NL8700127A - RECOMBINANT DNA, CODING FOR PHOE PROTEIN WITH AN INSERTED FOREIGN ANTIGEN DETERMINANT; USE OF ENTEROBACTERIACEAE BACTERIA OR THEIR PHOE PROTEIN AS A DIAGNOSTIC DEVICE OR VACCINE. - Google Patents

Description

• ^ \ ^. 4. ; VO 8492

Title: Recombinant DNA encoding PhoE protein with an inserted foreign antigen determinant; use of Erterobacfceriaceae bacteria or their PhoE protein as a diagnostic tool or vaccine.

The invention is in the fields of DNA recombinant technology, diagnostics and vaccine preparation.

Vaccines against pathogens are usually based on inactivated forms of the pathogens.

5 An example of this is the vaccine against the mouth-mouth disease (FMD) virus. FMD is responsible for large losses in livestock in solipeds, so that the prevention of FMD through vaccination is of great economic importance.

Current FMD vaccines are based on inactivated virus particles, which are grown in surviving bovine tongue epithelium or in baby hamster kidney cells.

Although these vaccines are satisfactory and vaccination programs have practically prevented disease 15 in Western Europe, these vaccines nevertheless have some drawbacks and risks, which make the development of alternative vaccines desirable: - the need for large-scale virus culture, and the associated risk of virus escape from the secured environment; - risk of surviving virus in vaccines; - the high production costs.

To a greater or lesser extent the same drawbacks also apply to the known vaccines against other pathogens, such as measles and polio virus. For example, a live attenuated vaccine is used against measles, which in itself is highly effective and has significantly reduced the number of measles cases in Western countries. However, the following disadvantages are associated with the vaccine: v 7

♦ 'V

-2- - the vaccine produces undesirable side effects ranging from pain and fever in a fairly large number of cases, to serious neurological complications in incidental cases; 5 - successful vaccination is only possible from 12-15 months of age due to interference by maternal antibodies when vaccinated at a younger age; - vaccination is excluded in persons who have immune system disorders or who are treated with 10 immunosuppressants.

Much research is being conducted to determine whether individual virus proteins or synthetic oligopeptides representing an antigenic determinant of such a protein are useful as a vaccine. Recent developments in molecular biology, in particular the recombinant DNA techniques, make it possible to easily produce these types of proteins or oligopeptides in large quantities.

The applicability of the recombinant DNA technique for the production of subunit vaccines (vaccines consisting mainly of proteins) strongly depends on the immunogenic effectiveness of the protein used.

Research into the possibility of eg.

However, using capsid proteins from viruses as a basis for a vaccine often shows that the immunogenicity of such proteins is much lower than that of the virus itself. Smaller peptides, which are composed of important antigenic determinants of about 30 virus, also appear to be less effective than the virus particle.

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

Foot-and-mouth disease virus is one of the picorna 35 viruses. It is relatively simple and consists of an RNA molecule, surrounded by 60 copies before r> y> B. * "· *« Λ - / 1 L / V 4 -3- of the capsid proteins, VP1, VP2, VP3 and VP4. Of these capsid proteins, VP1 is the most important since it contains the main antigen determinant of the virus. VP1, isolated from virus, has been shown to be capable of providing both a neutralizing response and protection in guinea pigs and bovine E. coli strains producing large amounts of VP1 have been constructed using recombinant DNA techniques VP1 isolated from such E. coli strain same properties as VP1, 10 isolated from the intact virus Also fragments of VP1, namely peptides corresponding to amino acid sequences 140-160 or 200-213, appear to give a neutralizing response in experimental animals, although both peptides have a clear neutralizing response For these proteins, as well as for VP1 itself, the effectiveness is much less than that of the virus itself, however, it is expected that by fusion of the determinant to another, larger protein molecule (a so-called carrier) the immunogenic effect can be increased.

For example, some hopeful results have already been achieved with the use of antigens organized in micelle or liposome structures, the use of fusion proteins containing multiple copies of the same antigen determinant in tandem, and the use of adjuvants , but a completely satisfactory solution has not yet been found.

For example, for FMD, the fusion of the 140-160 determinant to β-galactosidase appears to increase the immunogenic effectiveness of the antigen determinant.

However, the increase obtained here does not appear to be large enough yet. Further research into other, better carriers is therefore necessary.

Measurement of the effectiveness of measles vaccines has shown that for good immunity to measles infection, the presence of antibodies against both viral envelope glycoproteins - haemagglu- -4-

• «1 V

tinine (HA) and fusion factor (F) - is necessary. It may be expected that a subunit vaccine containing these two antigens or the immunologically important parts of these two antigens in biologically active form will be able to produce good and long-lasting immunity.

Such a vaccine would be free from the drawbacks associated with the live vaccine. Here too, however, the problem is to find a suitable carrier for the antigenic determinants of the measles virus.

Problems also arise in the preparation of antigens for diagnostic purposes. Some antigens are very difficult to isolate from pathogenic organisms, for example if the cultivation of these organisms in vitro is difficult or not feasible or very expensive. This problem occurs, for example, with the syphilis causative agent Treponema pallidum.

For the treatment of patients suffering from syphilis, it is particularly important to know at what stage of the infection the patient is.

The possibility of determining the stage of infection by detecting specific antibodies in the serum of patients is being investigated.

For this it is necessary to have specific antigens of the syphilis pathogen Treponema pallidum. Treponema cannot be cultured in vitro, and with the current culture method (in living rabbit testicles) it is impossible to isolate sufficient amounts of a specific antigen for routine diagnostic applications.

A number of Treponema antigens have already been donated in E.coli, which can be used diagnostically. The ultimate goal is to compile a 'kit' of different specific antigens with which sera of patients can be tested for the occurrence of antibodies. The prerequisite for this is that the antigen $ 7 f h * 5 y / * * -5- can be produced easily and reproducibly, which condition has not been met to date.

The invention now solves the above-mentioned problems by using PhoE protein as a carrier for foreign antigen 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 acts as a molecular sieve due to the pore-forming proteins, so-called porins, contained therein. The proteins form channels through the membrane, allowing small hydrophilic substances to pass through.

E.coli K12 has three proteins that function as pores in the outer membrane for small hydrophilic compounds. These proteins, called PhoE, OmpF and OmpC, occur in large numbers of copies, approximately 10 per cell. Of these proteins, OmpF and OmpC are constitutively expressed, while PhoE is induced under phosphate limitation. However, constitutive synthesis of PhoE protein can also be achieved by suitable mutations in one of the genes phoR, phoS, phoT or pst. Omp F and Omp C pores appear to be particularly efficient channels for positively charged compounds, while PhoE pores are particularly efficient channels for negatively charged compounds. The PhoE promoter is one of the strongest promoters of E.coli.

Like other exported proteins, PhoE protein is synthesized in a precursor form with an N-terminal signal sequence, which is essential and sufficient for transport through the membrane.

The structural genes for the above pore proteins have been cloned and the DNA sequences determined. For the DNA sequence of the structural gene for PhoE, see Overbeeke et al, J. Mol. Biol. 163 (1983), 513-532. A comparison r6 -6- of the sequences shows that these genes show a strong homology (ca. 60%) (Mizuno et al, J.Biol.Chem.-280 (1983), 6932-6940). Unlike most other membrane proteins, the porins lack hydrophobic sequences of sufficient length to bridge the membrane. The functional unit is a trimer with a compact structure without large domains outside the membrane.

PhoE protein is also found in Enterobac-10 teriaceae other than J2. coli. The phoE genes of several Enterobacteriaceae have been cloned, see, e.g., Verhoef et al, Gene 32 (1984) 107-115. Nucleotide sequence has been determined for the phoE genes of Enterobacter cloacae and Klebsiella pneumoniae (see Figure 3).

The observed homology was used to construct hybrid genes, replacing parts of one gene with corresponding parts of the other pore protein genes. Using those hybrid genes and specific monoclonal and polyclonal antibodies directed against the cell surface portion of PhoE, epitopes of PhoE located at the cell surface could be genetically localized. Insertions in these cell surface regions were found to be possible: recombinant DNA encoding PhoE protein but provided with an insert (a linker molecule or a DNA sequence encoding a foreign antigen determinant) inserted in a portion of the gene encoding a region of the PhoE protein exhibited at the cell surface, was found to be expressible in E. coli and the modified PhoE protein was found to be incorporated into the outer membrane. This has been demonstrated for both PhoE protein from _E. coli K12 as for Enterobacter 35 cloacae PhoE protein, the insert representing a length of 24 amino acids. The inserted polypeptide, such as *: '* n * ",?!. -!;" / Ƒ * * -7- a foreign antigen determinant, is thus produced by the bacterium and displayed on the outside of the cell, which both for the preparation of vaccines if for diagnostic purposes offers great advantages Cells carrying surface-exhibited foreign antigen determinants could be used per se as oral vaccines, in particular in veterinary medicine, one would preferentially enterobacteriaceae species, which is harmless to the host and able to survive in the gut, such as non-virulent Salmonella strains, such as the galE mutant typhimurium strain G30, which is not virulent but maintains sufficiently in the gut to support a local to elicit an immune response (Stevenson and Manning, FEMS Microbiol. Lett. 28 (1985) 317-321). Also EL coli strains could be used. Because E.coli K12 cannot maintain itself in the gut, the preference is given to wild type E. coli strains. Also, one could use killed cells, or PhoE protein derived from the cells, containing the foreign antigen determinant.

The large-scale production of vaccines, such as in particular an FMD vaccine, will be considerably cheaper than the current method of vaccine production.

Growing bacteria, also on a large scale, is very simple, so that large amounts of bacteria and / or PhoE / FMD fusion protein can be isolated.

Because coupling of the antigen determinants to PhoE protein results in the antigen being exposed on the surface of the bacterial cell and being easy to purify, the invention also offers great advantages for diagnostic purposes.

The invention now primarily relates to recombinant DNA comprising the genetic information for PhoE

i -8-

«V

protein from Enterobacteriaceae bacteria and, in a region encoding a cell surface located region of the PhoE protein, the genetic information for a foreign antigen determinant. In particular, the invention relates to the use of a gene encoding Escherichia coli PhoE protein, such as IS. coli K12 bacteria.

Preferably, the foreign antigen determinant is in a portion of the gene encoding one of the following amino acid regions of the PhoE protein: (a) amino acids 20 to 34; (b) amino acids 63 to 75; (c) amino acids 105 to 125; 15 (d) amino acids 154 to 163; (e) amino acids 189 to 205; (f) amino acids 229 to 245; (g) amino acids 269 through 283; (h) amino acids 307 through 320.

Very suitable sites for insertion of a foreign antigenic determinant are those parts of the gene encoding amino acids 158, 201, 238, 275 and their nearest neighbors.

Of course, the insertion (or substitution) should be such that the reading frame is not disturbed and expression in and incorporation into the outer membrane of the bacterial host is not prevented.

Although it is not excluded that longer DNA molecules can be introduced into the PhoE gene, it is preferred that the inserted genetic information for a foreign antigen determinant be no more than 90 nucleotide pairs in length.

For certain applications, it may be desirable to insert multiple foreign antigen determinants. In principle, this is possible, eg for the preparation of a measles vaccine by mixing in one ^ S .. yr- ». Of the said parts of the gene an antigen determinant of haemagglutinin and in one of the other parts of the gene mentioned an antigen determinant of the fusion factor.

The main advantages of the invention can be summarized as follows.

One of the strongest promoters of Enterobacteriaceae, such as Salmonella and E.coli, the phoE promoter is used, so that a high expression level can be achieved. Because the 'foreign' oligopeptide is on the cell surface, it is not accessible to proteolytic enzymes from the cell.

Since the foreign oligopeptide is on the cell surface, the antigen does not need to be purified for a number of applications, eg serum diagnostics.

To determine whether antibodies against a particular pathogen are present in a patient's serum, the Enterobacteriaceae, such as Salmonella and E. coli cells expressing such an antigen determinant, can be used directly in ELISA assays or agglutination reactions. In addition, such cells could be used as a live oral vaccine. Such an application also has the advantage that the conscious antigen determinant is presented as part of a natural immunogen, which increases immunity. The purification of PhoE protein is well described and fairly straightforward.

Thus, if the use of whole Enterobacteriaceae, such as E. coli cells for certain applications, is not desired, the antigen determinant coupled to the PhoE protein can be easily purified.

If the DNA encoding an antigen is available, the immunogenic epitopes can easily be determined by cloning fragments of this DNA into the vectors. The Enterobacteriaceae, ƒ -10-, such as E. coli clones obtained, are then tested for reaction with antibodies to the antigen.

Although the nature of the foreign antigen determinant is in principle not limited in any way, the invention is particularly suitable for antigen determinants of foot-and-mouth disease virus, in particular antigen determinants of the capsid protein VP1 thereof. Examples of strong antigenic determinants of this capsid protein VP1 are polypeptides corresponding to amino acids 140-160 or 200-213 of VP1. The genetic information for these antigen determinants is known and DNA encoding it can be isolated from suitable clones. Of course, synthetically prepared DNA molecules encoding the relevant antigen determinant can also be used. Given the known genetic code and preference of Enterobacteriaceae, such as EL coli for certain codons, synthetic DNA molecules can be composed of Enterobacteriaceae, such as JE. coli preferred codons for the same amino acid sequence. The nucleotide sequence of such synthetic DNA molecules may then deviate from the natural sequence while, nevertheless, the amino acid sequence of the resulting polypeptide is equal to that of the natural determinant.

Another preferred embodiment of the invention concerns the use of an antigen determinant of the syphilis causative agent Treponema pallidum, but also other antigen determinants such as that of poliovirus, measles virus, the leprosy causative agent Mycobacterium leprae, meningitis viral meningococcus, AIDS and hepatitis hepatitis. B virus can be used in the system according to the invention. The same applies to antigen determinants of organisms that cause intestinal diseases,, ei ** '*. .tj * r- ~ / 1 -¾ 4 -π- such as the cholera bacteria and corona viruses.

The invention not only relates to the above-described recombinant DNA, but also recombinant plasmids which, in addition to this recombinant DNA according to the invention, also contain a vector part which allows replication and expression in Enterobacteriaceae bacteria. Suitable vectors are known to those skilled in the art. Although the vector portion may contain one or more selection markers, this is not strictly necessary.

Indeed, transformants can be easily identified with the aid of specific antibodies to the inserted antigen determinant exhibited on the surface of the cells in the PhoE protein.

The invention also relates to Enterobacteriaceae bacteria containing such recombinant plasmids. By cultivating such bacteria, large amounts of the recombinant plasmids can be produced.

Preferably, hosts with a chromosomal deletion of the native phoE gene will be used to limit plasmid loss by recombination.

Particularly preferred are Enterobacteriaceae bacteria, which contain one or more such recombinant plasmids and which contain PhoE protein in their outer membrane, which carries a foreign antigen determinant according to the genetic information contained in said plasmids.

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

The invention also encompasses the use of transformed Enterobacteriaceae bacteria (optionally after killing) containing PhoE protein with a foreign antigen determinant incorporated therein, for vaccination or diagnostic purposes.

The PhoE protein can also be used instead of the Enterobacteriaceae cells.

Furthermore, the invention also relates to recombinant DNA comprising the genetic information for PhoE f r *? r% ..

-12- protein from Enterobacteriaceae bacteria and at least one restriction enzyme recognition sequence arranged in a region surface-coded for the cell surface of the PhoE protein, as well as on a recombinant plasmid containing such recombinant DNA in addition to a vector portion that allows replication and expression in Enterobacteriaceae bacteria, and transformed Enterobacteriaceae bacteria containing one or more such recombinant plasmids.

Recombinant DNA comprising the genetic information for PhoE protein with one or more restriction enzyme recognition sequences located in a region of the PhoE protein encoding a cell surface located region thereof forms a suitable starting material for preparing recombinant DNA, which in a portion of the genetic information for PhoE protein encoding a region of the PhoE protein located at the cell surface contains the genetic information for a foreign antigen determinant. The insertion of genetic information for a foreign antigen determinant is no problem for a person skilled in the art.

The invention will now be explained in more detail with reference to Examples.

Example I

Mutations in the phoE gene were introduced by insertion of linker molecules. Use was made of the plasmid pJP29 containing the phoE gene.

This plasmid is depicted in Fig.1. The construction of this plasmid has been described by Bosch et al, J. Mol Biol. 189 (1986), 449-455. It should be noted that not only 15. coli bacteria can be transformed with this plasmid. Thus, a successful transformation of the galE mutant S; has been performed. typhimurium strain G30, in which the EL coli phoE gene was expressed under phosphate limitation. The plasmid contains 5 unique restriction enzyme · * Λ. · O? 13 recognition sites used to insert oligonucleotides. To perform the insertions, 1 / mg of pJP29 was cut with one of these restriction enzymes and the protruding ends were either.

5 filled in with the Klenow fragment of E.coli DNA polymerase I or removed using the 3'-5 'exonuclease activity of T4 DNA polymerase, in the presence of 2mM dNTP.

The blunt-ended DNA thus obtained was ligated with T4 DNA ligase to 100 ng of one of the phosphorylated oligonucleotides dCGGATCCG (BamHI linker), dCGGGATCCCG (BamHI linker) or éCCAAGCTTGG (HindIII linker). By using oligonucleotides of either 8 or 10 base pairs in length, the correct reading frame could be ensured. After digestion with BamHI or HindlII and re-ligation with T4 DNA ligase, the DNA preparation was used to transform the E.coli strain CE1224, selecting chloramphenicol resistant colonies.

The E.coli K-12 strain CE1224 contains a deletion for the phoE gene and does not form OmpF and OmpC proteins due to ompR mutations. The strain has been described by Tommassen et al, EMBO journal 4 (1985) 1041-1047.

The plasmid DNA used in the experiments

was prepared on the by Birnboim and Doly, Nucleic

Acids Res.7 (1979), 1513-1524, as described, followed by CsCl ethidium bromide isopicnic centrifugation.

All constructs were verified by DNA sequence analysis. For this, the known method described by Sanger et al, Proc.Natl.

Acad. Sci. USA 74 (1977), 5463-5467.

In all cases, mutagenesis resulted in the addition of four amino acids into the PhoE protein, except in the case of an insertion at the Pst I site, which replaced one amino acid with {! [2 7-14 which led other amino acid residues. Results are summarized in Table A.

Table A

Derivatives of plasmid pJP29 with insertion mutations in the phoE gene instead of the mutagenesis DNA protein mutant plasmid Structure referred to as the insert

PstI 1 pJP29-Pl GGG'ATC CCG

Gly lie Pro

Mlul 74-75 pJP29-Ml CGC GGA TCC GCG

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-En TAC GGG ATC CCG

Tyr Gly lie Pro

BglII 279-280 pJP29-Bl HOLE CCG HOLE CCG

Asp Pro Asp Pro

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

In the right column the bases and amino acids are indicated which respectively. in the phoE gene and the PhoE

& ~ f \ ftr * IJ 7-15 protein have been inserted. In the mutant encoded by the plasmid pJP29-P1, the first amino acid residue of the wild type mature protein, an alanine residue, has been replaced by glycine, isoleucine 5 and proline residues.

Subsequently, the expression of products of the mutant genes was examined. In order to identify the polypeptides encoded by the mutants, cell envelope proteins were analyzed from CE1224 cells containing the insert plasmids after growth under phosphate limitation. The bacteria were cultured under aeration at 37 ° C in either L nutrient 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.Sei.USA 48 (1962), 1230-1237) in which the phosphate concentration is the growth limiting factor. Where necessary, the medium was supplemented with chloramphenicol (25 µg / ml) or ampicillin (50 µg / ml).

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

The possibility that the slower migrating proteins represented unprocessed mutant protein precursors was rejected on the basis of pulse-label and pulse-chase experiments. These were performed on the method described by Bosch et al., J. Mol. Biol. 189 (1986), pp. 449-455. When cells containing the plasmid pJP29 were pulse-labeled for 30 seconds under low phosphate conditions with -S-metbionine and the total of the cellular proteins on SDS polyacrylamide gels became 5. separated, two bands were detected on the autoradiogram of the gels, representing the precursor form and the mature form of the PhoE protein.

However, the precursor disappeared during a twenty-minute chase period. A transient precursor form could also be detected for all 10 mutant proteins.

Except in the case of the plasmid pJP29-C2, all mutant proteins were found to be constitutively expressed in the phoR mutant strain CE1248. This strain 15 has been described by Van der Ley et al, Eur.J.Biochem.147 (1985), 401-407, and, like the aforementioned strain CE1224, contains a deletion for the phoE gene as well as ompR mutations, making it incapable of is to produce OmpF and OmpC proteins. Thanks to a phoR 20 mutation, the strain CE1248 gives a constitutive expression of the pho regulon.

The latter strain CE1248 could not be transformed with the plasmid pJP29-C2. Apparently expression of the corresponding mutant PhoE 25 protein is lethal to the cells. This probably explains the smaller amount of mutant protein found in cells containing plasmid pJP29-C2.

The pulse-label experiments showed that all mutant proteins are processed normally, suggesting transport through the cytaplasmic membrane. In cell fractionation experiments, the mutant protein was located in the outer membrane, since the proteins were contained in the Triton X-100 insoluble fraction of the cell envelopes. However, since localization of mutant proteins by cell fractionation tests can be • unreliable, functional analyzes are 8 ƒ y!? , 4 / -17- 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 5 (1978), 164-170) and its host range derivative TC45 hrN3 described by Tommassen et al, Mol.Gen.Genet 197 (1984), 5Q3-508. Phage adsorption experiments were performed on phosphate-restricted CE1224 cells containing the mutant plasmids. The results are shown in Table B.

Table B

Interaction of cells containing the mutant plasmids with phages and monoclonal antibodies

Phage receptor activity Binding of monoclonal antibodies

Plasmid Percentage of in sensitivity PP1-4 PP2-1 PP3-4 the adsorption test for phages. bound phages PACYC184 <5 R 4 7 5 pJP29> 98 S 177 200 195 pJP29-Pl> 98 S 185 215 195 pJP29 — Ml <5 R 182 213 205 PJP29-C1> 98 S 196 212 221 PJP29-C2 <5 - 93 33 72 pJP29-En> 98 3 182 207 187 pJP29 — BI> 98 S 6 217 3> ƒ -18-

Strain susceptibility to phages was determined by cross-smear. Irreversible phage binding to whole cells was measured by the method described by Van der Ley et al, Eur. J. Biochem. 147, (1985) 5 401-407.

The two phages showed no differences in their interaction with the mutant cells. Phage susceptibility was tested with derivatives of the CE1248 strain, except in the case of the pJP29-C2 mutant which could not be introduced into this strain.

The binding of monoclonal antibodies was measured in CIRA experiments. This cell immunoradio analysis is described in the aforementioned publication by Van der Ley et al. However, the cells were grown in a phosphate limited medium. The cells were incubated with dilute ascites fluid and then with 125 I-labeled protein A. The amount of radioactivity found to be bound to the cells after washing is indicated in kcpm.

The phages were found to bind to cells containing the plasmids pJP29, pJP29-P1, pJP29-Cl, pJP29-N1 and pJP29-B1, but not to cells containing the plasmids pJP29-M1 and pJP29-C2. Consistent with these results is that derivatives of the phoR mutant strain CE1248, containing one of the former five plasmids, were found to be sensitive to the phages as opposed to the derivative containing pJP29-M1. In the CIRA experiments, the binding of three different monoclonal antibodies, namely PP1-4, PP2-1 and PP3-4, which recognize the cell surface portion of PhoE protein, was phosphate-limited CE1224 cells containing the insertion plasmids. , investigated.

These antibodies are described in the aforementioned article by Van der Ley et al.

55 It was found that only the binding of antibodies to cells containing pJP29-C2 and pJP29-Bl disrupts f * - * n * 2? · ··: v * y ί c / -19- was. The strain containing plasmid pJP29-C2 showed reduced binding of all antibodies, probably due to the relatively small amount of mutant PhoE protein produced by this strain.

The cells containing pJP29-B1 bound only the PP2-1 monoclonal antibody. Apparently, the insertion into this plasmid disrupts the antigen determinant recognized by the antibodies PP1-4 and PP3-4. Since in all cases at least one monoclonal antibody bound to whole cells producing the altered PhoE proteins, it appears that all proteins are taken up in the outer membrane.

As mentioned previously, the PhoE protein forms outer membrane pores with a preference for anionic compounds. The specificity and efficiency of the pores can be demonstrated by measuring the permeation rates of the beta-lactam antibiotics cephaloridine and cefsulodine in cells that produce only one type of porine. Cefsulodine is closely related to cephaloridin 20 but contains an additional negative charge and its molecular weight is higher. To measure the permeation rate of these antibiotics through the mutant pores, derivatives of the CE1248 strain containing pJP29 with the different phoE alleles and plasmid pBR322 were used to obtain a high beta-lactamase level. The method was originally described by Zimmerman and Rosselet (Antimicrob. Agents Chemother 12 (1977) 368-372), and modified by Overbeeke and Lugtenberg »Eur. J. Biochem. 126 (1982), 113-118.

The uptake rate of the beta-lactam compounds by pJP29-P1, pJP29-N1 and pJP29-B1 containing cells did not differ significantly from that of the strain producing wild type PhoE protein. See Table C showing the results. However, the uptake rate of cefsulodine by pJP29-Cl and pJP29-M1 containing -20 cells was found to be significantly slower, while cells containing the latter plasmid showed a higher uptake of cephaloridine. The specificity of the pores encoded by the latter two plasmids, expressed in the relative uptake rates of the two antibiotics, appeared to have changed. No pore activity could be observed in cells containing pJP29-C2.

10 Table C

Permeation rate of beta-lactam antibiotics through the mutant pores in intact cells 15 Absorption rate in intact cells

Pore-specific

Plasmid cefsulodine (Vu) Cephaloridine (Va) determined by

Vu / Va 20 pACYC184 0.6 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 25 PJP29-C1 5.6 5.8 1.0 PJP29-C2 0.6 1.5 PJP29-N1 11.5 6.1 1.9 30 PJP29-B1 11.7 6.3 1.8

In this table, the permeation rate of the beta-lactam compounds is expressed in nanomoles per minute per 35 JD 108 cells. The pore properties of mutant proteins encoded by pJP29-C2 were measured in CE1224 cells grown on Levinthal medium. No differences in 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 showed that the polypeptides encoded by the constructed insertion mutant alleles were all introduced into the outer membrane, as demonstrated 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, because expression of this protein was lethal to the cells and the protein did not function as a pore. In contrast, an insertion in the same place in PhoE with approximately the same amino acids but in a different order, ie in the plasmid pJP29-Cl, did not interfere with the normal uptake of the protein.

The insertion at the BglII site (corresponding to amino acid 280 of the mature PhoE protein) was found to disrupt the binding of two different monoclonal antibodies. Since the binding of another monoclonal antibody and of the phages was not disrupted, nor was the activity of this mutant protein disrupted as a pore, the overall conformation of the mutant protein does not appear to be drastically altered. The 30 amino acids in the vicinity of the amino acid residue 280 are apparently displayed on the surface of the cell and involved in the antigen determinant for the monoclonal antibodies PP1-4 and PP3-4.

The amino acid residue 75 also leads to exhibit on the cell surface and to be involved in the phage receptor activity. An insertion on the PstI

This does not appear to disturb the cell surface related properties of the PhoE protein, consistent with previous observations that the N-terminus of the protein is likely exhibited on the periplasmic side of the membrane.

In two cases, the pore properties of the mutant proteins were slightly altered.

The reduced rate of uptake of cefsulodine by cells containing pJP29-Cl can be explained by a narrowing of the channel due to the addition of four amino acids. The concomitant decrease in cefsulodine uptake and the increase in cephaloridin uptake in the case of pJP29-M1 is more difficult to explain; a possible explanation could be found in a shielding of amino acids involved in the anion specificity of the PhoE pores.

Example II

In this example, it was investigated to what extent extra amino acid residues could be included at the position of amino acid 158 (arginine).

The plasmid pJP29 was used as a starting point.

As indicated in Example I, this contains a normal phoE gene.

The Nrul site contained in this plasmid was removed by cutting the plasmid with Nrul and then treating with exonuclease Bal31. After ligating with T4 DNA ligase, the DNA was used to transform strain CE1224, selecting for chloramphenicol resistant colonies. Plasmid DNA was isolated from one of the transformants. This plasmid, which no longer contained a Nrul site, was designated pMR03.

To place a Nrul site in the phoE gene at the site corresponding to Argl58, the Pstl-35 BglII fragment of pJP29 was donated in the Pstl-BamHX digested M13 vector mp8. Site-directed f; Mutagenesis with a mutagenic oligonucleotide was performed according to the method described by Norris et al., Nucl. Acids Res. 11 (1983) 5103-5112. The desired Nrul site was created. From a obtained mutant plasmid, the Meul-Ndel fragment was then isolated bearing the Nrul site and this fragment was ligated into pMR03 from which the corresponding fragment had been removed. The resulting plasmid was designated pMR05.

This plasmid pMR05 was cut open by incubation with the restriction enzyme Nrul, after which small synthetic DNA linkers were ligated into the cut 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 resp. referred to as pMR08 (contains the BamHI linker), and pMR09 (contains the EcoRI linker). These plasmids each encode four additional amino acids, but in the case of the EcoRI left insert, the 3 bases GAC encoding asp were lost so that pMR09 encodes three additional amino acids net.

The plasmid pMR08 was then cut open with BamHI and a 60 base pair polylinker (corresponding to 20 amino acids) ligated into the cut plasmid which was cut from the plasmid pLL4 by BamHI. This polylinker contains the recognition sequences for many restriction enzymes, greatly simplifying further manipulations. The resulting plasmid is referred to as pMR10. It codes for 24 extra amino acids, compared to the normal phoE gene.

The mutant PhoE proteins, encoded by plasmids 35 pMR08, 09 and 10, were all found to still be transported normally to the cell surface of E. coli ♦ r -24 ”. This was determined by the binding of monoclonal antibodies directed against a portion of PhoE protein displayed on the cell surface. However, the binding of one of these 5 antibodies, namely PP2-1, was found to be disrupted, showing that the region around amino acid 158 (arginine) contains an immunogenic determinant of PhoE.

This example demonstrates that it is possible to insert 10 random peptides into this immunogenic determinant, which may be up to 24 amino acids in length, such that the incorporation of PhoE into the outer membrane is not disrupted.

The construction of these plasmids is shown in FIG. 2 is shown.

In another experiment, a synthetic oligonucleotide with the sequence dTTATAAACAGAAGATCATCGCCCCGGG, and the complementary oligonucleotide were incorporated into the Nrul site of the plasmid pMR05. The oligonucleotide encodes the octapeptide Tyr Lys Gin Lys Ile 20 He Ala Pro, requiring the first base (T) and the last two bases (GG) to maintain the correct reading frame. The PhoE protein in which this determinant of the FMD virus is incorporated was expressed and incorporated into the outer membrane of E.coli.

The E. coli cells were found to react in an ELISA with a monoclonal antibody directed against the FMD virus. In any case, this experiment has demonstrated that the invention can work for diagnostic purposes.

30 of the figures shows FIG. 1 the physical map of the plasmid pJP29. The thick line represents DNA, which is derived from the 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 encoding the signal sequence Ϋ ~ o / 'o 7 * <-25-. The plasmid contains a chloramphenicol marker (Cm), the location 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 corresponding amino acids in the PhoE protein. Furthermore, FIG. 2 the corresponding sections in piasmids pMR05, 08, 09 and 10, showing the changes made.

Fig. 3 shows the nucleotide sequences of the phoE 10 genes of Klebsiella pneumoniae and Enterobacter cloacae, compared to the phoE gene of Escherichia coli.

In the coding regions, only the nucleotides different from the E. coli gene are indicated. In the upstream, non-coding regions, the full sequences for all three genes are shown, regions of strong homology are outlined. The ribosome binding site is underlined, and translation start and stop signals are boxed.

The transcription terminators of E ^. coli and E. cloacae 20 are indicated by arrows.

* '2 y

Claims (22)

1. Recombinant DNA, comprising the genetic information for PhoE protein from Enterobacteriaceae bacteria and, in a region encoding a cell surface located region of the PhoE protein, the genetic information for a foreign antigen determinant. 2. Recombinant DNA according to claim 1, comprising the genetic information for PhoE protein from Escherichia coli bacteria and, in a region localized to the cell surface of the PhoE protein, the genetic information for a foreign antigen determinant. A recombinant DNA according to claim 1, comprising the genetic information for PhoE protein from Escherichia coli K12 bacteria and, in a portion of the gene encoding one of the following amino acid regions of the PhoE protein: (a) amino acids 20 to 34; (b) amino acids 63 to 75; 20 (c) amino acids 105 to 125; (d) amino acids 154 to 163; (e) amino acids 189 to 205; (f) amino acids 229 to 245; (g) amino acids 269 through 283; 25 (h) amino acids 307 through 320; the genetic information for a foreign antigen determinant. 4. Recombinant DNA according to claim 1, comprising the genetic information for PhoE protein from Escherichia coli K12 bacteria and, in a part of the gene encoding amino acid 158 and its nearest neighbors, the genetic information for a foreign antigen determinant. The recombinant DNA according to claim 1, comprising the genetic information for PhoE protein from Escherichia. r? e) J 35 -27- eoli K12 bacteria and, in a part of the gene encoding amino acid 201 and its nearest neighbors, the genetic information for a foreign antigen determinant » The recombinant DNA of claim 1, comprising the genetic information for PhoE protein from Escherichia coli K12 bacteria and, in a portion of the gene encoding amino acid 238 and its closest neighbors, the genetic information for a foreign antigen determinant. The recombinant DNA of claim I, comprising the genetic information for PhoE protein from Escherichia coli K12 bacteria and, in a portion of the gene encoding amino acid 275 and its nearest neighbors, * the genetic information for a foreign antigen determinant. 15 Recombinant DNA according to any one of the preceding claims, wherein the genetic information for a foreign antigen determinant has a length of at most 90 nucleotide pairs. 9. Recombinant DNA according to any one of the preceding claims, which contains the genetic information for an antigen determinant of foot-and-mouth disease virus. The recombinant DNA according to claim 9, which contains the genetic information for an antigen determinant of the capsid protein VP1 of foot-and-mouth disease virus. The recombinant DNA of claim 10, which contains the genetic information for a polypeptide corresponding to amino acids 140-160 or 200-213 of the capsid protein VP1 of foot-and-mouth disease virus. 12. Recombinant DNA according to any one of claims 1-8, which contains the genetic information for an antigen determinant of the syphilis causative agent Treponema pallidum. 13. Recombinant DNA according to any one of claims 1-8, which contains the genetic information for an antigen determinant of the poliovirus, of the measles virus, of the leprosy pathogen Mycobacterium leprae, of meningitis meningococcus and Haemophilus species, of the AIDS virus, : r: · /? -28- of the hepatitis B virus, of the cholera bacterium or of corona viruses. 14. Recombinant plasmid, comprising a vector portion permitting replication and expression in Enterobacteriaceae bacteria and recombinant DNA according to any one of claims 1-13. Enterobacteriaceae bacteria containing one or more recombinant plasmids according to claim 14. 16. Enterobacteriaceae bacteria, which contain one or more recombinant plasmids according to claim 14 and which contain in their outer membrane PhoE carrying a foreign antigen determinant according to the genetic information recorded in said plasmids. 17. PhoE protein carrying a foreign antigen determinant, obtained from Enterobacteriaceae bacteria according to claim 16. Diagnostic device comprising PhoE protein according to claim 17 or, optionally killed, Enterobacteriaceae bacteria according to claim 16. Vaccine comprising PhoE protein according to claim 17 or, optionally killed, Enterobacteriaceae bacteria according to claim 16. 20. Recombinant DNA comprising the genetic information for PhoE protein from Enterobacteriaceae bacteria and at least one restriction enzyme recognition sequence located in a region of the PhoE protein encoding portion of the cell surface. 21. Recombinant plasmid, comprising a vector portion permitting replication and expression in Enterobacteriaceae bacteria, and recombinant DNA according to claim 20. Enterobacteriaceae bacteria containing one or more recombinant plasmids according to claim 21. y 7> 0 7