AU673614B2 - Peptide for prevention of infection with bovine small piroplasma protozoa - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION NAME OF APPLICANT(S): Juridical Foundation The Chemo-Sero-Therapeutic Research Director of The National Institute of Animal Health Institute AND ADDRESS FOR SERVICE: DAVIES COLLISON CAVE Patent Attorneys I Little Collins Street, Melbourne, 3000.

INVENTION TITLE: Peptide for prevention of infection with bovine small piroplasma protozoa The following statement is a full description of this invention, including the best method of performing it known to me/us:o oo

I

la- The present invention relates to a peptide useful for prevention and treatment of bovine infectious diseases.

More particularly, the present invention relates to a peptide useful for prevention of an infectious disease caused by bovine small piroplasma protozoa, i.e. Theileria sergenti, a vaccine comprising said peptide, and a diagnostic method of said disease.

Prior Art Bovine small piroplasma disease is an ixodidmediated protozoiasis and exhibits anemia and emaciation as a main symptom. This disease, occurring in pasturage all over the world, shows a high mortality and hence is a substantial obstacle in pasturage management. For overcoming this problem, there have been employed an administration of a protozoacide to cattle and a scattering of miticides to cattle and pasturage for the purpose of killing the mediating mites. However, the administration of protozoacide is doubtful in its treating effect and has a problem of the residual drug in bovine milk or meat, and further the use of miticides provoke serious problem of environmental pollution.

For preventing this disease, there has been used a method of inoculating blood infected with bovine small piroplasma protozoa. However, due to direct administration of bovine blood, this method has a risk for spreading various diseases including bovine leukemia and nowadays has 2 come to be forbidden. Recently, it has also been reported that the onset of small piroplasma disease is reduced after grazing by inoculating a specified amount of a sporozoite of small piroplasma protozoa to cattle before grazing, said sporozoite being separated and purified from the salivary gland of mediating mites infected with small piroplasma protozoa [Kamio et al.: 113th Japan Veterinary Association excerpt, page 156 (1992)]. However, this method has not yet been put into practical use.

Recent studies reveal that a principal protein of piroplasm of small piroplasma protozoa (piroplasma protozoa at a growth phase which parasitizes bovine erythrocytes) has a molecular weight of 32 to 33 kd and is a main target antigen in humoral immunity of host cattle [Kawazu et al.: 111th Japan Veterinary Association excerpt, page 135 (1991); and Sugimoto et al.: 115th Japan Veterinary Association excerpt, page 143 (1993)]. The present inventors have isolated and purified a gene coding for the principal protein of piroplasm of one of small piroplasma protozoa, Theileria sergenti, analyzed said gene and determined a nucleotide sequence thereof [Kawazu et al.: 113th Japan Veterinary Association excerpt, page 155 (1992)].

On the other hand, in case of malarial protozoa, the same hematozoon as small piroplasma protozoa, a surface antigen of malarial protozoa has an amino acid sequence of repeating unit of several amino acids and a surface antigen protein of malarial protozoa at erythrocyte stage has pairs 3 of charged amino acids, lysine-glutamic acid at several portions within the molecule, among which amino acid sequences of lysine-glutamic acid-lysine (KEK), lysine-glutamic acid-valine (KEV) or lysine-glutamic acidleucine (KEL) were found to be involved in binding with a human erythrocyte receptor, and prevention of malaria by the use of a synthetic peptide comprising the KEK or KE sequence has been studied [Parasite Immunology, 14, page 111-124 (1992)].

However, a vaccine effective for prevention of bovine small piroplasma disease has not yet been developed S•and, in view of various problems associated with the conventional methods, there is a need for developing a means useful for prevention of bovine small piroplasma protozoa infection.

Brief Description of the Invention As mentioned above, upon determination of an amino acid sequence of the principal protein of Theileria serqenti, one of small piroplasma protozoa, by the present inventors, it was found that this amino acid sequence includes the KEK sequence like in the case of malarial protozoa as mentioned above. The present inventors have considered this fact and immunized cattle with a peptide including this sequence, and as a result, have found that said peptide including the sequence shows immunogenicity of the 33 kd peptide, the principal protein of bovine Theileria sergenti, and is extremely effective for prevention of bovine small piroplasma infection including 4 Theileria serqenti.

An object of the present invention is to provide a peptide effective for preventing bovine small piroplasmu protozoa infection, said peptide having a part of an amino acid sequence of a 33 kd peptide derived from bovine small piroplasma protozoa including an amino acid sequence of Lys-Glu-Lys.

Another object of the present invention is to provide the peptide which includes an amino acid sequence of: Glu-Val-Val-Trp-Lys-Glu-Lys-Lys-Glu-Val-Lys-Asp-Leu- S. Asp-Ala.

Further object of the present invention is to provide the peptide which consists of an amino acid sequence of: Glu-Val-Val-Trp-Lys-Glu-Lys-Lys-Glu-Val-Lys-Asp-Leu- Asp-Ala.

Still another object of the present invention is S. to provide a vaccine for prevention of bovine small piroplasma protozoa infection which comprises the peptide of the present invention and a substance having an adjuvant activity.

Still further object of the present invention is to provide a mixed bovine va-cine which comprises the vaccine of the present invention and one or more of vaccines selected from the group consisting of: a vaccine for infectious bovine rhinotracheitis, a vaccine for infectious bovine mucous diarrhea, il 5 a vaccine for bovine parainfluenza virus, a vaccine for bovine adenovirus, a vaccine for hemophilus, and a vaccine for clostridium.

Still another object of the present invention is to provide a method for assaying an antibody to bovine small piroplasma protozoa which comprises using the peptide of the present invention as an assay antigen.

These and other objects and the advantages thereof will be apparent to those skilled in the art from the following description.

Brief Description of the Drawing *000 Fig. 1 is a photograph showing the results of electrophoresis in Western blotting analysis conducted in Example 2, which depicts the reaction between sera immunized with the KEK peptide of the present invention and various piroplasms.

Detailed Description of the Invention The peptide of the present invention shows an immunogenicity of the 33 kd peptide, the principal protein of Theileria serqenti, and can be synthesized by a conventional solid phase method for preparing a peptide.

The peptide of the present invention has a part of an amino acid zequence of the 33 kd peptide of Theileria sergenti including a sequence of lysine-glutamic acid-lysine (KEK), said KEK sequence corresponding to the amino acid sequence of from No. 138 to No. 140 in Sequence Listing SEQ ID No: 2.

6 The peptide of the present invention, even if it consists of the smallest unit of only three amino acids, lysine-glutamic acid-lysine (KEK), can be used for prevention of bovine small piroplasma disease since it can be formulated into a peptide compound having an immunogenicity to bovine small piroplasma disease by devising a linkage with a carrier. In a preferable embodiment of the invention, in view of more sufficient immunogenicity, a longer peptide can be prepared by elongating the above smallest peptide both at N-terminus and C-terminus and thus prepared longer peptide exerts a higher effect. Size of the peptide of the present invention is 30 amino acids or less including the KEK sequence, preferably 20 amino acids or less including the KEK sequence. One example of such preferable peptides includes a peptide of the following amino acid sequence Glu-Val-Val-Trp-Lys-Glu-Lys-Lys-Glu-Val-Lys-Asp- Leu-Asp-Ala Referring to the above amino acid sequence as a standard, a peptide of any size including the Lys-Glu-Lys (KEK) sequence can suitably be prepared. If desired, a longer peptide than the above sequence can also be prepared. In this case, the original amino acid sequence of the 33 kd principal protein of Theileria sergenti (SEQ ID No: which the present inventors have previously determined, should be referred to and a peptide with elongated N-terminus and C-terminus can be prepared so that 7 the sequences at the elongated N- and C-termini correspond to those in the original amino acid sequence (The above peptide corresponds to an amino acid sequence of from No. 134 to No. 148 in SEQ ID No: 2).

It is also possible to add one or two amino acids at the N-terminus or C-terminus of the peptide of the present invention in order to facilitate the binding of the peptide of the invention with a carrier protein support.

An amino acid useful for this purpose includes, for example, tyrosine, lysine, glutamic acid, aspartic acid, cysteine, as well as a derivative thereof. A conventional Smethod for modification of a protein, such as acetylation of the amino terminus and amidation of the carboxylic terminus, can also be used in order to make easier the .*binding of the peptide of the present invention with a carrier protein support. It is also possible to bind the peptide of the invention with amino groups of MAP (multiple antigen peptide), said MAP being prepared by repeating procedures of binding lysine to P-alanine and then of binding lysine to both a and E amino groups of the lysine- 0-alanine.

The peptide of the present invention as prepared above can be used as an immunogen in combination with a suitable adjuvant such as aluminum gel or oil adjuvant.

The immunogen is then administered to cattle to raise an antibody against the peptide of the present invention in the cattle, whereby the onset of small piroplasma disease can be prevented or at least symptoms of the disease can 8 extremely be reduced even if the cattle is subject to infection via bloodsucking by mites infected with Theileria serqenti.

Considering that the peptide of the present invention plays an important role in prevention of bovine small piroplasma disease, immunological conditions of cattle against small piroplasma disease can be assessed by assaying an antibody against the peptide of the present invention. That is, bovine immunological conditions can be assessed by assaying an antibody specific for the peptide of the invention in bovine sera by utilizing the peptide as .an assay antigen. An assay system for assaying an antibody o can be constructed by using a known procedure. A typical example is an assay system utilizing an enzyme immunoassay (ELISA) which is simple and easy to handle.

The present invention is explained in more detail by the following Examples but should not be construed to be limited thereto.

Example 1: A peptide of the following amino acid sequence including the KEK (lysine-glutamic acid-lysine) sequence according to the present invention (hereinafter referred to as "KEK peptide") was synthesized by a solid phase method.

Glu-Val-Val-Trp-Lys-Glu-Lys-Lys-Glu-Val-Lys-Asp-Leu- Asp-Ala Using the thus prepared KEK peptide, a peptide in the form of the above-mentioned MAP was prepared wherein five KEK peptides are bound to each other via lysine to 9 form a molecule. The thus prepared peptide together with a Freund's complete adjuvant was intramuscularly injected to four Holsteins of about four months old at the hip three times at an interval of four weeks so that an amount of the peptide of each injection is 1 mg/animal.

After the third injection, the animals were attacked by bloodsucking by adult small piroplasm-infected mites on the third week. Four control Holsteins witho'. -nculation were also used. For sixty (60) days af- attack, there were conducted a clinical observation, a neasurement of body temperature, a blood test (red blood count, hematocrit value) and a test for parasitemia. The blood test was carried out by using Hematology Analyzer (PS-510V) manufactured by Nippon Denko K.K. and a parasitic percentage was obtained as a percentage of parasitized red e cells per 1000 red cells.

As a result, by administering as an immunogen the KEK peptide to the cattle, an antibody against the KEK peptide increased within the bovine body. At the time of attack, ELISA measuring a KEK antibody showed 2.0 or more of absorbance. On the other hand, the same ELISA value was 0.1 or less in the control group. A gel agar precipitation (hereinafter referred to as "AGP") test for measuring an antibody against the KEK peptide also revealed 4 to 8 times higher antibody titre in the group immunized with the KEK peptide of the present invention. On the other hand, the AGP antibody was negative in the control group.

Infectious conditions after the attack are shown 10 in the following Tables 1 and 2. After the attack, piroplasma protozoa appear around 9 to 15 days after the beginning of bloodsucking. A maximum parasitic percentage in the group immunized with the peptide of the present invention increased up to 9 in one of four cattle but was as low as 3.6 to 6.9 in the remaining three. On the other hand, the control group showed a maximum parasitic percentage of as high as 10.4 to 14.2 in three of four cattle. This result confirms that the immunized group shows significantly lower parasitic percentage than that of the control group.

Table 1 (Immunized group) e r o Antibody titre at the attack

AGP

ELISA

Clinical observation fever spirit appetite others Red blood cell count (minimum) x 104/mm 3 Hematocrit (minimum) Parasitemia Maximum parasitic percentage Duration Cattle No.

6 13 16 4 8 4 2.54 2.34 1.93 19 4 2.11 409 20.1 6.9 0 236 16.3 9.0 10 460 464 24.1 3.6 0 19.6 3.9 0 11 Table 2 (Control group) Cattle No.

8 15 Antibody titre at the attack AGP <2 <2 <2 <2 ELISA 0.14 0.02 0.03 0.10 Clinical observation fever spirit appetite others Red blood cell count (minimum) 303 388 236 189 x 104/mm 3 Hematocrit (minimum) 20.3 19.9 18.0 14.6 Parasitemia Maximum parasitic 14.2 7.0 10.4 10.4 percentage Duration 24 1 7 (Note)* Number of days which showed a maximum parasitic percentage more than that of the control cattle No. The red blood count in the group immunized with the peptide of the present invention decreased as the parasitic percentage increases with a minimum red blood count being 409, 460, 464 and 236 x 10 4 /mm 3 However, the group immediately showed recovery and the red blood count at the end of the observation was recovered to 600 to 800 4 3 x 104/mm 3 On the other hand, the control group showed a minimum red blood count of 189 to 388 x 10 /mm 3 which was recovered to 350 to 600 x 104/mm 3 at the end of the observation. As a result, it was found that the recovery of red blood count was more rapid in the immunized group than in the control group.

It was confirmed that the hematocrit values are consistent with a change in red blood count and decreased to a lower extent than that of the control group.

12 In summary, all the above results confirmed that bovine immunization with the KEK peptide of the present invention reduces the symptoms of small piroplasma disease.

Example 2: An antibody reactivity with the KEK peptide of the present invention was compared between field small piroplasma protozoa infection-resistant cattle and experimentally infected bovine sera. The reactivity was assessed by ELISA using the KEK peptide of the present invention as an assay antigen. The field infectionresistant sera tested were 11 serum samples taken more than V. two years after grazing and the experimentally infected sera were 18 serum samples taken 3 to 4 months after the infection. The results are shown in Table 3. The ELISA antibody titre to the KEK peptide of the present invention was evident in 8 among 11 samples of the field infected sera though at a low level with the absorbance being in a range of 0.15 to 0.32. On the other hand, all of the experimentally infected bovine sera showed the absorbance of 0.1 or less. This confirmed that an antibody against the KEK peptide of the present invention can be found in the field infection-resistant sera.

Table 3 Sera derived from: No. of samples O.D.

<0.15 >0.15 Field infection- 11 3 8 resistant sera Experimentally infected 18 18 0 sera Using sera immunized with the KEK peptide of the 13 present invention, the reactivity with piroplasms purified from various small piroplasma protozoa, Theileria sergenti Ikeda (hereinafter referred to as Theileria bufferi Warwick (hereinafter referred to as "TBW") and Theileria orientalis Essex (hereinafter referred to as was assayed with SDS polyacrylamide gel electrophoresis and Western blotting, which were conducted as described by Laemmli et al. [Laemmli et al., Nature, 227, page 680-685 (1970)] and Dunn [Dunn, Anal. Biochem., 157, page 144-153 (1986)], respectively. The results are shown in Figure 1.

Bovine sera immunized with the KEK peptide of the present invention recognized strongly the principal protein of TSI but did not reacted with the principal protein of TBW or :*too.

TOE, confirming that the KEK peptide of the present invention is specific for TSI.

In Figure 1, lanes 1, 2 and 3 show bovine sera infected with TSI, lanes 4, 5 and 6 bovine anti-peptide sera, erein 1 and 4 TSI, 2 and 5 TBW, and 3 and 6 TOE.

e• *f 14 SEQUENCE LISTING SEQ ID NO: 1 LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: peptide SEQUENCE DESCRIPTION: SEQ ID NO: 1: Glu-Val-Val-Trp-Lys-Glu-Lys-Lys-Glu-Val-Lys-Asp-Leu-Asp-Ala 1 5 10 SEQ ID NO: 2 LENGTH: 852 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear MOLECULE TYPE: cDNA to mRNA ORIGIN: Theileria sergenti SEQUENCE DESCRIPTION: SEQ ID NO: 2: S ATG TTG TCC AAG AGA ACG TTC AAC GTA CTT TGC CTA GGA TAC TTC CTT 48 Met Leu Ser Lys Arg Thr Phe Asn Val Leu Cys Leu Gly Tyr Phe Leu 1 5 10 ATC GTC TCT GCT ACC GCC GCA GAG GAA AAA AAA GAT GCA AAG GCT GAA 96 Ile Val Ser Ala Thr Ala Ala Glu Glu Lys Lys Asp Ala Lys Ala Glu 25 GAG AAG AAG GAC TTA ACT CTC GAA GTT AAC GCC ACC GCA GCC GAA CAT 144 Glu Lys Lys Asp Leu Thr Leu Glu Val Asn Ala Thr Ala Ala Glu His 40 TTT AAA GTC GAC GCC TCA AAC GCC AAC GAC GTC GTT TTT ACT GCC GAA 192 Phe Lys Val Asp Ala Ser Asn Ala Asn Asp Val Val Phe Thr Ala Glu 55 GAG GGA TAC CGC ATC AAG ACA CTC AAG GTC GGA GAT AAG AAC CTG TAT 240 Glu Gly Tyr Arg Ile Lys Thr Leu Lys Val Gly Asp Lys Asn Leu Tyr 70 75 ACC GTA GAT ACT TCC AAG TTC ACC CCA ACT GTC GCC CAC AGA CTG AAG 288 Thr Val Asp Thr Ser Lys Phe Thr Pro Thr Val Ala His Arg Leu Lys 90 CAT GCT GAC GAC CTG TTC TTC AAG CTC AAC CTG TCC CAC GCA AAG CCA 336 His Ala Asp Asp Leu Phe Phe Lys Leu Asn Leu Ser His Ala Lys Pro 100 105 110 15 TTG CTG TTC Leu Leu Phe 115 GCC CAG TAC Ala Gin Tyr

AAG

Lys

CTC

Leu 130 GAC CTC ASD Leu GAC GCA Asp Ala 145

GCT

Ala TTC GGT ACC Phe Gly Thr 9* AAG AAG GTC Lys Lys Val AAA TAC ACC Lys Tyr Thr 195 GTA AGA CTC Val Arg Leu 210 TAC TTC AAA Tyr Phe Lys 225 GCA AAC AAG Ala Asn Lys AAG CCT CTT Lys Pro Leu CTC ATC GCC Leu Ile Ala 275

ATG

Met 180

GCT

Ala

GAC

Asp

TTG

Leu

GAT

Asp

GTC

Val 260

TCC

Ser AAG AAG ACT GAC AAG GAT TGG GTT CAA TTC AGC TTC 384 Lys Lys Thr Asp Lys Asp Trp Val Gin Phe Ser Phe 120 125 GAT GAA GTT GTA TGG AAG GAG AAG AAG GAA GTA AAA 432 Asp Glu Val Val Trp Lys Glu Lys Lys Glu Val Lys 135 140 TCC AAG TTC GCA GAC GCA GGT CTT TTC GCC GCT GAG 480 Ser Lys Phe Ala Asp Ala Gly Leu Phe Ala Ala Glu 150 155 160 GGA AAG CTG TAC AAC TTC ATT GGA AAC TTC AAG GTC 528 Gly Lys Leu Tyr Asn Phe Ile Gly Asn Phe Lys Val 165 170 175 TTC GAG GAG AAG GAC GTT GGA GAT TCA AAC AAG GCC 576 Phe Glu Glu Lys Asp Val Gly Asp Ser Asn Lys Ala 185 190 GTC AAA GTT TAC GTC GGT TCC GAT GAG AAG AAA GTC 624 Val Lys Val Tyr Val Gly Ser Asp Glu Lys Lys Val 200 205 TAC TTC TAC ACT GGT GAT GAG AGA TTC AAG GAG GTT 672 Tyr Phe Tyr Thr Gly Asp Glu Arg Phe Lys Glu Val 215 220 GTA GAC GGA AAA TGG AAG AAG GTT GAG CAG AGC GAG 720 Val Asp Gly Lys Trp Lys Lys Val Glu Gin Ser Glu 230 235 240 TTG CAC GCC ATG AAC AGT GCT TGG CCT TCG GAC TAC 768 Leu His Ala Met Asn Ser Ala Trp Pro Ser Asp Tyr 245 250 255 GAC AAG TTC TCA CCA CTT GCC GTT CTC AGC GCG GTT 816 Asp Lys Phe Ser Pro Leu Ala Val Leu Ser Ala Val 265 270 CTC GCA GTA TTC TAT TAT CTC TAG 852 Leu Ala Val Phe Tyr Tyr Leu 280

Claims (1)

16- THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. A peptide effective for preventing bovine small piroplasma protozoa infection, said peptide having a part of an amino acid sequence of a 33 kd peptide derived from bovine small piroplasma protozoa of Theileria sergenti including an amino acid sequence of Lys-Glu-Lys. 2. The peptide of claim 1 which includes an amino acid sequence of: Glu-Val-Val-Trp-Lys-Glu-Lys-Lys-Glu-Val-Lys-Asp-Leu-Asp-Ala. 3. The peptide of claim 2 which consists of an amino acid sequence of: Glu-Val-Val-Trp-Lys-Glu-Lys-Lys-Glu-Val-Lys-Asp-Leu-Asp-Ala. 4. A vaccine for prevention of bovine small piroplasma protozoa infection which comprises the peptide of any of claims 1 to 3 and a substance having an adjuvant activity. 5. A mixed bovine vaccine which comprises the vaccine of claim 4 and one or more of vaccines selected from the group consisting of: a vaccine for infectious bovine rhinotracheitis, :a vaccine for infectious bovine mucous diarrhea, a vaccine for bovine parainfluenza virus, a vaccine for bovine adenovirus, a vaccine for hemophilus, and a vaccine for clostridium. 6. A method for assaying an antibody to bovine small piroplasma protozoa which comprises using the peptide of any of claims 1 to 3 as an assay antigen. 1't If it 111 94 11 ht 17 IA 17 7. The peptide of claim 1, a vaccine comprising said peptide, or a method of use of said peptide substantially as hereinbefore described with reference to the drawing and/or Examples. DATED this SEVENTEENTH day of SEPTEMBER, 1996 Juridical Foundation The ClP .,-'ro-Tlierapeutic Research Institute AND Director of The National Institv' Animal Health 10 by DAVIES COLLISON CAVE Patent Attorneys for the Applicants A'NT 0'~ Abstract of the Disclosure: A peptide effective for preventing bovine small piroplasma protozoa infection, said peptide having a part of an amino acid sequence of a 33 kd peptide derived from bovine small piroplasma protozoa including an amino acid sequence of Lys-Glu-Lys; a vaccine for prevention of bovine small piroplasma protozoa infection which comprises the peptide and a substance having an adjuvant activity; and a method for assaying an antibody to bovine small piroplasma protozoa which comprises using the peptide as an assay antigen. A I o.