JP2000072693A - Medicine to be injected into spleen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To induce immune response for preventing a disease by injecting a DNA encoding a protein functioning as a vaccine against the disease into spleen of a nonhuman vertebrate. SOLUTION: A DNA encoding a protein (preferably a tumor antigen) functioning as vaccine against a disease (preferably cancer) is injected into spleen of a nonhuman vertebrate. For example, a structural protein characteristic to a cell, a virus, a bacterium, a parasite or the like which is associated with the disease is cited as the protein functioning as the vaccine against these pathogens. The injection of the DNA into the spleen is usually carried out by using a syringe, and the dose of the DNA is generally about 1 μg to about 500 mg, preferably about 1 mg to about 10 mg per a body weight.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a DNA vaccine, and mainly belongs to the field of pharmaceuticals.

[0002]

2. Description of the Related Art A vaccine is a drug used to prevent the infection of pathogenic microorganisms and the onset of diseases caused by the infection by increasing the specific immunity against pathogenic factors. So far, inactivated vaccines that use inactivated vaccines while retaining the antigenicity of the pathogenic factor have been used. Live vaccines to be used, component vaccines using only a part of the structural proteins of pathogenic bacteria and viruses have been developed,
Targets for diseases such as rabies, diphtheria, yellow fever, cholera, tuberculosis BCG, rubella and hepatitis have been put to practical use (Nikkei Bio Latest Dictionary, Nikkei BP).

[0003] Recently, a DNA vaccine has been reported as a new form of vaccine. DNA vaccines are characterized by using DNAs encoding proteins that function as vaccines, instead of using some of the structural proteins of pathogenic bacteria and viruses as in conventional vaccines. Compared to conventional vaccines, DNA vaccines have various advantages, such as less risk of contamination and the like, induction of a long-term immune response, and low-cost preparation of vaccines. ,
It is receiving considerable attention and is being developed to target diseases such as malaria, acquired immunodeficiency syndrome (AIDS), herpes, and tuberculosis.

On the other hand, with respect to the invention relating to a DNA vaccine, a method for injecting DNA into muscle or skin of a mammal to induce an immune response (US Pat. No. 5,589,466) and DNA
Injecting DNA into mammalian muscle and producing a peptide corresponding to DNA in muscle cells (US Pat. No. 5,580,859), basic techniques for DNA vaccines, and retroviral infection DNA vaccine against U.S. Pat. No. 5,620,896; DNA vaccine against influenza virus infection (WO 94217)
No. 97 pamphlet), and DNA vaccines against various virus infections, such as a DNA vaccine against human immunodeficiency virus infection (JP-A-08-198774).

[0005] However, most of the conventional DNA vaccines are administered to the muscles and skin of a living body, and it is desired to develop a new DNA vaccine administration method capable of inducing an effective immune response. I was

[0006]

Accordingly, an object of the present invention is to provide a novel DNA vaccine administration method for effective prevention of disease and a DNA vaccine for use in such administration. I do.

[0007]

The present inventors have developed an effective
As a result of intensive research focusing on cancer to provide a method for administering DNA vaccines, DNA vaccine administration targeting the spleen is remarkable as compared to the conventional case of muscle administration. The present inventors have found that an excellent cancer cell growth inhibitory effect can be obtained, thereby completing the present invention.

[0008] That is, the present invention relates to a method for inducing an effective immune response in a living body by administering a DNA vaccine to the spleen, and a DNA vaccine for use in the method. A method for inducing an immune response to prevent a disease, wherein the DNA encodes a protein that functions as a vaccine against the disease
And (2) the method according to (1), wherein the disease is cancer.
(3) The method according to (2), wherein the protein that functions as a vaccine is a tumor antigen, and (4) for injecting into a spleen of a vertebrate, which contains a DNA encoding a protein that functions as a vaccine against a disease as an active ingredient. Drugs,
(5) The agent according to (4), wherein the disease is cancer, (6)
The drug according to (4), wherein the protein that functions as a vaccine is a tumor antigen.

[0009]

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for inducing an immune response for preventing a disease, which comprises injecting a DNA encoding a protein that functions as a vaccine into the spleen.

The diseases to be prevented by using the method of the present invention include, for example, diseases caused by infection with various viruses (eg, influenza, HIV, herpes, hepatitis virus, HPV, etc.) in addition to cancer. Diseases and parasites caused by bacterial (eg, Mycobacterium tuberculosis or Chlamydia) infections (eg, malaria, leishmania, etc.)
There is no particular limitation as long as the disease has a pathogenic immunogenicity. Examples of proteins that function as vaccines against these pathogens include, but are not limited to, structural proteins characteristic of disease-related cells, viruses, bacteria, parasites, and the like, or a part thereof. For cancer, for example,
P815A antigen gene may be used for immunization against P815 tumor cells (ROSATO, A. et al., Human Gene
Therapy 8: 1451-1458 (1997)). For more information on diseases and combinations of genes encoding antigens used as DNA vaccines, see http://www.genweb.com/Dnavax/Table.
s / infectious.html ".

[0011] DN used as a vaccine in the present invention
When A is administered to the spleen, a control region that ensures transcription from the DNA is ligated to the DNA. Various promoters and enhancers for gene expression in animal cells can be used as such control regions. Suitable control regions include, for example, CE promoter (a DNA molecule in which CMV · IE enhancer and human EF1αEF321 promoter are linked; Tak
ada et al., Nature Biotechnology 15 (5) 458-461 (1997)
), CAG promoter (a DNA molecule combining CMV / IE enhancer and chicken β-actin promoter; Niwa
et al., Gene (108) 193-200 (1991)) and CMV promoter.

[0012] When the DNA is administered to the spleen,
It can be administered in a mixture with liposomes, polylysine, a transport carrier such as polyethylene glycol, an adjuvant for enhancing an immune reaction, and the like, or can be administered in a naked state by mixing with physiological saline or the like. It is possible.

[0013] DNA is usually injected into the spleen using a syringe. The dosage of the DNA may vary depending on various factors such as the type and activity of the peptide encoded by the DNA, the condition of the patient, the weight, the age, the type of drug being taken, etc. It will range from about 1 μg to about 500 mg per body weight, preferably from about 1 mg to about 10 mg per body weight. In this example, 100 μg of DNA was administered to a mouse weighing 15 to 18 g.

[0014]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[Example 1] Construction of plasmid DNA Plasmid H52 (Hayashi et al., (1992) J. Immunol, (149) 122) was prepared by using NaeI and PstI to encode the coding region of the H52 gene.
3-1229) and inserted into the multiple cloning site of the pUC118 vector (Takara). This was then digested with EcoRI and BamHI, and the resulting fragment was inserted into the vector pCAGGS (Niwa et al., (1991) Gene, (108) 193-200) containing the CAG promoter and the SV40 early polyA signal (pCAGGS was , Provided by Dr. Saito). Thus, a plasmid expression vector pCAG-H52 was constructed.

Plasmid DNA was prepared by alkaline lysis and described in the literature (Maniatis et al., (1982) Molecular cloni.
ng), separation by cesium chloride gradient and
Purified by overnight centrifugation at 80,000 rpm.

Example 2 DNA Immunization For DNA immunization, plasmid DNA (100 μl of sterilized
100 μg in a PBS (-) solution was added to 6-week-old C57BL / 6 mice (SLC Ja
pan) were injected directly into the spleen and muscle. For injection into the spleen, anesthetize the mouse with pentobarbital and
A small incision was made to 5 mm. The spleen was exposed by gentle pull, and DNA was injected using a 27 gauge needle. The abdomen was immediately sutured with a non-absorbable suture. For intramuscular injection, mice were anesthetized by inhalation of ethyl ether and the area on quadriceps was wetted with ethanol before injection to visualize the muscle. The injection was performed using a 27 gauge needle. After performing DNA immunization on the spleen or muscle for the first time, the second into the muscle at weekly intervals,
A third DNA injection was performed. 5 × 10 5 tumor cells (MC-1 endothelial cell line) were inoculated one week after the final DNA injection.

As a result, in the case where the first DNA injection was performed into the spleen, when the vector alone (pCAGGS) was injected, tumor growth was observed in all eight mice by 20 days after tumor cell inoculation. Was observed (FIG. 1). On the other hand, H5
(2) In the case of those injected with the expression vector (pCAG-H52), tumor growth was observed from day 13 after tumor cell inoculation in only one of eight mice, whereas tumor growth was observed by day 34 in the remaining seven animals. No proliferation was observed (FIG. 1). In the same injection of the B7-1 expression vector also driven by the CAG promoter, tumor growth was observed by day 20 after tumor cell inoculation in 7 out of 8 mice, and 8
Tumors were observed in all animals (data not shown). On the other hand, when a vector that simultaneously expresses H52 and B7-1 using IRES (H52 is driven by the CAG promoter) was similarly injected, tumor growth was observed by 34 days out of 9 mice. None were found (data not shown). Therefore, it is considered that DNA immunization with the H52 expression vector was significantly effective.
In addition, when DNA was injected into the muscle three times, no matter whether the vector was injected alone (pCAGGS) or the H52 expression vector (pCAG-H52), all six animals were injected by day 23 after tumor cell inoculation. Tumor growth was observed in mice (Fig. 2). Therefore, it is considered that the immune reaction with the H52 expression vector was strongly induced by the first injection into the spleen.

[0019]

According to the present invention, a method for inducing an immune response in a living body by administering a DNA vaccine to the spleen, and a DNA vaccine for use in the method are provided. According to the method of the present invention, it is possible to effectively induce an immune response in a living body, and as a new DNA vaccine administration method in addition to conventional administration to muscles and skin, application to medicine and the like is possible. Be expected.

[Brief description of the drawings]

FIG. 1 is a graph showing the relationship between the growth of tumor cells inoculated in a living body and the number of days after inoculation when a DNA vaccine is administered to muscle. In the figure, "V" indicates a vector-only control, and "H" indicates a case where the H52 gene was administered.

FIG. 2 is a graph showing the relationship between the growth of tumor cells inoculated in vivo and the number of days after inoculation when the DNA vaccine was administered to the spleen. In the secondary diagram, "V" indicates a vector-only control, and "H" indicates the case where the H52 gene was administered.

Claims (6)

[Claims] 1. A method for inducing an immune response for preventing a disease, comprising injecting a DNA encoding a protein that functions as a vaccine against the disease into the spleen of a non-human vertebrate. 2. The method according to claim 1, wherein the disease is cancer. 3. The method according to claim 2, wherein the protein functioning as a vaccine is a tumor antigen. 4. An agent for injecting into the spleen of a vertebrate, comprising a DNA encoding a protein that functions as a vaccine against a disease as an active ingredient. 5. The drug according to claim 4, wherein the disease is cancer. 6. The agent according to claim 4, wherein the protein functioning as a vaccine is a tumor antigen.