WO1988005464A1

WO1988005464A1 - PhoE PROTEIN WITH AN INSERTED ANTIGENIC DETERMINANT

Info

Publication number: WO1988005464A1
Application number: PCT/NL1988/000002
Authority: WO
Inventors: Johannes Petrus Maria Tommassen
Original assignee: Rijksuniversiteit Utrecht; Stichting Centraal Diergeneeskundig Instituut
Priority date: 1987-01-20
Filing date: 1988-01-20
Publication date: 1988-07-28
Also published as: NL8700127A

Abstract

Recombinant DNA comprising the genetic information for PhoE protein of Enterobacteriaceae bacteria and, in a part thereof coding for a region of the PhoE protein localized at the cell surface, the genetic information for a foreign antigenic determinant. Enterobacteriaceae bacteria containing and expressing such recombinant DNA contain in their outer membrane PhoE with a cell-surface exposed foreign antigenic determinant. Such bacteria or the modified PhoE protein recovered therefrom can be used as a diagnostic aid or as a vaccine.

Description

PhoE protein with an inserted antigenic determinant.

This invention is in the fields of DNA recombi¬ nant technology, diagnostics and vaccine preparation. Vaccines against pathogens are mostly based on deactivated forms of the pathogens . An example thereof is the vaccine against the foot-and-mouth disease (FMD) virus. In one-hoofed agricultural domestic animals FMD is responsible for great losses in livestock, so that the prevention of FMD through vaccination is of great economic interest. The present vaccines against FMD are based on deactivated virus particles cultured in surviving bovine tongue epithelium or in baby hamster kidney cells.

Although these vaccines are quite satisfactory and the disease is of rare occurrence in Western Europe owing to vaccination programmes, these vaccines have some drawbacks and risks which render development of alternative vaccines desirable: the necessity of large-scale virus culture, and the attendant risk of escape of the virus from the protected environment; the risk of surviving virus in vaccines; the high cost of production.

T.he same drawbacks also apply more or less to the well-known vaccines against other pathogens, such as measles and polio virus. Against measles, for instance, a living attenuated vaccine. is used which is very effective per se and has considerably reduced the number of cases of measles in Western countries. However, the vaccine has the following drawbacks:

- the vaccine has undesirable side-effects varying from pain and fever in a rather large number of cases to serious neurologic complications in incidental cases; successful vaccination is only possible from the age of 12-15 months due to interference through maternal antibodies in case of vaccination at a younger age; vaccination is impossible with persons which have troubles with their immune system or are treated with im unosuppressants.

Much research is being conducted to find out whether isolated virus proteins or synthetic oligopeptides representing an antigenic determinant of such a protein are useful as a vaccine. Recent developments in molecular biology, especially .recombinant DNA techniques, render it possible to produce this type of proteins or oligopeptides easily in large amounts.

The suitability of the recombinant DNA technique for the production of subunit vaccines (vaccines consisting mainly of proteins) highly depends on the immunogenic effectiveness of the employed protein.

Research into the possibility of using, e.g. capsid proteins of viruses as a basis for a vaccine, often shows, however, that the immunogenicity of such proteins is much lower than that of the virus itself.

Smaller peptides composed of important antigenic determinants of such a virus also prove to be less effective than the virus particle.

One of the viruses with which the above problem occurs is the foot-and-mouth disease (FMD) virus.

Foot-and-mouth disease virus belongs to the picorna viruses. It is of a relatively simple structure and consists of an RNA molecule surrounded by 60 copies of the capsid proteins VP1, VP2, VP3 and VP4. Of these capsid proteins VPl is the most important because it comprises the most important antigenic determinant of the virus. VP1, isolated from virus, proves to be capable of giving both a neutralizing response and protection in guinea pigs and bovine. 12. coli strains producing large amounts of VP1 have been constructed by means of recombinant DNA techniques. VPl isolated from such an _E. coli strain has the same properties as VPl isolated from the intact virus. Fragments of VPl, namely, peptides corresponding to the amino acid sequences 140-160 or 200-213, also prove to give a neutralizing response in test animals. Although both peptides give a clear neutralizing response, it holds good for these proteins, like for VPl itself, that the effectiveness is much poorer than that of the virus itself. It is expected, however, that by fusion of the determinant to another, larger protein molecule (a so-called carrier) the immunogenic effect can be enhanced.

Thus, for instance, some hopeful results have already been obtained by using antigens which were organized in micelle or in liposome structures, using fusion proteins comprising several copies of the same antigenic determinant in tandem, and using adjuvants, but a fully satisfactory solution has not yet been found.

As far as FMD is concerned, for instance, the fusion of the 140-160 determinant to - β-galactosidase proves to give an increased immunogenic effectiveness of the antigenic determinant. The increase here obtained, however, is not yet large enough. Further research into other, better carriers is therefore necessary.

Research into the effectiveness of measles vaccines has made it clear that an effective immunity against measles- infection requires the presence of antibodies against both viral envelope glycoproteins : hemagglutinin (HA) and fusion factor (F) . It may be expected that a subunit vaccine comprising these two antigens or the immunologically important parts of these two antigens in biologically active form is capable of providing an effective and long-term immunity." Such a vaccine would be devoid of the drawbacks attendant upon the living vaccine. But also here the problem is how to find a suitable carrier for the antigenic determinants of the measles virus. Problems also occur with respect to the prepa¬ ration of antigens for diagnostic purposes. Some antigens are very hard to isolate from pathogenic organisms, e.g., if the in vitro culture of these organisms is difficult or unfeasible, or very expensive. This problem occurs, e.g., with Treponema pallidum which causes syphilis.

- For the treatment of patients suffering from syphilis it is very important to know at which stage of the infection the patient is. Research is being made into the possibility of establishing the stage of the infection by demonstrating specific antibodies in the serum of patients.

This requires the availability of specific antigens of Treponema pallidum causing syphilis. Treponema cannot be cultured in vitro, and with the present method of culturing (in testicles of living rabbits) sufficient amounts of a specific antigen cannot possibly be isolated for routine diagnostic applications.

There have already been cloned a number of Treponema antigens in E_. coli which can be used diag- nostically. The ultimate object is to compose a "kit" of different specific antigens with which sera of patients can be tested for the occurrence of antibodies. One condition therefor is that the antigen can be easily and reproducibly produced, which condition has not -5-

been met so far .

This invention provides a solution for the above-mentioned problems by using PhoE protein as a carrier for foreign antigenic determinants. The cell envelope of Gram-negative bacteria, such as Escherichia coli bacteria, consists of an inner membrane and an outer membrane, separated by the peptidoglycan layer and the periplasmic space. The outer membrane functions as a molecular sieve as a result of the presence of pore-forming proteins, so-called porins . These proteins form transmembrane channels, which allow the passage of small hydrophilic substances. E_. coli K12 has three proteins which function as pores in the outer membrane for small hydrophilic compounds. These proteins, called PhoE, OmpF and OmpC, occur in large numbers of copies, about 10⁵ per cell. Of these proteins, OmpF and OmpC are constitutively expressed, while PhoE is induced under phosphate limitation. By means of suitable mutations in one of the genes phoR, phoS, phoT or pst, however, there can also be obtained constitutive synthesis of

PhoE protein. Omp F and Omp C pores prove to be particularly efficient channels for positively charged compounds, whereas PhoE pores are particularly efficient channels for negatively charged compounds . The PhoE promotor is one of the strongest promotors of E_. coli.

Like other exported proteins, PhoE protein is synthesized in precursor form with an N-terminal signal sequence which is essential and sufficient for transport through the membrane. The structural genes for the above-mentioned pore proteins are cloned, and the DNA sequences are determined. For the DNA sequence of the structural gene for PhoE reference is made to Overbeeke et al, J.Mol.Biol.163 (1983) 513-532. From a comparison of _ the sequences it appears that these genes show a strong homology (about 60%) (Mizuno et al, J.Biol.Chem. 280 (1983), 6932-6940). In contrast to most of the other membrane proteins the porins lack hydrophobic sequences of sufficient length to span the membrane. The functional unit is a tri er having a compact structure without large domains outside the membrane.

PhoE protein Is also found in other Entero¬ bacteriaceae than E. coli. Of several Enterobacteriaceae the phoE genes have been cloned, see, e.g. Verhoef et al, Gene 32 (1984) 107-115. For the phoE genes of Enterobacter cloacae and Klebsiella pneumoniae the nucleotide sequence has been determined (see fig. 3).

The hqmology observed was utilized to construct hybrid genes, with replacement of parts of one gene by corresponding parts of the other pore-protein genes.

By means of those hybrid genes and of specific monoclonal and polyclonal antibodies, directed against the cell surface-exposed part of PhoE, ^"epitopes of PhoE located at the cell surface could be genetically localized. Insert into these regions located at the cell surface proved to be possible: it turned out that recombinant DNA coding for PhoE protein but provided with an insert (a linker molecule or a DNA sequence coding for a foreign antigenic determinant) located in a part of the gene coding for a cell surface-exposed region of the PhoE protein could be expressed in E_. coli, and the modified PhoE protein proved to be incorporated into the outer membrane. This has been shown for both PhoE protein of E_. coli 12 and PhoE protein of Enterobacter cloacae, with the insert representing a length of 24 a ino acids. The inserted polypeptide, such as a foreign antigenic determinant, is thus produced by the bacterium and exposed at the outside of the cell, which offers great advantages both for the preparation of vaccines and for diagnostic purposes. Cells carrying surface-exposed foreign antigenic determinants could by themselves be used as an oral vaccine, especially in veterinary medicine. Preferably, there will be used an Enterobacteriacea species which is innocuous to the host and can persist in the gut. In this connection reference may be made to non-virulent Salmonella strains, such as the galE mutant S_. typhimurium strain G30 which is not virulent but persists in the gut sufficiently to give a local immune response (Stevenson and Manning, FEMS Microbiol. Lett. 28 (1985) 317-321). E . coli^' strains could also be used. Because _E. coli K12 cannot persist in the gut, wild type E_. coli strains will be preferred. There could also be used killed cells or PhoE protein recovered from the cells and containing the foreign antigenic determinant.

The large-scale production of vaccines, such as in particular an FMD vaccine, will be considerably less expensive than the present way of vaccine production. The cultivation of bacteria, also on a large scale, is very simple, so that large amounts of bacteria and/or

PhoE/FMD fusion protein can be isolated.

Since the coupling of the antigenic determinants to PhoE protein results in that the antigen is exposed at the surface of the bacterium cell and can be easily purified, this invention also offers great advantages for diagnostic purposes.

The invention is first concerned with recombinant DNA comprising the genetic information for PhoE protein of Enterobacteriaceae bacteria and, in a part thereof coding for a region of the PhoE protein localized at the cell surface, the genetic information for a foreign antigenic determinant.

In particular, the invention relates to the use of a gene coding for PhoE protein of Escherichi coli, such as E_. coli K12 bacteria. The foreign antigen determinant is preferably located in a part of the gene coding for one of the following a ino acid regions of the PhoE protein:

(a) amino acids 20 through 34;

(b) amino acids 63 through 75;

(c) amino acids 105 through 125;

(d) amino acids 154 through 163; (e) amino acids 189 through 205;

(f) amino acids 229 through 245;

(g) amino acids 269 through 283; (h) amino acids 307 through 320.

Very suitable places for insertion of a foreign antigenic determinant are those parts of the gene which code for the amino acids 158, 201, 238, 275 and their nearest neighbours.

Of course, the insertion (or substitution) should be such that the reading frame is not disturbed and that expression in and incorporation into the outer membrane of the bacterial host is not prevented. Although it is not impossible that longer DNA molecules can be incorporated into the PhoE gene, it is preferred that the inserted genetic information for a foreign antigenic determinant has a length of not more than 90 nucleotide pairs»

For specific uses it may be desired to insert several foreign antigenic determinants. That is possible in principle, e.g., for the preparation of a measles vaccine by incorporating an antigenic determinant of hemagglutinin into one of said parts of the gene and an antigenic determinant of the fusion factor into one of the other said parts of the gene.

The most important advantages of the invention can be summarized as follows. One of the strongest promotors of Entero¬ bacteriaceae is used, such as Salmonella and E_. coli , the phoE promotor, so that a high expression level can be obtained. The "foreign" oligopeptide is at the cell surface, so it is not attainable by proteolytic enzymes from the cell.

Because the foreign oligopeptide is at the cell surface, the antigen need not be purified for a number of uses, e.g., serum diagnostics. In order to determine whether a patient's serum contains antibodies against a specific pathogen, the Enterobacteriaceae, such as Salmonella and E_. coli cells expressing such an antigenic determinant can be directly used in ELISA tests or agglutination reactions. Moreover, such cells could be used as a living oral vaccine. Such a use has the additional advantage that the antigenic determinant in question is offered as part of a natural immunogen, which increases immunity. The purification of PhoE protein is well documented and rather simple. If the use of whole Enterobacteriaceae, such as E_. coli cells, is not desirable for specific applications, the antigenic determinant, coupled to the PhoE protein, can therefore be easily purified.

If the DNA coding for an antigen is available, the immunogenic epitopes can be easily determined by cloning fragments of this DNA in the vectors. The resulting Enterobacteriaceae, such as E_. coli clones, are then tested for reaction with antibodies against the antigen. Although the nature of the foreign antigenic determinant is not subject to any restriction in principle, this invention is eminently suited for antigenic determi¬ nants of foot-and-mouth disease virus, especially antigenic determinants of the capsid protein VPl thereof . Examples of strong antigenic determinants of this capsid protein -10-

VP1 are polypeptides corresponding to the amino acids 140-160 or 200-213 of VPl. The genetic information for these antigenic determinants is known and DNA coding therefor can be isolated from suitable clones. Of course, synthetically prepared DNA molecules coding for the relevant antigenic determinant may also be used. Given the well-known genetic code and the preference of Entero¬ bacteriaceae, such as E_. coli, for specific codons, synthetic DNA molecules can be composed which are built up from codons preferred by Enterobacteriaceae, such as E_. coli, for the same amino acid sequence. The nucleo- tide sequence of such synthetic DNA molecules may then differ from the natural sequence, while notwithstanding the amino acid sequence of the resulting polypeptide is equal to that of the natural determinant.

Another preferred embodiment of the invention is related to the use of -an antigenic determinant of the Treponema pallidum causative agent of syphilis, but other antigenic determinants, such as those of polio virus, measles virus, the Mycobacterium leprae causative agent of leprosy, Meninqococcus and Haemophilus species causing meningitis, AIDS virus, and hepatitis B virus, can also be used in the system according to the invention. The same applies to antigenic determi¬ nants of organisms causing intestinal diseases, such as the cholera bacterium and corona viruses .

This invention not only relates to the above described recombinant DNA but also to recombinant plasmids containing, in addition to this recombinant DNA according to the invention, a vector part enabling replication and expression in Enterobacteriaceae bacteria. Suitable vectors are known to those skilled in the art. Although the vector part may contain one or more selection markers, this is not strictly necessary. In -fact, transformants can be easily Identified by means of specific antibodies -11-

against the inserted antigenic determinant exposed at the surface of the cells in the PhoE protein.

This invention also relates to Enterobacteriaceae bacteria containing such recombinant plasmids. By culturing such bacteria large amounts of the recombinant plasmids can be produced. Preferably, hosts will be used with a chromosomal deletion of their own phoE gene in order to limit plasmid losses through recombination.

Especially preferred are Enterobacteriaceae bacteria containing^' one or more of such recombinant plasmids and in their outer membrane PhoE protein carrying a foreign antigenic determinant corresponding to the genetic information laid down in said plasmids.

This PhoE protein, which can be easily recovered from the bacteria and purified, is a further object of the invention.

This invention also comprises the use of transformed Enterobacteriaceae bacteria (if required, after they have been killed) containing PhoE protein having included therein foreign antigenic determinant, for vaccination purposes or for diagnostic purposes. Instead of the Enterobacteriaceae cells, PhoE protein may be used.

This invention further relates to recombinant DNA comprising the genetic information for PhoE protein of Enterobacteriaceae bacteria and at' least one restriction enzyme recognition sequence arranged in a part thereof coding for a region of the PhoE protein localized at the cell surface, and to a recombinant plasmid containing such a recombinant DNA in addition to a vector part enabling replication and expression in Enterobacteriaceae bacteria, and to transformed Enterobacteriaceae bacteria containing one or more of such recombinant plasmids.

Recombinant DNA comprising the genetic infor- mation for PhoE protein and one or more restriction enzyme recognition sequences arranged in a part thereof coding for a region of the PhoE protein localized at the cell surface constitutes a suitable starting material for the preparation of recombinant DNA containing the genetic information for a foreign antigenic determinant in a part of the genetic information for PhoE protein coding for a region of the PhoE protein localizing at the cell surface. The insertion of genetic information for a foreign antigenic determinant is no problem to those skilled in the art.

The invention is illustrated in and by the following examples. Example 1

By insertion of linker molecules mutations were made in the phoE gene using plasmid pJP29 which contains the phoE gene. This plasmid is shown in Fig. ' 1.- The construction of this plasmid has been described by Bosch et al, J.MoI.BioI. 189 (1986), 449-455. Incident¬ ally,^" it is observed that not only E_. coli bacteria can be transformed with this plasmid. Thus, for instance, a successful transformation of the galE mutant S_. typhimurium strain G30 has been carried out, in which the E_. coli phoE gene was expressed under phosphate limitation.

The plasmid contains 5 unique restriction enzyme recognition sites used for the insertion of oligonucleotides. For carrying out the insertions 1/mg of pJP29 was cut with one of these restriction enzymes, and the protruding ends were either filled by means of the Klenow fragment of _E. coli DNA polymerase I or removed by using the 3 '-5' exonuclease activity of T4 DNA polymerase, in the presence of 2mM dNTP. The DNA thus obtained with a blunt end was ligated with T4 DNA ligase to 100 ng of one of the phosphorylated oligonucleotide dCGGATCCG (BamHI linker), dCGGGATCCCG (BamHI linker) or dCCAACGTTGG (HindIII linker) . By using either 8 or 10 base pairs long oligonucleotides the correct reading frame could be ensured. After digestion with BamHI or HindiII and renewed ligation with T4 DNA ligase the DNA preparation was used to transform E_. coli strain CE1224, selecting for chloramphenicol resistant colonies. The E_. coli K-12 strain CE1224 contains a deletion for the phoE gene and forms no OmpF and OmpC proteins as a result of ompR mutations. The strain has been described by Tommassen et al, EMBO journal 4 (1985) 1041-1047.

Plasmid DNA used in the experiments was prepared as described by Birnboim and Doly, Nucleic Acids Res. 7 (1979), 1513-1524, followed by CsCl ethidium bromide isopicnic centrifugation. All constructions were verified by DNA sequence analysis using the known method described by Sanger et al, Proc. Natl. Acad. Sci. USA 74 (1977), 5463-5467.

In all cases, the mutagenesis resulted in the addition of four amino acids in the PhoE protein, except for the insertion at the PstI site, which resulted in the replacement of one amino acid by three other amino acid residues. The results are summarized in Table A.

Table A

Derivatives of plasmid pJP29 carrying insertion mutations in the phoE gene

site of mutagenesis mutant plasmid structure of

DNA protein designated as the insertion

PstI 1 pJP29-Pl GGG ATC CCG Gly He Pro

Mlul 74-75 pJP29-Ml CGC GGA TCC

Arg Gly Ser Ala

Clal 142-143 pJP29-Cl GCG GGA TCC CGC Ala Gly Ser Arg

Clal 142-143 pJP29-C2 GCC AAG CTT GGC Ala Lys Leu Gly

Ndel 173-174 pJP29-Nl TAC GGG ATC CCG Tyr Gly Ile Pro

Bglll 279-280 pJP29-Bl GAT CCG GAT CCG Asp Pro Asp Pro

In the table, under "DNA", the restriction site in plasmid pJP29 used for the insertion mutagenesis has been indicated. The number of the corresponding amino acid residues in the mature PhoE protein is indicated in the column "protein". As is known, wild type mature PhoE protein consists of 330 amino acid residues.

In the right-hand column the bases and amino acids inserted in the phoE gene and the PhoE protein, respectively, are indicated. In the plasmid pJP29-Pl encoded mutant, the first amino acid residue of the wild type mature protein, an alanine residue, is replaced by glycine, isoleucine and proline residues. Subsequently, the expression of products of the mutant genes was studied. To identify -the poly- peptides encoded by the mutants, cell envelope proteins were analyzed from CE1224 cells carrying the insertion plasmids after growth under phosphate limitation. The bacteria were always grown under aeration at 37°C in either L-broth (described by Tommassen et al. in EMBO journal 4 (1985), 1041-1047) or in a medium described by Levinthal et al. (Proc. Natl . Acad. Sci . USA 48 (1962), 1230-1237), in which the phosphate concentration is limiting for growth. Where necessary, the medium was supplemented with chloramphenicol (25 /ug/ml) or ampicillin (50 /ug/ml).

As compared to the wild type PhoE protein, the mutant proteins showed an identical or a lower electrophoretic mobility. For the isolation and character--" ization of cell fractions the cell envelopes were isolated by ultrasonic disintegration of cells, followed by centrifugation. A fraction containing proteins of the outer membrane was isolated by extraction of cell envelopes with Triton X-100. The protein patterns of the cell fractions were analyzed by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis .

The possibility that the slower migrating proteins represented unprocessed precursors of the mutant proteins, was ruled out on the basis of pulse-label and pulse-chase experiments. These were conducted as described by Bosch et al., J. Mol. Biol. 189 (1986), pages 449-455. When cells containing the plasmid pJP29 were pulse-labelled for 30 seconds, during phosphate starvation conditions, with 35g__methionine and total cellular proteins were separated on SDS-polyacrylamide gels, two bands, representing the precursor form and the mature form of PhoE protein, were detected on the autoradiogram of the gels. The precursor disappeared during a 20 minute chase period. Similarly a transient precursor form could be detected for all mutant proteins.

Except in the case of plasmid pJP29-C2, all mutant proteins proved to be constitutively expressed in phoR mutant strain CE1248. This strain has been described by Van der Ley et al., Eur.J.Biochem. 147 (1985), 401-407, and contains, like the earlier mentioned strain CE1224, a deletion for the phoE gene as well as ompR mutations, so that it is not capable of producing OmpF and OmpC proteins. Because of a phoR mutation the strain CE1248 constitutively expresses the pho reguloπ.

The latter strain CE1248 could not be transformed with the plasmid pJP29-C2. Apparently, expression of the corresponding mutant PhoE protein is lethal to the cells. This probably explains the observed decreased amount of mutant protein in cells containing plasmid PJP29-C2.

From the pulse-label experiments, it appeared that all mutant proteins are normally processed, suggesting transport through the cytoplasmic membrane. Cell fraction- ation experiments localized the mutant protein in the outer membrane, since the proteins were present in the Triton X-100 insoluble fraction of the cell envelopes. However, since localization of mutant proteins by cell fractionation experiments may be unreliable, functional assays are required to confirm the presence of these proteins in the outer membrane.

PhoE protein serves as (part of) the receptor for phage TC45 (described by Chai and Foulds, J. Bacteriol. 135 (1978), 164-170) and its host range derivative TC45 hrN3 described by Tommassen et al. , Mo1.Gen.Gene . 197 (1984), 503-508. Phage adsorption experiments were performed with phosphate limited CE1224 cells carrying the mutant plasmids. The results are listed in Table B. Table B

Interaction of cells carrying the mutant plasmids with phages and monoclonal antibodies.

phage receptor activity monoclonal antibody bindi

percentage of phages sensitivity plasmid bound in to phages PP1-4 PP2-1 PP3- adsorption test

PACYC184 < 5 R 4 7

PJP29 >98 S 177 200 19

PJP29-P 1 >9 Θ S 185 ^• 215 19

PJP29-M1 < 5 R 182 2 1 3 20

PJP 29-C 1 >98 S 196 21 2 22

PJP29-C2 < 5 9 3 _. 33 7

PJP 29-N 1 >98 S 182 207 18

PJP29-B 1 >98 S 6 2 17

The sensitivity of strains to phages was determined by cross-streaking. Irreversible phage binding to whole cells was measured with the method described by Van der Ley et al., Eur.J.Biochem.147, (1985), 401-407.

The two phages showed no mutual differences in their interaction with the mutant cells. The sensitivity to phages was tested with derivatives of strain CE1248, except for the case of pJP29-C2 mutant which could not be introduced into this strain. Monoclonal antibody binding was measured in CIRA experiments. This cell immuno radio assay is described in the earlier mentioned publication by Van der Ley et al. The cells, however, were grown in a phosphate limited medium. The cells were incubated with diluted ascites fluid and subsequently with 125j_ labelled protein A. The amount of radio activity bound to the cells after washing is indicated as kcpm.

The phages proved to bind to cells carrying plasmids pJP2 , pJP29-Pl, pJP29-Cl, pJP2^"9-Nl and pJP29-Bl, but not to cells carrying plasmids pJP29-Ml and pJP29-C2. Consistent with these results, derivatives of phoR mutant strain CE12 8 carrying either one of the former five plasmids provedto be sensitive to the phages in contrast to the derivative carrying pJP29-Ml. The binding of three different monoclonal antibodies, namely, PP1-4, PP2-1 and PP3-4, which recognize the cell-surface exposed part of PhoE protein, to phosphate limited CΞ1224 cells carrying the insertion plasmids, was studied in sera experiments. These antibodies are described in the earlier mentioned article by Van der Ley et al.

It turned out that only- the binding of antibodies to cells carrying pJP29-C2 and pJP29-Bl was disturbed. The strain carrying plasmid pJP29-C2 showed a reduced binding of all the antibodies, probably because of the relatively low amount of mutant PhoE protein produced by this strain. The pJP29-Bl containing cells bound only monoclonal antibody PP2-1. Apparently, the insertion in this plasmid disturbs the antigenic determinant recognized by the antibodies PPl-4 and PP3-4. Since in all cases at least one monoclonal antibody bound to whole cells producing the altered PhoE proteins, it turns out that all these proteins are incorporated into the outer membrane. As stated before, the PhoE protein forms pores in the outer membrane with a preference for anionic compounds. The specificity and the efficiency of the pores can be demonstrated by measuring the rates of permeation of the beta-lactam antibiotics cephaloridine and cefsulodin in cells producing only one porin species. Cefsoludin is closely related to cephaloridine but contains an additional negative charge and its molecular weight is higher. The rate of permeation of these anti¬ biotics through the mutant pores was measured in derivates of strain CE1248 carrying pJP2 with the different phoE alleles and plasmid pBR322 to provide a high beta-lactama level. The method was originally described by Zimmerman and Rosselet (An imicrob.Agents Che other 12 (1977) 368-372), and modified by Overbeeke and Lugtenberg, Eur.J.Biochem.126 (1982), 113-118.

The rate of uptake of^' he beta-lactam compounds by pJP29-Pl, pJP29-Nl and pJP29-Bl carrying cells did not significantly deviate from that of the strain producing wild type PhoE protein. See Table C listing the results. The rate of uptake of cefsulodin by pJP29-Cl and pJP29-Ml carrying cells, however, proved to-be significantly decreased, whereas cells carrying the latter plasmid showed an increased uptake of cephaloridine. The speci¬ ficity of the pores encoded by the latter two plasmids, expressed by the relative rates of uptake of the two antibiotics, also appeared to be changed. No pore activity could be detected in pJP29-C2 carrying cells. Table C

Rate of permeation of beta-lactam antibiotics through the mutant pores in intact cells

Rate of uptake in intact cells

Pore specificity plasmid cefsoludin (Vu) Cephaloridine (Va) determined by Vu/Va

PACYC184 o:6 1.8

PJP29 11.2 6.2 1.8 PJP29-P1 11.5 6.2 1.8

PJP29-M1 5.5 9.7 0.6

PJP29-C1 5.6 5.8 1.0

PJP29-C2 0.6 1.5

PJP29-N1 11.5 6.1 1.9

PJP29-B1 11.7 6.3 ^' 1.8

In this table the rate of permeation of the beta-lactam compounds is expressed in nmole per minute per 10⁸ cells. The pore characteristics of pJP29-C2 encoded mutant proteins were measured in CE122 cells grown on Levinthal medium. No differences of beta-lactam permeation were observed between the wild type PhoE producing CE1224 cells grown on this medium and the wild type PhoE producing phoR strain CE1248 grown on L-broth .

Therefore, the above experiments accordingly showed that the polypeptides encoded by^' the constructed insertion mutant alleles were all incorporated into the outer membrane, as shown by the binding of phages, the binding of monoclonal antibodies or both. In one case, namely, that of pJP29-C2, the mutant protein was found not to be normally incorporated into the outer membrane, since expression of this protein was lethal to the cells and since the protein did not function as a pore. However, an insertion at the same site in PhoE of approximately the same amino acids but in a different order, i.e. in plasmid pJP29-Cl did not interfere with the normal incorporation of the protein. The insertion at the Bglll site (corresponding to amino acid 280 of mature PhoE protein) proved to interfere with the binding of two different monoclonal antibodies. Since the binding of^* another monoclonal antibody and of the phages, nor the pore functioning of this mutant protein were disturbed, the overall conformation of the mutant protein is probably not drastically changed. The amino acids in the environment of amino acid residue 280 are apparently cell-surface exposed and involved in the antigenic determinant for monoclonal antibodies PP1-4 and PP3-4.

Amino acid residue 75 is also likely to be cell-surface exposed and involved in the phage receptor activity. Insertion at the PstI site does not appear to interfere with the cell-surface related properties of the PhoE protein, which is in agreement with earlier observations that the N-terminus of the protein is probably exposed at the periplasmic side of the membrane. The pore characteristics of the mutant proteins were slightly modified in two cases. The decreased rate of uptake of cefsulodin by cells carrying pJP29-Cl may be explained by a narrowing of the channel due to the addition of four amino acids. The simultaneous decrease of cefsulodin uptake and the increase of the cephaloridine uptake in the case of pJP29-Ml are more difficult to explain; shielding of amino acids Involved in the anion specificity of the PhoE pores might be an explanation. Example II

In this example it was investigated to what extent additional amino acid residues could be introduced into the position of the amino acid 158 (arginine) .

Plasmid pJP29 was used as the starting point. As indicated in Example I, it contains a normal phoE gene. The Nrul site present in this plasmid was removed by cutting the plasmid with Nrul and then treating it with exonuclease Bal31. After ligation with T4 DNA ligase the DNA was used to transform strain CE1224, selecting chloramphenicσl resistant colonies. Plasmid DNA was isolated from one of the transformants. This plasmid, which no longer contained an Nrul site, was called pMR03.

In order to introduce an Nrul site into the phoE gene at the position corresponding to Argl58 the Pstl-Bglll fragment of pJP29 was cloned in the Pstl-BamHI digested M13 vector mp8. Site-directed mutagenesis with a mutagenic oligonucleotide was conducted according to the method described by Norris et al. , Nucl. Acids. Res. 11 (1983) 5103-5112, creating the desired Nrul site. Of a resulting mutant plasmid the Meul-Ndel fragment was then isolated with the Nrul site thereon, and this fragment was ligated in pMR03 , from which the corresponding fragment was removed. The resulting plasmid was called pMR05. This plasmid pMR05 was cut open by incubation with the restriction enzyme Nrul, after which small synthetic DNA linkers were ligated in the cut-open DNA. A first linker had the nucleotide sequence CATGGATCCATG and contained a BamHI recognition sequence. The second linker consisted of the nucleotide sequence CCGGAATTCCGG and contained an -EcoRI recognition sequence. The resulting plasmids were designated as pMR08 (contains the BamHI linker) and pMR09 (contains the EcoRI linker), respectively. These plasmids each code for four additional amino acids, but in the case of the EcoRI linker insertion the 3 bases GAC coding for asp were lost, so that pMR09 net coded for three additional amino acids.

Plasmid pMR08 was then cut open with BamHI, and in the cut-open plasmid a polylinker of 60 base pairs (corresponding to 20 amino acids) was ligated, which was cut from plasmid pLL4* with the aid of BamHI. This polylinker contains the recognition sequences for many restriction enzymes, so that further manipulations are considerably simplified. The resulting plasmid is designated as pMRlO. It codes for 24 additional amino. acids, as compared with the normal phoE gene.

The mutant PhoE proteins encoded by plasmids pMR08, 09 and 10 all proved to be normally transported to the cell surface of E_. coli. This was established by means of the binding of monoclonal antibodies directed against a cell-surface exposed part of PhoE protein. The binding of one of these antibodies, namely, PP2-1, however, proved to be disturbed, which demonstrates that the region around amino acid 158 (arginine) contains an immunogenic determinant of PhoE.

This example shows that it is possible to insert in this immunogenic determinant new arbitrary peptides which may have a length of at least up to 24 amino acids, such that the incorporation of pHoE into the outer membrane is not disturbed. The construction of these plasmids is shown in Fig. 2.

In another experiment a synthetic oligonucleotide having the sequence dTTATAAACAGAAGATCATCGCCCCGGG and the complementary oligonucleotide were incorporated into the Nrul site of plasmid pMR05. The oligonucleotide codes for the octapeptide Tyr Lys Gin Lys He He Ala Pro, the first base (T) and the last two bases (GG) being required for maintaining the correct reading frame. The PhoE protein into which this determinant of the FMD virus is incorporated was expressed and was incorporated into the outer membrane of E_. coli. The E_. coli cells proved to react in an ELISA with a monoclonal antibody directed against the FMD virus. This experiment shows anyhow that the invention can serve for diagnostic purposes.

Of the figures Fig. 1 shows the physical map of plasmid pJP29. The thick line represents DNA derived from cloning vector pACYC 184. The thin line represents chromosomal DNA. The position and direction of transcription of the phoE gene is indicated by an arrow. The wavy line represents DNA coding for the signal sequence. The plasmid contains a chloramphenicol marker (Cm) , the position of which is indicated in the figure. Fig. 2 shows the part of the phoE gene relevant to the experiments^" described in Example II, as well as the pertinent amino acids in the PhoE protein. Fig. 2 further shows^' the corresponding parts in plasmids pMR05, 08, 09 and 10, so that the modifications made are rendered visible.

Fig. 3 shows the nucleotide sequences of the phoE genes of Klebsiella pneumoniae and Enterobacter cloacae, as compared with the phoE gene of Escherichia coli. In the coding regions, only- those nucleotides which differ from the J3. coli gene are indicated. In the upstream noncoding regions, complete sequences for all of the three genes are given, with regions of high homology indicated by boxes. The ribosome binding site is underlined, and the translation start and stop signals are boxed. The transcriptional terminators from E_. coli and E . cloacae are indicated by arrows.

Claims

-26-C L A I M S

1. Recombinant DNA comprising the genetic infor¬ mation for PhoE protein of Enterobacteriaceae bacteria and, in a part thereof coding for a region of the PhoE protein localized at the cell surface, the genetic information for a foreign antigenic determinant.

2. Recombinant DNA according to claim 1 comprising the genetic information for PhoE protein of Escherichia coli bacteria and, in a part thereof coding for a region of the PhoE protein localized at the cell surface, the genetic information for a foreign antigenic determinant,

3. Recombinant DNA according to claim 1 comprising the genetic information for PhoE protein of Escherichia coli K12 bacteria and, in a part of the gene coding for one of the following amino acid regions of ,the PhoE protein:

(a) amino acids 20 through 34;

(b) amino acids 63 through 75;

(c) amino acids 105 through 125;

(d) amino acids 154 through 163

(e) amino acids 189 through 205;

(f) amino acids 229 through 245,

(g) amino acids 269 through 283 (h) amino acids 307 through 320 the genetic information for a foreign antigenic determinant.

4. Recombinant DNA according to claim 1 comprising the genetic information for PhoE protein of Escherichia coli K12 bacteria and, in a part of the gene coding for amino acid 158 and its nearest neighbours, the genetic information for a foreign antigenic determinant.

5. Recombinant DNA according to claim 1 comprising the genetic information for PhoE protein of Escherichia coli K12 bacteria and, in a part of the gene coding for amino acid 201 and its nearest neighbours, the genetic information for a foreign antigenic determinant.

6. Recombinant DNA according to claim 1 comprising the genetic information for PhoE protein of Escherichia coli K12 bacteria and, in a part of the gene coding for amino acid 238 and its nearest neighbours, the genetic information for a foreign antigenic determinant.

7. Recombinant DNA according to claim 1 comprising the genetic information for PhoE protein of Escherichia coli K12 bacteria and, in a part of the gene coding for amino acid 275 and its nearest neighbours, the genetic information for a foreign antigenic determinant.

8. Recombinant DNA according to any of the preceding claims wherein the genetic information for a foreign antigenic determinant has a length of not more than

90 nucleotide pairs.

9-. Recombinant DNA according to any of the preceding claims containing the genetic information for an antigenic determinant of foot-and-mouth disease virus.

10. Recombinant DNA according to claim 9 containing the genetic information for an antigenic determinant of the capsid protein VPl of foot-and-mouth disease virus.

11. Recombinant DNA according to claim 10 containing the genetic information for a polypeptide corresponding to amino acids 140-160 or 200-213 of the capsid protein VPl of foot-and-mouth disease virus.

12. Recombinant DNA according to any of claims 1-8 containing the genetic information for an 'antigenic determinant of the Treponema pallidum causative agent of syphilis.

13. Recombinant DNA according to any of claims 1-8 containing the genetic information for an antigenic determinant of the poliomyelitis virus, the measles virus, the Mycobacterium leprae causative agent of leprosy, of Meningococcus and Haemophilus species causing meningitis, the AIDS virus, the hepatitis B virus, the cholera bacterium or of corona viruses.

14. Recombinant plasmid comprising a vector part- enabling replication and expression in Enterobacteriaceae bacteria, and recombinant DNA according to one of claims 1-13.

15. Enterobacteriaceae bacteria containing one or more recombinant plasmids according to claim 14.

16. Enterobacteriaceae bacteria containing one or more recombinant plasmids according to claim 14 and, in their outer membrane, PhoE carrying a foreign antigenic determinant corresponding to the genetic information laid down in said plasmids-.^*

17. PhoE protein carrying a foreign antigenic determinant and being recovered from Enterobacteriaceae bacteria according to claim 16.

18. Diagnostic aid comprising PhoE protein according to claim 17 or, if desired killed, Enterobacteriaceae bacteria according to claim 16.

19. Vaccine comprising PhoE protein according to claim 17 or, if desired killed, Enterobacteriaceae bacteria according to claim 16.

20. Recombinant DNA comprising the genetic informa¬ tion for PhoE protein of Enterobacteriaceae bacteria and at least one restriction enzyme recognition sequence arranged in a part thereof coding for a region of the PhoE protein localized at the cell surface.

21. Recombinant plasmid comprising a vector part enabling replication and expression in Enterobacteriaceae bacteria, and recombinant DNA according to claim 20.

22. Enterobacteriaceae bacteria containing one or more recombinant plasmids according to claim 21.