WO2002017960A1

WO2002017960A1 - Vaccines against toxoplasma gondii

Info

Publication number: WO2002017960A1
Application number: PCT/JP2001/001472
Authority: WO
Inventors: Takeshi Mikami; Masayuki Mishima
Original assignee: Chugai Seiyaku Kabushiki Kaisha
Priority date: 2000-08-31
Filing date: 2001-02-27
Publication date: 2002-03-07
Also published as: JPWO2002017960A1

Abstract

Vaccines against Toxoplasma gondii containing an antigenic protein SRS1, P22 or P54 of Toxoplasma gondii. These vaccines protect animals from the infection with Toxoplasma gondii and inhibit the formation of cysts.

Description

Description Vaccine technology for Toxoplasma gondii

The present invention relates to an antigen using an antigen derived from Toxoplasma gondii, a method for producing the same, and use thereof. Background art

Toxoplasma gondii is a parasite that can infect humans and many warm-blooded animals. Toxoplasmosis in healthy people is rarely fatal, but cysts form in various organs of the host as the infection continues for life. Symptoms of toxoplasma infection in humans include abortion, severe encephalitis in the fetus, and visual impairment, and various clinical symptoms appear especially in persons with immune disorders. Opportunistic infection of the central nervous system with Toxoplasma in AIDS patients is often fatal. The risk of T. gondii infection is increasing due to the spread of AIDS infection and immunosuppression in organ transplantation. On the other hand, T. gondii transmission to livestock causes abortion, resulting in significant economic losses.

To date, much effort has been devoted to the development of vaccines to prevent transmission to livestock, and live vaccines using T. gondii varieties have achieved some success. However, to date, live vaccines have not been widely accepted in the market due to their side effects, short shelf life and high cost. Raw vaccines also pose the risk of dangerous reverse mutations, unexpected mass transmission and accidental transmission to humans. Therefore, in recent years, there has been much interest in developing subunit vaccines and DNA vaccines against T. gondii to avoid such dangers. Many antigens have been isolated from T Gondi, and the genes encoding them have been sequenced and cloned. One of the surface antigens, SAG1 (P30), is the most dominant surface antigen. For this reason, studies on subunit pectin against Toxoplasma gondii have mainly been performed on the antigen SAG1 (P30).

However, the antigen P30 was not necessarily effective as a vaccine immunogen against Toxoplasma gondii. Disclosure of the invention

Therefore, an object of the present invention is to provide a novel vaccine against Toxoplasma gondii using a subunit antigen protein derived from Toxoplasma gondii.

The present inventors have conducted various studies to solve the above-mentioned problems. As a result, among the protein subunits derived from Toxoplasma gondii, SRS1, P22 and P54 were replaced by another protein subunit P30. And found that they have much higher immunogenicity than P43 and completed the present invention.

Accordingly, the present invention provides a pectin against Toxoplasma gondii comprising the antigen SRS1, P22 or P54 derived from Toxoplasma gondii.

The present invention also comprises the antigen SRS1, wherein the antigen SRS1 has the amino acid sequence shown in SEQ ID NO: 2, or one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2. Amino acid sequence modified by acid deletion, addition and substitution by amino or other amino acids A vaccine that has the SRS1 antigen and maintains the immunogenicity of the SRS1 antigen.

The present invention also comprises the antigen P22, wherein the antigen P22 has the amino acid sequence shown in SEQ ID NO: 4, or 1 or 2 in the amino acid sequence shown in SEQ ID NO: 4. It has an amino acid sequence modified by deletion or addition of a plurality of amino acids and / or is substituted by another amino acid, and maintains the immunogenicity of the P22 antigen. Provide a protein, Pectin.

The present invention also comprises the antigen P54, wherein the antigen P54 has an amino acid sequence shown in SEQ ID NO: 6, or one or more amino acids in the amino acid sequence shown in SEQ ID NO: 6. A protein having an amino acid sequence modified by deletion, addition and / or substitution of another amino acid by amino acids and maintaining the immunogenicity of the P54 antigen, Provide Pectin.

The present invention also includes a protein which is coded by a DNA that hybridizes under stringent conditions to a DNA having the nucleotide sequence of SEQ ID NO: 1 and has immunogenicity equivalent to that of the SRS1 antigen. A pectin comprising:

The present invention also includes a protein encoded by a DNA that hybridizes to a DNA having the nucleotide sequence of SEQ ID NO: 3 under stringent conditions and having an immunogenicity equivalent to that of the P22 antigen. Comprising a vaccine.

The present invention also relates to a protein which is coded by DNA that hybridizes under stringent conditions to DNA having the nucleotide sequence of SEQ ID NO: 5 and has immunogenicity equivalent to that of the P54 antigen. The present invention provides a pectin comprising:

The present invention further provides, in the method for producing a vaccine, the antigen A method comprising culturing a host transformed with an expression vector containing DNA encoding a protein and collecting the antigen protein from the culture is provided.

The present invention further provides a method of immunizing a mammal other than human against Toxoplasma gondii, wherein the vaccine is administered to a mammal other than human. I do. Embodiment of the Invention

The antigen protein of the present invention is conveniently obtained by a gene recombination method and by expression of a gene encoding the same. Since the antigen gene used in the present invention does not contain intron, genomic DNA can be used as the DNA encoding the antigen.For example, using genomic DNA of Toxoplasma gondii, The genomic DNA can be cloned by PCR or the like using the above genomic DNA as a type II, using a primer pair designed based on the genomic DNA. In addition, according to a conventional method, the cDNA encoding the antigen can be cloned using the cDNA library. Specific examples of cloning are described in Example 1.

As an example of a typical sequence, the nucleotide sequence of DNA encoding the SRS1 antigen protein is described in SEQ ID NO: 1, and the amino acid sequence encoded thereby is described in SEQ ID NO: 2. In addition, the nucleotide sequence of DNA encoding the P22 antigen protein is described in SEQ ID NO: 3, and the amino acid sequence encoded thereby is described in SEQ ID NO: 4. Further, the nucleotide sequence of DNA encoding the P54 antigen protein is described in SEQ ID NO: 5, and the amino acid sequence encoded thereby is described in SEQ ID NO: 6.

Thus, as a typical example, the SRS1 antigen protein of the present invention SEQ ID NO: 2 has the amino acid sequence shown in SEQ ID NO: 2, the P22 antigen protein has the amino acid sequence shown in SEQ ID NO: 4, and the P54 antigen protein is the amino acid sequence shown in SEQ ID NO: 6. Has an array. However, it is well known that the amino acid sequence of a protein having a certain biological activity retains its natural biological activity even when modified by deletion, addition or substitution of another amino acid.

Accordingly, the antigenic protein of the present invention includes not only the protein having the amino acid sequence shown in SEQ ID NO: 2 but also the deletion of one or more amino acids from the amino acid sequence shown in SEQ ID NO: 2. And modified by addition and / or substitution with another amino acid, and which maintains the same immunogenicity as the protein having the amino acid sequence shown in SEQ ID NO: 2. . In the present invention, such modified antigen proteins are also referred to as SRS1 antigen proteins.

Similarly, the antigenic protein of the present invention includes not only the protein having the amino acid sequence shown in SEQ ID NO: 4 but also one or more amino acids with respect to the amino acid sequence shown in SEQ ID NO: 4. And proteins which have been modified by deletion, addition and substitution with Z or another amino acid, and which maintain the same immunogenicity as the protein having the amino acid sequence shown in SEQ ID NO: 4. It is. In the present invention, such a modified antigen protein is also referred to as a P22 antigen protein.

Similarly, the antigenic protein of the present invention includes not only the protein having the amino acid sequence shown in SEQ ID NO: 6 but also one or more amino acids with respect to the amino acid sequence shown in SEQ ID NO: 6. Proteins modified by acid deletion, addition and / or substitution with other amino acids, and maintaining the same immunogenicity as the protein having the amino acid sequence shown in SEQ ID NO: 6 included. In the present invention, such modified antigen proteins are also referred to as P54 antigen proteins. In the above modification, the number of amino acids to be modified is in a range that can be modified by a conventional amino acid sequence modifying means such as site-directed mutagenesis, PCR, and the like, and is preferably, for example, 1 to 20. Ranges from 1 to 15, more preferably 1 to 10, such as 1 to several amino acids.

DNA encoding such a modified antigenic protein can be prepared by, for example, using a DNA having the nucleotide sequence shown in SEQ ID NO: 1, 3, or 5 by a site-specific mutagenesis method, a PCR method, or other conventional means. It can be obtained by modification. Furthermore, DNA encoding an antigen protein having a shortened C-terminal can also be obtained by introducing a termination codon at a predetermined position in the above-mentioned nucleotide sequence.

If the DNA encoding a protein having a biological activity is cloned, the DNA that hybridizes to the DNA under stringent conditions can encode the protein having the biological activity described above. Often there is.

A DNA that can hybridize with a base sequence of a certain DNA is at least about 70%, preferably at least about 80%, more preferably at least about 90%, and most preferably at least about 95% with the base sequence. For example, DNA containing a nucleotide sequence having homology to the DNA is used. The hybridization is performed according to a known method, for example, the method described in Molecular Cloning (MoMoecu 丄 ar Cloning) 2nd (J. oambrook et al., Old Spring Harbor Lab. Press, 1989). be able to . When a commercially available library is used, the procedure can be performed according to the method described in the attached instruction manual. Hybridization under stringent conditions is usually carried out in a hybridization solution of 5 XSSC or equivalent salt concentration at a temperature of 37-42 ° C in a hybridization solution of 5 XSSC or equivalent. 1 2 hours, 5 XSSC Alternatively, it can be carried out by preliminarily washing with a solution or the like having a salt concentration equivalent thereto, followed by washing with 1 XSSC or a salt concentration equivalent thereto. Further, in order to obtain a higher stringency, washing can be performed by performing washing in a solution having a salt concentration of 0.1 XSSC or an equivalent thereof.

Therefore, the antigenic protein of the present invention hybridizes not only to the protein encoded by the nucleotide sequence shown in SEQ ID NO: 1 but also to DNA having the nucleotide sequence shown in SEQ ID NO: 1 under stringent conditions. Also included in the present invention are proteins encoded by the mutated DNA and having the same immunogenicity as the protein having the amino acid sequence shown in SEQ ID NO: 2. This is called the SRS1 antigen protein.

The antigenic protein of the present invention also hybridizes under stringent conditions not only to the protein encoded by the nucleotide sequence shown in SEQ ID NO: 3 but also to DNA having the nucleotide sequence shown in SEQ ID NO: 3. Also included in the present invention are proteins encoded by DNAs that have the same immunogenicity as the proteins having the amino acid sequence shown in SEQ ID NO: 4, and the P22 antigen including such proteins. It is called protein.

The antigenic protein of the present invention may further hybridize not only to the protein encoded by the nucleotide sequence shown in SEQ ID NO: 5 but also to DNA having the base sequence shown in SEQ ID NO: 5 under stringent conditions. A protein encoded by soybean DNA and having the same immunogenicity as the protein having the amino acid sequence shown in SEQ ID NO: 6 is also included in the present invention. It is called 54 antigen protein.

For reference, Toxoplasma gondii P30 antigen protein The nucleotide sequence of DNA encoding the protein and the corresponding amino acid sequence are shown in SEQ ID NOs: 7 and 8, respectively, and the nucleotide sequence of DNA encoding the P43 antigen protein and the corresponding amino acid are shown in SEQ ID NOs: 7 and 8, respectively. The acid sequences are shown in SEQ ID NOs: 9 and 10, respectively.

The antigen protein of the present invention can be produced by expressing the DNA encoding it in a conventional manner. Once the DNA encoding a given protein has been cloned, expressing it to produce the given protein is only a routine technique. That is, the antigen protein of the present invention may be obtained by culturing a host transformed by an expression vector containing DNA encoding the antigen protein according to a conventional method, and collecting the antigen protein from the culture. . A specific example is shown in Example 1. Example

Next, the present invention will be described more specifically with reference to examples.

Example 1. Production of antigen protein

Using the genomic DNA of Toxoplasma gondii RH strain as type I, DNA encoding five types of antigen proteins was amplified using the primers shown in Table 1 below.

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Group ¹ J group UW Κύ 丄 a C gat [C Γ (J AA \ iA ΤiΓlrΓuΤ 丄 Γ （JA ΓJΓA 丄丄

Shun ^ ~ force tr s tsu tsu oma <ー _ ac gaa t CCA shi 丄 IAUA L Lrir A A 丄 z 0 oo

IJ no no ^ c gaa X then Cili \ then 1Λ 丄 LrAlri Q

丄丄丄丄 3 Shun Kanofumer acgaatic 丄丄丄 1A A

A then AAA then u 14

OC A

Eye IJ force Zofimer acgaattC Α1 J JAAAA 11Ι 丄丄 Back primer acgaattcTACTCAGAAGTCTC 16

P30 Forward primer acgaattcTTGTATGTCGGTT 17

Rear primer acgaattcGACGAGTATGTTT 18

P43 Forward primer acgaattcCGAGATGCAGCTGT 19

Back primer acgaattcCATTTAGGCAGCCA 20 Each of the above cloned DNAs was then inserted into the EcoRI site of the E. coli expression vector pGEM (obtained from Promega, New York, USA), and E. coli was transformed with this vector. After transformation, the Escherichia coli was cultured according to a conventional method, and the antigen protein was expressed as a fusion protein with the gene 10 leader peptide. The cultured E. coli cells are collected by centrifugation, and cell lysates are prepared by sonication (Ultra Shomogenizer UP-158, Taitech), and the fusion protein is precipitated by centrifugation at 180,000 XG. I let you go.

The resulting pellet is washed, resuspended in phosphate buffer, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE), and extracted with Coomassie brilliant blue; CBB). The fusion protein content is determined by the concentration of SDSPAGE—CBB band corresponding to the antigen Specified. Similarly, a 10-peptide gene constituting a part of the fusion protein was prepared.

Each of the coding genes from the E. coli was subcloned into pUC19 (Toyobo) and sequenced using PRISM Cycle Sequencing Systems (PE Applris Biosystems, Japan) by standard methods. SEQ ID NO: 1 shows the nucleotide sequence of the coding region of the obtained DNA.

(SRS 1), SEQ ID NO: 3 (P22), SEQ ID NO: 5 (P54), SEQ ID NO: 7 (P30), and SEQ ID NO: 9 (P43), and these The amino acid sequence encoded by SEQ ID NO: 2

Example shown in (SRS1), SEQ ID NO: 4 (P22), SEQ ID NO: 6 (P54), SEQ ID NO: 8 (P30), and SEQ ID NO: 10 (P43) 2. Immunization experiment

For this experiment, the above five antigen proteins SRS1, P22, P54, P30 and P43, and a mixture of the above five antigen proteins for comparison (mixed antigen and ), A gene 10 peptide (referred to as Gene O) constituting a part of the fusion protein, and a cell lysate of Toxoplasma gondii (referred to as TL) were used. The total protein content in the cell lysate TL was measured Ri by the absorbance at 5 6 2 nm after 18 Temple I Nkyupe preparative at 3 7 ⁰ 〇 with Coomas sie Protein Assay Reagent (Pierce Co.) TL. Experimental Special Pathogen Free (SPF) female BALB / C mice were purchased from C1ea Japan (Shizuoka). The mice were tested at the age of 8 weeks and 7 mice. Mice ^ 3, all antigens treated according to animal experiment guidelines approved by the Japanese Association for Laboratory Animal Science. The concentration was adjusted to 0.1, and 0.1 ing of the protein was intraperitoneally administered to mice twice at two-week intervals. The first was administered with Freund's complete adjuvant, and the second with Freund's incomplete adjuvant. Two weeks after the second antigen administration, a challenge with Toxoplasma was performed. That is, 300 fresh cells of brady zoite of Toxoplasma gondii Be Very strain were suspended in 0.5 mL of a phosphate buffer and inoculated intraperitoneally into mice. Four months after the challenge, observations were made regarding survival and clinical findings. Mice that survived during this period were killed 4 months later, and then tissue histology in the brain was observed. Sterile phosphate buffer was added to the excised brain to make 3 mL, homogenized with a glass homogenizer, and kept on ice. 10 lOiiL spots of the brain suspension were observed under a microscope.

In addition, all homogenates were approximately aliquoted and injected intraperitoneally into three SPF mice. The mice were tested by clinical observation of Toxoplasma infection, serum analysis by ELISA, and observation of brain histology by microscopy.

In all mice, apparent experimental toxoplasma infection was observed. Approximately one week after the challenge, over a period of several days, the mouse activity decreased and the hair grew. Thereafter, some of the mice receiving P22, SRS1, P54 or the mixed antigens disappeared with the symptoms disappeared, and the other mice died. Mice immunized with Toxoplasma cell lysate (TL) had milder symptoms than the other groups, but the survival rate gradually declined during the experiment. Cumulative mortality in the groups receiving P22, SS1, P54 or the mixed antigens was significantly lower than that in Packard. Table 2 shows the results. Table 2

Survival after immunized mice were infected with Toxoplasma gondii Beverley strain Bradeley

Survival rate (%)

Immunogen n

73) 8 9 10 11 12 15 60 90 120

P30 12 100 100 33 0 0 0 0 0 0 0

P22 12 100 100 58 25 17 * 17 * 17 * 17 * 17 * 17 *

P43 12 100 92 50 17 0 0 0 0 0 0

SRS1 12 100 92 50 25 ** 25 ** 25 ** 25 "25 ** 25 ** 25 **

P54 12 100 100 42 25 17 * 8 8 8 8 8

Mix 12 100 100 33 25 '17 * 17 * 17 * 17 * 17 * 17 * genelO ¹ ' 12 100 100 25 8 0 0 0 0 0 0

TL ²⁾ 6 100 100 100 ** 100 ** 100 ** 100 ** 83 ** 50 ** 33 ** 33 **

1) Gene 10 leader peptide

2) Toxoplasma cell lysate

3) The number after the challenge

*: P-0.05, **: P-0.01; significant difference (chi-square test) compared to background data (Day 11, n = 37)

Tissue cysts in the brains of surviving mice 4 months after challenge were not detected in mice receiving the P22, SRS1 or P54 antigens, but were not detected in toxoplasma cell lysates ( In TL) -administered mice, more than 2000 cis were observed. The results for each mouse are shown in Table 3 below.

Table 3

Number of tissue tissues in the brain of each mouse that survived 4 months after challenge Number of immunogens Surviving mice Number of cysts

P 2 2 2 N D, N D

S R S 13 N D, ND, ND P 54 N D

Mixed antigen 2 N D, N D

Toxoplasma cell lysate 2 2 0 0 0, 2 7 0 0

ND: Not detected.

In order to test the formation of cysts by other methods, P22 or SRS1 was administered, and the brain homogenate of mice that survived four months after challenge was injected into other mice. There were no significant clinical findings 2 months after injection, no serum positive response in the ELISA, and no tissue cysts in the brain.

As described above, the vector for Toxoplasma gondii of the present invention protected animals from Toxoplasma gondii infection and prevented the generation of cysts.

Claims

The scope of the claims

1. A vaccine against Toxoplasma gondii, comprising the antigen SRS1, P22 or P54 derived from Toxoplasma gondii.

2. It comprises the antigen SRS1, and the antigen SRS1 has the amino acid sequence shown in SEQ ID NO: 2 or the deletion of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2. 2. The vaccine according to claim 1, which is a protein having an amino acid sequence modified by addition, substitution by Z or another amino acid, and maintaining the immunogenicity of the SRS1 antigen.

3. It comprises the antigen P22, wherein the antigen P22 has the amino acid sequence shown in SEQ ID NO: 4, or one or more amino acids in the amino acid sequence shown in SEQ ID NO: 4 2. A protein having an amino acid sequence modified by acid deletion, addition and substitution with amino acid or another amino acid and maintaining the immunogenicity of the P22 antigen. The note in the note.

4. An antigen P54 comprising the amino acid sequence shown in SEQ ID NO: 6 or one or more amino acids in the amino acid sequence shown in SEQ ID NO: 6 A protein having an amino acid sequence modified by deletion, addition and substitution of Z or another amino acid, and maintaining the immunogenicity of the P54 antigen. The vaccine according to claim 1.

5. SEQ ID NO: 1 is coded by DNA that hybridizes to DNA having the nucleotide sequence of SEQ ID NO: 1 under stringent conditions, and contains a protein having immunogenicity equivalent to that of SRS1 antigen. The vaccine according to claim 1, comprising:

6. SEQ ID NO: a protein that is coded by DNA that hybridizes under stringent conditions to DNA having the nucleotide sequence of SEQ ID NO: 3 and has immunogenicity equivalent to that of the P22 antigen The actin of claim 1 comprising:

7. SEQ ID NO: encoded by DNA that hybridizes under stringent conditions to DNA having the nucleotide sequence set forth in 5, and has immunogenicity equivalent to that of P54 antigen The vaccine according to claim 1, comprising a protein having

8. The method for producing a vaccine according to claims 1 to 7, wherein the transformed host is cultured by an expression vector containing a DNA encoding the antigen protein, and the antigen protein is isolated from the culture. A method comprising the step of harvesting.

9. A method for immunizing mammals other than humans against Toxoplasma gondii, wherein the vaccine according to any one of claims 1 to 7 is administered to mammals other than humans. And how.