AU654392B2 - Enzymatically-inactive phospholipase D of corynebacterium pseudotuberculosis - Google Patents

Description

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A UST R ALI A PATENT ACT 1990 COMPLETE SPECIFICATION Original Name of Applicant/Nominated Person: Actual Inventor (s) CSL LIMITED Jill A. HAYNES; Jo TKALCEVIC; Ian T. NISBET DAVIES COLLISON CAVE, Patent Attorneys 1 Little Collins Street, Melbourne. Vic. 3000.

Address for Service: Invention Title: C C C C I I "Enzymatically-inactive phospholipase D of Cortjnebacterium pseudotubercuZos1is The following statement is a full description of this invention, including the best method of performing it known to us: 920922Zp:\operims,PK8484/91,2 la- "ENZYMATICALLY-INACTIVE PHOSPHOLIPASE D OF CORYNEBACTERIUM PSEUDOTUBERCULOSIS' This invention relates to enzymatically-inactive analogues of phospholipase D (PLD) of Corynebacteriun pseudotuberculosis, and to methods for the production thereof, particularly by mutagenesis of the PLD gene followed by expression of the mutated gene, for example in Escherichia coli ii, ;-i ir I Ii Corynebacterium pseudotuberculosis is the aetiological agent of caseous lymphadenitis (CLA), a significant and common disease of sheep and goats. It produces an exotoxin, phospholipase D (PLD), which is an important factor in the dissemination of the organism (Hsu et al, 1985; Batey, 1986). This is exemplified by the fact that sheep can be vaccinated against caseous lymphadenitis using detoxified PLD, either as a crude culture supernatant or in a purified form (Batey, 1986; Eggleton et al, 1991).

The gene coding for PLD has recently been cloned, sequenced and expressed in E.coli [International Patent Application No. PCT/AU90/00121 (WO 90/11351); see also Hodgson et al, 1990; Songer et al, 1990]. The gene has been shown to encode a 24 amino acid residue signal sequence and a 259 amino acid residue (31.4 kDa) polypeptide. The nucleotide and predicted amino acid sequences of PLD are shown in Figure 4 of the International Patent Application No. PCT/AU90/00121, and are incorporated herein by reference. The numbering of amino acid and nucleotide positions used throughout this specification is in accordance with Figure 4 of International Patent Application No. PCT/AU90/00121. This work has made it feasible to consider vaccines based upon genetically-toxoided PLD molecules. Such an approach has been demonstrated with other bacterial toxins, such as diphtheria and pertussis toxins, where changing one or two amino acids at specific sites 920922,jmsspe.001,PLD,1 t .Cb P -'w d i; r* -2has been shown to produce nontoxic molecules which are still immunogenic (Tweten et al, 1985; Pizza et al., 1989). In work leading to the present invention, random chemical mutagenesis of the PLD gene has been used as a means of producing enzymaticallyinactive PLD analogues.

In accordance with the present invention, there is provided a mutant Corynebacterium pseudotuberculosis phospholipase D which is enzymatically-inactive and which exhibits the immunogenicity of phospholipase D, wherein in said mutant phospholipase D at least one of the histidine residues by wild-type phospholipase D is replaced by a different amino acid residue, for example the histidine residue at position is replaced by a tyrosine residue.

In another aspect, this invention provides a vaccine composition for use against caseous lymphadenitis (CLA) in sheep, which comprises a mutant phospholipase D as described above, and optionally an adjuvant in an acceptable carrier or diluent.

In such vaccine composition, the mutant phospholipase D may be one component of a multi component vaccine. For example, the formulation may include additional vaccine antigens such as those involved in the protection against clostridial diseases.

*0 i ft This invention also extends to a method of immunising sheep against CLA which comprises administration of an effective amount of a vaccine composition as described above.

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940823,q:\oper\jms,25290-p.234,2 3 -3- In yet another aspect, this invention provides an isolated nucleotide sequence coding for a mutant Corynebacterium pseudotuberculosis phospholipase D preduc which is enzymatically-inactive and which exhibits the immunogenicity of phospholipase D.

In one embodiment of this aspect of the invention there is provided an isolated nucleotide sequence coding for a mutant phospholipase D pr-edet in which at least one histidine residue in wild-type phospholipase D, for example, the histidine residue at position 20, is replaced by a different amino acid residue, for example a tyrosine residue. In such a nucleotide sequence, the cytosine base at nucleotide position 129 of the PLD gene is replaced by a thymine base resulting in an amino acid change at amino acid 20 from histidine to tyrosine.

This invention also extends to recombinant DNA molecule including the nucleotide sequence described above operatively associated with an expression control sequence, to a cloning vector or vehicle including such a recombinant DNA molecule, and to a host cell (such as Escherichia coli) transformed with such a vector or vehicle. Expression of the recombinant DNA molecule in such a host cell enables production of the mutant phospholipase D preodct as a synthetic or recombinant product suited for use in a vaccine composition as described above.

In addition, other forms of CLA vaccine may be based upon the mutant 25 phospholipase D product of this invention. For example, a live attenuated Svaccine may be produced by the expression of the mutant phospholipase D pr- t in a non-toxigenic strain of Corynebacterium pseudotuberculosis of any other non pathogenic host organism. The host organism would thus serve to produce the deliver the mutant PLD preduet within the vaccinated animal, thereby resulting in a positive immune response.

%j :920922,jmsspe.001,PLD,3 :21= -4- In this embodiment, this invention provides a live vaccine composition comprising a live vaccine vector having inserted therein a nucleotide sequence coding for a mutant Corynebacterium pseudotuberculosis phospholipase D -proeduct as described above, together with an expression control sequence for in vivo expression of said mutant phospholipase D.pr- det.

The live vaccine vector may be a non-pathogenic viral or bacterial vector, for example an attenuated organism.

The invention further extends to a method of immunising sheep against CLA which comprises administration of an effective amount of a live vaccine composition as described above.

00:0 In the case of expression of the mutant PLD product by non-toxigenic Ce.o Corynebacterium pseudotuberculosis host cells, for use as a live attenuated vaccine as described above, several suitable vectors are available. These include, but are not limited to, vectors based upon or derived from the plasmids pCSL17 (International Patent Application PCT/AU90/00121), pHY416 (Yoshihama et al., J.Bacteriol 162: 591-597; 1965) and pNG2 20 (Serwold-Davies et al. Proc. Natl. Acad. Sci. (USA) 84, 4964-4968; 1987).

Alternatively, mutant PLD gene(s) may be retained within the non-toxigenic host by insertion into the host cell chromosome.

In the work leading to the present invention, the gene encoding the 25 phosphclipase D of Corynebacterium pseudotuberculosis was mutagenised using j formic acid and then expressed in Escherichia coli Mutagenesis was targeted L-at the region coding for mature PLD under conditions designed to produce only one or a limited number of point mutations. Transformants were screened for the enzymetic and immunological properties of their PLD products. One clone was found to produce a protein which was enzymaticallyinactive but which was of equivalent size and antigenicity as PLD. The modified PLD gene was completely sequenced, revealing a single base change 920922,jmsspe.001,PLD,4 from the native PLD gene. As a consequence, the codon for histidine 20 was converted to a tyrosine codon. These results suggest that histidine 20 forms part of the active site of the PLD molecule.

Further details of the present invention will be apparent from the following Example, and from the accompanying drawings, in which: Figure 1 shows Western blots comparing mutated PLD with wildtype PLD. Proteins bound to nitrocellulose were probed with (A) hyperimmune rabbit antisera against PLD or high titre antisera from vaccinated sheep. Samples: Al, purified PLD; A2, Bl, Corynebacterium pseudotuberculosis culture supernatant; A3, B3, periplasmic extract from CSL170 (wild-type); A4, B2 periplasmic extract from CSL315 (mutant).

S 15 Figure 2 shows the nucleotide and predicted amino acid sequence of wild-type and mutated PLD gene around the codon for amino acid DNA sequencing was carried out using double-stranded DNA, primers based upon the PLD sequence and the Sequenase procedure (United States Biochemicals). The mutated sequences are underlined.

EXAMPLE 1 *Production and screening of mutants, In order to restrict the screening process to mutations in the mature S 25 PLD product, the following mutagenesis and screening process was devised.

I 'Single-stranded DNA from plasmids pCSL39 and pCSL40 (pUC118 containing either orientation of the PLD gene on a 1.5 kb SacI fragment, as described in Hodgson et al, 1990) was prepared by infecting plasmid transformed cells with M13K07 helper phage. Formic acid mutagenesis was performed basically as per Myers et al, 1985. Single-stranded DNA was treated with 12M formic acid for 5 mins in a total volume of 20ul at room temperature. The reaction was then stopped and the DNA precipitated by the addition of 20ul of 920922,jmsspe.001,PLD,5 -6sodium acetate, pH 7; 5ul of 20mg/ml glycogen: 55ul of distilled water; and 200ul of ethanol. The DNA was precipitated twice more with ethanol, then dried, Using the universal sequencing primer, a second DNA strand was filled in.

The double stranded, modified PLD gene was cleaved out by restriction enzyme digestion with PvuIT/SacI, yielding a 0.8kb fragment. PvuII cleaves the PLD gene within the coding region for the signal sequence, immediately upstream of the signal cleavage site. Thus, the PvuII/SacI fragment contained just the coding sequence for the mature PLD product. This fragment was cloned via a three-way ligation into pCSL33 (Hodgson et al, 1990) in place of the existing PvuII/Xbal fragment, thus restoring an unmodified PLD promoter and signal sequence and an intact plasmid backbone.

15 The DNA was transformed into E.coliDH5a and transformants were o 4 S' screened for toxin production using the synergistic haemolytic assay. In this assay, plates were prepared by adding sheep erythrocytes and filtered culture supernatant from Rhodococcus equi, which had been cultured in brain heart infusion broth (Difco), to Luria agar, as per Egen et al, 1989.

20 Transformants producing functional PLD were seen surrounded by zones of hemolysis, following 24 to 48 h of incubation at 35 OC. Around 80% of transformant colonies were lysis negative and 4000 were chosen for further screening. Approximately 200 of these transformants were screened by colony hybridisation with a labelled oligonucleotide to the PLD gene, thereby S 25 confirming that the PLD gene was present in most of lysis negative clones (results not shown).

All lysis negative colonies were then immunoscreened using hyperimmune rabbit sera obtained by dosing a rabbit with purified PLD (Hodgson et al, 1990). Around 500 of lysis negative colonies were positive by immunoscreening. These clones were further assayed for PLD functional activity using a modified Zaki test and a sphingomyelinase assay, 92092 2 nws._OO,PI),W ~7I -7and PLD antigenic activity using an enzyme immunoassay (EIA) and a Western blot using hyperimmune rabbit sera. One isolate was obtained which exhibited no enzymatic activity but still had immunological activity. The plasmid coding for this protein was designated pCSL70 and the transformed host strain CSL315. Table I shows a comparison between the activity of th,; mutant PLD and wild-type PLD.

The mutated protein reacted as strongly in an EIA as wildtype but had no detectable enzymatic activity in a Zaki test or sphingomyelinase assay. The Western blots in Fig 1. show that the mutated PLD was indistinguishable from wildtype PLD by Western blot in terms of size or reactivity with hyperimmune rabbit sera. The mutant protein also reacted with high titre sheep sera, although it did not appear to react as strongly as the wild-type.

t r S' 15 Sequence analysis of mutated PLD.

cQ The PLD gene contained on pCSL70 was sequenced concurrently with the nonmutated gene from pCSL33. The only difference detected between the two sequences was a transition at position 129 (relative to the of the 'ATG' being nucleotide 1) where a cytosine in the wild-type sequence was changed to a thymine in the mutant sequence. This mutational event creates an amino acid change at position 20, from a histidine in the wild-type to a tyrosine in the mutant (Fig 2).

Given that the mutant PLD appears to be structurally-intact but S 25 enzymatically-inactive, this result provides strong evidence that His-20 is part of the active site of the PLD molecule. Such a situation is consistent with observations that histidine has a major role as a catalytic residue in enzymes.

According to Marketa et al, 1988, histidine represents 33% of all known catalytic residues although overall it represents only 2.3% of residues in the same enzymes. F"'rthermore, they suggest that ten percent of all histidine residues in enzymes ere involved in the catalytic mechanism, and another bind the essential metal ion. As PLD contains 9 histidines in all, and is known 920922,jmsspe.001,PLD,7

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-8to require Mg2+ for activity, it may be that other histidines as well as are required for PLD enzymatic activity.

With regard to other phospholipases, a functional role for histidine has also been observed. Phospholipase A 2 molecules contain a conserved histidine residue at position 48 which is involved in catalytic activity (Waite, 1987). As previously noted (Hodgson et al, 1990), there is a minor degree of homology between C. pseudotuberculosis PLD and mammalian PLA. However, this does not encompass the region of His-20 in PLD or His-48 in PLA.

TABLE I. Comparison of the activity of periplasmic extracts a from CSL170 and CSL315.

Assay CSL170(wildtype) CSL315 Zaki test 640 <2 t Sphingomylinase assayc 4ug/ml

ND

S: EIA d 64 64 a. Periplasmic extracts were performed as described by Kendall et al, 1986.

b. The Zaki test, which is an anti-haemolysin inhibition assay, was performed as por Hodgson et al, 1990. The titre shown is the inverse of the greatest dilution which prevents red cell lysis by staphylococcal Blysin.

c. The sphingomyelinase y was based on Gatt et al, 1978, using the chromogenic substrate trinitro-phenylaminolauryl sphingomyelin as substrate. This was able to detect down to 50ng sphir.gomyelinase activity. ND Not detectable. d. The EIA used a high titre polyclonal sheep antiserum as the capture antibody. Controls consisting of knov., quantities of purified PLD from C. pseudotuberculosis were included in all assays. The titre shown is the 920922,jmsspe.001,PLD,8 L

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REFERENCES

Batey, Pathogenesis of caseous, lymphadenitis in sheep and goats. Aust. Vet.

J. 63 (1986) 269-272 Egen, Cuevas, McNamara, Sammons, Humphrey, R. and Songer, Purification of the phospholipase D of Corynebacterium pseudotuberculosis by recycling isoelectric focusing. Am.. J. Vet. Res. 50 (1989) 1319-1322.

Eggleton, Haynesj.A., Middleton, H.D. and Cox,,I,C.: Immunisation against caseous lym-phadenitis: Corr-elation between Corynebacterium pseudotuberculosis toxoid content and protective efficacy in combined clostridial- 0 corynebacterial vaccines. Aust. Vet. J. (i'991) in press.

2' Gatt, Dinur, T.,and Barenholz, A spectrophotometric method for determination of sphingomyelin ase. B iochim. B iophys. Acta 530 (1978) 503-507.

Hodgson, Bird, P. and Nisbet, Cloning, nucleotide sequence, and expression in Escherichia coli of the phospholipase D gene from C'orynebactedriu pseudotuberculosis J. Bact. 172 (1990) 1256-1261.

Hsbu, T.Y.,Renshaw, Livingston,C'.L., Augustine, Zink, D.L. and Gauer, B Corynebacterium pseudotuberculosis exotoxin: fatal h aemolytic aemia induced in gnotobiotic neonatal small ruminants by parenteral administration of preparations containing exotoxin. Am. J. Vet. Res. 46 (1985) 1206-1211.

Kendall, Bock, S.K. and Kaiser, Idealisation of the hydrophobic segment of the alkaline phosphatase signal peptide. Nature ('Iondon) 321 (1986) 706-708.

920922,jmsspe.001,PLD,10 .1X (a member of the firm of DAVIES COLLISON CAVE for and on behalf of the Applicant).

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is',) $4 00 40 4 VI 4 V $4 V $4 Marketa, Zvelebil, M.J. and Sternberg, Analysis and prediction of the location of catalytic residues in enzymes. Protein Engineering 2 (1988) 127-138.

Muckle, and Gyles, Exotoxic activities of Corynebacterium pseudotuberculosis. Current Microbiol. 13 (1986) 57-60.

Myers, Lerman, L.S. and Maniatis, A general method for saturation mutagenesis of cloned DNA fragments. Science 229 (1985) 242-247.

Pizza, CGacci, Bartoloni, A.,Perugini, Nencioni, De Magistris, Villa, Nucci, Manetti, Bugnoli, Giovannoni, Olivieri, R., Barbieri, Sato, H. and Rappuoli, Mutants of pertussis toxin suitable for vaccine development. Science 246 (1989) 497-500.

Porro, Saletti, Nencioni, Tagliaferri, L. and Marsibi, Immunogenic correlation between cross-reacting material (CRM197) pL'oduced by a mutant of Corynebacteriunt diphtheriae and diphtheria toxoid. J. Tnfect. Dis. 5 (1980) 716- 724.

Songer, Libby, landolo, J.J. and Cuevas, Cloning and expression of the phospholipase D gene from Corynebacterium pseudotuberculosis in Escherichia coli Infect. Immun. 58 (1990) 131-136.

Tweten Barbieri, J.T. and Collier, Effect of substituting aspartic acid for glutamic acid 148 on ADP-ribosyltransferase activity. J. Biol. Chem. 260 (1985) 10392-10395.

Waite, The phospholipases. Plenum Press, New York, 1987.

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Claims (10)

12- THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. A mutant Corynebacterium pseudotuberculosis phospholipase D which is enzymatically-inactive and which exhibits the immunogenicity of phospholipase D, wherein in said mutant phospholipase D at least one histidine residue in wild-type phospholipase D is replaced by a different amino acid residue. 2. A mutant phospholipase D according to claim 1, wherein the histidine residue at position 20 in wild-type phospholipase D is replaced by a different amino acid residue. 3. A mutant phospholipase D according to claim 2, wherein said histidine residue at position 20 is replaced by a tyrosine residue. 4. A vaccine composition for use against caseous lymphadenitis (CLA) in sheep, which comprises a mutant phospholipase D according to any one of claims 1 to 3, and optionally an adjuvant, in an acceptable carrier or diluent. 5. A vaccine composition according to claim 4, wherein said composition contains at least one other active immunogen. 6. A vaccine composition according to claim 5, wherein said other active immunogen is a clostridial antigen. 7. A method of immunising sheep against CLA, which comprises administration of an effective amount of a vaccine composition according to any one of claims 4 to 6. 8. An isolated DNA molecule having a nucleotide sequence coding for a mutant Corynebacteriumpseudotuberculosis phospholipase D which is enzymatically-inactive and w1" ,h exhibits the immunogenicity of phospholipase D, wherein in said mutant phospholipase D at least one histidine residue in wild-type phospholipase D is replaced by a different amino acid residue. -1 i I II 11 i 1 940823,q:\oper\jms,25290-po.234,12 -I 13 9. An isolated DNA molecule having a nucleotide sequence according to claim 8 coding for a mutant phospholipase D wherein the histidine residue at position 20 in wild- type phospholipase D is replaced by a different amino acid residue. An isolated DNA molecule having a nucleotide sequence according to claim 9 coding for a mutant phospholipase D wherein said histidine residue at position 20 is replaced by a tyrosine residue. 11. An isolated DNA molecule having a nucleotide sequence according to claim wherein the cytosine base at nucleotide position 129 of the wild-type phospholipase D gene is replaced by a thymine base. C 12. A recombinant DNA molecule comprising a DNA molecule having a nucleotide S* sequence according to any one of claims 8 to 11, operatively associated with an i expression control sequence.

13. A recombinant cloning vehicle or vector comprising a recombinant DNA molecule according to claim 12.

14. A host cell transformed with a recombinant cloring vehicle or vector according to claim 13.

15. A host cell according to claim 14, said host cell being E.coli.

16. A method for the production of a mutant phospholipase D which comprises expression of the recombinant DNA molecule coding for said product in a host cell according to claim 14 under suitable conditions, and isolating and if desired purifying the expressed product.

17. A mutant phospholipase D produced by the method according to claim 16.

18. A live vaccine composition comprising a live vaccine vector having inserted 17.~b A muatpopoiaeDprdcdb h ehdacodn ocam1.1 940823,q:\oper\jms,25290-po.234,13 92o922,p:\oper\jms,PK8484/91, 2 i 1 I 0. i w 14- therein a DNA molecule having a nucleotide sequence according to any one of claims 8 to 11, together with an expression control sequence for in vivo expression of the mutant phospholipase D encoded by said nucleotide sequence.

19. A live vaccine composition according to claim 18, wherein said vaccine vector is a non-pathogenic viral or bacterial vector. A live vaccine composition according to claim 19, wherein said non-pathogenic vector is an attenuated organism.

21. A live vaccine composition according to claim 20, wherein said attenuated organism is a non-toxigenic strain of Corynebacterium pseudotuberculosis.

22. A method of immunising sheep against CLA which comprises administration of an effective amount of a live vaccine composition according to any one of claims 18 to 21. Dated this 23rd day of August, 1994 CSL Limited By its Patent Attorneys Davies Collison Cave C tr C Ct Ctr i( .4 4 CC 1; a ,J lr 1_L12~ 1 i f"' ii l 940823,q:\oper\jms,25290-po.234, 14 -L i i i i iL -::M%:isli r ~rsc ABSTRACT A mutant Corynebacterium pseudotuberculosis phospholipase D product which is enzymatically-inactive and which exhibits the immunogenicity of phospholipase D. A vaccine composition comprising the mutant phospholipase D product is used against caseous lymphadenitis in sheep. Nucleotide and amino acid sequences of the mutant phospholipase D product are disclosed, and a method of production by expression of the mutant gene. A live vaccine composition for in vivo expression of the mutant gene is also disclosed. 41r t t r litt I* C Ce C C I tr I CI- i 1 B 'I I: f 920922jmsspe.001,PLD,15