WO1996012808A1

WO1996012808A1 - Nucleic acid preparation for immunization and method for immunization using the same

Info

Publication number: WO1996012808A1
Application number: PCT/JP1995/002134
Authority: WO
Inventors: Masashi Sakaguchi; Kengo Sonoda; Kazuo Matsuo; Fukusaburo Hamada
Original assignee: Juridical Foundation The Chemo-Sero-Therapeutic Research Institute
Priority date: 1994-10-20
Filing date: 1995-10-18
Publication date: 1996-05-02
Also published as: AU3709495A; JPH08116976A

Abstract

The present invention relates to a nucleic acid preparation for immunization of an animal, comprising a linearized DNA which comprises a promoter for expression of a gene functioning in an animal cell and a gene coding for an immunizing antigen derived from a pathogen linked to the downstream of said promoter; and a method for immunizing an animal utilizing said nucleic acid preparation. Specifically, this invention relates to the immunization of poultry with a DNA coding for Newcastle disease virus (NDV)-F protein under the control of an improved chicken beta-actin promoter.

Description

D E S C R I P T I O N Nucleic Acid Preparation for Immunization and Method for Immunization Using the Same

Technical Field

The present invention relates to a nucleic acid preparation for immunization of an animal, including humans, comprising a linearized DNA and use thereof, more particular¬ ly, to a nucleic acid preparation for immunization of an animal comprising a linearized DNA linked with a gene coding for an immunizing antigen derived from a pathogen, and to a method for immunization of an animal by introducing said linearized DNA expressing said gene coding for an immunizing antigen (foreign gene) to allow for expression of said foreign gene product within the living body of the animal so that said animal is endowed with immunity against the pathogen.

Background Art

Hitherto, a vaccination has been accomplished either by a live vaccine using an attenuated live pathogen or by an inactivated vaccine using an inactivated pathogen. In general, an inactivated vaccine is superior to a live vaccine from the viewpoint of safety, but has disadvantageously a short duration of efficacy. On the other hand, though a live vaccine bears more concern about adverse side effects in view of safety, it is excellent in both effectiveness and duration of efficacy. Under such circumstances, in case that a pathogen is successfully attenuated and safety is also confirmed, a live vaccine is generally widely used in order to attain effectiveness. However, as far as a live vaccine is concerned, safety concern always sticks around and there still remains a possibility that even those vaccines autho¬ rized by the government provoke great problems with respect to safety like, for example, in the case of the adverse side effects of viral meningitis induced by mumps virus vaccine in recent years.

Recently, in order to make use of the advantages that a live vaccine has an excellent effectiveness and duration of efficacy, it has been attempted to prepare a polyvalent live vaccine which utilizes as a vector a live vaccine to which a gene coding for an antigen derived from another pathogen is incorporated for protection of infection.

However, since such viral vector vaccine is also based on a live vaccine, there still remains apprehension for pathogenicity in compensation for effects thereof . Further¬ more, in addition to the above safety concern, a live vaccine has another disadvantages: (i) in case that a host has already acquired an immunity against a vaccine strain or a pathogen of the same type as that of the vaccine strain, the vaccine strain inoculated to the host is easily eliminated from the host, and hence, the vaccine does not effectively work; (ii) by the same reason as mentioned in (i), a vaccine does not effectively work in the presence of a maternal antibody; and (iii) in view of interferential action between different kinds of live vaccines, it is hard to prepare a polyvalent live vaccine, i.e. a mixture of plural live vaccines.

Under such circumstances, as an unprecedented immunological procedures, it has been attempted to directly administer a gene (plasmid) encoding a pathogen antigen into the living body for expressing said antigen within the living body, and as a result, successful results have been obtained in an experiment with a heterogeneous animal in chicken influenza and rabies viruses (Montgomery, DNA Cell.Biol. , 12, 777-783 (1993); Influenza/mouse, and Robinson, Vaccine, 11, 957-961 (1993); Influenza/mouse, chicken).

Such a vaccine using a plasmid (hereinafter refer to as "DNA vaccine") is expected to facilitate a construction of a polyvalent vaccine since, unlike a live vaccine, decrease in effects of vaccine due to interference between plural plasmids does not occur theoretically. Such DNA vaccine is also advantageous in that it can solve the problem posed by a conventional live vaccine or a newly developed recombinant vaccine using a live vaccine as a vector, that is, there is no such a problem that an individual inoculated with said live vaccine excretes the live microorganism and it becomes newly an infectious source to other individuals.

However, as reported previously, for example in case of chicken influenza, only 50% protection could be attained by simultaneously administering each 100 μg plasmid via three routs (i.e. intravenous, subcutaneous and intraperitoneal) twice (Robinson, Vaccine, 11, 957-961 (1993)). In an experiment using mice, the desired protection from influenza could be obtained by administering 200 μg plasmid three times (Montgomery, DNA Cell.Biol. , 12, 777-783 (1993)).

According to another report, it has been attempted to administer a plasmid in the form of being adhered to a precious gold powder in order to decrease a dose of a plasmid (E.F.Fynan, ProNAS, 90, 11478-11482 (1993)), this is not economically practical in the field of poultry (e.g. chicken) vaccine where a vaccine is available with a low cost.

As mentioned above, although an immunization procedure by a direct administration of a plasmid has already been attempted in several cases, an animal immunization method actually practical in view of effectiveness and dosage form has not yet been reported, and hence, there is a need to develop a practically effective method for immunization in lieu of the conventional vaccine.

Object of the Invention

Under such circumstances, the present inventors have intensively studied an improved method for immunizing an animal with a plasmid and have now succeeded in effectively protecting an animal from a targeted infectious disease by constructing a plasmid wherein a gene coding for an antigen for immunization is incorporated into the downstream of a promoter, and directly administering said plasmid, not in the form of a natural circular DNA but after being linearized by restriction enzyme treatment, to said animal such as young chicken.

An object of the present invention is to provide a nucleic acid preparation for immunization of an animal, comprising a linearized DNA which comprises a promoter for expression of a gene functioning in an animal cell and a gene coding for an immunizing antigen derived from a pathogen linked to the downstream of said promoter.

Another object of the present invention is to provide a method for immunizing an animal which comprises preparing a linearized DNA which comprises a promoter for expression of a gene functioning in an animal cell and a gene coding for an immunizing antigen derived from a pathogen linked to the downstream of said promoter, and introducing said DNA into the living body of the animal.

Brief Explanation of Drawing

Fig. 1 shows a construction of NDV-F protein- expressing plasmid pCAGF in Example 1.

Disclosure of the Invention

Hitherto, a linearization of a plasmid has been utilized in a homologous recombination or integration of a foreign DNA into a genome. However, the effects of a DNA vaccine have been confirmed only in the form of a usual circular DNA, and hence, the improvement of the vaccinal effects due to linearization is entirely unexpected. One of the reasons why a linearized DNA has not been utilized in an experiment of a DNA vaccine is that a linearized DNA has been considered to be labile to a DNase. However, as mentioned hereinbelow, the vaccinal effects could have been exhibited only by a linearized plasmid whereas a significant increase in an antibody titer or the vaccinal effects could not have been shown with an administration of a circular plasmid. This shows, on the contrary to the previous prediction, that a DNA vaccine in a linearized form is far more effective than that in a circular form.

Hereinbelow, the present invention is explained in more detail.

According to the present invention, firstly (1) a gene encoding various antigens for protection of infection is cloned into the downstream of a promoter in an expression plasmid; then (2) said plasmid is linearized with a restric¬ tion enzyme at an appropriate site that does not affects an expression of said gene; and (3) the resulting linearized plasmid is administered to an individual. The promoter used in the procedure (1) includes most preferably a chicken β- actin gene promoter. Especially, when the DNA vaccine of the present invention is used as a vaccine for chicken, the chicken β-actin gene promoter, the original promoter in chicken, is the most preferable. For the chicken β-actin gene promoter, a variety of improved promoters have been known for the purpose of enhancement of expression efficiency (Japanese Patent First Publication (Kokai) No. 2-156891 and Japanese Patent First Publication (Kokai) No. 3-168087), and such improved promoters based on the chicken β-actin gene promoter can also be suitably employed.

A gene coding for an antigen for protection of infection to be incorporated into the plasmid includes a gene coding for an antigenic protein for immunization which can be a vaccinal antigen for various chicken diseases such as viral diseases, bacterial diseases or parasitic diseases.

Construction of a polyvalent vaccine can easily be accomplished by mixing plural plasmids, each plasmid having a gene coding for a different antigen for protection of infection linked to the downstream of a promoter, or by constructing a plasmid in which multiple sets of a gene coding for an antigen for protection of infection linked to the downstream of a promoter are ligated in tandem. When a commercially available phagemid vector, pUC119, which allows for cloning of DNAs of up to about 20kb and stable replication thereof, is used, a promoter, e.g. a chicken β-actin gene promoter (about 1.4 kb) is cloned into pUC119, and at the downstream of said promoter, any gene coding for an antigen for protection of infection and a poly A signal can stably be incorporated. A length of about 20 kb of a gene which can be incorporated into this vector is large enough for any gene coding for an antigen for protection of infection. For example, in case of infectious bursal disease virus (IBDV), a total necessary gene is of 3.2 kb including genes coding for all the capsid proteins, VP243. An F gene of a chicken Newcastle disease virus (NDV) is of about 1.7 kb, and a gene coding for a spike protein of infectious bronchitis virus (IBV) is of about 4.2 kb, and therefore, these genes can be well incorporated into the phagemid vector pUC119 to prepare the linearized DNA of the present invention.

An animal to be inoculated in accordance with the present invention includes any animal to which a conventional vaccination has been conducted, including porcine, bovine, birds including poultry such as chicken, and mammals including humans. Although a chicken is used for assessing the effects of the present invention in the following Examples, the present invention is not limited to such Examples but various other animals can also be expected to exert the effects of the present invention. Especially in case of a chicken, those immediately after hatching were inoculated in the following Examples, but it should be understood that the DNA vaccine of the present invention is effective regardless of a specific site for inoculation or age of an animal.

Although the effectiveness of the linearized DNA alone is shown in the experiment herein, it is expected that a more effective vaccine can be prepared in combination with gold particles or a carrier such as a liposome as reported previously, in case of a vaccine for other animals such as bovine, porcine, canine, feline or human where a vaccine can be rated at a higher price.

Best Mode for Carrying out the Invention

The present invention is explained in more detail by the following Examples with a DNA vaccine prepared from a plasmid expressing an F protein gene of chicken Newcastle disease virus (NDV) but it should not be construed to be limited thereto. Example 1 (Construction of F protein-expressing plasmid)

A plasmid pCAGGS bearing a chicken β-actin gene promoter (Japanese Patent First Publication (Kokai) No. 3- 168087) was cut at the Hindlll site at the 3' (downstream) of said promoter, and after a gel electrophoresis, a 3.8 kb fragment containing said promoter was collected from the gel. After this fragment was subjected to end-filling treatment and dephosphorylation, a 1.7 kb NDV-F protein gene (H.Sato et al., Virus Research, 7, p241-255 (1987)) was incorporated into said site to construct an NDV-F protein gene-expressing plasmid pCAGF. A scheme for construction of the NDV-F protein gene- expressing plasmid pCAGF is shown in Fig. 1. Example 2 (Immunization test with F protein-expressing DNA)

The NDV-F protein gene-expressing plasmid pCAGF was linearized with a restriction enzyme Seal by cutting a single site derived from the plasmid pUC119 (cf. Fig. 1). The linearized DNA was collected in a usual manner by a phenol treatment and ethanol precipitation. Then, each 100 μg of a circular or linearized pCAGF was administered intramuscularly to the crus muscle of one week old SPF (specific pathogen free) chicken with a syringe. In order to attain efficient incorporation of DNAs into cells, the effects of liposome, one of drug delivery systems (DDS), was also examined. That is, in accordance with protocol of LIFE TECHNOLOGIES, 500 μg of the linearized pCAGF was mixed with Lipofectin or Lipofectamine, and after the mixture was allowed to stand for a period as instructed in the protocol, they were inoculated to two groups of chickens, each group consisting of 5 chickens.

In each group, chickens were bled at 3 weeks up to 9 weeks after the inoculation, an anti-F protein antibody titer was measured by ELISA with passage of time. The ELISA used herein is such a method for measuring an antibody utilizing as an antigen cells which continuously produces F protein (mouse myeloma cells P3-X63-Ag8.653 transformed with NDV-F gene under regulation of the above β-actin gene promoter) and which is immobilized onto 96-well plate for tissue culture as described in more detail in Japanese Patent Application No. 5-96727.

As a result, among the groups to which the linearized DNA is administered, a significant increase in an antibody titer was observed both in the group without DDS and in the group with Lipofectin wherein the latter group showed a lower titer than that of the former group as shown in Table 1, which confirms that the linearization of the vaccinal plasmid exhibits equivalent to or more than the use of Lipofectin as DDS from the viewpoint of antibody titer. Table 1

Individual Form of F-ELISA value (weeks after

No. plasmid DDS inoculation1 I

3 4 5 6 9

81 Circular None -0.02 -0.02 0.05 -0.01 -0.06

82 -0.02 0.08 0.12 0.16 0.04

83 0.00 0.00 0.00 0.02 0.01

84 -0.03 0.00 0.06 0.04 0.04

85 -0.04 -0.03 0.00 -0.01 -0.05

86 Linear None 0.01 0.01 0.03 0.03 -0.09

87 -0.03 -0.03 0.01 0.00 0.01

88 -0.04 -0.01 0.00 -0.03 0.00

89 0.23 0.59 0.39 0.32 0.13

90 0.16 0.96 1.43 1.19 0.33

91 Linear Lipo¬ 0.13 0.50 0.57 0.57 0.17 fectin

92 -0.02 -0.03 0.04 0.04 -0.01

93 -0.03 0.16 0.34 0.20 0.08

94 " -0.08 -0.04 0.04 0.01 0.13

95 0.00 0.26 0.56 0.55 0.29

96 Linear Lipo- -0.08 -0.07 -0.01 0.00 -0.01 fectamine

97 -0.02 -0.03 0.03 0.02 0.00

98 ^» -0.03 -0.02 0.01 0.00 0.01

99 ^» ^» -0.06 -0.03 0.02 0.00 0.01

100 ^•• -0.06 -0.03 0.00 0.00 0.00

101 No Ino¬ None -0.03 -0.01 0.01 0.02 -0.01 culation

102 0.03 -0.03 0.01 0.00 0.01

103 -0.04 -0.02 0.00 -0.01 -0.01

104 -0.03 0.01 -0.04 0.00 0.01

105 0.00 -0.03 0.00 0.01 -0.03

The antibody titer was measured by the method for measuring an antibody by ELISA as described in Japanese Patent Application No. 5-96727. From these results, it was assumed that those individuals to which the preparation for immuniza- tion of the present invention was administered could well protect from challenge with virulent NDV. In order to confirm this, a test was carried out by challenge with virulent NDV. That is, after bleeding at the 9th week from the inoculation, the individuals were subjected to intramuscular challenge with 10^A minimum lethal dose of virulent NDV Sato strain in accordance with the National Assay Standard for Newcastle disease virus vaccine, and were observed for 2 weeks.

As a result, as shown in Table 2, it was confirmed that the individuals showing increase in antibody titer exhibited a sufficient protection even at the 9th week after inoculation of the DNA vaccine, proving the protective effects assumed by the assessment of antibody titer. In case of the individuals who were expected not to exhibit protection in view of the prior data (No. 89 with antibody titer at challenge being 0.13; No. 93 with antibody titer at challenge being 0.08; and No. 94 with antibody titer at challenge being 0.13), it was assumed that a cellular immunity provoked by inoculation of the DNA vaccine is involved in protection.

According to the above results, there is possibility to provide immunity for a period of time well sufficient for breeding broiler with one inoculation.

Surprisingly, some individuals did not exhibited increase in antibody titer until the 9th week like No. 94, and hence, it was assumed that antibody titer may be increased after longer period of time and the increased antibody titer be continued thereafter in some individuals. Table 2

Indivi .dual Form of Antibody titer Assessment

No. plasmid DDS at Attack of Results

81 Circular None -0.06 Death

82 0.04 Death

83 0.01 Death

84 0.04 Death

85 -0.05 Death

86 Linear None -0.09 Death

87 " 0.01 Death

88 0.00 Death

89 0.13 0

90 " 0.33 0

91 Linear Lipo¬ 0.17 0 fectin

92 -0.01 Death

93 0.08 0

94 0.13 0

95 0.29 0

96 Linear Lipo- -0.01 Death fectamine

97 0.00 Death

98 0.01 Death

99 0 01 Death

100 0 00 Death

101 No Ino¬ None -0.01 Death culation

102 0.01 Death

103 -0.01 Death

104 0.01 Death

105 -0.03 Death

Claims

C L A I M S

1. A nucleic acid preparation for immunization of an animal, comprising a linearized DNA which comprises a promoter for expression of a gene functioning in an animal cell and a gene coding for an immunizing antigen derived from a pathogen linked to the downstream of said promoter.

2. The nucleic acid preparation for immunization of claim 1 wherein said promoter for expression of the gene is a chicken β-actin gene promoter or an improved chicken β- actin gene promoter.

3. The nucleic acid preparation for immunization of claim 1 wherein said gene coding for an immunizing antigen derived from a pathogen is a gene coding for a chicken Newcastle disease virus F protein.

4. The nucleic acid preparation for immunization of claim 1 wherein said animal is a poultry.

5. The nucleic acid preparation for immunization of claim 1 wherein said poultry is a chicken.

6. A method for immunizing an animal which comprises preparing a linearized DNA which comprises a promoter for expression of a gene functioning in an animal cell and a gene coding for an immunizing antigen derived from a pathogen linked to the downstream of said promoter, and introducing said DNA into the living body of the animal.

7. The method of claim 6 wherein said promoter for expression of the gene is a chicken β-actin gene promoter or an improved chicken β-actin gene promoter.

8. The method of claim 6 wherein said gene coding for an immunizing antigen derived from a pathogen is a gene coding for a chicken Newcastle disease virus F protein.

9. The method of claim 6 wherein said animal is a poultry.

10 The method of claim 9 wherein said poultry is a chicken.

11. The method of claim 10 wherein said chicken is a young bird.