LU87207A1 - VACCINE DERIVED FROM BACTERIA OF THE GENUS YERSINIA PROCESS FOR ITS PREPARATION AND APPLICATIONS - Google Patents

Description

VACCINE DERIVED FROM BACTERIA OF THE GENUS YERSINIA, PROCESS FOR ITS PREPARATION AND APPLICATIONS.

Technological background

The bacteria of the genus Yersinia are Gram-negative bacteria belonging to the family of enterobacteria. Three species are pathogenic for humans: Y. pestis is the agent of plague; Y. pseudotuberculosis is the agent of pseudotuberculosis and Y. enterocolitica is an agent of gastroenteritis. Y. enterocolitica is a relatively common enteropathogenic agent. Its distribution is cosmopolitan and it includes ubiquitous strains and strains pathogenic for humans and animals. Due to its phenotypic heterogeneity, the species is subdivided into serogroups on the basis of somatic antigens (Ag 0) lipopolysaccharides. Only a few serogroups, including 0: 3, 0: 5.27, 0: 8 and 0: 9, prove to be pathogenic for humans and animals. The 0: 8 serogroup strains, isolated exclusively in North America, appear more virulent than the others.

The pathogenic power of Y, enterocolitica is of an invasive type. After oral inoculation, the bacteria adhere to the surface of the intestinal epithelium. By an endocytosis phenomenon, they enter the enterocytes and cross the cytoplasm, enclosed in vacuoles. The presence of Y. enterocolitica in the lamina propria causes an inflammatory reaction there. Bacteria capable of neutralizing the host's immune system mainly multiply in mesenteric lymph nodes.

30 Y. enterocolitica infections result in an enteric syndrome consisting of abdominal pain sometimes accompanied by diarrhea, mesenteric adenitis or acute terminal ileitis.

The pathogenic power of Y. enterocolitica is close to that of Y. pseudotuberculosis, responsible for pseudotuberculosis, and strongly recalls that of Y. pestis, plague agent. Indeed, in all cases, we observe a capacity to cross the epithelial barrier 2 and cause adenitis. The relatively low virulence of Y. enterocolitica has led many researchers to choose this bacterium as a model for the study of invasiveness.

5 The invasiveness of Y. enterocolitica is associated with the presence of a plasmid (pYVe, Virulence-associa-ted plasmid of Y. enterocolitica) of approximately 70 kilobases (kb). DNA-DNA hybridizations show a sequence homology of 90% between the plasmids of the strains of sero-00 groups 0: 3, 0: 5.27 and 0: 9, and 55% between these and the plasmid of Y , enterocolitica of the serogroup 0: 8. Plasmids structurally and phenotypically close to the latter are present in Y. pseudotuberculosis and Y. pestis. They are highly related to each other and are 55% homologous with the plasmid of Y. enterocolitica 0: 8.

The functions allowing the propagation of the plasmids (replication functions) and the functions determining its capacity of coexistence with other plas-20 mides in the same bacterium (incompatibility function) were located on the Yersiniae plasmids.

The virulence plasmid of Y, enterocolitica confers on the bacterium several properties.

The virulent strains of Y. enterocolitica 25 require calcium ions for their multiplication at 37 ° C. This phenotype of calcium dependence (Ca D) is found in Y. pseudotuberculosis and Y. pestis. The plasmid region which determines this property extends over approximately 20 kb and is particularly well conserved among the 3 species of Yersinia (FIG. 1). The mechanisms of calcium dependence are not yet explained. The fact that the plasmids of the three Yersinia species determine a calcium requirement for growth at 37 ° C. and that the region of the plasmid conferring this phenotype is highly conserved suggests that a strong selection pressure plays in favor of this phenotype.

The plasmid of Y. enterocolitica codes for at least twenty polypeptides including a cytoplasmic protein 3, the antigen V, and proteins of external membrane which were designated by the initials YOP (for Yersinia Outer membrane Protein) or POMP (for Plasmid encoded Outer Membrane Protein) by the authors who studied them in Y. pseudotuberculosis. In Y. enteroco-litica, seven YOP (or POMP) have been detected which are identified by the acronym YOP (or POMP) followed by the molecular weight in thousands of daltons. For two of these proteins, however, the numbers introduced by other authors have been kept because they are relatively well characterized or because their structural gene has been localized. They are the protein YOP1 (product of the yopA gene) which forms external appendages, and the protein YOP4, product of the gene yopD which has been localized on the plasmid of Y ^ enterocolitica and Y. pseudotuberculosis. In Y. entero-colitica, Y0P1 has a PM of 2 40 kilodaltons (Kda) while it is 150 Kda in Y, pseudotuberculosis. The PM of YOP4 is 37.5 kda in Y. enterocolitica and 36 kda in Y. pseudotuberculosis. The structural genes of other Y. enterocolitica YOPs were located on the plasmid pYVe.

Antibodies to YOPs are present in the serum of patients with yersiniosis. The proteins of Y. enterocolitica are immunologically related to the outer membrane proteins detected in Y. pseudotuberculosis and Y. pestis.

The expression of the outer membrane proteins of Y. enterocolitica depends on the temperature and the calcium concentration in the culture medium. These proteins appear only after incubation of the bacteria at 37 ° C. in a medium deficient in calcium.

The invasiveness of Y. enterocolitica is a multi-stage phenomenon. Several of them whose adhesion to epithelial cells and resistance to phagocyto-35 se by polymorphonuclear cells seem to require the presence of proteins from the outer membrane.

The phenomenon of dependence on calcium is associated with the pathogenic power of Y. enterocolitica.

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In animal models, the strains independent from calcium (Ca I) appear to be avirulent and only produce the protein Y0P1. Analysis of insertion mutants in the plasmid of Y. enterocolitica 0; 9 made it possible to define three transcription zones (virA, B and C) in the region which codes for the phenomenon of Ca D (FIG. 1). The temperature controls their expression at the level of transcription. On the contrary, calcium ions have little influence on it. These experiments also show that the expression of most of the outer membrane proteins encoded by the plasmid is controlled by the transcription units virA, B and C.

Results obtained in Y. pseudotuberculosis and Y · pestis show that the regions determining the phe-

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notype Ca D in the three species are structurally and functionally very close. The structural genes of the YOPs are located on the pYV plasmids of Y. pseudotuberculosis and Y. pestis outside the region governing the Ca D phenotype. It therefore appears that the Ca 2 0 D region contains the control genes of a regulon, induced by temperature and controlling the expression of outer membrane proteins.

The proteins YOPs have been reported to appear in the outer membrane of Y. enterocolitica in-2 R

cubed at 37 ° C. in the absence of calcium. It has also been shown that, under the same conditions, Y. enterocolitica releases a certain number of proteins into the culture supernatant. The released proteins have also been shown to include the YOPs protein and at least one other protein which had not been detected in the outer membrane.

The form in which proteins are present in the culture supernatant is unknown. However, it appears that these proteins are not released in a soluble form but are assembled in the form of vesicles or aggregates. Curiously, the YOPs appear in the culture supernatant before appearing in the outer membrane. It is therefore clear that the export of these proteins implies the existence of a particular secretion system. This system could be coded by the region determining the phenotype of Ca D.

5 Object of the invention

Bacteria of the genus Yersinia and in particular of the species Y. enterocolitica exhibit a series of interesting properties, in particular those of (1) crossing the intestinal barrier without damaging the mucosa and activating the immune system; (2) export in large quantities proteins encoded by the plasmid pYV into the external membrane and even the culture medium; (3) have a regulatory system which allows the production of these proteins in vivo, in particular during contact with the immune system.

The invention proposes to take advantage of these properties for the construction of an immunogenic system. The aim is in particular to use Y. enterocoliti-20 ca as a vector for the propagation of protective antigens by subjecting them to the regulation and export characteristic of the proteins encoded by the virulence plasmid of this bacterium. According to the invention, this can be achieved by inserting the genes of the protector antigens into the plasmid of Yersinia and in particular of Y. enterocolitica. Conceptually, this project is in line with vaccines based on the vaccinia system.

Characteristic Elements of the Invention The invention relates more particularly to a vaccine comprising bacteria of the genus Yersinia in which at least one of the plasmid genes coding for an outer membrane protein is replaced by at least one bacterial or viral gene coding for a protein or an epi-35 tope against which one wishes to vaccinate.

More particularly, Yersinia enterocolitica or Yersinia pseudotuberculosis is used.

In the following, when the description refers to the use of Yersinia enterocolitica, the Yersinia pseudotuberculosis can also be substituted for it.

To make such a vaccine, it is generally necessary to attenuate the virulence of Y. enterocolitica and to insert the foreign genes into the plasmid. This attenuation can be obtained by any conventional attenuation technique.

However, it is observed that generally the replacement of one or the other protein encoded by the plasmid by an immunogen already results in a notable reduction in the virulence of Y. enterocolitica. This therefore constitutes an important additional advantage obtained by the technique of the invention.

Examples of vaccines which can be produced in this way include those obtained with the genes for the B subunit of the CT toxin from Vibrio cholerae and the LT enterotoxin from E. coli, the hepatitis B virus surface antigen (HBsAg) and the CS surface protein of Plasmodium falciparum. This list is of course in no way limiting and other possibilities such as the genes which have already been studied for vaccinia can be envisaged such as those of HBsAg, 1 hemagglutinin of the influenza virus and herpes simplex virus glycoprotein D which have been integrated into the genome of vaccinia virus. These genes are expressed by infected cells in vitro and their products are integrated into the cell membrane or secreted into the surrounding environment. Tests on laboratory animals (rabbits, hamsters, mice) show the appearance of a high level of antibodies and protection against the disease. The envelope gene for the AIDS virus has also been integrated into the vaccinia genome. Mice infected with these recombinant viruses produce antibodies specifically recognizing the envelope proteins of the AIDS virus.

The operating techniques to be used are generally those of biological engineering well known to those skilled in the art. They will however be 7 illustrated below in the examples.

According to another aspect of the present invention, it also relates to a process for the preparation of such a vaccine, in which at least one of the plasmid genes coding for an outer membrane protein is replaced in bacteria of the genus Yersinia by at least minus a bacterial or viral gene encoding a protein or an epitope against which it is desired to vaccinate.

In another aspect, the invention also relates to the use of antigens purified from 10 strains of recombinant Y. enterocolitica or their culture supernatant for the production of diagnostic kits for detecting specific antibodies. Example

In the plasmid of Y. enterocoli-15 tica 0: 9 (pYVe09), the genes of the proteins exported in the outer membrane and the culture medium (yop) are replaced by the genes of protective antigens (qap). In this way, the virulence of Y, enterocolitica will be attenuated and the expression of the qap gene will be under the control of the plasmid pYVeO: 9.

at. The study of yop genes

By mutagenesis, the yop structural genes can be located on pYVe09. Virulence tests make it possible to choose one or more of these genes. The 25 genes whose inactivation results in an attenuation of the pathogenic power are selected.

In order to locate the regulatory signals and the open reading phase of these genes, they are cloned and sequenced.

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This step determines where and how to insert the qap gene into the yop gene.

b. Placing the qap gene under the control of yop signals.

The yop gene cloned onto a mobilizable plasmid, is replaced by the gap gene, while retaining the regulatory signals of the yop gene. The plasmid is next. i 8 introduced into a strain of Y. enterocolitica 0: 9 and the expression of qap analyzed in vitro.

vs. The placement of the qap gene in pYVe09.

In order to eliminate unnecessary plasmids and antibiotic resistance, the qap gene is introduced into pYVe09 according to the method of Ruvkun and Ausubel (1981).

d. The in vivo study of recombinants.

The in vivo study of recombinants begins with the analysis of the humoral response of animals that have received the recombinant bacteria per 10 bones. The presence in their serum of antibodies directed against the protective antigen propagated by Y. enterocolitica attests at first, to the validity of the system. These antibodies are detected by ELISA or RIA tests, 1) Virulence test.

The plasmid pGB63 is a conjugate cointegrate containing the plasmid R388 and the virulence plasmid of Y. enterocolitica W22 708 (serogroup 0: 9) labeled with the transposon Tn3 (pYVe22 708 :: Tn3). Mutagenesis by insertion of a Mini-Mu dlac into the plasmid pGB63 makes it possible to obtain various mutants. Two of these transposants are affected in the yop 51 and yop2 5 genes of pYVe22708, respectively. These 2 mutations result in the absence of the corresponding protein in the culture supernatant. The virulence of these two strains is tested on mice according to the method of Robins-Brown and Prpic (1985). The strain with the best attenuation of pathogenicity is kept for the following experiments. 2) Exact location of the yop gene.

The genes yop51 and yop2 5 were located respectively on the EcoRI E3 and E2 fragments. These fragments were cloned into the vector pACYC194 and introduced into the strain Y. enterocolitica W22708 cleared from its virulence plasmid. The clones obtained do not express any YOP. However, when the cointegrate containing the virulence plasmid mutated at the level of the yop51 gene is introduced into Y. enterocolitica (pACYCI84-E3) by conjugation, the 9 * ‘t conjugates release the yop51 protein into the culture medium.

The plasmid of the mutated strain is introduced at the level of yop51, into Y. enterocolitica (pACYCI84-E3). Y0P51 is then searched by electrophoretic analysis of the proteins of the culture supernatant (SDS-PAGE).

Then, in order to locate the yop51 and yop2 5 genes more precisely on the EcoRX E2 and E3 fragments, these fragments are cloned onto a mobilizable type 10 plasmid (see below) and subjected to mutagenesis by the pMBLG21 transposition system ( see below).

The mobilizable plasmids in question have a mobilization site (mob). Introduced into certain strains of E. coli, they become mobilizable thanks to the trans complementation of the RP4 transfer genes. To do this, it is possible to use the plasmid RP4 itself or one of its derivatives, one or the other unwanted resistance gene of which has been inactivated by insertion of transposon lactose Tn 951.

Plasmid pMBLG31 is a heat-sensitive replicating conjugate plasmid which contains the depressable transposable element Tn2506 conferring resistance to chloram-phenicol. The transposon is defective and complemented by a transposition gene located on the vector plasmid, outside of the transposon. The mutations generated are therefore stable.

Cloning and mutagenesis are carried out as follows: - cloning of the E2 and E3 fragments at the EcoRI site located in the chloramphenicol resistance gene of the mobilizable plasmids; 30 - transformation of E. coli S17-1 with the recombinant plasmids and then introduction by conjugation of the plasmid 'pMBLG31; incubation of the transformants at 42 ° C. on medium containing chloramphenicol in order to eliminate pMBLG31 and to select for the insertion of Tn2506; - introduction by conjugation of the transposants into the strain of Y. enterocolitica having the virulence plasmid mutated at the level of the corresponding yop gene; 4 ♦ 10 - analysis by electrophoresis (SDS-PAGE) of the culture supernatants of the conjugates to seek the expression of the proteins YOPs; - study by restriction of transposants whose yop 5 gene has been altered (gene localization). The gene is sequenced by the method of Sänger and Coulson (1977) in order to search for the regulatory signals of the yop gene (promoter, signal sequence ...) and the open reading phase.

A recombinant plasmid carrying the yop gene is cleaved at the level of the yop gene, either downstream of the presumed regulatory signals, or even downstream of the first codons of the structural gene; exposed to exonuclease activity

Bal 131 so as to obtain ends allowing subsequent cloning in the three phases and supplemented with linkers adapted to the gap gene to be cloned.

The gap gene is then inserted into this plasmid.

The new plasmid obtained is introduced into the strain of Y. enterocolitica containing the plasmid of pYVe09 mutated at the level of the yop gene.

Among the recombinant plasmids introduced into Yersinia, those in which the cloning was carried out in phase are sought. To do this, we look for the presence of protective Ag by "immunoblotting" on colonies. The subsequent analysis of the clones obtained consists of; - locate the insertion of gap in the yop gene by enzymatic restriction or sequencing; - study in vitro the conditions of expression (temperature-30 re, concentration of calcium ions) and the cellular localization of the product obtained (cytoplasm, external membrane, extracellular environment). These analyzes are carried out by electrophoresis (SDS-PAGE) and "Western blot".

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Claims (14)

11 “CLAIMS. 1. Vaccine characterized in that it comprises bacteria of the genus Yersinia in which at least one of the plasmid genes coding for an outer membrane protein is replaced by at least one bacterial or viral gene coding for a protein or an epitope against which wish to vaccinate. 2. Vaccine according to claim 1 characterized in that the bacteria of the genus Yersinia are the bacteria Yersinia enterocolitica or Yersinia pseudotuberculosis. 1 0 3. Vaccine according to any one of claims 1 or 2 characterized in that the virulence of bacteria of the genus Yersinia is attenuated in a conventional manner. 4. Vaccine according to any one of claims 1 or 3 characterized in that the virulence of bacteria of the genus Yersinia is attenuated by the replacement of one or the other protein encoded by the plasmid by an immunogen. 5. Vaccine according to any one of the preceding claims, characterized in that the bacterial or viral genes are the subunit B of the CT toxin of Vibrio cholerae and of the enterotoxin LT of E. coli, hepatitis B virus surface antigen (HBsAg) or CS surface protein of Plasmodium falciparum. 6. Vaccine according to any one of the claims 2 5 1 or 4 characterized in that the bacterial or viral genes are those of HBsAg / hemagglutinin of the influenza virus and glycoprotein D of the virus l herpes simplex which have been integrated into the genome of the vaccinia virus, 30,, 7. Method for preparing a vaccine according to any one of the preceding claims, characterized in that at least one of the plasmid genes coding for an outer membrane protein is replaced in bacteria of the genus Yersinia by at least one bacteria gene -35 nothing or viral encoding a protein or an epitope against which one wishes to vaccinate. 8. Method according to claim 7 characterized "'’' -f 12 in that the bacteria Yersinia enterocolitica or Yersinia pseudotuberculosis are used. 9. Pharmaceutical preparation containing at least the vaccine according to any one of claims 1 to 6. 10 Purified antigens obtained from a recombinant y.enterocolitica strain or its culture supernatant. 11. Use of purified antigens according to claim 10 for the production of diagnostic kits. 10 12. Yersinia modified by action on the plasmid suitable as a vaccine. 13. A method for modifying bacteria of the genus Yersinia, characterized in that one of the plasmid genes coding for an outer membrane protein is replaced by at least one bacterial or viral gene coding for a protein or an epitope. 14. Use of modified Yersinia in vaccines. 2 0 2 5 30 35