CA2086761A1 - Production of outer membrane (om) proteins in gram-positive bacteria and recovery of protective epitopes - Google Patents

Abstract

ABSTRACT
The invention provides a method for producing cloned outer membrane (OM) protein from pathogenic gram-negative bacteria. The invention also provides a method for renaturing the cloned outer membrane protein thus produced so the cloned OM protein regains immunologically active epitopes which are capable of eliciting production of antibodies, in mammals and other animals, that are bactericidal and can provide protection against infection by the pathogenic gram-negative bacteria. According to the method, DNA encoding outer membrane protein from gram-negative bacteria, known to be pathogenic in humans and animals, is expressed in a gram-positive bacterial host. The recombinant or cloned OM protein thus produced is then renatured so as to regain biologically or immunologically active epitopes which are capable of eliciting production of antibodies in animals and humans; the antibodies are bactericidal and protect the animals and humans from infection by the pathogenicgram-negative bacteria from which the gene encoding the cloned OMP's was derived. The method of the invention is exemplified in part by the production ofcloned and renatured class 1 outer membrane protein from Neisseria meningitidis, class 3 OM protein of Neisseria meningitidis and the OM protein OmpA of Escherichia coli.

Description

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PRODUCTION OF OUTER MEMBRANE (OM) PROT~INS IN
GRAM-POSITIVE BACTERIA AND RECOVERY OF PROTECTIVE-EPITOPES

S Background of the In~entioll The surface layer of gram-negative bacteria is composed of the outer membrane (OM). The bulk of OM is composed of so-called outer membrane proteins (OMP's). OMP's are proteins of unique structure and properties. In their native state the OMP's of grarn-negative bacteria are intimately bound to lipopolysaccharide (LPS) and other membrane components.
The con~ormation of the OMP's seems to depend on their association with LPS~s and other specific factors in the environmeint. What these other factors are, and how they affect conformation, are not well understood.
X-ray diffraction studies indicate that the epitopes on native proteins comprise about 15-25 amino acid residues, which are made up of two or three discontinuous surface loops. See for example, Laver e~ al., ~ell 61:553-556 (1990). The protective epitopes contained in the outer membrane proteins are exposed on tbe cell surface of the bacterium, where they are capable - of inducing antibodies that can protect an animal against infection by that strain of bacteria. Energetic calculations suggest that a smaller subset of 5-6 amino acid residues of an epitope contributes most of the binding energy lo the antibodies, with the surrounding residues merely aiding in complementarity. The residues proposed to contribute most of the binding energy are not arranged in alinear sequence but are scattered over the epitope surface.
` 25 In the OMP's, the epitope regions are loops connecting so-called - beta-regions which protrude out of the membrane. These loops are hydrophilic and may bei locally water soluble, even though this is not true of the whole ~. OMP.
In denatured proteins, the conformation of the native (protective) - ~ 30 epitopeis is usually lost,~and antibodies formed against such denatured proteins are not protective against organisms from which such proteins were derived. It is, however, sometimes possible to regain the proper conformation of the , .
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proteins (and their epitopes). Much experimental work has been done using heat, acid, alkali, urea and guanidine hydrochloride denatured proteins to elucidate the process of refolding. Small proteins refold fast ~e.g., 0.1 s) unless the ci*-trans isomeAzation of proline residues or the fonnation of disulfide bonds is involvedS (Kim, P. and Baldwin, R. Ann. Rev. Biochem. 59:631-660 (1990)). The refolding of large proteins is naturally more complicated and very much more unpredictable. Proteins denatured with urea, guanidine hydrochlvride or SDS
easily loose the original cis-trans configuration of their proline residues. Theoriginal configuration is not easily regained by chemical methods, but can some~mes be catalyzed by peptidyl-prolyl cis-~rans isomerase enzymes (Fisher,G. and Bang, H. BA 828:39-42 (1985)). Restoration of the original disulfide bonds is also often very problematic (~wbank, J. and Creighton, T.
Nan~re 350:518-520 (1991~). This can be true even in cases where the native proteins are water soluble. (See generally, Richard, F. Scientific Ame~?can 264:34-41 (January, 1991), for more about the protein folding problem.) Purified outer membrane proteins could be used in medicine as vaccines to prevent diseases caused by pathogenic gram-negative bacteria or as reagents to diagnose such diseases by immunological methods. For example, Neisseria meningitidis bacteria (meningococci) cause a s~ious human infection, purulent meningitis (Peltola, H. Rev. Infect. Dis. J. 5:71-91 (1983)), and the need for a vaccine to pre~ent meningococcal infections has existed for a long time. In ~he e~ly 1970's, capsular polysaccharide vaccines were shown to be efiScacious against two meningococcal serogroups, A and C (YVorld Health Organization Study Group. Technical Report Series No. 588. World Health Organization, (ieneva, 1976). The capsule of the third major serogroup, B
(hereinafter MenB) proved, however, to be structurally closely similar to the saccharide part of glycoproteins in some human tissues (Finne, J., Leiinonen, M., and Makela, P.H. Lancet ii:355-357 (1983)). The resulting strong immunological cross-reactivity may explain why attempts to produce a capsular polysaccharide vaccine for MenB have failed.
Alternative candidates for a MenB vaccine have been sought. The outer membMne of Neisseria meningitidis bacteria, as the surface structure of the - - , '-.
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- 2~67~1 bacteria in immediate contact with the environment, is one such candidate.
Protection afforded by monoclonal antibodies directed to different OM
components has been studied in an infant rat model (Saukkonen, K. Microbial.
Pathogenesis 4:203-212 (1988)). Antibodies dir~ted to OM proteins were ~ound S protective. OM comple~c vaccines, consisting of semi-purified OM components, have been prepared and tested in several field trials (Frasch, C.E. Vaccine 5:3~(1987); Froholm, L.O., Berdal, B.P., Bovre, K., Gaustad, P., Halstensen, A.I., lHarboe, A., Harthug, S., Holten, E., Hoiby, E.A.~ Lystad, A., Omland, T., Rosenqvist, E., Viko, G., Frasch, C.E., and Zollinger, W.D. Antonie van Lee~wenhoek (52:239-241 (1986)).
Although there is a need for OMP's such as OMP's from Neisseria meningitid~is, and other pathogens such as Neisseria gonorrhoeae, Haemophilus influenzae, Yersinia sp., and Brucellu sp., their preparation and purification from gram-negative bacteria is difficult with conventional 15 biochemical methods. A special problem is the tight association of the OMP's with lipopolysaccharide (Hitchcock, P.J., and Morrison, D.C. In E.T. Rietschel (ed.~ ~Iandbook of Endotoxin, vol. I. Chem_ry of Endotoxin, Elsevier Science Publishing, Inc. New York, 1984.~, which is toxic. The problem of removing toxic LPS has so far not been solved in a satisfactory manner, even using harsh 20 methods.
Both the production and purification of outer membrane proteins would be simplified and more e~fective if the OMP's were produced using the methods of genetic engineering, in gram-positive bacteria, which are devoid of lipopolysaccharide.
When proteins are produced using the methods of genetic engineering, it is often a goal to produce the recombinant proteins in large amounts. Sometimes when large amounts of recombinant proteins are produced in bacteria, the proteins form insoluble aggregates. These insoluble aggregates are referred to as inclusion bodies. In inclusion bodies the proteins are in an 30 unnatural state devoid of their authentic configuration and epitopes. Before restoration of the biological activity (e.g., enzymatic or receptor activities, or . .

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protective epitopes) the recombinant proteins must returned to their native conformations, i.e., need to be coIIectly folded.
Chaotropic agents and detergents can be used to solu~ilize some proteins and return them to their native conformations. (Chaotropic agents S pre~ent the random coils among hydrophic regions, which are characteristic of denatured lipophilic p~oteins in aqueous solutions, from occurring.) Unfortunately, the proteins produced in bacteria as inclusion bodies are very resistant to many chaotropic agents and detergents (see generally, Methods of Enymology, 1990) and are soluble, if at all, only in high concentrations of ureaor guanidine }lydrochloride, or SDS. This is true even though the native proteins were water soluble.
In some eases the activity of an enzyme protein can be regained after removal of the chaotropic agent, e.g., active prourokinase is formed from inactive prourokinase inclusion bodies by solubilization with 6 M guanidine hydrochloride and 2-mercaptoethanol and subsequent ~efolding of the protein for ~4 h at 15C in a buffer containing 2 M urea (Orsini, G. et al., Eur. J.
- Biochem. 195:691-697 (1991)). Some recombinant proteins are soluble in sarkosyl (Puohiniemi, R., M. Karvonen, J. Vuopio-Varkila, A. Muotiala, I.M.
Helander and M. Sarvas. Inf: Imm. 58:1691-1696 (1990); Frankel, S. et al., Proc. Natl Acad. Sci 88:1192-1196 (1991)) and may regain their biological activi~ after removal of the detergent. Alkali may also induce native conformation of inclusion body proteins. Prochymotrypsin, solubilized in aLkali-urea, folds in a proper native conformation and is subsequently able to aut~process at an acidic pH (Marston, F. et al., FEMS Microbiol. Lett~N
77:243-250 (1984)).
Less is known about the restoration of epitopes on denatured antigens than is known about the recovery of enzymatic activity in proteins thatfunction as enzymes. What little knowledge there is has come from studies involving denatured native proteins. In one study outer membrane proteins, OmpA and OmpF of E. cvli, were first purified by preparative SDS-gel electrophoresis (Dornmair, K. et al., J. Biol. Chem. 265:18907-18911 (1990));
in another these same OMP's were extracted with octyl-POE extraction (Eisele ;, . . .

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20~6~1 and Rosenbusch, 1990). Lipopolysaccha~ide (LPS) was not required to restore activity in ei~her case. T~his is not in accordance with a study w;th E. coli spheroplasts (Sen, K. and Nikaido, H. J. Bacteriol. 173:g26-928 (1991)), which showed that trimerization of OmpF protein takes place only in the presence of LPS.
While the knvwledge of the renaturing of Escherichia coli OmpA
and OmpF is a useful addition to the store of overall knowledge of renaturation of native proteins, it does not teach or predict what is required for renaturation of Iecombinantly produced OMR's that never were in tight association with native LPS's or other membrane and/or environmental components.
As indicated above, the outer membrane proteins of gram-negative bacteria are intimately bound to lipopolysa~ccharide (LPS) and perhaps other membrane components; their proper conformation is dependent on this association with such components of the membrane environment. Thus if LPS
and possibly other membrane components are not present in preparations of isolated OMP's, these stabilizing components must be replaced by other cornponents that mimic their function, so as to ensure the stability of the OMP in its native beta-barrel conformation. (The beta structure is the well-ordered part of the OMP that is complexed with LPS.) Thus renaturation of OMP's is more complicated than merely solubili~ing a recombinantly produced OMP and dialyzing out the chaotropic agent. To restore immunogenic function, i.e., to re-stabilize the membrane-bound regions that normally exist in a beta-configuration so as to expose the epitopic loops, it is ne~ssary to replace the detergent or other agent with an the environment that mimics the natura1 one. While it may not be necessary to recover 100% of the native OMP conformation, this replacement must permit (a) proper refolding of the epitopic loops (which are a relatively small part of thetotal peptide), and (b) their accessibility to the immune system, i.e., the epitopic loops must protrude into the primarily aqueous environment after injection into an animal or human and subsequent dilution of the preparation.
It is an object of the present invention to provide a method for producing pure cloned outer membrane proteins, and to provide a method for . .
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their renaturation so as to regain biologically or immunologically active epitopes which are capable of eliciting ~he production of antibodies in animals that are (a) bactericidal and (b~ protect the animals against infection by the original infectious agent.
lt is a further object of the present invention to use these pure cloned and renah~red OMP's as diagnostic antigens for the identification of infections caused by gram-negative bacteria.
It is a further object of the present invention to use these pure cloned and renatured OMP's in vaccines to protect animals and humans against infection by that s~ain of bacteria fsom which the gene encoding the cloned OMP's was derived.

D~finitions In the present specification and claims, reference is made to phrases and terms of art which are expressly defined for use herein as follows:
By "regulation and expression sequence" it is meant to include within the scope of the instant invention the DNA sequence of a gene preceding the DNA sequence encoding a polypeptide; the DNA sequence is needed for the transcription and translation of the DNA sequence encoding that polypeptide.
Such sequence typically includes the promoter and ribosomal binding site and possibly binding sit~s for regulatory proteins. The "regulation and expression sequence" may be any biologically active fragment thereof. "The regulation and expression sequence" may include also a DNA sequence encoding an N-terminal fragment of the polypeptide, if that fragment of the polypeptide is not a functional signal sequence ~or export.
By "outer membrane protein" and by "mature outer membrane protein" it is meant an outer membrane protein, which is devoid of a signal peptide for export, or devoid of a functional signal peptide.
By "vector" it is meant any autonomous element capable of replicating in a host independently of the host's chromosome, into which additional sequences of DMA may be incorporated. Such vectors include, but are not limited to, bacterial plasmids and phages.

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20~67~1 By "operationally linked" it is meant that the regulation and expression sequence, including the promoter, controls the initiation of expression of the polypeptide encoded by the structural gene; there may be a DNA sequence derived îrom the same gene as the promoter or any other DNA sequence between 5 the promoter and the initiation of the polypeptide to enhance the expression of the polypeptide. This DNA sequence may encode a peptide that remains fused to the polypeptide, but the said peptide must not be a ~unctional signal for export.
As used herein, "recombinantly producedn, when referring to the production of OMP, means that the OMP's of one species of bacteria, or DNA
10 encoding for such protein, ale expressed in the cells of a bacterium from another species. Recombinantly produced OMP will have been produced using the techniques of gene expression and genetic engineering. Recombinantly produced OMP's are cloned OMP's, and are to be distinguished ~rom OMP's that are extracted from the natural outer membranes of bactena.
Summary of the ~vention The invention provides a method for producing cloned outer membrane (OM) protein from pathogenic gram-negative bacteria. The invention also provides a method for renaturing the cloned outer membrane protein thus 20 produced so the cloned OM protein regains immunologically active epitopes which are capable of eliciting production of antibodies, in mammals and other animals, that are bactericidal and can provide protection against infection by the pathogenic gram-negative bacteria. According to the method, DNA encoding outer membrane protein from gram-negative bacteria, known to be pathogenic in 25 humans and animals, is expressed in a gram-positive bacterial host. The recombinant or cloned OM protein thus produced is then renatured so as to regain biologically or immunologically active epitopes which are capable of eliciting production of antibodies in animals and humans; the antibodies are bactericidal and protect the animals and humans from infection by the pathogenic30 gram-negative bacteria from which the gene encoding the cloned OMP's was derived. According to the invention, a Bacillus or other suitable gram-positive bacterial host containing a recombinant DNA molecule comprising the regulation . .. . . . - . . ~ , . ... . ... .... . .. . ..

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and expression sequence of a gene expressed well in the host is operationally linked to a DNA sequence encoding an outer membrane protein from a gram-negative bacteria known to be pathogenic to animals and humans. The regulation and expre~sion signal is typically an effective promoter and ribosomal binding S site. In preferred forrn, the DNA sequence encoding the outer membrane proteinis devoid of functional signal sequence, as is the regulation and expression sequence. The presence of signal sequences decreases the amount of recombinant outer membrane protein expressed in the gram-positive host.
According to the invention, the recombinant DNA molecule may 10 be introduced into the Bacilll~s or other suitable gram-positive host by transforming the host with a vector that is capable of replicating in several copies in the host strain. Alternatively, the recombinant DNA molecule may be integrated into the chromosome of the Bacillus or other host strain. Using the method, more than one hundred milligrams OI product per liter of culture are 15 obtained when ordinary laboratory media are used. The amount can be higher inhigh density cultures. The product may be aggregated intracellularly or ~ound inthe form of inclusion bodies.
According to the teaching of the invention, the conditions for refolding the OM proteins are such that allow the OM proteins to take the same 20 or partially the same conformation as they have in their natural environment, in the outer membrane. While such conditions can be achieved by using the same amphiphilic compounds present in OM, e.g., LPS, use of LPS is not preferred since LPS is toxic. Non-toxic derivatives of LPS can used. Other non-toxic lipids or their derivatives or analogs, may also be used, alone or in conjunction 25 with detergents, variations in pH and/or temperature, the addition of chemicals, e.g., sugars and amino acids, as well as other conditions which may favor refolding of protective epitopes. Any of these methods and agents, or combinations of agents, may be used as long as they permit ~a) proper refolding of the epitopic loops and (b) their aecessibility to the immune system, i.e., the 30 epitopic loops must protrude into the primarily aqueous serum of an animal or human after injection and subsequent dilution.

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7 ~ 1 The method of the invention is exemplified by the production of cloned and renatured class 1 outer membrane protein from Neisseria meningitidis, class 3 OM protein of Neisseria meningitidis and the OM protein OmpA of Escherichia coli, all in Bacillus subtilis. However, as those skilled in5 the art will appreciate, as a result of the teaching tllat it is possible to renature recombinant OM proteins, the method can be used to produce other outer membrane proteins, including, but not limited to, other OM proteins of Ncisseriameningi~idis9 and the OM proteins of ~eisseria gonorrhoeae, Haemophilus influen2ae, Yersinia sp. and Brucella sp.
One objective of the invention is to use the method for the pr~duction of safe and effective vaccines. Another objective is to provide diagnostic antigens for the identification of infections caused by gram-negativebacteria.

Brief Description of Drawings and Nucleotide Sequences Figure 1. Construction of pKTH288 and 289.
Figure 2. Construction of pKTH290.
~ igure 3. The protein pattern of whole cells of Bacillus subtilis strains. Lane a, low molecular weight standards, lanes b, c, d and f control 20 strains, lane e, IH6627.
Figure 4. The protein pattern of inclusion bodies ~=2,000xg pellets) derived from 0.5 mg wet weight of bacteria. Lane a, low molecular weight standards, lanes b, c, d and f, control strains, lane e, IH6627.
Figure 5. The sodium dodeculsulphate polyacrylamide gel 25 electrophoresis (SDS-PAGE, Coomassie Blue staining) of 12 transformant colonies tested (see Example 3). Lane 1, the molecular weight standard; Lane 2, Sarkosyl solubilized Bac-OmpA produced from pKTH217 (Puohiniemi, R., M.
Karvonen, J. Vuopio-Varkila, A. Muotiala, I.M. Helander and M. Sarvas. In~
Imm. 58:1691-1696 (19~0); Lane 3, Mock preparation made similarly to the 30 OmpA preparation in lane 2 from IH6418, a strain containing the secretion vector without any insert. Lanes 4-15, samples of separate transformant : , ~ , ~ . ; . . . . . . .

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colonies. The figures to the left show the position and size (kDa) of molecular weight markers. Symbol on the right indicate position of OmpA.
Figure 6. SDS-PAGE and Coomassie Blue staining of different steps of particulate centrifugation of 500 ml of IH6649 (wet weight of cells 5-7S g). Lane 1, the molecular weight standards, 5 ~1 (suspended in 500 ,ul of sample buffer) was applied; Lane 5, the supernatant after 2,000xg centrifugation, 2 ,~11 of 20 ml was applied; Lane 6, the pellet after 2,000xg centrifugation (wet weight 1.88 g), the pellet was resuspended in 10 rnl of 50 mM TrisHCI (pH 83 and 0.3 ~11 was applied, Lane 13, the 2,000xg pellet after washing (wet weight 0.38 g), the final pellet was resuspended in 4 ml of the above buffer and 3 ~1 of a 1 to 10 dilution was applied; Lane 14, the pellet after 5,000xg centrifugation (wet weight 0.45 g), the pellet was resuspended in 5 ml of the above buffer and 3 ~1 of a 1 to lû dilution was applied; Lane 11, the supernatant after 5,000xg centrifugation, 2 ~ul of the 20 ml supernatant was applied. I~e figures to the left show the 15 p~sition and size ~kDa) of molecular weight markers. Symbols on the right indicate ~osition of OmpA.
Se~quence ID Numbers 1 and 2 (Seq. ID 1 and Seq. ID 2).
Oligonucleotides used to amplify the DNA coding for the class 1 proteins in a PCR reaction. The oligonucleotide of Seq. ID 1 consists of 4 nucleotides 20 (AACC), a Hi~ldIII site, and nucleotides 125-155 of Barlow, et al., Mol.
Microbivl. 3:131-139 (1989) coding for the first 10 amino acids of the mature protein. The oligonucleotide of Seq. ID 2 consists of 4 nucleotides (AACC), the reverse sequence of the following: a HindIII site (including part of the stop codon), stop codon and nucleotides 1246-1217 of Barlow, et al., A~ol. Microbiol.25 3:131-139 (1989). The sequences are included in the speci~lcation, just preceding the claims.
Sequence ID Number 3 (Seq. ID 3). DNA sequence of the pKTH250 insert; Pl.7,16. Nucleotide 1 of the sequence shown, correspondls to nucleotide 125 of the sequence from Barlow e~ al., Mol. Microbiol. 3:131-139 30 (1989).
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Detailed Description cf tbe Yn~ention Production of OM[ proteins As those skilled in the art will appreciate, the methods described in the following paragraphs for Bacillus can be applied with appropriate S modifications, if needed, and without undue e7cperimentation, for other gram-positive bacteria.

The Production of OM Protei~s by Transforming a Bacillus or Other Gram-Positive IIost A wide variety of suitable expression vectors may be used in the present invention, and are known to those of skill in the art of recombinant genetics. A preferred vector is disclosed in PCT WO 90FI41, filed 2 Feb 1990 and published 23 Aug 1990 as WO 9009448: New Recombinant DNA Molecules fior Producing Proteins and Peptides in Bacillus Strains.
The transfer vector may be any plasmid or phage capable of replicating in several copies in a Bacillus strain or other gram-positive bacterium.
A multitude of such vectors are availa~le, the most representative of them beingthe plasmids isolated from Staphylococcus, Bacillus or Strep~ococcus or their de~ivatives.
The regulation and expression sequence is in most cases first ligated to the transfer vector to be used and is therea~ter modified for example by ~e aid of DNA-linkers so that the genes to be expressed may be joined downstream from the regulation and expression sequence of the vector.
A number of methods to clone OM protein genes can be used in the ~resent invention. Those methods are known to those of skill in the art of recombinant genetics.
Before transformation of the host, a DNA sequence encoding the OM protein (or epitopically functional portion~s) thereof) is ligated to a suitable vector.
The DNA sequences need not be identical to the DNA sequences encoding a particular OM protein as found in a particular natural gene. They may be derived from the sequences of natural genes of OM proteins, but . ... . ............ .. .. . . . . . . . . .. . . . . .

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modified in ways that may alter the properties of the resulting protein. Suitable sequences may also be made synthetically or semi-synthetically.
The selected host may be transformed and cultivated by conventional methods. The choice of suitable transformation systems and cultivation conditions depends on the selected host.

Purification of OM Proteins Produced l~tracellu1arly in Bacillus Ho~,t or ial Other G~m-Positive Bacteria The OM proteins produced with the method of fhe invention often form int~acellular inclusion bodies. The main advantage of producing inclusion bodies (or intracellular aggregates of overproduced protein~ is that they are easy to purify (Marston and Hartley, Me~hods of En~ymology 182:264-276 (1990~).
The bacteAal cells are disrupted by sonication, passage through French pressure cells, lysozyme-treated or by other suitable means. From this suspension the indusion bodies can be pelleted with low speed centrifugation and usually further washed with a mild detergent. Usually (however, depending on the protein~ the inclusion bodies are soluble only in chaotropic agents like urea or guanidine hydrochloride or strong detergents like SDS. In the solubiliæd form, the proteins can be fi~rther purified with conventional purification methods.
If the OM protein produced in Bacillus is not present in inclusion bodies, modifications of the methods above may be applied. The purification may also involve solubilization and differential extraction with various types of detergents, and chromatography and electrophoresis in the presence and absence of detergents.
There are several different ways known to those skil1ed in the art, in which a composition of the recombinant polypeptides produced by the method of the invention may be prepared. The purified OM proteins or their fragments may be used alone to prepare a pharmaceutically-acceptable dosage form and they may be mixed together in any combination. The recovery of the native epitopes may involve addition of solubilization and/or denaturing agents such asurea, guanidine hydrochloride and SDS, which may be later removed. It may also involve addition of compounds like phospholipids and/or sarkosyl or their ~ , , , : ~ :
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derivatives and analogs. The preparation may be in a form of liposomes or in another forrn. Immunoadjuvants such as aluminium hydroxide and pharmacologically-acceptable preservatives such as thiomersal may be added to the composition. These methods are described, for example, in Remington S Pharmaceutical Science, 16th Ed., Mac. Eds. (1980).
Without further elaboration, it is believed that one of ordinary skill in the art can, using the preceding description, and the fol10wing Examples, utiliæ the present invention to the fullest extent. The m~terial disclosed in the examples is for illuskative pu~poses and therefore should not be construed as being limiting in any way of the appended claims.

Examples E~xample 1 Cloning of Pl.7,16 (cl~ss 1) OM Pr~tein of N. mening~idis in IH6627 Cloning of the l:~NA Sequence Coding for the Class 1 Protein The DNA fragment coding ~or the mature protein was acquired as follows: using the published nucleotide sequence of meningococ~al class 1, Pl.7,16 protein (Barlow et al., Mol Microbiol 3:131-139 (1989)) two oligonucleotides (~rimer 1 and primer 2; primer 1 is shown herein as Se~q. ID l;primer 2 is Seq. ID 2) wer~ synthesi~ed and used to amplify the DNA coding for ` the mature class 1 protein in a polymerase chain reaction (PCR) with chromosomal meningococci DNA. The meningococcal DNA was isolated from strain IH5341 (Pl.7,16) grown on proteose-peptone plates containing 1.5%
agarose in lieu of agar. After treatment with a zwitterionic detergent in citrate buffer (Domenico et al., J Microbial Methods 9:211-19 (1989)) to remove e.g., capsule, the DNA was isolated. The PCR reaction was performed using a GeneAmp~ kit using the methods described by the manufacturer (Perkin Elmer Cetus). The amplified DNA fragments were of two sizes when separated by agarose gel electrophoresis. The bigger fragment seemed to be the expected size for class 1, i.e., about llOO bp whereas the other fragment was smaller, about 900 bp. The amplified DNA mixture was purified by phenol extraction, ethanol precipitated, resuspended in TE (10 mM Tris-HCl pH8, 1 mM EDTA) and ., , . . . , . . .. . ~ . . ...
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digested with the restriction enzyme HindIlI. Conventional methods of DNA
technology and microbiology used here and in the following examples are described, e.g., in Sambrook, J., E. F. Fritsch~ and T. Maniatis. (1989) MQ~cular Clonin~. A Laboratory Manual, Second Edition. Cold Spring Harbor Laboratory, N.Y.
The plasmid vector pUC18 was digested with the restriction enzyme HindIII. The linearized vector DNA was ligated with the HindIII-digested amplified DNA. The ligation mixture was used to transform competent Escherichia coli K12 TGI cells which were grown, after transformation, on Luria plates containing 100 ~ug/ml ampicillin, 40 ~g/ml Xgal and 0.5 Mm IPIG. About 10% of the colonies grown overnight were blue, thus representing the background caused by the vector. In the case of the amplified Pl.7,16 DNA, 90 white colonies were tested to check the size of the putative insert. Of these, 11 contained a plasmid with an insert of the expected size.
One of these strains, EH1563, con~ining plasmid pKTH250, was further characterized. It was shown to give the expected sized fragments after treatmentwith the restriction enzymes HindIII, EcoRI or ~pnl. The insert was sequenced with the Sanger dideoxy sequencing method after subcloning into M13. The sequence of the PKI'H250 insert is shown as Seq. IlD 3. As expected it shows very few changes compared to the published Pl.7,16 sequence (Barlow et al., Mol. Microbiol. 3:131-139 (1989).
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Constrlaction of pK1~290 for Intracellular Production of Class 1 OM Protein in Bacill~s subtilis Construction of the plasmid pKTH290 is shown schematically in Fig. 3. Plasmid pKTH250 (pUC18 containing DNA coding for the cloned class 1 protein Pl.7,16) was digested with HindIlI to release the cloned class 1 gene.Also plasmid pKl'H289 was digested with HindIII to release the extra adapter copy and to linearize the vector (see Fig. 1). The two ~indIII digested plasmidswere ligated and the ligation mixture was used to transform IH6140. Cells that had received at least pKTH289 were selected on the basis of kanamycin resistance. The size of p]asmids present in the colonies whieh grew on Luria ,., ~, ,.

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plates containing kanamycin was checked by cell lysis and running samples in an agarose gel by standard methods. Colonies which contained piasmids of the expected size were analyæd for class 1 protein expression by sodium dodeculsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and SDS-PAGE
S followed by immunoblotting (Western blot). One strain IlH6627 containing plasmid pKTH~90 which expressed the class 1 protein was further analy~ed.
The cloning site was checked by Sanger dideoxy sequencing of pKTH290 to ensure that only one copy of the adapter was present.

Screening for the Expr~ision of Class 1 Proteins Prodllced Intracellularly in Bacillus subtilis The expression of class 1 Pl.7,16 protein in Bacillus transformants was screened using SDS-PAGE in the following way: a loopful of bacteria grown on LuAa agar plates was suspended in Laemmli sample buffer and after heating 15 at 100C, a sample was applied to SDS-PAGE. The class 1 protein was visualized with Coomassie Blue staining (Fig. 3) and with immunostaining of the SDS-PAGE after blotting the proteins onto millipore filtPr (Western blot).
Antisera KH 1110 prepared by immunization of a rabbit with an extract of MenB:15:Pl.7,16 bacte~ia, was used in immunostaining. The result was 20 confirmed with Pl.7,16 a specific monoclonal antiserum, obtained from a commercial kit for serotyping meningococci (RIVN, Box 457, 3720 AL
Bilthoven, The Netherlands).
One transformant expressing Pl.7,16 protein (strain IH6627) was chosen for further studies.
Similar constNctions were also made with DNA coding several other class 1. protein subtypes (Pl. 15, P1.2, Pl.l, Pl.9) and meningococcal class 3 protein serotypes 9 and 15.

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Example 2 Production of BacP1.7,16 Protein, the P1.7,16 ~class 1) OM Protein of N. meningitidis in D~6627 S l'reparation of Inclusion Bodies (IB) Contai~ g BacP1.7,16 Protein The bacteria we~e grown either in liquid or on solid Luria medium . containing 10 - 30 ~g kanamycin per liter. When growing one li~r of Luria broth about 10 g bacte~ial cells (wet weight) were obtained~
The bacteria were disrupted with Iysozyme in the following way:
one gram of bacteria grown on Luria agar plates containing kanamycin was suspended in S ml of 20 sucrose in buffer (25 mM TrisHCl, pH 8.0; 15 mM
MgCl2 containing 1 rng Iysozyme/ml). After an incubation of 30 min at 37C
the protoplasts were collected by centrifugation (lO,OOOxg) and Iysed by suspending them in S ml of 50 mM TrisHCl, pH 8,0. DNAse (I mg/ml) was added and after five minutes inclusion bodies were collected by centrifugation lO,OO~g for 10 min. They were further suspended (washed) in 5 ml of 2 %
NP-40 in 50 mM TrisHCI, pH 8.0, and collected by centrifugation lO,OOOxg f.or 10 min. A sample of the product was electrophoresed in SDS-PAGE (Laemmli).
Flg. 4 shows the protein pattern of this 5DS-PAGE stained with Coomassie Blue.
BacPl.7,16 protein isolated as inclusion bodies was called BacPl.7,16-IB.
The inclusion body fraction derived from 10 g of bacteria (about 500 mg of protein) contained 300 mg pr~tein, as measured by the Lowry method (Lowry, O.H. et al., J. Biol. Ch~m. 193:265-275 (1951)). The amount of BacPl.7,16 protein in this fraction was roughly estimated by visual inspection of the SDS-PAG}i (Fig. 6) to be at least 120 mg. That means that more than 1/3 of the protein present in the inclusion bodies from IH6627 is BacPl.7,16 protein.
It can be calculated that BacPl.7,16 protein comprised at least 25% of the totalcel1ular protein of IH6627. It can also be calculated that there was more than one hundred mg of BaçPl.7,16 protein per liter of culture.
The size of the BacPl.7,16 protein is roughly the same as that of the authentic protein of N. meningitidis in SDS-Page.

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3~ample 3 Intracellular Production of Outer Membrane ~rotein OmpA of Eschenc~ia coli in B~cillus subtzlis S Construction of the l~xpression Yector pKTH3125 The plasmid pKTH 217 (described in Puohiniemi, R., M.
Karvonen, J. Vuopio-Varkila, A. Muotiala, I.M. Helander and M. Sarvas. Infi Imm. 58:1691-1696 (1990)) contains a 2.5 Kb HindIII-BamHI fragment which encodes, starting at the HindIII telminus, the 8 to 325 amino acid residues of the OmpA protein of E. coli. The fragment is flanked at the HindllI terminus by a unique ClaI-HindIII fragment. To construct the plasmid P~TH3125, this ~laI-HindIlI fragment was replaced by the ~laI-~lindIII ~ragment of the plasmid pKTH288 shown in Figure 1. The latter fragment contains the promoter and t~uncated, nonfimctional signal sequence of cY-amylase. In pKTH3125 they were fused to the DNA fragment encoding the OmpA protein. The plasmids pKTH217 and 288 were digested with endonucleases ClaI and HindIII, phenol extracted, ethanol precipitated and resuspended in water. Then 1.2 ~g of digest~d pKTH217 was mixed with 2.5 ~g of digested pKTH28X, treated with polynucleotide kinase and then ligated. All DNA manipulations were performed as described in Maniatis et al., (Maniatis, T., E. F. Fritsch, and J. Sambrook, Molecular Clonin~. A Laboratory Manual. Cold Spring Harbor Laboratory, N.
Y. 1982.) Competent cells of B. subtilis strain IH6140 were then transformed with the ligation mixture as described in Maniatis et al., and plated on Luria-plates containing 10 ~g of kanamycin. The expression of OmpA was 2~ tested as follows from 24 of more than 104 transformant colonies obtained.
About half of the colony was mixed with 10 ~1 of SDS-PAGE sample buffer, hea~ed to 100C for S minutes and the sample was electrophoresed in SDS~PAGE (Laemmli, U. K. Nature, (London) 227:680-658 (1970)). As judged by the presence of a major band of the size of the mature OmpA protein (Fig. 4, lanes 4, 6-9, 13, 14), several colonies contained OmpA protein. This band also reacte~ with OmpA serum in immunoblotting. One of them was designated IH6649, and the plasmid in this strain pKTH3125.

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- Analysis and ~ri~ tion of OmpA Made in I~I6649 IH6649 was grown overnight at 37C with shaking (250 rpm) in liquid culture in twofold-concentrat~d L-broth containing 10 mg s~f NaCI per liter, 10 ~Lg of kanamycin per ml, and 30 ~ul of potato extract per ml (Kallio, P., M. Simonen, I. Palva, and M. Sarvas, J. Gen. Microbiol. 132:677-~78 (1986)).
Cells from 500 ml of culture were collected by centrifugation (wet weight 5.7 g), protoplasted with lysozyme and dis~upted with osmotic shock in the presence of DNase and RNase (about 5 ,ug/ml) (Schnaitman3 C., Manual of Methods for General Bacteriology ASM, Washington DC (1981)). The breakage of cells was monitored by phasç contrast microscopy. The particulate material was then pelleted by centrifugation at 16,000xg, for 10 min. OmpA was not a major band in the supernatant, as analyzed with SDS-PAGE ~Fig. 5, lane 4). The pellet (1.9 g wet weight) was resuspended in 50mM TrisHCI, pH 8 and centrifuged at 2,000xg, for 5 min. The pellet (0.9 g wet weight) was resuspended in 10 ml of washing buffer containing 5 mM EDTA, 150 mM NaCI, 1% NP-40, 50 mM
TrisHCI, pH 8. The SDS-PAGE of the suspens;on showed that it contained OmpA as a major band (liig, 5, lane 6). The suspension was then centrifuged at S,OOOxg, for 10 min. The pellet was washed with PBS and pelleted again (S,OOOxg, 10 min). The resulting pellet, 0.38 g (wet weight), was resuspended into 4 ml 50 mM TrisHCl, pH 8 (Fig. S, lane 13). The supernatant, after the above 2,000xg centrifugation (Fig. 5, lane 5) also contained OmpA as a major band and that is why it was further centrifuged at 5,000xg, for 10 min. This resultant supernatant contained only traces of the original OmpA (Fig. S, lane 11) and the PBS-washed pellet (0.45g wet weight) was resuspended in 5 ml of 50 mM TrisHCI, pH 8 (Fig. 5, lane 14), also contained OmpA but a lesser amount ~compare lanes 13 and 14 in Fig. 5 in which the same amount of sample was applied). As a conclusion, surprisingly, when OmpA is expressed in B. subtilis IH6649 it forms aggregates or inclusion bodies that can be eollected by centrifuging at 2,000-51000xg.
The amount of OmpA in the 2,000xg pellet after washing and the 2,000xg supernatant after pelleting at 5,000xg and washing was estimated visually by comparing the intensity of the OmpA bands in SDS-PAGE (Fig. S, .. . .. . . .
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20~7~1 lanes 13 and 14) with the intensity of the molecular weight standard bands (when5 ~41 of the standard is applied the 67 Kd and 30 Kd band contain 0.83 ~g of protein, the 43 Kd band contains 1.47 ,ug protein according to the manufacturer,Pharmacia). Into both lanes were applied 3 ~ul of 1:10 diluted sample. The S band in lane 13 was estimated to contain 1.5 ~4g of OmpA which makes the total amount 20 mg in the 2,0~xg pellet. The band in lane 14 was estimate~ to contain 0.8 ,~g of OmpA which makes the total amount 13 mg of OmpA ;n the 5,000xg pellet. The total yield of purified OmpA was thus about 60 mg/l of culture.
, 10 Regolding of OM Proteins Produc~d in Bacillus sl~btilis and Recovery of Prntective Epitopes from BacP107,16 Protein Produced in IH 6627 The presence of protective epitopes in refolded BacPl.7,16 protein was analyzed by immunizing mice and analyzing the immune sera in enzyme 15 immunoassay (EIA), and in bactericidal and protection assays. In the case of OmpA the refolding of native epitope was analyzed by a bacteriophage inhibition ass~y (Example 8).
., ' .
~nunizatioll of Mice The recovery of protective epitopes was tested by immunizing groups of ten mice with 20 ~g of BacPl.7,16 protein treated in various ways9 given in two injections. The immunizing injection was either subcutaneous or intraperitoneal (i.p.) with 0.1 ml of antigen diluted in PBS. The interval betwe n the two doses was six weeks. The first injection contained an adjuvant as 25 indicated in Tables 1-4. Ten days after the second injection the mice were bled, and the pooled sera analyzed.

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2~751 Analysis of the Immune Se~a Eozyme Immunoassay ~EIA~
Anti-meningoco~al antibodies were measured by EIA (Jalonen et S al., J. Infect. 19:127-134 ~1989)) using Pl.7,16 meningococcal OM preparation or BacPl.7,16 protein as the antigens. The optimal dose for coating was in both case 5 ~g protein/ml.

Bactericid~l As.say (Goldschneideretal.,J.~p.Med. 129:1307-1323(1969)) N.
meningihdis group B:l5:Pl.7,16 strain H44/76 (frorn E. Holten, Norway) was used in the bactericidal assay. Meningococci of other subtypes (strains MenB:2b:Pl.2:L2 and MenB:15:Pl.15:Ll,8 from J. T. Poolman, The Netherlands) were used to assess the specificity of the bactericidal reaction.
Fresh quina pig serum was used as complement souræ. The highest s~rum dilution that gave 50% killing was taken as the end point titer. It is known that the bactericidal activity of serum correlates with protection. Thus all sera, which were positive in this assay, were also tested in protection assays.
.
Assay for Protection Against Men~Inf~tion The ability of the sera t~ protect in~ant rats from bacteremia and meningitis was tested in the experimental infection of 5 day old outbred Wistar rat pups (Saukkonen, Micrvb. Pathog. 4:203-212 ~1988)). A mouse serum (HH209) obtained by immunization with a Pl.7,16 meningococcal OM
preparation was used as a positive control.
The pups were randomized in groups of 6 pups each and injected i.p. with 100 ~1 of the immune sera in several dilutions (1:10, 1:100, 1:1000).
One hour later a bacterial challenge 106 bacteria/mV was injected i.p. in a volume of 100 ~1. The development of bacteremia and meningitis was assessed by taking the appropriate samples 6 hours after the challenge to enumerate the viable bacteria by culture.

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Protective antibodies reduce the bacterial numbers, which fully correlates with protection from death within 48 hours (Sauldconen et al., Microb.
Pathog. 3:261-267 (1987)).

Example 4 ~nmunization of Mice with BacPl.7,16 Protein Treated Witlb Detergent and Guanidine Hydrochloride (see Table 1) Solubilization of BacP1.7916-IB Protein BacPl.7,16-IB protein is wholly soluble in SDS, guanidine hydrochloride and urea, but only partially in sarkosyl or cetylammonium bromide. The protein precipitates if the solubilizing agent is removed. If the agent was guanidine hydrochloride, the precipitated protein is referred to as BacPl.7,16-Gu. In detail BacPl.7,16-Gu is prepared from BacPl.7,16-IB in the following way: 5 mg of BacPl.7,1~IB protein was solubilized with 1 ml of 6 M
guanidine hy~rochloride. After centrifugation (for S min 5,000xg) the clear supernatant was diluted 1:6 in water and dialyzed against water. The precipitatewas collected by centrifugation. BacPl.7,16-Gu differs from BacPl.7,16-IB in being fairly soluble both in sarkosyl and cetylammonium bromide (CTB).
Mice were immunized with BacPl.7,16-IB and -Gu preparations, and with prepa~ations dissolved in above the mentioned detergents (1 mg of ~ -protein/l ml of 2% detergent in 10 mM TrisHCl, pH 8.0 containing 5 mM
BDTA) and diluted 1:5 in PBS.
The immune sera were analyzed as above. The result of the tests are shown in Table 1. All the sera contained antibodies against BacPl.7,16 protein, but only one of them, HH249 prepared with sark~syl-solubilized BacPl.7,16-Gu protein, had antibodies against MenB:Pl.7,16 protein and was slightly bactericidal an~ protective.

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E~xample 5 l~muni:zation of mice with BacPl.7,16-LPS Comple~es a) HH175. One mg of BacPl.7,16-IB was solubilized in 250 ~13 M guanidine hydrochloride (GuHCI) in 50 mM TrisH(:l, pH 8.0, and dialyzed twice against 10 ml of 50 mM TrisHCI, S mM EDTA, pH 8.0 The precipitate was collected by low speed centrifugation and dissolved in 225 yl of 1% SDS in 50 mM TrisHCI, S mM EDTA, pH 8. 250 ~u~ of Salmonella 0-6.7 LPS in 125 ~1 distilled water was added. The sample was dialyzed twice against 40 ml of 50 nM TrisHCI, S mM EDTA, pH 8.0 and appropriately diluted in 0.9% NaCl.
b) HH178. One mg of BacPl.7,16-IB was solubilized in 250 ~13 M guanidine hydrochloride (GuHCI) in 50 mM TrisHCI, pH 8.0, and sequentially dialyzed against 6 ml of lM and 0.6 M GuHCI, S mM EDTA, pH
8Ø 250 ~g of LPS in 125 ~1 distilled water was added, and further dialyzed against 40 ml of 0.6 GuHCl in 50 mM TrisHCl, S mM 13DTA, pH 8.0 and appropriately diluted in 0.9% NaCl.
c) HH187. One mg of BacPl.7, 16-IB was dissolved in 170 ~1 of 3 M guanidine hydrochloride (GuHCl), 1 mM EDTA. After extensive dialysis against 50 mM sodium borate, 1 mM EDTA, pH 8.3, 200 ~g Salmonella Rb2 LPS in 100 ~1 distilled water and 30 ~1 of 20% SDS was added to the suspension. The sample was extens;vely dialyæd first against 10% glycerol in 20 mM TrisHCI, 0.1 mM EDTA, pH 8.0, containing 0.1% SDS and next against the same solution, but omitting SDS. The sample was appropriately diluted in PBS.
d~ HH220. One mg of BacPl.7,16-Gu was solubilized in 0.1 ml of 1% SDS. 250 ,ug LPS and 0.9 RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% Doc, 0.1% SDS in 50 mM TrisHCl, pH 8) were added. Before immunization the sample was diluted 1:5 in PBS. -The immune sera were analyzed as using the methods discussed above. The result of the tests are shown in Table 2. All the sera contained -substantial amounts of antibodies against BacPl.7,16 protein. They also had antibodies against MenB:Pl.7,16-membranes, were bactericidal and protective, which indicates that protective epitopes were obtained by this refolding method.

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Examp1e 6 ~nunization of Mice with BacP1.7,1~Lecithil~ Complexes a) 1 mg BacPl.7.1~Gu was dissolved in 1 ml of 2% SDS in 100 mM TrisHCI, pH 8, and heated for 5 min. at 100C. The clear supematant was S diluted 1:5 either with 2% octylglucoside or 2% octyloligooxyethylene (octyl-POE), in 100 mM TrisHCI, pH 9, and incubated overnight at room temperature.
b) Preparation of a lecithin~etergent ~llm on glass tube. 100 mg of octylglucoside or octyl-POE was dissolved in 2.5 ml of chloroform:metbanol 10 (2:1) and 20 mg of soybean lecithin in chloroform was added. The chloroform was evapo~ated away under N2 Solution (a) was added onto the film. After thorough mixing the suspension was dialyzed against PBS for 2 days with 4 exchanges. Before immunization the sample was diluted 1:5 in PBS.
The immune sera were analyzed using the methods disclosed 15 above. The result of the tests are shown in Table 3. All the sera contained antibodies against BacPl.7,16 protein and MenB:Pl.7,16-membranes, were bactericidial and protective, which indicates that protective epitopes were obtained by this refolding method.
. .

Example 7 ~nmunization of Mice with BacP1.7,16 Protein-I~cithin-Sarkosyl Complexes One mg of BacPl.7,1~IB or BacPl.7,16-Gu was suspended in 1 ml of 2% sarkosyl, and 0.1 ml of soybean lecithin (25 mg/ml of 2% sarkosyl) was added. Before immunization the samples were diluted 1:5 in 0.9% NaCI.
In each case the mice received 0.2 ml of the antigen preparation.
The immune sera were analyzed using the methods disclosed above. The result of the tests iare shown in Table 4. One of the two sera, prepared with BacPl.7,16-IB protein, contained antibodies against BacPl.7,16 protein and MenB:Pl.7,16-membranes, was bactericidial and protective. This indicates that protective epitopes were obtained by this refolding method.

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,: . ' 2~6761 Example 8 Refolding of OmpA by Addition of Lipopolysaccharide Datta et al., J. Bact. 131:821-829 (1977)) have shown that the bacteriophage K3 receptor loop, in the purified OmpA of Escherichia coli, can be refolded with the aid of LPS (lipopolysaccharide) and magnesium. This loop consists the amino acid residues centered in amino acid number 70 and is supposed to be located outs;de the outer membrane. The refolding was measu~ed by phage-binding inhibition assay; e.g., decrease of the number of plaques titrated on indicator E. coli bacteria indicates presence of native epitopes in the refolded BacOmpA.
It has been shown that BacOmpAn80mpA228-ss ~a tandem duplication of amino acid 8-228 of OmpA with complete signal se~quence of Bacillus amyloliquefacien (produced in the strain IH6443) can be refolded similarly as purified OmpA of E. coli. The confonnation of OmpA
(BacOmpA-ss~) by LPS was studied using BacOmpA228OmpA22g-ss protein as a positive control. The BacOmpA-ss~, as shown in Table S, was able to inhibit the binding of K3 phages to the indicator E. coli. The mass needed was, however, more than that of BacOmpA228OmpA228-SS, as 15 ~g of BacOmpA228OmpA228-ss(~LPS) inhibited 83% of the phage binding, whereas 75 ,ug of BacOmpA~+LPS) was needed to inhibit 70% of the phage binding. Hence binding by BacOmpA+LPS is less e~ficient. It can be said that the phage binding capacity of BacOmpA-ss can be restored with LPS to a limited extent.

Deposits Plasmid pKTH290 in Bacillus subtilis in the parent strain IH6140 (the recombinant strain denoted as strain IH6627) was deposited on July 5, 1990 with the Deutsche Sammlung von Mikroorganismen (DSM), Mascheroder Weg 1, D-3300 Braunschweig, Federal Republic of Germany, under the terms of the Budapest Treaty on the lnternational Recognition of Deposits of Microorganisms for Purposes of Patent Procedure and the Regulations promulgated under the Treaty. The deposit has been accorded DSM Deposit No. DSM 6089. Samples of Bacillus subtilis containing plasmid pXTH290 are and will be available to - . .

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industrial property offices and other persons legally entitled to receive them under the terms of the Treaty and Regulations and otherwise in compliance with the patent laws and regulations of all nations or international organizations inwhich this application, or an application claiming priority of this application, is S filed or in which any patent granted on any such application is granted.

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Su~nary It may be seen that the invention provides a method for producing cloned and renatured outer membrane (OM) protein from pathogenic gram-negative bactena. The renatured (OM) proteins produced by the rnethod of the S invention have immunologically active epitopes which are capable of elicit;ng production of antibodies, in mammals and other animals, that are bactericidal and can provide protection against in~ection by pathogenic gram-negative bacteria.
These cloned renatured OM proteins are useful as diagnostic antigens ~or the identification of infections caused by gram-negative bacteria. They are also 10 useful as vaccines or as connponents of vaccines.
Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

.

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2~67~1 :
~ EQUENCE LI8TI~G8 ~1) GENER~L INFORMATION:
(i~ APPLICANT: Sarvas, Matti Butcher, Sarah Kalliokoski-Nurminen, Narjatta Runeberg-Nyman, Xate Muttilainen, Susanna Wahlstrom, Eva Idanpaan-Heikkila, Ilona Pouhiniemi r Ritvalenna (ii) TITLE OF INVENTION: Production of Outer Nembrane (OM) Proteins in Gram-Positive Bacteria and Recovery of Protective Epitopes (iii) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS: :
(A) ADDRESSEE: Ms. Virginia ~. Meyer c/o McCubbrey, Bartels, Meyer &
Ward :
(B) STREET: One Post Street, Suite 2700 (C) CITY: Sa~ Francisco (D) STATE: California ~E) COUNTRY: USA
j (F) ZIP: 94104 5231 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 -(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: To Be Assigned (B) FILING DATE: To Be Accorded ~C) CLASSIFICATION~
(viii) ATTORNEY/AGENT INFORMATION:
:

: . . .: : : , . . ' . . , ' .

~ 0 8 6 rl~

(A) NAME: Meyer, Virginia H.
(B) REGISTRATION NUMBER: 30,089 (C) REFERENCE/DOCRET NUMBER: 51268 (ix) TELECONMUNICATION INFORMATION:
(A) TELEPHONE: (415)391-6665 (B) TELEFAX: (415)391-6663 ~2) ~FORMa~ION FO~ 8EQ ID ~O.1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) l'OPOLOGY: linear (ii) MOLECULE TYPE. DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI~SENSE: NO
(v~ FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:
; (A) ORGANISM: Neisseria meningitidis (B) STRAIN: IH5341 (xi) SEQU~NCE DESCRIPTION: SEQ ID NO:1:
~ , ~ACCAAGCTT GATGTCAGCC TGTACGGCGA AATCAAAGCC 40 (2) XNFO~A~IO~ FO~ 8EQ ID NO:2:
' (i) SEQUEMCE CHARACTERISTICS:
- (A) LENGTH: 41 base pairs - (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomi~) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE~
(A) ORGANISM: Neisseria meningitidis (B) STRAIN: IH5341 -. , .. : .. .: .. , . . :

.
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, ~ . ., .: : . ,,:, - ' ' ~67~1 (xi) S~QUENCE DESCRIPTION: SEQ ID NO:2:
AACCAAGCTT AGAATTTGTG &CGCAAACCG ACGGAGGCGG C 41 (2) INFORMA~ION FO~ 8BQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1122 base pairs (B) TYPE- nucleic acid (C) STRANDEDN~SS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(V) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:
(A) ORGANISM: Neisseria meningitidis (B) 5TRAIN: IH5341 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
: .
~' GATGTCAGCC TGTACGGCGA AATCAAAGCC GGCGTGGAAG GCAGGAACTA CCAGCTGCAA 60 GCCATTGATC CTTGGGACAG C~ATAATGAT GTGGCTTCGC AATTGGGTAT TTTCAAACGC 420 GGCGACAAAA CCAAAAACAG TACGACCGAA ATTGCCGCCA CTGCTTCÇTA CCGCTTCGGT 900 AATGCAGTTC CACGCATCAG CTATGCCCAT GGTTTCGACT TTATCGAACG CGGTAAAAAA 960 ~ :

~'. .: ,~ :. ' ; ' " , ' .' ' .

- .; , ' :

.
.

Claims (25)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method for producing cloned outer membrane protein from pathogenic gram-negative bacteria and renaturing the cloned outer membrane protein thus produced so as to regain immunologically active epitopes which are capable of eliciting production of antibodies, in mammals and other animals, that are bactericidal and capable of providing protection against infection by the original infectious agent, said method comprising: (a) expressing in a gram-positive bacterial host DNA encoding outer membrane protein from gram-negative bacteria known to be pathogenic in humans and animals, (b) renaturing said outer membrane protein from step (a) so as to regain biologically or immunologically active epitopes which are capable of eliciting production of antibodies in animals and humans that are bactericidal and protect said animals and humans from infection by said gram-negative bacteria known to be pathogenic in humans and animals.

2. The method of Claim 1 wherein said bacterial host is any bacterium of the genus Bacillus.

3. The method of Claim 1 wherein said host is Bacillus subtilis.

4. The method of Claim 1 wherein said DNA encoding said outer membrane protein is selected from the group consisting of DNA sequences encoding outer membrane proteins of Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae, Yersinia sp. and Brucella sp.

5. The method of the Claim 1 wherein said DNA sequence encoding said outer membrane protein is a DNA sequence encoding class 1 outer membrane protein of Neisseria meningitidis.

6. The method of Claim 1 wherein said outer membrane protein from step (a) is renatured with an agent or combination of agents selected from the group consisting of SDS, guanidine hydrochloride, cetylammonium bromide, phospholipids, lecithin, sarkosyl and urea.

7. Outer membrane protein of pathogenic gram-negative bacteria prepared by any of the methods of Claims 1-6.

8. Outer membrane protein of pathogenic gram-negative bacteria, wherein the outer membrane protein is class 1 outer membrane protein of Neisseria meningitidis, prepared by any of the methods of Claims 1-6.

9. Outer membrane protein of pathogenic gram-negative bacteria, wherein the outer membrane protein is class 1 outer membrane protein of Neisseria meningitidis, and further wherein said class 1 outer membrane protein is from a strain of Neisseria meningitidis which belongs to serogroup B, prepared by any of the methods of Claims 1-6.

10. A composition comprising cloned and renatured outer membrane protein produced by any of the methods of Claims 1-6.

11. A composition comprising cloned and renatured outer membrane protein produced by any of the methods of Claims 1-6, wherein said con3position is in a pharmaceutically acceptable dosage form.

12. A vaccine comprising cloned and renatured outer membrane protein produced by any of the methods of Claims 1-6.

13. A vaccine according to Claim 12 wherein said vaccine also contains immunoadjuvants and pharmacologically-acceptable preservatives.

14. Use of outer membrane proteins of any of Claims 7-10 as antigenic diagnostic reagents for detecting antibodies against pathogenic gram-negative bacteria with immunological methods.

15. Use of outer membrane proteins of any of Claims 7-10 as a vaccine or component thereof.

16. A method for producing cloned outer membrane protein from pathogenic gram-negative bacteria and renaturing the cloned outer membrane protein thus produced so as to regain immunologically active epitopes which are capable of eliciting production of antibodies, in mammals and other animals, that are bactericidal and capable of providing protection against infection by the original infectious agent, said method comprising: (a) expressing in a gram-positive bacterial host DNA encoding outer membrane protein from class 1 outer membrane protein of Neisseria meningitidis (b) renaturing said outer membrane protein from step (a) so as to regain biologically or immunologically active epitopes which are capable of eliciting production of antibodies in animals and humans that are bactericidal and protect said animals and humans from infection by Neisseria meningitidis.

17. The method of Claim 16 wherein said bacterial host is any bacterium of the genus Bacillus.

18. The method of Claim 16 wherein said host is Bacillus subtilis.

19. The method of Claim 16 wherein said outer membrane protein from step (a) is renatured with an agent or combination of agents selected from the group consisting of SDS, guanidine hydrochloride, cetylammonium bromide, phospholipids, lecithin, sarkosyl and urea.

20. Class 1 outer membrane protein of Neisseria meningitidis, prepared by any of the methods of Claims 16-19.

21. Class 1 outer membrane protein of Neisseria meningitidis, wherein said class 1 outer membrane protein is from a strain of Neisseria meningitidis which belongs to serogroup B, prepared by any of the methods of Claims 16-19.

22. A composition comprising cloned and renatured outer membrane of Neisseria meningitidis produced by any of the methods of Claims 16-19.

23. A composition comprising cloned and renatured outer membrane protein of Neisseria meningitidis produced by any of the methods of Claims 16-19, wherein said composition is in a pharmaceutically acceptable dosage form.

24. A vaccine comprising Class 1 outer membrane protein of Neisseria meningitidis, prepared by any of the methods of Claims 16-19.

25. A vaccine according to Claim 24 wherein said vaccine also contains immunoadjuvants and pharmacologically-acceptable preservatives.