asp Leu Gly Ala Leu Thr Ala Glu gtg Val aac Asn tac Tyr 240 ctg Leu tct Ser atc Ile gtt Val gat Asp 320 aaa Lys 624 672 720 768 816 864 912 960 1008 1035 <210> 2 <211> 344 <212> PRT <213> Klebsiella pneumoniae <400> 2 Met Lys Ala Ile Phe Val Leu 1 5 Tyr Ala Gly Gly Lys Leu Gly Tyr Gly Asn Gly Phe Gin Asn Leu Gly Ala Gly Ala Phe Gly 55 Phe Glu Met Gly Tyr Asp Trp 70 Val Asp Asn Gly Ala Phe Lys Leu Gly Tyr Pro Ile Thr Asp 100 Gly Met Val Trp Arg Ala Asp 115 Val Ser Arg Ser Glu His Asp 130 135 Gly Val Glu Trp Ala Val Thr 145 150 Trp Phe Gin Gly Ser Lys Gly Gly Gly Tyr 160 3 Gin Trp Val Asn Asn Ile Gly Asp Ala Gly Thr Val Gly Thr Arg Pro 165 170 175 Asp Asn Gly Met Leu Ser Leu Gly Val Ser Tyr Arg Phe Gly Gin Glu 180 185 190 Asp Ala Ala Pro Val Val Ala Pro Ala Pro Ala Pro Ala Pro Glu Val 195 200 205 Ala Thr Lys His Phe Thr Leu Lys Ser Asp Val Leu Phe Asn Phe Asn 210 215 220 Lys Ala Thr Leu Lys Pro Glu Gly Gin Gin Ala Leu Asp Gin Leu Tyr 225 230 235 240 Thr Gin Leu Ser Asn Met Asp Pro Lys Asp Gly Ser Ala Val Val Leu 245 250 255 Gly Tyr Thr Asp Arg Ile Gly Ser Glu Ala Tyr Asn Gin Gin Leu Ser 260 265 270 Glu Lys Arg Ala Gin Ser Val Val Asp Tyr Leu Val Ala Lys Gly Ile 275 280 285 Pro Ala Gly Lys Ile Ser Ala Arg Gly Met Gly Glu Ser Asn Pro Val 290 295 300 Thr Gly Asn Thr Cys Asp Asn Val Lys Ala Arg Ala Ala Leu Ile Asp 305 310 315 320 Cys Leu Ala Pro Asp Arg Arg Val Glu Ile Glu Val Lys Gly Tyr Lys 325 330 335 Glu Val Val Thr Gin Pro Ala Gly 340 <210> 3 <211> 303 <212> DNA <213> Respiratory syncytial virus, G2Na <220> <221> CDS <222> <400> 3 acc gtg aaa acc aaa aac acc acg acc acc cag acc cag ccg agc aaa 48 Thr Val Lys Thr Lys Asn Thr Thr Thr Thr Gin Thr Gin Pro Ser Lys 1 5 10 ccg acc acc aaa cag cgt cag aac aaa ccg ccg aac aaa ccg aac aac 96 Pro Thr Thr Lys Gin Arg Gin Asn Lys Pro Pro Asn Lys Pro Asn Asn 25 gat ttc cat ttc gaa gtg ttc aac ttc gtg ccg tgc age ate tgc age 144 Asp Phe His Phe Glu Val Phe Asn Phe Val Pro Cys Ser Ile Cys Ser 40 aac aac ccg acc tgc tgg gcg ate tgc aaa cgt atc ccg aac aaa aaa 192 Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys Arg Ile Pro Asn Lys Lys 55 ccg ggc aaa aaa acc acg acc aaa ccg acc aaa aaa ccg acc ttc aaa 240 Pro Gly Lys Lys Thr Thr Thr Lys Pro Thr Lys Lys Pro Thr Phe Lys 70 75 acc acc aaa aaa gat cat aaa ccg cag acc acc aaa ccg aaa gaa gtg 288 Thr Thr Lys Lys Asp His Lys Pro Gin Thr Thr Lys Pro Lys Glu Val 90 ccg acc acc aaa ccg 303 Pro Thr Thr Lys Pro 100 <210> 4 <211> 101 <212> PRT 4 <213> Respiratory syncytial virus, G2Na <400> 4 Thr Val Lys Thr Lys Asn Thr Thr Thr Thr Gin Thr Gin Pro Ser Lys 1 5 10 Pro Thr Thr Lys Gin Arg Gin Asn Lys Pro Pro Asn Lys Pro Asn Asn 25 Asp Phe His Phe Glu Val Phe Asn Phe Val Pro Cys Ser Ile Cys Ser 40 Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys Arg Ile Pro Asn Lys Lys 55 Pro Gly Lys Lys Thr Thr Thr Lys Pro Thr Lys Lys Pro Thr Phe Lys 70 75 Thr Thr Lys Lys Asp His Lys Pro Gin Thr Thr Lys Pro Lys Glu Val 90 Pro Thr Thr Lys Pro 100