NL9001404A - NON-TOXIC PSEUDOMONAS EXOTOXINE A. - Google Patents

Description

Title: Non-toxic Pseudomonas exotoxin A.

The present invention relates to a non-toxic Pseudomonas exotoxin A, its preparation and its use in the preparation of a PE vaccine.

Pseudomonas aerucrinosa are gram-negative bacilli. They are widely distributed in nature and comprise about 10% of the normal gut bacteria. Such bacteria rarely infect healthy individuals, but infect those with deficient immunity, such as patients suffering from burns, cystic fibrosis, malignancies, or patients taking immunosuppressive drugs. Pseudomonas aeruginosa produce Pseudomonas exotoxin A (PE) after infection. PE toxin is effective against liver cells and destroys liver function, leading to death. Pseudomonas aeruqinosa are the major pathogens in hospitals. In addition, they have a strong tolerance for most antibiotics; infections usually result in therapeutic difficulties. Vaccination is the best method to solve this problem.

PE is a single polypeptide chain comprising 613 amino acids. The three-dimensional molecular structure of PE is divided into the three structural domains as shown below, namely domain I, which includes domain Ia (residues 1-252) and domain Ib (residues 365-404), domain II (residues 253-364) and domain III (residues 405-613)

Ia II Ib III

0 252 364 404 613 amino acids

Domain Ia is the domain in which the toxin can recognize its receptor on the membrane of toxin-sensitive cells. Domain II is the translocation domain of PE that moves the toxin from the endosome to the cytoplasm. Domain III and the adjacent 20 amino acids (residues 385-404) of domain Ib are the ADP ribosylation enzymatic domains of PE (Gray et al. (1984) Proc. Natl. Acad. Sci. USA £ 1,2645-2649; Hwang et al. (1987) Cell 4, 129-136; Douglas and Collier (1987) J. Bacteriol. 169, 4967-4971; and Chen et al. (1987) J. Gen. Microbiol. 133, 3081-3091).

The mechanism of the PE intoxication process begins with the binding of PE to its specific receptor on the surface of toxin-sensitive cells. The PE receptor complex then enters the cell through receptor-mediated endocytosis. Finally, the PE is translocated to the cytosol where it catalyzes the transition of the ADP ribose unit from NAD + to elongation factor 2. This renders EF-2 inactive in protein synthesis and leads to death of affected cells (Fitz Gerald et al. (1980) Cell 21, 867-873; Fitz Gerald et al. (1982) Infect. Immunol. 35r 715-720;

Morris et al. (1983) Infect. Immunol. 40,806-811;

Eidels et al. (1983) Microbiol. Rev. 47, 596-620; and Middlebrook and Dorland (1984) Microbiol. Rev. 48, 199-221).

The following shows the balance of ADP-ribosylation of EF-2 with PE: EF-2 + NAD + (abundant in cells) === ADP-ribose-EF-2 + nicotinamide + H +

Traditional vaccine preparation methods include detoxification of the pothogens by heat or chemicals, or purification of the surface antigen from the pathogens. Such methods have flaws; for example, toxins remain, the antigenicity is not strong enough, or there is a possibility that those preparing the vaccines may become infected.

Marburg et al. Proposed in 1983 that the PE toxoid prepared by photoaffinity inactivation can be used as a vaccine. The principle consists in producing a covalent bond between NAD + and PE by UV irradiation to cause the loss of ADP ribosylation enzymatic activity of PE (Marburg et al. (1983) Proc. Natl. Acad. Sci. USA 80, A lack of this method, however, is that such a reaction cannot ensure that all PE molecules are photoaffinitively inactivated and therefore toxins remain. Therefore, the PE toxoid prepared by such a method is not suitable.

Today, the best method involves ligating the structural genes of the pathogen's surface antigens into a vector by genetic engineering, transforming the vector into E. coli. yeast or mammalian cells for expression followed by purification. In this invention, non-toxic PE fragments are prepared by this technique and used to prepare a new PE vaccine. The use of such non-toxic PE as a vaccine is absolutely safe and has a strong antigenicity.

This invention relates to non-toxic PEs. Such modified PEs completely lose their ADP ribosylation activity and have no cytotoxicity due to the removal of 36 or more carboxy terminal amino acids.

Such non-toxic PE is isolated from that expressed in the host in which the plasmid with sequential deletions of the structural genes of the carboxy terminus of PE is expressed.

Since the PE without cytotoxicity can block the cytotoxicity of intact PE, it can be used as a vaccine against the diseases caused by PE.

Accordingly, it is an object of the present invention to provide a non-toxic PE with a deletion of 36 or more carboxy terminal amino acids.

It is another object of the present invention to provide a plasmid with sequential deletions of the structural genes of the PE carboxy terminus.

It is yet another object of the present invention to provide a preparation process for the PE.

It is a further object of the present invention to provide non-toxic PE for the preparation of the new PE vaccine.

These and other objects, features and advantages of the present invention will become apparent from the following description, which, together with the accompanying drawings, discloses the presently preferred embodiment of the present invention. It is to be understood that this description is illustrative rather than exhaustive, the scope of the present invention being defined by the claims.

The present invention will be further explained with reference to the pending drawings in which: Fig. 1 (Ά) shows the properties of the structural PE gene; and (B) constructs of plasmids used for the expression of PE with various deletions at the carboxy terminus. pJH4 was cut partially with Hhal. Linearized 5.9 kilobase DNA was then completely digested with EcoRI. DNA fragments ranging from 4.4-5.9 kilobases were isolated and ligated with a synthetic oligonucleotide linker containing stop codons in all three reading frames; Figure 2 shows PE deletone clones expressed by the modified plasmids; Figure 3 shows the ADP ribosylation enzymatic action of the PE of the expression of the modified plasmid in host BL21 (DE3). Full length PE obtained from BL21 (DE3) / pJH4 was used as a positive control and represents 100% ADP ribosylation activity. BL21 (DE3) / - was used as a negative control. The relative ADP ribosylation activities of all other PE fragments obtained from BL21 (DE3) / pJJ1 to BL21 (DE3) / pJJ14 were measured. The specific ADP ribosylation activities of PE deletions were compared to those of full-length PE obtained from BL21 (DE3) / pJH4.

According to the present invention, the non-toxic PE in which 36 or more amino acid residues at the carboxy terminus of PE polypeptide have been removed by recombinant DNA technique retains toxin binding activity, but loses ADP ribosylation enzymatic activity and loses according to its cytotoxicity .

The non-toxic PE of the present invention can be prepared by transforming the plasmid with deletions of the structural PE gene of 36 or more amino acids from the carboxy terminus into a host and expressing the transformed plasmid in the host . The construction of the plasmid involves partial digestion of a plasmid containing the complete coding sequence of mature PE with Hhal and isolation of the linearized DNA; complete digestion of the linearized DNA with EcoRI; and ligation of the DNA fragment with a linker.

The domains of the ADP-ribosylation enzymatic action of PE have been identified as domain III and part of domain Ib. To further determine the portion of the carboxy terminus of the toxin necessary for ADP ribosylation activity, the plasmids constructed according to the present method were individually transferred into E. coli for expression. The size of the deletion in the plasmid was resolved by restriction analysis and size mapping.

The transformants obtained from the expression of the deletion plasmids in hosts were cultured appropriately and the ADP ribosylation activities of the obtained PE clones were determined in an assay (Collier and Kandel (1971) J. Biol. Chem. 246. 1496- 1503). The results indicate that clones with deletions of the carboxy-terminal amino acid residues from Arg-609 to Lys-613 and replaced by Arg-Asn retained a wild-type PE ADP ribosylation activity. Deletion of the terminal amino acid residues from Ala-596 to Lys-613 and replaced by Val-Ile-Asn reduced ADP ribosylation activity by 75%, while deletions of 36 or more amino acids from the carboxy terminus completely lost their ADP ribosylation activity. .

These modified PEs were also tested for their cytotoxicity and ability to block PE cytotoxicity. The results show that modified PEs that lost their ADP ribosylation activity lost correspondingly to their cytotoxicity. In addition, extracts containing PE fragments without ADP ribosylation activity were able to block the cytotoxic activity of intact PE. Based on this principle, the use of the PEs with deletions of 36 or more amino acids from the carboxy terminus is the best choice for new PE vaccines against the diseases caused by PE. In addition, the amount of the modified PE produced by the expression of the 36 amino acid deletion of the carboxy terminus of the PE gene is in β. coli abundant. With regard to safety, the vaccines prepared by this genetic engineering method are absolutely safe, while they are absolutely effective with regard to antigenicity. With regard to production, the costs are not high and the process is easy.

The following examples are given for a better understanding of the present invention and are not intended to limit the scope thereof.

EXAMPLE 1

Construction of plasmids with serial deletions at the 3 'end of the structural PE oen

Plasmids (pJJ1-pJJ14) with serial deletions at the carboxy terminus of the structural PE gene were constructed from the plasmid pJH4, which contains the complete coding sequence of mature PE with an additional methionine at the amino terminus using the method as shown in fig. 1.

Using the sequence data reported by Gray et al. (1984) Proc. Natl. Acad. Sci. USA 81, 2645-2649 we first partially digest pJH4 with Hhal. The linearized 5.9 kilobase DNA was isolated and then completely digested with EcoRl. DNA fragments ranging from 4.4 to 5.9 kilobases were isolated. A synthetic oligonucleotide duplex with cohesive ends for Hhal and EcoR1 was used to ligate the linearized fragments. The synthetic oligonucleotide duplex, used to link the two non-complementary ends, included a universal sequence for the termination of protein synthesis. It contained stop codons in all three reading frames.

pJJ1 to pJJ14 were individually transferred to the host (£. coli) and the deletion size in each plasmid was resolved by restriction analysis and size mapping. The results are shown in Fig. 2.

EXAMPLE 2

The preparation of non-toxic PEs

Plasmids pJJ1 to pJJ14 were individually transferred to host BL21 (DE3), which was lysogenized with a phage (DE3) carrying the T7 RNA polymerase gene under the control of a lacUVS promoter, according to the method of Davis et al. (1986) Basic Methods in Molecular Biology, pp. 90-92, Elsevier Scientific Publishing Co., New York. The resulting transformants were immediately grown in LB broth with 50 μg / ml ampicillin at 37 ° C. When the absorbance at 650 nm reached 0.3, isopropyl-1-thio-BD-galacto-pyranoside (IPTG) was added to a final concentration of 0.5 mM to synthesize T7 polymerase, which is lysogenic on the BL21 (DE3) was a phage to induce. The cells were harvested 90 minutes later to recover the PE clones with serial deletions at the carboxy terminus expressed in BL21 (DE3).

For an assay to ADP ribosylation activity, the general procedure of Collier and Kandel was followed (J. Biol.

Chem. 246, 1496-1503, 1971). Wheat germ extracts, enriched for EF-2, were used as a source of EF-2. Assays (500-μ1 total volume) contain approximately 10 pmol EF-2, 37 pmol [U-14C] NAD + (0.06 μΟΙ), lysate of BL21 (DE3) with various PE clones and buffer (40 mM dithiothreitol, 1 mM EDTA) , and 50 mM Tris, pH 8.1). The cells were lysed with 2 mg / ml lysozyme at room temperature in 50 mM Tris, pH 8.0, and 50 mM EDTA, followed by centrifugation to separate supernatant from pellet. The pellet was then dissolved in 8 M urea. Activity in the supernatant and pellet was measured in picomole ADP-ribose transferred to EF-2 over 30 minutes in separate reactions. After incubation for 30 minutes, 37 ° C, 0.5 ml of 12% trichloroacetic acid was added to each assay mixture. The assay mixtures were then placed in an ice bath for 15 minutes followed by centrifugation at 4 ° C, 3000 g for 10 minutes. The pellet was washed with 1 ml of 6% trichloroacetic acid and centrifuged as above. The 14C content of the pellet was measured in a liquid scintillation counter as an index for the ADP ribosylation activity.

Full-length PE obtained from BL21 (DE3) / pJH4 was used as a positive control and represents 100% ADP ribosylation activity. BL21 (DE3) / - was used as a negative control. The relative ADP ribosylation activity of all other PE fragments obtained from BL21 (DE3) / pJJ1 to BL21 (DE3) / pJJ14 was measured. The specific ADP ribosylation activities of PE deletions were compared to that of PE of full length obtained BL21 (DE3) / pJH4. The results are shown in Fig. 3.

The PE fragment of plasmid pJJ1, which contains a deletion of the carboxy-terminal amino acid residues from Arg-609 to Lys-613 and is replaced by Arg-Asn, retained wild-type PE ADP ribosylation activity. However, a deletion of carboxy-terminal amino acid residues from Ala-569 to Lys-613 and replaced with Val-Ile-Asn dramatically reduced ADP ribosylation activity by 75%. Deletions of 36 or more amino acids from the carboxy terminus all reduced enzymatic activity to the level of BL21 (DE3) host cells exposed to equal induction conditions.

EXAMPLE 3 Cell Protection Assay

Testing of the PE fragments to protect the PE cytotoxicity was performed in Swiss 3T3 cells.

Swiss 3T3 cells were "seeded" 24 hours before the protection assay. Each well contained 2 x 104 cells. After incubation for 72 hours in the presence of different concentrations of PE alone or with BL21 (DE3) cell extracts containing PE fragments, the monolayers were then stained with methylene blue to detect the surviving cells.

The pJJ3 and pJJ4 deletion fragments, which did not retain any ADP ribosylation activity, were also found to have lost their cytotoxicity. However, these deletion fragments retained their ability to competitively block the cytotoxicity of native PE on Swiss 3T3 cells. At higher concentrations, the modified fragments produced by plasmids pJJ3 and pJJ4 could effectively prevent intoxication by PE.

While the invention has been described with respect to certain preferred examples and embodiments, the invention is not intended to be limited to this scope, but only to the claims which follow hereafter.

Claims (9)

Non-toxic Pseudomonas exotoxin A (PE), characterized in that it is a PE polypeptide chain with a deletion of 36 or more amino acid residues from the carboxy terminus, and is capable of blocking the cytotoxicity of intact PE . The use of a PE according to claim 1 in the preparation of a PE vaccine. 3. Plasmid with deletions of the structural PE gene of 36 or more amino acids from the carboxy terminus. The method of constructing the plasmid according to claim 3, comprising: (A) partial digestion of a plasmid containing the complete coding sequence of mature PE with Hhal and isolation of linearized DNA; (B) complete digestion of the linearized DNA with EcoRI; (C) Ligation of the DNA fragment with a linker to obtain the desired plasmid. The method of claim 4, wherein the plasmid containing the fully encoding sequence of mature PE is pJH4. The method of claim 4, wherein the linker is a synthetic oligonucleotide duplex comprising a universal protein synthesis termination sequence. The method of claim 6, wherein the linker is 31TAATTAATTAG GCATTAATTAATCTTAA 5 '. A method of preparing a non-toxic PE comprising transforming the plasmid of claim 3 into a host and expressing the transformed plasmid in the host to obtain the deleted PE. The method of claim 8, wherein the host is E. coli.