KR100377948B1 - Novel plasmid dna used as immunogen of aids virus - Google Patents

Abstract

PURPOSE: Provided is a novel plasmid DNA used as an immunogen of AIDS(acquired immunodeficiency syndrome) virus. Also, provided is a method for increasing immunity using the same. CONSTITUTION: A novel plasmid DNA is characterized by containing gag, env and rev genes of HIV-1(human immunodeficiency virus) and interleukin-2 of a mammal to be immunized. It produces HIV-1 specific antibody when used as an immunogen.

Description

Novel plasmid DNA used as an immunogen for AIDS virus

The present invention relates to a novel plasmid DNA used as an immunogen of AIDS (acquired immunodeficiency syndrome) virus and an immune enhancing method using the same. More specifically, the present invention includes the gag, env and rev genes of human immunodeficiency virus (HIV-1), and the interleukin-2 gene of a mammal to be immunized, and when used as an immunogen in a mammal, Plasmid DNA to produce specific antibodies and methods for increasing the immune response to HIV-1 using the same.

Plasmid DNA has been reported to form antibodies specific for antigens expressed in plasmid DNA and to induce protective immune responses when injected into mice by themselves without the adjuvant addition of any immune enhancer (see: Ulmer et al. 1993 Science 259. 1745). Influenza virus (Ulmer et al. 1994 Vaccine 12. 1541), Hepatitis B virus (Chow et al. 1997 J. Virol 71. 169), and Lavis Rabies virus (Xiang et al. 1994 Virol. 199. 132), Herpes Simplex virus (Mcclements et al. 1996 Proc, Natl. Acad. Sci. (USA) 93 11414), Hepatitis C virus (Lagging et al. 1995 J. Virol. 69. 5859), HIV-1 (Wang et al. 1995 Virol 211. 102) Injecting plasmid DNA into animal models reported that humoral and cellular immune responses were induced. In particular, some viruses such as influenza virus and Rayvis virus have been reported to induce a protective immune response.

Plasmid DNA is much more preferable than the conventional four vaccines or subunit vaccines not only in terms of safety, convenience of manufacture, storage and transportation, but also in terms of its effect as a vaccine. Inoculated plasmid DNA vaccines can synthesize immunized viral proteins in the host, which not only produce antibodies that inactivate the virus and antibodies specific for the expression protein, but also kill cytotoxic cells. T lympocytes) resulting in an increase in response (MacDonnell et al. 1996. New Engl. J. Med. 334, 42). In addition, since the plasmid DNA injected once exists in the host for a long time, the immune response can also be sustained, which is suitable as a prophylactic vaccine and a lifetime vaccine.

Plasmid DNA vaccines having the above-mentioned advantages are attracting attention for the improvement of HBV, influenza virus vaccines that are already in use, or for the development of new vaccines against Hepes simplex virus, HCV, HIV, etc., in which vaccine manufacture has failed.

Since the initial announcement that DNA vaccines against NP genes of influenza viruses induce protective immune responses against other strains of influenza virus (Ulmer et al. 1993. Science 259. 1745), HIV-1, which also contains various strains, DNA vaccines have been developed with great interest. That is, plasmid BNAs expressing the envelope proteins (gp160, gp120, gp41) and rev of HIV-1 (see Wang et al. 1993 Proc, Natl. Acad. Sci (USA) 90. 4156) and monkeys. (In Coney et al. 1994 Vaccine 12. 1545), or plasmid DNA expressing fragments of coat proteins and non-infectious viral proteins (Lu et al. 1995 Virol. 209.174). When injected into, it has been shown that humoral and cellular immune responses are induced.

However, these AIDS virus vaccines show only about 10 times the amount of antibody production compared to the standard group inoculated with plasmids expressing other antigens (Wang et al. 1995 AIDS 9, S159). There is an urgent need for new plasmid DNA that can provide positive antibodies.

Therefore, the present inventors have made intensive studies to develop a novel plasmid DNA that induces a better immune response against the AIDS virus, including the gag, env and rev genes of HIV-1 and the interleukin-2 gene of rats. When the plasmid DNA is injected into mice as an animal model, the production of HIV-1 specific antibody is not less than three times higher than that of the conventional AIDS virus vaccine, and the immune response is high even with a small number of injections. It confirmed that a ratio was high and came to complete this invention.

After all, it is an object of the present invention to provide novel plasmid DNA for use as an immunogen for AIDS viruses.

It is another object of the present invention to provide a method of increasing the immune response to HIV-1 by inoculating mammalian plasmid DNA to produce HIV-1 specific antibodies.

1 is a schematic diagram illustrating the cloning process of plasmids pCD-HIV / GE and pCD-HIV / GE-IL2 of the present invention.

FIG. 2 is a graph comparing the amount of HIV-1 specific antibodies generated by two injections of plasmids pCD-HIV / GE and pCD-HIV / GE-IL2 into mice in a serial dilution method.

3 is a graph showing the degree of generation of HIV-1 specific antibodies according to the elapsed time after injection of plasmids pCD-HIV / GE and pCD-HIV / GE-IL2 into mice.

Hereinafter, the present invention will be described in more detail.

We have prepared plasmid DNA that can be used as an immunogen for AIDS virus, including plasmid DNA that can include and express gag, env and rev genes of HIV-1, and gag, env of HIV-1. And a plasmid DNA capable of expressing the rev gene and the interleukin-2 gene of a mammal to be immunized. In particular, in one preferred embodiment of the present invention, plasmid DNA comprising gag, env and rev genes of HIV-1 and interleukin-2 gene of rats has not been applied as an animal model to HIV-1 DNA vaccine research so far. rats) were injected to determine the production of antibodies specific for HIV-1.

As a result, when the mice were inoculated with plasmid DNA containing the gag, env and rev genes of HIV-1 as an immunogen, HIV-1 specific antibodies were generated. Slightly higher or equal to the amount of antibody induced (Wang et al. 1995 AIDS 9. S159). However, the amount of antibody produced in mice vaccinated with plasmid DNA containing the gag, env and rev genes of HIV-1 and the interleukin-2 gene of mice was three times higher than that of plasmid DNA containing the gag, env and rev genes of HIV-1. The above high antibody amount was shown. In addition, plasmid DNA containing the gag, env and rev genes of HIV-1 and the interleukin-2 gene of rats increased the number of immune responses and immune responses with a small number of injections. Therefore, the use of plasmid DNA expressing interleukin-2 together was found to increase efficiency as an AIDS virus vaccine.

Thus, even if the plasmid containing the gag, env and rev genes of HIV-1 and the interleukin-2 gene at the same time, both plasmids containing the former two groups of genes were injected into the mammal, May increase the immune response.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only to specifically describe the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Example 1 Preparation of Plasmid DNA for Use as Immunogen of AIDS Virus

To prepare plasmid DNA that can be used as an immunogen for the AIDS virus, 2.5 kb DNA fragments obtained by cleaving plasmid pEX5496 (Sodroski et al. 1986 Nature 321 412) with SalI and BamHI were first cleaved with SalI and BamHI. Plasmid pHXB-Δtat was prepared by inserting the plasmid pHXB-CAT1 (Terwilliger et al. 1989 Nature 321. 412). The 8.0 kb DNA fragment obtained by cleaving the pHXB-Δtat plasmid with NarI and XbaI restriction enzymes was then linked to the pBluescriptSK- (Stratagene, USA) plasmid digested with ClaI and XbaI to obtain a pSK-HIV / GPE plasmid. Subsequently, the pSK-HIV / GPE plasmid was digested with BalI to remove 1.9 kb DNA fragments and then self-linked to prepare plasmid pSK-HIV / GE.

Meanwhile, the HindIII-BamHI DNA fragment of pUC19 was inserted between the HindIII and BamHI cleavage sites of plasmid pCDNA1 (Invitrogen, USA) to obtain plasmid pCDNA1-1.

The plasmid pCDNA1-1 obtained above was digested with XhoI and XbaI to obtain a DNA fragment, which was then inserted into the plasmid pSK-HIV / GE digested with XhoI and XbaI to prepare a pCD-HIV / GE plasmid.

In addition, the plasmid pCD-HIV / GE was cleaved with XbaI, followed by inserting a murine IL-2 gene obtained by amplification by polymerase chain reaction (PCR), thereby inserting pCD-HIV / GE-IL2. Plasmids were prepared. At this time, PCR was performed by using pSK-mIL-2 as a template and using T3 (5'-ATTAACCCTCACTAAAG-3 ') and IL-2A (5'-GGTCTAGAGTTATTGAGGGCTTGTTG-3') as primers, respectively. Plasmid pSK-mIL-2 was a 0.5 kb DNA fragment (mIL) obtained by digestion of an MFG-mIL-2 plasmid (see, Dranoff et al. 1993. Proc. Natl Acad. Sci. (USA) 90. 3539) with XbaI and BamHI. -2 gene) was prepared by inserting into the pBluescriptSK- (Stratagene, USA) plasmid digested with restriction enzymes XhaI and BamHI.

The construction of the plasmids pCD-HIV / GE and pCB-HIV / GE-IL2 described above is schematically shown in FIG.

Escherichia coli MC1061 was transformed with the two plasmid DNAs prepared above, and the transformed Escherichia coli were called Escherichia coli MC1061 (pCD-HIV / GE) and Escherichia coli MC1061 (pCD-HIV / GE-IL2), respectively. It was deposited on May 30, 1997 with the deposit numbers KCTC 8802P and KCTC 8803P, respectively, to the Genetic Bank (KCTC) affiliated with the Korea Institute of Science and Technology.

On the other hand, the plasmid DNA used in the Examples to be described later is a plasmid purified by CsCl-EtBr method after culturing using colon group MC1061 as a host.

Example 2 Comparison of Antibody Production Amount Using pCD-HIV / GE and pCD-HIV / GE-IL2 as Immunogen

As a first step to determine whether the plasmid DNA prepared in Example 1 functions as an immunogen of the AIDS virus, purified plasmid DNA was inoculated into a rat population of 8 weeks old obedient (BUF / n). At this time, in order to enhance the delivery effect of the plasmid DNA, three days before the inoculation of the plasmid DNA, the muscle cell destroying agent Bupivacaine was diluted to 0.25% (v / v) of the mouse in an amount of 300 ul per individual. The bilateral tibialis anterior (TA) muscles were injected into four sites (Domini 1995. FEBS lett. 332. 179). In addition, plasmid DNA was also divided into four sites in the T.A muscle, and 600 ug of each individual was suspended in saline (0.85% NaCl) to inoculate.

Next, in order to compare the amount of antibody produced when plasmids pCD-HIV / GE and PCD-HIV / GE-IL2 were used as immunogens, plasmids pCD-HIV / GE and pCD-HIV / GE-IL2 were used as described above. Was initially inoculated. Serum at 14 dwells was serially diluted after inoculation, injected into an ELISA plate pre-reacted with HIV-1 viral lysate, added peroxidase-conjugated anti mouse-IgG secondary antibody, and then peroxidated. Reaction with the substrate of the enzyme was observed to change the OD value (see Figure 2). At this time, the O.D value is higher as the amount of anti-HIV-1 antibody is higher, and the standard serum represents the serum of standard group rats inoculated with plasmids expressing other antigens (pCI-HCV / NS).

As shown in FIG. 2, the sera of mice inoculated with pCD-HIV / GE showed 1/100 dilution of the standard serum and comparable OD values at 1/900 dilution and were immunized with pCD-HIV / GE-IL2. The rat serum was higher than the OD value when diluting 1/100 of the standard serum even though it was diluted 1/3000. The amount of antibody produced in mice vaccinated with plasmid pCD-HIV / GE was slightly higher than or equal to that of mice derived from conventional AIDS DNA vaccines (Wang et al. 1995 AIDS 9. S159), and plasmid pCD-HIV. The amount of antibody produced by mice inoculated with / GE-IL2 was three times higher than that of pCD-HIV / GE.

Example 3 Comparison of the Number of Immune Responses Using pCD-HIV / GE and PCD-HIV / GE-IL2 as Immunogen

In order to compare the number of immune responses when plasmids pCD-HIV / GE and pCD-HIV / GE-IL2 were used as immunogens, pCD-HIV / GE and pCD-HIV / GE-IL2 were respectively injected into mice as in Example 2. Three injections were performed and the number of immune responses was compared (see Table 1), i.e. the serum of rats 2 weeks after the 2nd and 3rd inoculation (14 weeks and 26 weeks after the first inoculation) Diluted by 1/100, the change in OD value was observed by the same ELISA method as in Example 2. At this time, mice that appeared 3 times higher than the serum of the standard group mice inoculated with plasmids expressing other antigens (pCI-HCV / NS) were included in the immune response population.

Table 1: Immune Response Population by Immunogen

As shown in Table 1, in the case of plasmid pCD-HIV / GE-IL2, two of the inoculations showed the number of four immune-responsive individuals, which was compared with the three injections of plasmid pCD-HIV / GE. The same result was found. Therefore, it was confirmed that the plasmid pCD-HIV / GE-IL2 of the present invention can be used as an excellent AIDS virus vaccine by increasing the population of the immune response even with a small number of injections.

Example 4 Comparison of Immune Reactivity with pCD-HIV / GE and pCD-HIV / GE-IL2 as Immunogen

To compare the immunoreactivity (degree of antibody production) when plasmids pCD-HIV / GE and pCD-HIV / GE-IL2 were used as immunogens, pCD-HIV / GE and pCD-HIV / GE- as in Example 2 IL2 was initially inoculated into rats, blood was drawn from the eyes of rats 8, 14, 20, and 26 weeks after inoculation, and serum-specific antibodies were obtained using the same ELISA method as in Example 2. The amount of was measured as the average OD value (see Figure 3).

As shown in FIG. 3, the two-time inoculation of plasmid pCD-HIV / GE showed an average OD value three times higher than that of the antibody of standard group mice inoculated with plasmid pCI-HCV / NS, and the plasmid pCD-HIV. In the rat group using / GE-IL2 as the immunogen, the single vaccinated group showed an average OD value similar to that of two pCD-HIV / GE inoculations. Higher values than one case. Two doses of plasmid pCD-HIV / GE-IL2 and three doses of PCD-HIV / GE showed higher OD mean values despite the same number of immune responders (see Table 1). This is because the mice inoculated with pCD-HIV / GE-IL2 showed higher OD values.

As described and demonstrated in detail above, in the present invention, novel plasmids pCD-HIV / GE and pCD-HIV / GE-IL2, which can be used as immunogens of the AIDS virus, were prepared, in particular pCD-HIV expressing interleukin-2. / GE-IL2 caused a decrease in the number of inoculations, an increase in immune response population, and an increase in immune responsiveness, thereby further enhancing the effect as an AIDS DNA vaccine.

Claims (5)

Plasmid pCD-HIV / GE (KSTC 8802P) containing gag, env and rev genes of human immunodeficiency virus-1 (HIV-1). Plasmid DNA capable of expressing the gag, env and rev genes of HIV-1 (human immunodeficiency virus-1) and interleukin-2 genes of mammals to be immunized. The method of claim 2, The plasmid DNA, characterized in that the mammal is a human. The method of claim 2, The plasmid DNA, characterized in that the mammal is a rat. Immunizing a mammal other than human by injecting a plasmid simultaneously or separately with gag, env and rev genes of HIV-1 (human immunodeficiency virus-1) and the interleukin-2 gene of the mammal to be immunized, How to increase immune response to HIV-1.

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