NZ264325A - Bovine small piroplasma antigen, assay and vaccine - Google Patents

Description

264325 Patents Form 5 | ! Date-;?.); ^>P.\s\S.3.

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! ' C.Q1 K.Q.e.QTl ; .So!.^ G.(o.\K3^|1OO7 I • 2 6 MAR 1996 i 1 • ■ l 4_o~2_ .

N.Z. No.

NEW ZEALAND Patents Act 1953 COMPLETE SPECIFICATION PEPTIDE FOR PREVENTION OF INFECTION WITH BOVINE SMALL PIROPLASMA PROTOZOA We, JURIDICAL FOUNDATION THE CHEMO-SERO-THERAPEUTIC RESEARCH f\ t-OmPA*.A-1 or- INSTITUTE," 668 Okubo, Shimizu-machi, Kumamoto-shi, Kumamoto-ken, Japan and DIRECTOR OF THE NATIONAL INSTITUTE OF ANIMAL HEALTH, 3-1-1, Kannodai, Tsukuba-shi, Ibaraki-ken, Japan do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- - 1 - (Followed by 1A) - i A- Peptide for Prevention of Infection with Bovine Small Piroplasma Protozoa 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 serqenti, a vaccine comprising said peptide, and a diagnostic method of said disease.

Prior Art Bovine small piroplasma disease is an ixodid-mediated 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 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 Veterinc^y 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 serqenti, 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 of charged amino acids, lysine-glutamic acid (KE), 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 acid-leucine (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 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 serqenti. and is extremely effective for prevention of bovine small piroplasma infection including 264325 4 Theileria serqenti.

An object of the present invention is to provide 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, said peptide including an amino acid sequence of Lys-Glu-Lys.

Another object of the present invention is to provide a peptide which includes an amino acid sequence Glu-Val-Val-Trp-Lys-Glu-Lys-Lys-Glu-Val-Lys-Asp-Leu- 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 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 vaccine which comprises the vaccine of the present invention and one or of: Asp-Ala.

Further object of the present invention is to vaccines selected from the group consisting of: a vaccine for infectious bovine rhinotrachei a vaccine for infectious bovine mucous diarrl a vaccine > p : ■ • 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 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 sequence of the 33 kd peptide of Theileria serqenti 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.

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 (A): (A) Glu-Val-Val-Trp-Lys-Glu-Lys-Lys-Glu-Val-Lys-Asp-Leu-Asp-Ala Referring to the above amino acid sequence (A) 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 (A) can also be prepared. In this case, the original amino acid sequence of the 33 kd principal protein of Theileria serqenti (SEQ ID No: 2), 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 the sequences at the elongated N- and C-termini correspond to those in the original amino acid sequence (The above peptide (A) 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 method 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 j3-alanine and then of binding lysine to both a and 6 amino groups of the lysine-j3-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 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 diseasee 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 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-Va 1 -Va 1 -Trp-Ly s -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 (5) KEK peptides are bound to each other via lysine to form a molecule. The thus prepared peptide together with a Freund's complete adjuvant was intramuscularly injected to four (4) Holsteins of about four months old at the hip three times at an interval of four (4) 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 (4) control Holsteins without inoculation were also used. For sixty (60) days after the attack, there were conducted a clinical observation, a measurement 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 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 ^ame 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 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) Cattle No. JL 13 16 19 (1) Antibody titre at the attack AGP 4 8 4 4 ELISA 2.54 2.34 1.93 2.11 (2) Clinical observation fever + + + + spirit - - - - appetite - - - - others — — - — (3) Red blood cell count (minimum) x 10A/mm3 409 236 460 464 (4) Hematocrit (minimum) .1 16.3 24.1 19.6 (5) Parasitemia Maximum parasitic 6.9 9.0 3.6 3.9 percentage (%) Duration (*) 0 0 0 - 11 -Table 2 (Control group) Cattle No. 1 1 15 20 (1) Antibody titre at the attack AGP <2 <2 <2 <2 ELISA 0.14 0.02 0.03 0.10 (2) Clinical observation fever + + + + spirit - - appetite - - others - - - - (3) Red blood cell count (minimum) 303 388 236 189 x loVmm (4) Hematocrit (minimum) 20.3 19.9 18.0 14.6 (5) Parasitemia Maximum parasitic 14.2 7.0 10.4 10.4 percentage (%) Duration (*) 24 1 7 10 (Note)* Number of days which showed a maximum parasitic percentage more than that of the control cattle No. 5 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 loVmm3. However, the group immediately showed recovery and the red blood count at the end of the observation was recovered to 600 to 800 A ^ x 10 /mm'. On the other hand, the control group showed a minimum red blood count of 189 to 388 x 104/mm3 which was recovered to 350 to 600 x 104/mm3 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.

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 infection-resistant sera tested were 11 serum samples taken more than two (2) years after grazing and the experimentally infected sera were 13 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 h; nd, 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 P.P. <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 present invention, the reactivity with piroplasms purified from various small piroplasma protozoa, Theileria serqenti Ikeda (hereinafter referred to as "TSI"), Theileria bufferi Warwick (hereinafter referred to as "TBW") and Theileria orientalis Essex (hereinafter referred to as "TOE"), was assayed with SDS polyacrylamide gel electrophoresis and Western blotting, which were conducted as described by Laemmli et al. [Laenvmli 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 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, wherein 1 and 4 TSI, 2 and 5 TBW, and 3 and 6 TOE.

SEQUENCE LISTING SEQ ID NO: I 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 15 10 15 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: 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 1U ATC GTC TCT GCT ACC GCC GCA GAG GAA AAA AAA GAT GCA AAG GCT GAA 96 He Val Ser Ala Thr Ala Ala Glu Glu Lys Lys Asp Ala Lys Ala Glu 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 45 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 50 55 60 GAG GGA TAC CGC ATC AAG ACA CTC AAG GTC GGA GAT AAG AAC CTG TAT 240 Glu Gly Tyr Arg lie Lys Thr Leu Lys Val Gly Asp Lys Asn Leu Tyr 65 70 75 30 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 85 90 95 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 op/ -L 0 v , TTG CTG TTC AAG AAG AAG ACT GAC AAG GAT TGG GTT CAA TTC AGC TTC 384 Leu Leu Phe Lys Lys Lys Thr Asp Lys Asp Trp Val Gin Phe Ser Phe 115 120 125 GCC CAG TAC CTC GAT GAA GTT GTA TGG AAG GAG AAG AAG GAA GTA AAA 432 Ala Gin Tyr Leu Asp Glu Val Val Trp Lys Glu Lys Lys Glu Val Lys 130 135 140 GAC CTC GAC GCA TCC AAG TTC GCA GAC GCA GGT CTT TTC GCC GCT GAG 480 Asp Leu Asp Ala Ser Lys Phe Ala Asp Ala Gly Leu Phe Ala Ala Glu 145 150 155 160 GCT TTC GGT ACC GGA AAG CTG TAC AAC TTC ATT GGA AAC TTC AAG GTC 528 Ala Phe Gly Thr Gly Lys Leu Tyr Asn Phe lie Gly Asn Phe Lys Val 165 170 175 AAG AAG GTC ATG TTC GAG GAG AAG GAC GTT GGA GAT TCA AAC AAG GCC 576 Lys Lys Val Met Phe Glu Glu Lys Asp Val Gly Asp Ser Asn Lys Ala 180 185 190 AAA TAC ACC GCT GTC AAA GTT TAC GTC GGT TCC GAT GAG AAG AAA GTC 624 Lys Tyr Thr Ala Val Lys Val Tyr Val Gly Ser Asp Glu Lys Lys Val 195 200 205 GTA AGA CTC GAC TAC TTC TAC ACT GGT GAT GAG AGA TTC AAG GAG GTT 672 Val Arg Leu Asp Tyr Phe Tyr Thr Gly Asp Glu Arg Phe Lys Glu Val 210 215 220 TAC TTC AAA TTG GTA GAC GGA AAA TGG AAG AAG GTT GAG CAG AGC GAG 720 Tyr Phe Lys Leu Val Asp Gly Lys Trp Lys Lys Val Glu Gin Ser Glu 225 230 235 240 GCA AAC AAG GAT TTG CAC GCC ATG AAC AGT GCT TGG CCT TCG GAC TAC 768 Ala Asn Lys Asp Leu His Ala Met Asn Ser Ala Trp Pro Ser Asp Tyr 245 250 255 AAG CCT CTT GTC GAC AAG TTC TCA CCA CTT GCC GTT CTC AGC GCG GTT 816 Lys Pro Leu Val Asp Lys Phe Ser Pro Leu Ala Val Leu Ser Ala Val 260 265 270 CTC ATC GCC TCC CTC GCA GTA TTC TAT TAT CTC TAG 852 Leu lie Ala Ser Leu Ala Val Phe Tyr Tyr Leu 275 280 264325 16 What we claim is: 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, said peptide including an amino acid sequence of Lys-Glu-Lys. 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 one of claims 1 to 3 and a substance having an adjuvant activity.

Claims (1)

1. 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 one of claims i to 3 as an assay antigen. - " 1 : ' " 2. A peptide of claim 1 which includes an amino acid 5. A mixed bovine vaccine which comprises the vaccine of 264325 - 17 - 7. A peptide according to claim 1 substantially as herein described or exemplified. 8. A method according to claim 6 substantially as herein described or exemplified. JURIDICAL FOUNDATION THE CHEMO-SERO-THERAPEUTIC RESEARCH INSTITUTE and DIRECTOR OF THE NATIONAL INSTITUTE OF ANIMAL HEALTH By their attorneys HENRY HUGHES Per :