JP2003321391A - Hiv vaccine using recombinant vaccinia virus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a vaccine having a strong HIV immune response and a sufficiently low toxicity. <P>SOLUTION: A useful HIV vaccine is composed by using vaccinia virus LC16m8 and incorporating, into it, a gene for coding the envelope protein of HIV virus; that is, the HIV vaccine of the invention comprises recombinant vaccinia virus LC16m8 having a gene or its part for coding the envelope protein of HIV and expresses the gene or its part. The safety can be enhanced by further introducing an antibiotics-sensitive gene into the vaccinia virus. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an HIV (human immunodeficiency virus) vaccine using recombinant vaccinia virus LC16m8.

[0002]

2. Description of the Related Art Currently, more than 40 million people worldwide are hi
It is infected with V, most of which is in developing countries. Due to recent advances in anti-HIV drugs and their combination therapy, HI
Mortality in V-infected patients was dramatically reduced. However, implementing such treatments in developing countries is economically difficult. Therefore, in order to prevent the spread of HIV / AIDS, it is essential to develop a safe, effective and inexpensive infection protective vaccine. A vaccine using a live virus as a vector can be expected to highly express the antigen in the body and induce strong immunity. However, the vaccinia virus currently under study is a growth-defective strain such as Ankara strain. HIV-1 antigen expression plasmid, so-called HIV
Co-administration method with DNA vaccine is HI in primates
There is a report that it is possible to induce V infection protection. However, it is considered that the growth-deficient strain cannot sufficiently protect against infection by its single immunization.

[0003] The vaccinia virus strain LC16m8 is known to be an attenuated strain capable of proliferating in humans and mammals but having low proliferative ability, and particularly low reproducibility in the central nervous system. Effort ",
Clinics and viruses vol.24, No.1, 41-47 (1996.3)). It was reported that this LC16m8 strain was inoculated to about 50,000 children in 1974 and did not encounter any serious side effects (Ministry of Health and Welfare Smallpox Research Group research report, clinical and virus vol.3,
No. 3, 269, 1975). In addition, studies on human hepatitis B vaccine using the LC16m8 strain as a vector have been found to strongly induce an antigen-specific immune response (Hashizume Takeshi, Basic, clinical and viral vol. Vol. Of the new attenuated smallpox strain LC16m8 strain).
3, No. 3, 229, 1975). On the other hand, a vaccine in which a vaccinia virus such as NYCBH is recombined to express an HIV envelope protein (gp-120 or gp-160) has been developed (Table 2000-504326). However, since the toxicity of such vaccinia virus is not weak enough,
There was a problem with the safety of this vaccine itself.

[0004]

It is known that recombinant vaccinia virus can induce a strong immune response (Belyyakov IM et al., J. Virology vol.72, 82).
61-8772, 1998), there is no report that vaccinia virus currently available for humans can completely protect against HIV infection. The present invention aims to provide a vaccine with a strong HIV immune response and a sufficiently low toxicity.

[0005]

[Means for Solving the Problems] To solve the above problems, the inventors of the present invention have a vaccinia virus LC16m8, which is known as an attenuated and effective expression vector.
By incorporating a gene encoding an envelope protein of HIV virus into a useful HIV
Configured the vaccine. Further, the present inventors, since this vaccinia virus is a smallpox virus originally, in case of emergency, by introducing a gene more sensitive to antibiotics to this vaccinia virus, when this virus grows, By adding a means (safety key) that can be treated by the administration of this antibiotic, safety has been improved.

[0006] That is, the present invention is an HIV vaccine comprising a recombinant vaccinia virus LC16m8 having a gene encoding HIV envelope protein or a portion thereof and expressing the gene or a portion thereof. The vaccine of the present invention is mainly composed of the recombinant vaccinia virus LC16m8 described above, and may optionally contain a pharmaceutically acceptable carrier, adjuvant, antiviral chemotherapeutic compound and the like.

This gene or a part thereof may be incorporated into the thymine kinase (TK) gene of the vaccinia virus LC16m8. Further, the envelope protein is preferably gp160 (GenBank No. U21135).
It is preferable to further incorporate a gene sensitive to antibiotics or a portion thereof into the vaccinia virus LC16m8. As a gene susceptible to this antibiotic, the herpes simplex virus TK (HSV-TK) gene (GenBank
No. AF05731) is simple and preferable.
Ganciclovir is preferred as the antibiotic.

[0008]

EXAMPLES The present invention will be illustrated below with reference to Examples.
It is not intended to limit the light.Example 1 In this example, the HIV virus envelope protein
Is HIV_89.6Recombinant vaccinia expressing full-length gp160
A virus was made. pSC65 plasmid (molecular weight 1
0243 bp, provided by Dr. B. Moss, NIH, MD, USA. )
At the positions of Sal 1 and Sam 1 of gp160 gene
By incorporating (GenBank No. U21135)
Kinase (TK) gene, gp160 full-length gene and LacZ gene
Was constructed (Fig. 1). one
However, TK (-) cells (CRL143 cells were obtained from ATCC)
Chisinia virus (LC16m8, from Chiba Serum Research Institute)
Obtained) and transferred to the transfer vector
Was introduced by electroporation and cultivated in the presence of BudR.
This recombinant vaccinia by culturing (MEM medium)
Only the virus was amplified. X-gal containing agar
Select blue plaques on (MEM medium) and
3 times and partially purified by sucrose density gradient centrifugation
did. Hereinafter, the recombinant vaccinia virus obtained in this way
Lusu is called "vLC16m8-HIV".

[0009]Example 2 The vLC16m8-HIV prepared in Example 1 was treated at 37 ° C. for 1 hour,
Infected CV-1 cells (obtained from ATCC) at 24:00
After a while, the infected cells were collected with a scraper, and 3,000 r
It was centrifuged at pm for 10 minutes. Pellet the NP-40 lysis buffer
(50 mM Tris-HCl, 150 mM NaCl, 1% (wt / vol) NP-40, 1 mM
PMSF) and vortex on ice for 30 minutes
It was Centrifuge at 10,000 rpm for 10 minutes and collect the supernatant.
It was Add equal amount of supernatant and 2xSDS sample buffer,
It was dissolved and boiled for 5 minutes. This is density gradient SDS-PA
Separated by GE. Using this as an antigen, HIV infected humans
Serum (Ortho HIV Western Blot Kit, Dia
Western blot using Gnostic Co., Ltd. as an antibody
Was placed (Fig. 2). From Figure 2, the incorporated gp
Strong expression was observed around 160 kd with a size of 160 (res.
1). Vaccinia virus LC16m8 only (below
It is called "vLC16m8-empty". ) Do the above with
The obtained soluble protein (lane 2)
No band was observed.

[0010]Example 3 10 of vLV16m8-HIV obtained in Example 1⁶pfu each 7-8 weeks old B
ALB / c mice were intramuscularly administered on days 0 and 10. 96w
Coat the gp120 purified protein onto an ell plate.
And react with a diluted solution of mouse serum after administration.
Compared with before, the 3rd, 6th, 19th and 29th week after the first immunization
The HIV-1 specific IgG antibody titer during exsanguination was measured by ELISA.
It was The results are shown in Fig. 3. HIV after vLC16m8-HIV administration
-1 Antigen-specific blood antibody titer of vLV16m8-empty
While the antibody titer was constant in the control, vLC1
In the 6m8-HIV administration group, HIV-1 antigen-specific
There was a marked elevation of the body.

Fo of the above mice 13 days after the first immunization
The peptide (SEQ ID NOS: 1 to 5) was administered to both feet of the OT Pad at 4 μg each, and the delayed type hypersensitivity reaction was measured by comparing the thickness of the Foot Pad after 24 hours with that before the administration (Foot
pad swelling method). The results are shown in FIG. 4. In the control group (vLC16m8-empty), almost no reaction was observed, whereas in the vLC16m8-HIV administration group, a high swelling reaction was confirmed by HIV-1 antigen stimulation.

[0012] In addition, 2 times from the initial immunization of the above mice.
Three days later, spleen cells were separated, and cells stained with CFSE were subjected to antigen stimulation with peptides (SEQ ID NOS: 1 to 5) for 5 days and those without antigen stimulation and analyzed by flow cytometry (T cell proliferation assay). The result is shown in Fig. 5.
Shown in. The numbers in the table indicate the percentage of dividing T cells. Right side is vLC16m8-HIV immunized, left side is vL
It represents a control to which C16m8-empty was administered. The upper part shows the one which was not stimulated and the lower part shows the one which was stimulated for 5 days. In the figure, it is considered that the M1 region is a divided cell group, and 4% (16% → 20%) divided T cells were detected by antigen stimulation culture in mouse spleen cells immunized with vLC16m8-HIV.

Further, intracellular IFN-γ was measured for the above mice. That is, the intracellular cytocan IFN-γ was analyzed by flow cytometry 24 hours after the administration of the mouse spleen cells after administration with or without antigen stimulation. The result is shown in FIG. The numbers in the table indicate the percentage of CD8 ⁺ Twell producing IFN-γ. Right side is vLC16m8-HIV immunized, left side is vLC16m8-empt
Represents a control dosed with y. The upper part shows the spleen cells 24 hours after the peptide stimulation, and the lower part shows the 24 hours after the peptide stimulation. IFN in spleen cells at the same time
−γ-producing CD8 ⁺ T cells were treated with control (vLC16m8-em
The pty) dose was 0.01% even after the antigen-stimulated culture, whereas 0.12% was detected by the antigen-stimulated culture in the vLC16m8-HIV-administered.

In addition, about the above-mentioned mouse, after 7 months, Elis
HIV-1 antigen stimulation-specific cellular immune response was measured by the pot method. 6w each 10 ⁶ Cell / well of spleen
Elispot IP pl coated with IFN-γ mAb diluted 100 times for each well and left for 24 hours
The number of IFN-γ-producing cells was counted 24 hours after the addition of the antigen to 3 wells, and the number of IFN-γ producing cells was counted, and the average value of those not stimulated was calculated. The results are shown in FIG. 7. HIV-1 antigen-specific IFN in spleen cells at the 7th month of immunization using the Elispot method.
-Γ-secreting cells were compared with the control group at 7 months
High detection was observed in the vLC16m8-HIV administration group.

[0015]Example 4 In this example, recombinant vaccinia virus (vLC16m8-HI
V) Immunity induction of dendritic cell DCs
did. First, mouse bone marrow-derived dendritic cells (DCs) are removed.
Put it out, 10 cm Petri dish 4 × 10⁶/ 10m 10%
FCS RPMI mouse culture with IL-4 and GMCSF
Then, after 3 days, add another 10 ml medium for 3 months
Later, only floating cells were taken and selected again with medium,
Two days later, vLC16m8-HIV was infected. P
After washing with BS, X-gal staining was performed. Figure of the situation
8 shows. Vaccinia virus is stained blue inside the cell
It is thought that DCs can infect DCs.

Next, vLC16m8-HIV obtained as described above
Infected DCs were intraperitoneally administered to mice, and 1 week later, INF-γ-producing CD8 ⁺ T produced by antigen stimulation of splenocytes.
The cells were analyzed by flow cytometry. The result is shown in FIG. DCs infected with vLC16m8-HIV were intraperitoneally administered to mice 1 week after, INF-γ producing CD8 ⁺ T cells in spleen cells were hardly detected in the control, whereas DC was administered. In the case of the antigen stimulation, a high value of about 0.5% (0.17% → 0.68%) was detected. This makes vaccinia virus DC
It is believed that s may present the antigen.

[0017]Example 5 Genes sensitive to antibiotics (herpes simplex virus T
K (HSV-TK) gene, GenBank No. AF05731)
The expression vector pSC65 / TK having
Of recombinant vaccinia virus vLC16m8-HIV TK
To produce a drug-sensitive virus
It was In this way, HSV-TK was introduced in FIG.
Virus vLC16m8-HIV strain with different acyclovir concentration
The state when it culture | cultivated at 37 degreeC for 10 days is shown. Acyclo
The plaque decreased as the building concentration increased,
At μg / ml, virus growth was completely blocked.

In addition, virus vL introduced with HSV-TK
C16m8-HIV strain (displayed as "HSV-TK"), vLC16
m8-HIV strain (HSV-TK is not introduced) or WR
CV-1 cells were infected with the strain (HSV-TK was not introduced), and an antiviral agent (acyclovi
After culturing at 37 ° C. for 10 days in a culture solution containing r)), the numbers of plaques of CV-1 cells were compared. The result is shown in FIG. From FIG. 11, when the drug (acyclovir) concentration increased, the number of plaques of HSV-TK introduced virus decreased, but vLC16m8-HIV strain and WR strain showed almost the same number of plaques regardless of the drug concentration. Was done.

From Examples 1 to 4, vLC16m8-HIV of the present invention
However, it was confirmed that it induces both long-term antigen-specific humoral immunity and cell-mediated immunity. It was also suggested that DCs might be involved in the induction of immunity by vaccinia virus. The vLC16m8-HIV of the present invention is considered to be an effective vaccine due to its high antigenicity and low pathogenicity. Further, in Example 5, the vaccinia virus (vLC16m
8-HIV) was confirmed to be sensitive to drugs such as antiviral agents (acyclovir). It has been shown that even if the vaccinia virus of the present invention is slightly pathologically increased, it is possible to take measures to treat it.

[0020]

[Sequence list]     SEQUENCE LISTING <110> Japan Science and Technology Corporation <120> HIV vaccine using recombinant vaccinia virus <130> PS02-1112 <160> 5 <210> 1 <211> 19 <212> PRT <213> HIV <400> 1 Thr Arg Lys Ser Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Thr Thr   1 5 10 15 Gly Glu Ile <210> 2 <211> 15 <212> PRT <213> HIV <400> 2 Phe Pro Val Arg Pro Gln Val Pro Leu Arg Pro Met Thr Tyr Lys   1 5 10 15 <210> 3 <211> 11 <212> PRT <213> HIV <400> 3 Arg Leu Ile Asp Leu Leu Leu Ile Ala Lys Arg   1 5 10 <210> 4 <211> 15 <212> PRT <213> HIV <400> 4 Lys Ile Arg Leu Arg Pro Gly Gly Asn Lys Lys Tyr Lys Leu Lys   1 5 10 15 <210> 5 <211> 11 <212> PRT <213> HIV <400> 5 Ser Pro Ala Ile Phe Gln Ser Ser Met Thr Lys   1 5 10

[Brief description of drawings]

FIG. 1 pSC in which the HIV gp160 gene was integrated.
It is a cleavage map of 65 plasmid.

[Figure 2] vLC16m8-HIV (lane 1) or vLC16m8-empty
It is a figure which shows the western blotting which used the HIV infected human serum as an antibody about the protein which the CV-1 cell infected by (lane 2) expresses. Lane 3
Indicates the envelope protein (HIV-1 gp120).

FIG. 3 is a graph showing IgG antibody titers of mice administered with vLC16m8-HIV or vLC16m8-empty.

FIG. 4 is a diagram showing Footpad swelling of mice administered with vLC16m8-HIV or vLC16m8-empty.

FIG. 5 is a diagram showing a T cell proliferation assay of mice administered with vLC16m8-HIV or vLC16m8-empty.

FIG. 6 shows flow cytometry of mice administered with vLC16m8-HIV or vLC16m8-empty.

FIG. 7 is a diagram showing the HIV-1 antigen-stimulation-specific cellular immune response by the Elispot method in mice administered with vLC16m8-HIV or vLC16m8-empty.

FIG. 8 is a view showing a photograph of bone marrow-derived dendritic cells (DCs) (X-gal staining) of a mouse infected with vLC16m8-HIV.

FIG. 9 is a diagram showing flow cytometry of mice administered with DCs infected with vLC16m8-HIV.

FIG. 10: Virus vLC16m8-HI introduced with HSV-TK
It is a figure which shows a mode when culture | cultivating the V strain by changing the density | concentration of acyclovir. The numbers are the concentration of acyclovir (unit: μ
g / ml).

FIG. 11: HSV-TK-introduced virus vLC16m8-HI
V strain (“HSV-TK”), vLC16m8-HIV strain (“vLC16m8-HI
V ") or WR strain (" WR ") is infected with CV-1 cells and cultured in a culture medium containing acyclovir.
It is a figure which shows the plaque number of -1 cell.

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Claims (5)

[Claims] 1. An HIV vaccine comprising a gene encoding a HIV (human immunodeficiency virus) envelope protein or a portion thereof and comprising recombinant vaccinia virus LC16m8 expressing the gene or a portion thereof. 2. The HIV vaccine according to claim 1, wherein the gene or a part thereof is incorporated into the thymine kinase (TK) gene of the vaccinia virus LC16m8. 3. The envelope protein is gp16.
The HIV vaccine according to claim 1 or 2, which is 0 (GenBank No. U21135). 4. The vaccinia virus LC16m8
The HIV vaccine according to any one of claims 1 to 3, further comprising a gene sensitive to an antibiotic or a portion thereof. 5. The herpes simplex virus TK (HSV-TK) gene (GenBan) is a gene sensitive to the antibiotic.
HIV vaccine according to claim 4, which is k No. AF05731).