KR20020060100A - HIV Like Particles And The Use Thereof - Google Patents

Abstract

PURPOSE: Provided is HIV like particle which is composed of HIV-1 Gag, Env, Pol and/or Pro proteins expressed by an expression vector containing HIV-1 env, gag, pro, pol genes. The HIV like particle can be used as an antigen in a diagnosis kit of HIV infection and for HIV vaccine composition. CONSTITUTION: Alphavirus based expression vectors contain HIV-1 env, gag, pro and pol genes and they are as follows: pSFV/env-gag, pSFV/gag-env, pSFV/env-gag-pro, pSFV/env-gag-gagpro, pSFV/env-gag-gagΔpro, and pSFV/gagpol-env. The expression vectors further contains CTE(constitutive transport element) gene and they are as follows: pSFV/env-gag-gagpro-CTE, pSFV/env-gag-gagΔpro-CTE, pSFV/gagpol-env-MCTE, and pSF/gag-env-MCTE. HIV-like particle is manufactured by introducing one of the above vectors or its RNA transcript into an animal cell then expressing it.

Description

H I V Similar Particles and Their Uses {HIV Like Particles And The Use Thereof}

HIV-1 (Human Immunodeficiency Virus-1) is the pathogen of the Acquired Immunod eficiency Syndrome (hereinafter referred to as "AIDS"). HIV-1, along with HIV-2, belongs to the lentiviridae of the retrovirus, and the infectious virion has an RNA (+) genome consisting of two identical single-stranded. When HIV-1 infects a cell, the RNA is converted into double-stranded DNA by reverse transcriptase, which is encoded by the RNA genome, which is inserted into the host cell's chromosome. This forms a provirus.

The HIV-1 genome (9.2kb) is composed of structural genes of gag, pol, and env and accessory genes such as tat, rev, nef, vif, vpu, and vpx. 1 shows the genomic structure of HIV-1.

Gag protein of HIV is expressed as 55 kDa proprotein (Pr55gag) in gag-pol full-length RNA. It is prestostolation at the N-terminus of the protein, thereby targeting the inside of the plasma membrane to form viral particles. Pr55gag is processed and matured into proteins of matrix (MA, p17), capsid (CA, p24), nucleocapsid (NA, p9) and P6 by viral protease (Pro) expressed by the pol gene (Gheysen). D. et al, Cell , 59, 103-112 (1989)). (Bray, M. et al, Proc. Natl. Acad. Sci. USA 91, 1256-1260 (1994); Cann, AJ, and J. karn, AIDS 3 (Suppl. 1), S19-S34 (1989); Ratner, L., W. et al., Nature 313, 277-284 (1985); Wain-Hobson, S. et al., Cell 40, 9-17 (1985)).

Envelope glycoprotein Env is expressed as a precursor called gp160 in spliced RNA. It is cleaved into the N-terminal gp120 and the C-terminal gp41 protein by the host or viral protease in the cell, and the gp120 thus formed forms glycoprotein subunits present in the external surface domain. gp41 constitutes the transmembrane domain. In mature virions, gp120 and gp41 are noncovalently linked to form a heterodimer, which is known to be directly involved in the infectivity, cellular tropism and cytopathogenicity exhibited by HIV (Robey, E.). and Axel, R., Cell 60, 697-700 (1990)). As such, the Env protein of HIV is closely related to antigenicity and thus is the most important research subject in the manufacture of HIV vaccines.

Protease (Pro) is produced by autologous cleavage from the precursor protein (Pr160gag-pol), which is produced by the ribosomal frame shift at 3 'of gag mRNA (Jacks, T. and Varmus, HE Sience , 230 1237-1242 (1985); Jacks, T. et al, Nature (Lodon) , 331, 280-283 (1988); Sonigo, P. et al, Cell , 45, 375-385 (1986)). Pro is involved in cleavage of Pr160gag-pol polyprotein and processing of Env and has been reported to play an important role in the maturation and infectivity of viral particles (Kohl, N. et al., Proc . Natl. acad. Sci. USA., 85 , 4686-4690 (1988)).

Previous developments in AIDS vaccines include inactivated vaccines, attenuated vaccines, recombinant live vaccines, virus-like particle vaccines, subunit vaccines, and the like. The following techniques have been tried in each vaccine field.

"Inactivated whole virus vaccine" refers to the treatment of isolated virus particles by chemical or physical treatment that results in loss of infectivity. While it is easy to manufacture and has the advantage of using almost all epitopes, there is a risk of causing a disease if the virus is not completely inactivated, and conversely, if a strong treatment is done to completely inactivate it, the epitope is destroyed. have. There have been reports of successful reports of infection of SIV in the literature (Scott EJ Nature, 253, 393 (1991); Le Grand R. et al., Nature, 355, 684 (1992); Crange MP et al., Nature, 355, 685-686 (1992); Arthur L. 0., et al., J virol, 69, 3117-3124 (1995); Neidrig M., et al., Vaccine, 11, 67-74 (1993)) As a result, the result is questioned that the result is due to xeno-antigen of the cultured host cell, and thus cannot be regarded as a vaccine that is still in the successful stage.

"Live attenuated vaccine" is a deficiency of some of the HIV genes, for example, the nef gene or the simultaneous deprivation of the nef and vpr genes (Descrosiers RC., AIDS Res Hum Restroviruses, 8, 411). -421 (1992); Gibbs JS et al., AIDS Res Hum Retroviruses, 10, 607-616 (1994); Cranage MP et al., Virology, 229, 143-154 (1997); Wyand MS, et al., Hature Med, 3, 32-36 (1997)), a virus is a relatively early stage of development that has a problem in stability because the virus becomes pathogenic when re-acquire the missing gene in the animal body.

"Live recombinant vaccine" refers to a vaccine in which the non-essential genes of non-pathogenic viruses are replaced with HIV antigens. In the literature, gag, Pol or env from vaccinia virus, poxvirus, canarypox virus, avidox virus, polio virus, influenza virus, etc. Examples of expression of genes such as or expression of antigenic sites containing HIV V3 loops and the like in the form of chimeric proteins have been reported (Tartaglia J. et al., Virology, 188, 217-232 (1992); Abimiku A., et al., Nature Med, 1, 321-329 (1995); Natuk RJ. Et al., AIDS Hum Retroviruses, 9, 395-404 (1993); Li S. et al., J Virol, 67, 6659-6666 (1993); Muster T. et al., J Virol 69, 6678-6686 (1995). This technique generally does not provide a good immunogen because only a portion of the total antigen is used as a vaccine, and the disadvantage is that the constructed vector is often unstable (Morrow CD et al., AIDS Res Hum Retroviruses, 10, S61-). S66 (1994); Anderson MJ et al., AIDS Res Hum Retroviruses, 13, 53-62 (1997)).

Subunit vaccines, which use subunits of HIV that are naturally isolated or genetically engineered as vaccines, have the advantage of being relatively easy to make, but they also use only a portion of the HIV antigen site. Vaccines have limitations in that they take different conformation from viral particles. Recombinant Env glycoproteins gp120 and gp160 have been expressed in mammalian cells or insect cells (Lasky, LA, et al., Science, 249, 932-935 (1986); Barr, PJ et al., Vaccine , 5, 90-101 (1987); Hu, SL. Et al., J. Viol ., 61, 3617-3620 (1987); Rusche, JR et al., Proc. Natl. Acad. Sci., 84, 6924-6928 (1987); Berman, PW, et al., J. Virol., 63, 3489-3498 (1989); Ivey-Hoyle, M., and Rosenberg, M., Mol. Cell. Biol., 10, 6152- 6159 (1990); Lasky, LA et al., Cell, 50, 975-985 (1987)), gp120 expressed in CHO cells failed in challenge experiments in chimpanzees (Berman, P. W et al. , Proc. Natl. Acad. Sci., 85, 5200-5204 (1988)).

Providing the vaccine in the form of viral particles is more immunogenic and stable than providing subunit antigens. Therefore, a method has recently been developed using non-infectious "virus-like particles" (VLPs) that do not contain genes as vaccines. An example is the production of VLPs without genomic RNA as a result of mutations in the packaging gene, or the production of VLPs consisting of unprocessed Gag proteins using recombinant baculovirus or vaccinia virus (Gorelink RJ et al. , J Virol, 64, 3207-3211 (1990); Gheysen DE et al., Cell, 59, 103-112 (1989) .Haffar 0. K. et al., Virology, 183, 487-. 495 (1991)) reported the simultaneous infection of two recombinant vaccinia viruses containing the gag-pol and env genes to produce a virus-like particle without genes. Although it has been successful in producing neutralizing antibodies, syncitium-inhibiting, env-CD4 protective antibodies, etc. in the body, there are limitations that they are not mature viral particles and lack infectivity.

VLP-type vaccines also include hybrid particles such as polio virus capsids, HBV core particles, HBsAg particles or yeast retrotransposon virus-like particles constructed to contain antigenic groups of HIV. The limitations of the antigenic sites that can be inserted into pseudo-viral particles were not overcome (Grene E. et al., AIDS Res Hum Retroviruses, 13, 41-51 (1997); Schlienger K., et al. , J Virol, 66, 2570-2576 (1991); Adams SE et al., Nature 329, 68-70 (1987); Layton GT et al., J Immunol, 151, 1097-1107 (1993); Eckart L et al., J Gen Virol, 77, 2001-2008 (1996).

The present inventors have attempted to develop a vaccine in the form of mature virus-like particles using an alpha viral vector system in an attempt to develop a new AIDS vaccine, thereby providing a method for preventing AIDS disease.

The present invention is an expression vector characterized by expressing Gag protein of HIV, an expression vector characterized by expressing Env protein of HIV and an expression vector characterized by simultaneously expressing Gag and Env protein of HIV, HIV An expression vector characterized by expressing a Gagpol polyprotein and an expression vector characterized by simultaneously expressing Gagpol polyprotein and Env protein of HIV.

In addition, the present invention relates to a cell line, characterized in that transformed with the expression vector.

In addition, the present invention relates to a method for producing HIV-like particles (HIVLP) characterized in that the recombinant expression vector or its RNA transcript is introduced into an animal cell and expressed by the method. .

The present invention also relates to a kit for diagnosing HIV infection, comprising a mature HIV-like particle, and a method for diagnosing an HIV disease by using the kit.

The present invention also relates to a vaccine composition comprising a mature HIV-like particle or an AIDS vaccine composition comprising the expression vector and / or its RNA transcript.

In addition, the present invention comprises (a) taking a sample that is expected to contain HIV specific antibodies, and (b) using the sample taken from step (a) as the antigen, the mature HIV-like particles of the present invention, the antigen and the antibody. Contacting under conditions that allow binding of the antigen to form an antigen-antibody immune complex, and (c) detecting the formation of the immune complex, specifically binding to the HIV retroviral antigen in the sample. It relates to a method of determining the presence of a reacting antibody.

1 is a schematic diagram showing the genomic structure of HIV-1.

2 is a schematic diagram showing the structure of a pSFV vector and a pSFV-helper.

3 is a schematic diagram illustrating a process of preparing a pSFV / gag expression vector.

4 is a schematic diagram illustrating a process of preparing a pSFV / gagpro expression vector.

5 is a photograph showing the results of Western blot of virus particles obtained by transfection of SFV-helper and pSFV / gag or SFV-helper and pSFV / gagpro RNA transcripts to BHK-21 cells.

FIG. 6 shows BHK infected with BHK-21 cells after infection with BHK-21 cells by activating virus particles obtained after transfection of pSFV-helper and pSFV / gag or pSFV-helper and pSFV / gagpro RNA transcripts to BHK-21 cells. A photograph showing the results of Western blot of the lysates of -21 cells with AIDS patient serum.

7 is a photograph confirming expression of Gag by transfecting pSFV / gag or pSFV / gagpro RNA transcripts to BHK-21 cells and immunochemically staining the cells with a polyclonal antibody against p24 (capsid). .

Figure 8 shows transfection of RNA transcripts of pSFV / gag (A) or pSFV / gagpro (B) to BHK-21 cells, followed by electron staining of the VLP obtained from the supernatant of the cell culture medium. Observed photograph (X140,000).

9 is a schematic diagram illustrating a process of preparing a pSFV / env expression vector.

10 is a photograph confirming the expression of gp160 by transfecting pSFV / env RNA transcript with pSFV-helper RNA transcript to BHK-21 cells and immunochemically staining with monoclonal antibody against gp160.

FIG. 11 shows that defective SFV particles obtained after transfection of pSFV / env RNA transcripts with BSFK-helper RNA transcripts to BHK-21 cells were re-in vitro activated to infect BHK-21 cells, and infected BHK-21 cells. It is a photograph showing the result of Western blot of the cell lysate with AIDS patient serum.

12: Immunohistochemical staining of cells with monoclonal antibodies against gp160 (B) or AIDS patient serum (C) after transfecting pSFV / gag and pSFV / env RNA transcripts simultaneously to BHK-21 cells. This shows that HIV-1 Env and Gag proteins were simultaneously expressed.

Figure 13 shows the transfection of pSFV / gag RNA transcripts or pSFV / gag and pSFV / env RNA transcripts into BHK-21 cells at the same time to obtain VLPs, respectively, and the RNA packaged in these particles was analyzed by RT-PCR and PCR. It is a photograph showing the result of the analysis.

Figure 14 shows COS- by in vitro activation of respective defective SFV particles obtained by co-transfection of pSFV-helper and pSFV / gag RNA transcripts, or pSFV-helper and pSFV / env RNA transcripts to BHK-21 cells. Re-infection of 1 cell resulted in Western blots showing the production of VLP consisting of Gag and Env of HIV-1.

15 is a schematic diagram illustrating a process of preparing a pSFV / pro expression vector.

Figure 16 is a schematic diagram showing the manufacturing process of the pSFV / env-gag expression vector.

Figure 17 is a schematic diagram showing the manufacturing process of the pSFV / env-gag-pro expression vector.

18 is a photograph showing the results of Western blot of VLP or cell lysate obtained after transfection of RNA transcript of pSFV / env-gag to BHK-21 cells with AIDS patient serum.

19 is an immunocytochemical picture showing that the RNA transcript of pSFV / env-gag-pro was transfected into BHK-21 cells to express Gag, Env and Pro of HIV-1 in cells.

20 is a schematic diagram illustrating a process of preparing a pSFV / CTE expression vector.

FIG. 21 is a schematic diagram illustrating a process of preparing a pSFV / gagpro-CTE expression vector. FIG.

22 is a schematic diagram illustrating a process of preparing a pSFV / env-gag-gagpro-CTE expression vector.

Figure 23 is a schematic diagram showing the insertion of gag ?? pro to pSFV / env-gag removing the frame shift sequence occurring at the 3 'end of the gag in the process of creating a pSFV / env-gag-gag ?? pro-CTE expression vector to be.

24 is a schematic diagram illustrating a process of manufacturing pSFV / gagpol.

25: Transfection of pSFV / gagpol to BHK-21 cells followed by immunocytochemical staining of cells with polyclonal antibodies against p24 and polyclonal antibodies against proteases to express Gag and Gagpol polyproteins. This is a confirmed picture.

FIG. 26 is a photograph showing the results of Western blots using the ECL method for transfecting RNA transcripts of pSFV / gagpol to BHK-21 cells using polyclonal antibodies against p24.

27 is a schematic diagram illustrating a process of manufacturing pSFV / envMCTE.

28 is a schematic diagram illustrating a process of manufacturing pSFV / gag-envMCTE.

29 is a schematic diagram illustrating a process of manufacturing pSFV / gagpol-envMCTE.

FIG. 30 shows Gag and Env proteins as transfecting pSFV / gag-envMCTE to BHK-21 cells, and then the cells with polyclonal antibodies against p24, polyclonal antibodies against proteases and monoclonal antibodies against env. It is a photograph confirming the expression of.

Figure 31 Immunocells after transfecting pSFV / gagpol-envMCTE to BHK-21 cells, then cells with polyclonal antibodies against p24, polyclonal antibodies against protease and monoclonal antibodies against env. Chemical staining is a photograph confirming the expression of Gagpol polyprotein and Env protein.

FIG. 32 shows polyclonal antibodies against p24 and monoclonal antibodies against env after 48 hours after transfecting BHK-21 cells with RNA transcripts of pSFV / gag-envMCTE and pSFV / gagpol-envMCTE The photograph shows the result of Western blot using the ECL method.

The present invention provides alpha virus-based expression vectors that express structural proteins of HIV. HIV of the present invention includes all human retroviruses such as HIV-1, HIV-2, HTLV-1 or HTLV-2.

"Alpha virus" refers to viral species and subtypes commonly classified in the art as alpha viruses, for example Eastern Equine Encephalitis virus (EEE), Venezuelan Equine Encephalitis virus (VEE), Everglades virus, Mucambo virus, Pixuna virus, Western Encephalitis virus (WEE), Sindbis virus, South African Arbovirus No. 86, Girdwood SA virus, Ockelbo virus, Semliki Forest virus, Middleburg virus, Chikungunya virus, 0'Nyong-Nyong virus, Ross River virus, Barmah Forest virus, Mayaro virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Whataroa virus, Babanki virus, Kyaylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus, Buggy Creek virus and other viruses classified as alpha viruses by the International Committee on Taxonomy of Viruses These include this.

Alphaviruses are encapsulated positive-strand RNA viruses belonging to Togaviridae that can infect a wide range of cells (insects, birds, mammals). It is an efficient system for RNA replication and transcription because the viral RNA genome itself is infectious and codes for itself RNA replicators (Liljestrom, P. and H. Garoff, Biotechnology, 9, 1356-1361 (1991)). ).

Alpha viruses of the present invention are preferably "Semliki Forest virus (SFV) virus" and "Sindbis virus". More preferably, the alpha virus of the present invention is SFV. The SFV-based expression vector pSFV used in the embodiment of the present invention inserts the cDNA genome of SFV into a plasmid with an SP6 promoter, and is capable of in vitro transcription using SP6 polymerase, and inserts a foreign gene instead of a structural gene. Is an expression vector transformed to be expressed by the 26S subgenomic promoter.

In order to introduce recombinant RNA into cells by infection, it is necessary to use an in vivo packaging system. pSFV-helper contains information on the RNA replication signal and structural gene expression of SFV, but lacks the genomic RNA packaging signal. Co-translation of pSFV-helper with recombinant pSFV vector results in SFV. Virus particles are produced in which only recombinant RNA is packaged within the structural protein. The viral particles infect cells and express recombinant genes but do not form viral particles again. The structure of pSFV and pSFV-he1per used in the embodiment of the present invention is shown in FIG. 2. DNA vectors and RNA transcripts thereof used in the present invention are all included in the scope of the present invention.

One aspect of the invention provides that the gag, gagpro, env, env and gag, env and gag and gagpro or gagΔpro, gagpol or gagpol and env genes of HIV-1 are operably linked under the subgenomic promoter of the alpha virus. It is to provide an expression vector.

pro or gene is inserted under a 26S subgenomic promoter to provide an alpha virus-based expression vector expressing Gag, Env, Pol or Pro, respectively. Expression of Pro is thought to be dependent on the amount of Gag protein (Velissarios K. et al., Virology, 193, 661-671 (1993); Magdeleine H. et al., J Virol. 72, 4819-1824 ( Joel G. et al., Virology, 244, 87-96 (1998). In the present invention, in order to maximize the expression of Pro for the processing of Gag, the gagΔpro gene, in which the frame shifted codon sequence is deleted at the vicinity of gagpro or gagpol after the env gene, is linked to the 26S promoter, respectively, or env after the gagpol gene. A vector linked to the gene 26S promoter was constructed.

The present invention provides an alpha virus-based expression vector in which the gag gene of HIV-1 is operably linked under a promoter. "Operably linked" in the art generally means that the linked DNA sequences are in contact with each other and are present in the reading frame, and this meaning applies equally here. pSFV / gag is an example of such a vector (Example 1). The present invention provides an alpha virus-based expression vector to which the gagpro gene of HIV-1 is operably linked under a promoter. pSFV / gagpro is an example of such a vector (Example 1). The present invention provides an alpha virus-based expression vector to which the env gene of HIV-1 is operably linked under a promoter. pSFV / env is an example of such a vector (Example 4).

The present invention relates to an alpha viral vector, preferably an env gene, which expresses the env and gag genes of HIV-1 simultaneously, so that the env gene is operable under a first subgenomic promoter, and the gag gene is a second subgenomic promoter. Alpha virus-based expression vectors operably linked to are provided. pSFV / env-gag is an example of such a vector (Example 9). The present invention provides an alpha virus-based expression vector linked so that the pro gene of HIV is operable under a promoter. pSFV / pro is an example of such a vector (Example 9).

The present invention further relates to an alpha viral vector, preferably an env gene, which simultaneously expresses the env, gag and pro genes of HIV-1 in such a way that it is operable under a first subgenomic promoter, and the gag gene is linked to a second subgenomic. An alpha virus-based expression vector is provided that is operably linked under a genomic promoter and that the pro gene is operably linked under a third subgenomic promoter. pSFV / env-gag-pro is an example of such a vector (Example 9).

The present invention relates to an alpha virus vector expressing a gagpro gene and a Constitutive Transport Element (CTE) gene of HIV-1, preferably an alpha virus-based expression in which a gagpro gene is operably linked under a promoter, and to which a CTE gene is further linked. Provide a vector. pSFV / gagpro-CTE is an example of this vector (Example 12).

The present invention relates to an alpha viral vector expressing the env, gag, gagpro and CTE genes of HIV-1, preferably the env gene is operably linked under a first subgenomic promoter, and the gag gene is linked to a second sub Operably linked under a genomic promoter, and gagpro gene is operably linked under a third subgenomic promoter and provides an alpha virus-based expression vector further comprising a CTE gene.

pSFV / env-gag-gagpro-CTE is an example of this vector (Example 12).

The present invention provides a gagΔpro gene having a deletion of a nucleic acid sequence at a junction of a gag and a Pro gene such that Pro protein can be produced without a ribosome frame shift occurring at the 3 ′ end of an env, gag and gag mRNA of HIV-1. Alpha viral vectors expressing the CTE gene, preferably the env gene, are operably linked under the first subgenomic promoter, the gag gene is operably linked under the second subgenomic promoter, and the gagΔpro gene Provides an alpha virus-based expression vector operably linked under a third subgenomic promoter and further comprising a CTE gene. pSFV / env-gag-gag Δpro-CTE is an example of the vector (Example 13).

The present invention provides an alpha virus vector expressing the gagpol full length gene of HIV-1, preferably an alpha virus-based expression vector linked to the gagpol full length gene to be operable under a subgenomic promoter. pSFV / gagpol is an example of such a vector (Example 14).

The present invention relates to an alpha viral vector that expresses the gag and env genes of HIV-1 simultaneously, preferably the gag gene is operably linked under a first subgenomic promoter and the env gene operates under a second subgenomic promoter. Provided are alpha virus-based expression vectors that are possibly linked and further comprise an MCTE gene.

pSFV / gag-envMCTE is an example of such a vector (Example 16).

The present invention relates to an alpha viral vector that expresses the gagpol and env genes of HIV-1 simultaneously, preferably the gagpol gene is operably linked under a first subgenomic promoter and the env gene operates under a second subgenomic promoter. Provided are alpha virus-based expression vectors that are possibly linked and further comprise an MCTE gene. pSFV / gagpol-envMCTE is an example of such a vector (Example 16).

Vectors of the present invention can be prepared by those skilled in the art based on the genetic recombination techniques known in the art by adding modifications appropriate to the present invention.

The present invention provides a method for producing HIV-1 structural proteins Gag, Pro and / or Env (precursor protein and processed subunits) using the above expression vector. Alpha viral vectors of the invention can express proteins in a wide range of host cells. The present invention provides a method for producing Gag, Pro and / or Env proteins of HIV-1 by expressing the above expression vector or its RNA transcript in a host cell, preferably an animal cell. The animal cell of the invention is preferably a bird, mammal, reptile, amphibian, insect or fish cell. Examples of mammalian cells include human, monkey, hamster, rat and pig cells. Examples of specific preferred host and vector systems are BHK / pSFV and COS / pSFV, although the expression system of the present invention is not so limited.

The alpha virus expression vector or RNA transcript thereof is directed to animal cells, for example BHK-21 cells, by conventional transfection methods such as DEAE-Dextran, calcium phosphate method, preferably electroporation. Can be introduced. In the case of the pSFV vector illustrating the invention, RNA transcripts can be introduced into the host cell in a very high yield by electroporation. The methods can also be used to cotransfection one or more vectors. Techniques for transfection, including electroporation, are known in the art.

Transformation of cells increases their expression efficiency when infected with infectious viral particles. The present invention provides a method for preparing said protein by infecting a host cell with an infectious viral particle comprising an RNA transcript according to the invention made using a helper virus. The above methods are illustrated in Examples 2 and 6. HIV-1 proteins produced by infection of a defective virus particle (Defective Virus Particle) made using the vector transcript or helper virus of the present invention can be isolated by various methods. As the separation method, various methods known in the art may be used, for example, ion exchange chromatography, affinity column chromatography, gel permeation chromatography, and the like.

The present invention provides an RNA transcript of the vector, a host cell or cell line that is transiently transformed with or permanently transformed with the vector containing the RNA. The stably transformed cell line, for example, the cytomegalovirus (CMV) promoter required for insertion of the cDNA genome of HIV into the chromosome of the host cell and the CTE (constitutive transport element) gene for rev-independent expression. , Animal cell line with anti-neomycin gene as selection marker. The CTE gene is preferably a gene of MPMV (Mason-Pfizer monkey virus).

The present invention provides a method for producing infectious HIVLP using the expression vector and the host cell. The present invention provides a method of introducing immature virus-like particles (HIVLP) consisting of Gag of HIV by introducing a vector expressing Gag protein into a host cell. For HIV, Gag not cleaved by protease does not form mature particles and forms immature VLPs. "Virus-like particles (VLPs)" refer to particles that are not identical to wild-type viruses but have similar physicochemical properties. An “immature virus-like particle (immature VLP)” refers to the formation of a particulate structure as a virus that has not undergone the processing necessary to become a wild virion. The term "replicon" refers to a genome containing genes essential for gene replication, and may be used as an expression vector that binds a foreign gene to propagate a virus and express a foreign gene.

HIV forms viral particles with Gag alone, but it is immature and not infectious. Epitopes of neutralizing antibodies to HIV are known to be located in the coat protein, which has the most diverse amino acid sequence (variable sequence). Therefore, incorporating Env proteins into Gag-only recombinant virus particles is one of the important techniques for making HIV vaccines. The present invention co-transduces the expression vector containing the gag gene of HIV and the expression vector containing the env gene, thereby simultaneously expressing two proteins in one host cell, thereby being immature composed of Gag and Env proteins of HIV. Provided are methods for making virus-like particles (HIVLP).

The production of HIVLP by co-transfection is cumbersome and inefficient. Therefore, in the present invention, by using a recombinant plasmid cloned with two or three genes of interest in a single vector, HIVLP can be produced and recovered efficiently, economically and efficiently. Therefore, the present invention clones an alpha virus replicon of gag and env genes of HIV under respective promoters, and transfects the vector into cells, thereby immature HIV-like particles (HIVLP) consisting of Gag and Env proteins of HIV-1. It provides a method of manufacturing.

The HIV virus cleaves Gag by protease during or after the assembly or budding process and recombines the cut structural proteins to form complete mature particles. HIVLP, which consists only of Gag proteins, differs greatly from natural HIVLP in terms of size and shape, and HIVLP, which consists of Gag and Env, does not form mature virus particles unless processed by protease. In order to elicit an effective immune response, it is important to provide immunogens in the form most similar to those present in nature in nature, thus making mature HIVLP provide the most powerful means with HIV vaccines. In the present invention, by simultaneously expressing Gag and Pro proteins of HIV-1 or by simultaneously expressing Gag, Env and Pro proteins, a method for producing mature HIVLP of HIV has been developed. “Mature VLP” refers to a virus particle whose HIV structural proteins have been fully processed by Pro for HIV, and whose shape and size are substantially similar to wild type HIV. In the present invention, when the gagpol gene is expressed simultaneously with the env gene, it was found that a lot of mature HIVLP is generated.

HIV-like particles can be said to be most preferred as vaccines or antigens for diagnosing HIV infection. However, expressing Gag, Env and / or pretease necessary for making HIV-like particles by each expression vector is not only cumbersome but also poor in efficiency, in which case a stable cell line producing HIV-like particles is obtained. It is also difficult. In the present invention, an alpha virus-based expression vector capable of simultaneously expressing Gag, Env, and protease of HIV in an amount sufficient to form HIV-like particles was successfully prepared, and infectious virus-like particles were prepared and separated therefrom. Succeeded.

Accordingly, the present invention further provides a vaccine composition for preventing AIDS comprising the expression vector, an RNA transcript of the vector or HIVLP in an amount sufficient to cause an immune response, and a method for preventing or treating AIDS disease by administering the same. . The HIVLP includes both mature and immature HIVLP, infectious and non-infectious HIVLP. Particularly preferred is mature HIVLP. The use of mature HIVLP as a vaccine can induce an excellent immune response because it can provide the conformation of HIV.

The vaccine composition of the present invention is administered to a subject in a form suitable for a living body. Here, the form suitable for the living body refers to the form of the substance to be administered in a form in which the prophylactic or therapeutic effect of the disease is higher than any toxic effect. The material may be administered to any animal, preferably a human.

The vaccines of the present invention may further comprise suitable diluents, excipients and / or carriers. Preferably, the vaccine includes excipients that can enhance the immunogenicity of the vaccine in vivo. The excipients include the lipid-A portion of the endotoxin of gram negative bacteria, trehalose dimycolate of mycobacteria, phospholipid lysolecithin, dimethyl dictadecyl ammonium bromide (DDA), any linear polyoxypropylene-polyoxyethylene (POP- POE) may be selected from known excipients in the art, including block polymers, aluminum hydroxide and liposomes. The carrier may be used as long as it does not produce an undesirable antibody in the subject to which the vaccine composition is administered. Many suitable carriers or diluents can be used in the present invention, particularly saline, buffered saline, and saline mixed with nonspecific serum albumin. Pharmaceutical compositions may include water, saline, glycerol, ethanol, etc. or emulsifiers, wetting agents or pH adjusters in addition to adjuvant, buffers, antioxidants, carbohydrates such as glucose, sucrose or dextrin, and chelating agents such as EDTA. Can be. The vaccine composition may comprise an adjuvant to enhance the immune response. Examples of the adjuvant include aluminum hydroxide (alum), thr-MDP, nor-MDP, MPT-PE and the like. The vaccine may also comprise cytokines known to enhance immune responses including GM-CSF, IL-2, IL-12, TNF and IFNγ.

The dosage of the vaccine can vary depending on factors such as the disease state, age, sex and weight of the patient and the ability of the antibody to elicit the desired response to the patient. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the urgency of the therapeutic situation. The vaccine may be administered by conventional methods such as injection (eg subcutaneous, intravenous and intramuscular), oral administration, inhalation, transdermal administration, or suppositories. Booster immunization can be braked after 4-6 weeks.

The present invention provides a diagnostic kit for diagnosing an infection of HIV and a method for diagnosing a disease using the same. The present invention provides a kit for diagnosing HIV infection by (a) contacting a biological sample obtained from an animal, in particular a human, with (b) detecting the binding of an antibody in the sample to HIVLP and the kit. Provides a method for diagnosing HIV infection. Methods of determining the extent of antigen-antibody binding are already well known in the art. ELISA is one method that can be used for this. Such diagnosis can also be performed by direct or competitive binding.

In view of the above, the present invention relates to an HIV prepared by the method of claim 18, which comprises (a) taking a sample that is expected to contain HIV specific antibodies, and (b) using the sample taken from step (a) as an antigen. Contacting the analogous particle with the antigen and antibody under conditions such that the antigen-antibody immune complex is formed, and (c) detecting the formation of the immunocomplex. Provided are methods for determining the presence of antibodies that specifically react with HIV retroviral antigens.

Mature HIV-like particles of the present invention may be immunoassay or anti-retrovirus (e.g. HIV) HIV antibodies and retroviral antigens (e.g., enzyme-linked immunosorbent assays (ELISA), RIA and other non-enzyme linked antibody binding assays). , As an immunogen, ie as an antigen, in procedures known in the art for the detection of HIV). In an ELISA assay, mature HIV-like particles are immobilized on a specific surface, for example, a surface capable of binding to a protein, such as a well of a polystyrene microtiter plate. After washing to remove incompletely adsorbed mature HIV-like particles, test samples and non-specific proteins such as bovine serum albumin (BSA) or casein solutions known to be antigenically neutral can be bound onto specific surfaces. have. This blocks the non-specific adsorption sites on the surface where immobilization occurs, thereby reducing the background caused by non-specific binding of antiserum on that surface.

The surface on which immobilization is then made is contacted with a sample, such as a clinical material or a biological material, to be examined in such a way that immune complex (antigen / antibody) formation occurs. This may include diluting the sample with a diluent such as a solution of BSA, bovine gamma globulin (BGG) and / or phosphate buffered saline (PBS) / twin. The samples are then incubated for about 2-4 hours in turn, for example at a temperature of about 25 ° C. and then 37 ° C. After incubation, the surface to which the sample contacted is washed to remove non-immunocomplexed material. Washing procedures may include washing with a solution such as PBS / Twin or borate buffer.

After forming a specific immunocomplex between the mature HIV-like particles of the present invention in combination with a test sample and subsequently washing, the frequency and in some cases the amount of the immunocomplex formation is determined by the agent having specificity for the first antibody. It can determine by applying to 2 antibodies. If the test sample is from a human, the second antibody is an antibody that is specific for human immunoglobulins, generally IgG. To provide a means of detection, the second antibody may have associated activity, such as, for example, an enzyme activity that develops color when incubated with a suitable chromogenic substrate. It can then be quantified by measuring the degree of color development using, for example, a visible light spectrophotometer.

As one diagnostic aspect where it is desired to identify antibodies that recognize multiple HIV isolates, the immunologically distinct plurality of mature HIV-like particles of the present invention is immobilized on a specific surface. Alternatively, one or a limited number of mature HIV-like particles can be immobilized when the anti-HIV antibody recognizes an epitope that is highly conserved between several HIV isolates. As another diagnostic aspect where specific identification of antibodies recognizing a single HIV isolate (eg, LA1, MN, SF2 or HXB2) is required, a single specific mature HIV-like particle of the invention can be immobilized. . This additional diagnostic aspect has particular use in the fields of medicine, clinical trials, and forensics, which may be important for determining specific HIV isolates responsible for inducing immune responses, including antibody responses.

As a further diagnostic aspect, it may be necessary to specifically identify immunologically distinct HIV, eg, HIV isolates belonging to different classes. Immunologically distinct HIV isolates may include, for example, LA1, MN, SF2, HXB2 or native HIV-1 isolates. In such diagnostic embodiments certain mature HIV-like particles of the invention are useful for producing antibodies, including monoclonal antibodies that specifically recognize such immunologically distinct HIV isolates.

Of course, immunologically distinct mature HIV-like particles can be used as immunogens, for example in vaccines or diagnostic agents. There may be situations where a mixture of mature HIV-like particles is used to provide cross-isolate protection and / or diagnosis. In such cases, the mixture of immunogens is often referred to as a "cocktail" agent.

In a further diagnostic embodiment, the immunogenic compositions of the invention, either individually or as a mixture comprising a cocktail formulation, can be used to detect HIV or antigen or neutralize HIV in a sample comprising a biological sample. Useful for the production of antibodies (including monoclonal antibodies).

In another diagnostic embodiment, mature HIV-like particles of the invention can be used to specifically stimulate HIV specific T-cells in biological samples taken from, for example, an HIV-infected person for diagnosis or treatment.

The following examples are intended to illustrate the invention. Therefore, the scope of the present invention should not be limited by the following examples.

<Example 1>

Preparation of pSFV / gag and pSFV / gagpro Expression Vectors

Gag protein is the major component of virions expressed in the full-length RNA of gagpol of HIV-1. Gag proteins by themselves are known to assemble virus-like particles (VLPs) without other viral proteins. Gag proteins are processed into proteins of the matrix (MA, p17), capsid (CA, p24), nucleocapsid (NA, p9) and p6 by viral protease expressed by the pol gene. (Gheysen D. et al, Cell , 59, 103-112 (1989)). In the present invention, an expression vector expressing Gag was prepared by inserting a gag gene of HIV-1 into alpha viral replicon.

The HIV clade E gene (Genbank accession number U51188, Journal of Virologly, 1996) was used as a template. To make pSFV / gag, the nucleotide sequence of HIV-1 (832-2328 bp) corresponding to the full-length gene of Gag was amplified by PCR using primers 1 and 2 below. PSFV / gagpro was prepared to express Protease (Pro) with Gag. Full length genes (832-2577 bp), including gag, frame shift site and protease site of HIV-1, were amplified by PCR using primers 1 and 3. 4 μl of 2.5 mM dNTP (Boehringer Mannheim, Germany), 1 μl of sense primer (100 pmol / μl), 1 μl of antisense primer (100 pmol / μl) in pUHD (100 ng / μl) with HIV-1 clade E full-length gene as template Add 5 μl of Taq polymerase 10X buffer and 37 μl of DW to make a mixed solution. The series of reactions kept in minutes was repeated 30 times. The amplified genes were inserted into BamHI sites of pSFV, respectively, to create pSFV / gag (FIG. 3) and pSFV / gagpro (FIG. 4).

Primer 1: Sense (SEQ ID NO: 1)

5'-GCGGATCCCGGGATGGGTGCGAGAGCGTCAATATTAAGT-3 '

Primer 2: Antisense (SEQ ID NO: 2)

5'-CGCGGATCCCTGTTACTGTGACAAGGGGTCGTTGCCAAA-3

Primer 3: Antisense (SEQ ID NO: 3)

5'-CGCGGATCCCTGCAGTTAAAGTACAACCAATCTGAGTCAACA-3 '

<Example 2>

Preparation of VLPs Using pSFV-helper and pSFV / gag or pSFV-helper and pSFV / gagpro

Protease of HIV-1 (Pro) is activated during or after budding of virions and is involved in processing structural proteins by cleaving Gag or Gag-Pol polyproteins. In this experiment, pSFV / gag or pSFV / gagpro prepared in Example 1 together with pSFV-helper were expressed in host cells.

pSFV / gag or pSFV / gagpro were treated with restriction enzyme Pvu I to make linear DNA followed by P / C treatment. 10 μl of DNA, 5 μl of 1 × SP6 buffer (TakaRa), 5 μl of 10 mM m ⁷ G (5 ′) ppp (5 ′) G (BM), 2.5 μl of 100 mM DTT, rNTP mix (10 mM ATP, 10 mM CTP, 10 mM 5 μl of UTP, 5 mM GTP, BM), 2 μl of RNasin (BM), 1.5 μl of SP6 RNA polymerase (TakaRa), and 19 μl of DW were prepared, followed by reaction at 37 ° C. for 1 hour 30 minutes. mRNA transcripts (50 μl of pSFV / gag or pSFV / gagpro RNA transcripts, respectively, in 25 μl of pSFV-helper RNA transcript) were added to PBS (Phosphate buffered saline, 1.37 mM Sodium Chloride, 0.02 mM Potassium Chloride, 0.1 mM Phosphate buffer /). ml) BHK-21 cells (10 ⁷ cells / ml) suspended in buffer were added to 800 µl and then pulsed twice at 830 V / 25 Hz in a 0.4 cm electroporation cuvette (Bio-Rad Gene Pulser). After 48 hours of transfection, the supernatant was centrifuged at 3500 rpm for 15 minutes, followed by ultrafast centrifugation (SW41 rotor, 2 hr, 30,000 rpm, 4 ° C, BECKMAN) to obtain virus particles. This pellet was suspended in TNE (50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 0.5 mM EDTA) buffer and stored at -70 ° C. Defective VLPs collected from the supernatant were subjected to SDS-PAGE, followed by Western blot using AIDS patient human serum (FIG. 5) (lane M: negative control; lane 1: pSFV-helper). VLP generated from pSFV / lacZ; lane 2: VLP generated from pSFV-helper and pSFV / gag; lane 3: VLP generated from pSFV-helper and pSFV / gagpro).

Gag protein was detected in lane 2 of FIG. This indicates that Gag protein was expressed by pSFV / gag RNA transcripts entering the cytoplasm of BHK-21, and HIVLP consisting of Gag protein was released into the cell supernatant with SFVLP particles. In Western blots of the pSFV / gagpro expression system constructed to express Gag protein and Pro together, less Gag protein was detected compared to the case of pSFV / gag (FIG. 5, lane 3).

5 μl of chymotrypsin (10 mg / mℓ) and 5 μl of 50 mM CaCl ₂ were added to 100 μl of virus particles suspended in TNE buffer and reacted for 30 minutes on ice. Then, 45 μl of aprotinin (2 mg / mℓ) was added to activate the VLP. I was. The activated particles were infected with 70% monolayered BHK-21 cells and incubated for 2 days after adding fresh medium after 1 hour and 30 minutes. After 2 days, 1 hour NP40 (Nonidet P-40), 50 mM Tris-HCl (pH7.6), 150 mM NaCl, 2 mM EDTA, 1 μg / mPMPMSF) was reacted for 1 hour and centrifuged after 2 days. Sample buffer was added to the supernatant obtained by separation (12000 rpm, 4 ° C., 5 minutes) and boiled for 5 minutes, followed by electrophoresis on 12% SDS-PAGE. Electrophoresis gels were transferred to PVDF western blotting membrane (BM) at 15 V, 7 mA for 2 hours and then Western blotted with specific antibodies. The primary antibody used is AIDS patient serum and the secondary antibody is biotinylated anti-human IgG. Blots were reacted with AB solution and then developed with DAB solution (FIG. 6) (lane 1: BHK-21 cells infected with VLP obtained from pSFV / gag; lane 2: BHK-21 infected with VLP obtained from pSFV / gagpro). cell). A band of 55 kDa indicates that Gag protein was produced in the cytoplasm of BHK-21 cells as a result of reinfection. Thus, it can be seen that the VLP obtained from pSFV / gag or pSFV / gagpro is an infectious VLP with a gag gene.

<Example 3>

Preparation of VLPs Using pSFV / gag or pSFV / gagpro

Since the VLP made in Example 2 is a mixture of VLP (SFVLPgag or SFVLPgagpro), which is a SFV core, and VLP (HIVLPgag or SFVLPgagpro), which has an HIV-1 core, to express only a VLP made of an HIV-1 core. PSFV / gag and pSFV / gagpro constructed in 1 were transcribed in vitro and transfected into BHK-21 cells without pSFV-helper RNA transcript, respectively, by the method of Example 2 above. Transfected cells were fixed for 4-6 minutes with cold 100% MtOH at 4 ° C. and −20 ° C. and blocked for 1 hour in 1% gelatin before immunization with polyclonal antibody (rabbit) against p24 (capsid) of HIV. Chemical staining. Secondary antibodies are biotinylated anti-rabbit IgGs. As a result, it was confirmed that Gag protein was expressed in the transformed BHK-21 cells (FIG. 7) (A: negative control group, untransfected BHK-21 cells; B: BHK- transformed with pSFV / gag). 21 cells; C: BHK-21 cells transformed with pSFV / gagpro).

Following detection of Gag protein in the cytoplasm, VLPs were isolated from cell culture medium supernatants according to the method of Example 2 and observed by EM (FIG. 8) (A: VLP obtained from pSFV / gag; B: pSFV / VLP obtained from gagpro). When pSFV / gag or pSFV / gagpro RNA transcripts were expressed without pSFV-helper transcripts, infectious VLP (HIVLPgag) consisting only of Gag of HIV-1 was produced.

<Example 4>

Preparation of pSFV / env Expression Vectors

To amplify the gp160 gene of HIV-1, the HIV clade E gene (Genbank accession number U51188, Journal of Virologly, 1996) was used as a template and the base sequence (6264-8879 bp) was amplified by PCR using primers 4 and 5. It was.

Primer 4: Sense (SEQ ID NO: 4)

5'-CGCGCCTCGAGCGGGATCCCATGAGAGTGAAGGGGACACGGA-3 '

Primer 5: Antisense (SEQ ID NO: 5)

5'-AATGGATCCTATAGCAAAGCCCTTTCCAAGCCC-3 '

PUHD (100 ng / μl) with template HIV-1 clade E full-length gene 4 μl of 2.5 mM dNTP (Boehringer Mannheim, Germany), 1 μl of sense primer (100 pmol / μl), 1 μl of antisense primer (100 pmol / μl), Add 5 μl of Taq polymerase 10X buffer and DW37 μl to make a mixed solution, and warm for 5 minutes at 98 ° C., then add 1 μl of Taq polymerase, and then for 1 minute at 94 ° C., 2 minutes at 55 ° C., and 3 minutes at 72 ° C. The series of reactions to keep was repeated 30 times. The amplified gene was inserted into the BamHI site of the pSFV vector to prepare a pSFV / env expression vector (FIG. 9).

<Example 5>

Preparation of VLPs with pSFV-helper and pSFV / env

Since Env alone does not form VLPs, VLPs were prepared using pSFV-helper and pSFV / env. The pSFV / env expression vector prepared in Example 4 was treated with restriction enzyme Sph I to make linear DNA, followed by P / C treatment. 10 μl of this DNA, 5 μl of 10 × SP6 buffer (TaKaRa), 5 μl of 10 mM m ⁷ G (5 ′) ppp (5 ′) G (BM), 2.5 μl of 100 mM DTT, rNTP mix (10 mM ATP, 10 mM CTP, 10 mM 5 μl of UTP, 5 mM GTP, BM), 2 μl of RNasin (BM), 1.5 μl of SP6 RNA polymerase (TaKaRa), and 19 μl of DW) were reacted at 37 ° C. for 1 hour 30 minutes. mRNA transcripts (25 μl of pSFV / helper RNA transcript and 50 μl of pSFV / env RNA transcript) were transfected into the BHK-21 cells suspended in PBS by the method of Example 2 above. After 48 hours of transfection, the supernatant was centrifuged at 3500 rpm for 15 minutes, followed by ultrafast centrifugation (SW41 rotor, 2hr, 30,000rpm, 4 ° C, BECKMAN) to obtain virus particles. This pellet was suspended in TNE (50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 0.5 mM EDTA) buffer and stored at -70 ° C. Transfected cells were fixed for 4-6 minutes with cold 100% MtOH at 4 ° C. and −20 ° C., blocked for 1 hour in 1% gelatin and immunochemically stained with specific monoclonal antibodies (FIG. 10). (A: negative control, untransfected BHk cells; B: BHK cells transfected with pSFV / env). The primary antibody was a monoclonal antibody to gp160 (1 mg / ml), a biotinylated anti-mouse IgG (5 μl / ml) as the secondary antibody and AB solution (avidin 5 μl, biotin 5 μl / PBS I). ML, VECTOR) for 30 minutes and then color development with DAB (3,3'diaminobezidine) solution (VECTOR).

Env protein was expressed in cells transfected with pSFV / env.

5 μl of chymotrypsin (10 mg / mL) and 5 μl of 50 mM CaCl ₂ were added to 100 μl of virus particles suspended in TNE buffer and reacted for 30 minutes on ice. Then, 45 μl of aprotinin (2 mg / mL) was added to activate VLP. . The particles were infected with virus monolayered BHK-21 cells and 1 hour and 30 minutes later in fresh medium and incubated for 2 days. After 2 days, 1 hour NP40, 50 mM Tris-HCl (pH7.6), 150 mM NaCl, 2 mM EDTA, 1 μg / ml PMSF) was reacted for 1 hour, followed by centrifugation (12000 rpm, 4 ℃, 5 minutes) was added to the sample buffer into the supernatant obtained by boiling for 5 minutes and electrophoresed in 12% SDS-PAGE. Electrophoresis gels were transferred to PVDF western blotting membrane (BM) at 15 V, 7 mA for 2 hours and then Western blotted using specific antibodies. The primary antibody used was AIDS patient serum, the secondary antibody was biotinylated anti-human IgG, and the blot was developed with DAB solution after reaction with AB solution (FIG. 11) (lane 1: negative control, BHK-21). Cells; lane 2: BHK-21 cells infected with VLP). After collecting VLPs and activating them in vitro and infecting BHK-21 cells, we found that infectious SFVLPenv was produced because Env protein was produced in the cytoplasm of BHK-21 cells.

<Example 6>

Preparation of VLPs Using pSFV / gag and pSFV / env

It is known that immature VLPs are assembled when only the gag gene of HIV is expressed and that the Env protein plays an important role in forming neutralizing antibodies of HIV (Gheysen D. et al., Cell, 59, 103-112). (1989); Lasky, LA et al., Science, 249, 932-935 (1986); Lasky, LA et al., Cell, 50, 975-985 91987)), thus inserting Env into Gag particles. In order to perform the experiment to simultaneously transfect the RNA transcripts of pSFV / gag prepared in Example 1 and pSFV / env prepared in Example 4 to.

pSFV / gag and pSFV / env the second embodiment according to the methods described for the transfer in vitro, respectively (without pSFV-helper) nose to BHK-21 cells transfected and after incubation for 48 hours, the Gag and Env Cells were immunostained with a monoclonal antibody against gp160 that recognizes both AIDS patient serum and Env that recognizes. Gag and Env of HIV-1 in cells were found to be stained more than when stained with patient sera (FIG. 12C) with monoclonal antibodies against gp160 (FIG. 12B). It was found that they were all expressed (FIG. 12) (A: negative control group; B: monoclonal antibody against gp160; C: serum of AIDS patient). This result shows that HIVLP consisting of Gag and Env of HIV-1 is being produced.

<Example 7>

Analysis of RNA Packaged in VLPs

To determine whether the VLPs of Example 3 obtained by expressing only pSFV / gag RNA transcripts or the VLPs of Example 6 obtained by simultaneously transfecting RNA transcripts of pSFV / gag and pSFV / env contain nucleic acid, The VLP was passed through a 10% sucrose cushion. In order to prevent contamination of other RNA or DNA, 1 mg RNase A and 70 U RNase-free DNase I were reacted in MgSO ₄ -acetate buffer for 1 hour at room temperature. RNA was isolated using a Viral RNA purification kit (Viogene) to perform RT-PCR and PCR. For the gag gene, we amplified a p650 gene of about 650 bp and a portion of gp41 about 350 bp for the env gene.

4 μl of 5-fold concentrated Superscript II reverse transcription buffer (250 mM Tris-HCl [pH 8.5], 375 mM KCl, 15 mM MgCl ₂ ), 2 μl 0.1 M DTT, 4 μl deoxynucleoside triphosphate After adding buffer (dNTP; 25 mM; TaKaRa) and a primer, a total of 20 µl was kept at 42 ° C for 2 minutes. 1 μl of SuperSklypt II reverse transcriptase (reverse transcriptase (Gibco BRL) derived from pol gene of Molony Murine Leukemia Virus) was added to the solution and reacted at 42 ° C. for 50 minutes to make cDNA. 1 μl of 100 pmol of Primer 6 was used for gag RNA and Primer 7 was used for env RNA.

Primer 6: Antisense (SEQ ID NO: 6)

5'-CCCAAGCTTTTAGCATGCTGTCATCATTTC-3 '

Primer 7: Antisense (SEQ ID NO: 7)

5'-GGTTCTGCAGAAGCTTCCTTGTTATTTCAAACCA-3 '

The denatured RNA / DNA hybrid was denatured and the reverse transcriptase activity was inhibited for 15 minutes at 70 °. The cDNA thus produced was amplified using Z tag DNA polymerase (TaKaRa) and a primer (primer 8 and primer 6, gag RNA search; primer 9 and primer 7, env RNA search).

Primer 8: Sense (SEQ ID NO: 8)

5'-CCCAAGCTTCATCAGGCCTTATCACCT-3?

Primer 9: Sense (SEQ ID NO: 9)

5'-TCCCCCGGGAAGCTTGCGCAGCAGCATCTGTTG-3?

As a template, 10 μl of cDNA generated from the above RT-PCR, 4 μl of 2.5 mM dNTP (Boehringer Mannheim, Germany), 1 μl of sense primer (100 pmol / μl), 1 μl of antisense primer (100 pmol / μl), Z taq polymerase 10X 5 μL buffer, DW 282 μl, 1 μl of Z taq polymerase (TaKaRa) was added to form a mixed solution. After denaturation at 94 ° C. for 5 minutes, 1 minute at 94 ° C., 1 minute at 55 ° C., and 1 minute at 72 ° C. 35 It was carried out twice and reacted at 72 ° C. for 7 minutes. The amplified product was observed by UV illumination after electrophoresis on agarose gel.

The p24 gene was amplified in both HIVLP obtained by expressing only pSFV / gag RNA transcript or HIVLP obtained by simultaneously transfecting RNA transcripts of pSFV / gag and pSFV / env (lanes 1 and 2 in FIG. 13). However, the gp41 gene was not amplified in any VLPs (lanes 1 to 3 of FIG. 13). The results indicate that only RNA with a gag gene is packaged and budded in the VLP. (VLP obtained from lane 1: pSFV / gag; lanes 2 and 3: VLP by co-transfection of pSFV / gag and pSFV / env; lanes 1 and 2 were amplified using p24 primer, and lane 3 was amplified using p41 primer, where 650 bp represents gag gene region and 350 bp represents env gene region).

<Example 8>

Production of VLPs by Infecting Defective SFVLPs

The VLPs obtained in Examples 2 and 5 were activated in vitro and infected with COS-1 cells to confirm the production of VLPs. VLPs from infected COS-1 cell lysates and supernatants were western blotted with AIDS patient sera, polyclonal antibodies against p24 (rabbits) and monoclonal antibodies against p24 (FIG. 14). (A: AIDS patient serum; B: polyclonal antibody against p24; C: monoclonal antibody against p24; negative controls (lanes 1, 4, 7, 10, 13); infection with SFVLPgag (lanes 2, 5 , 8, 11, 14); simultaneous infection with SFVLPgag and SFVLPenv (lanes 3, 6, 9, 12, 15); cell lysate (lanes 1, 2, 3, 7, 8, 9, 10, 11, 12) VLP (lanes 4, 5, 6, 13, 14, 15)).

In FIGS. 14A and 14B, Gag protein was detected in intracellular cells (lanes 2 and 8) and the supernatant VLPs (lane 5) when infected with a defective SFVLP containing a gag gene. In the case of simultaneous infection with SFV replicons containing gag and env genes, both Gag and Env proteins were detected in the intracellular cells (lanes 3 and 9) and the supernatant VLPs (lane 6). Thus, when Gag and Env proteins are expressed in one cell at the same time, it was confirmed that VLPs composed of these two proteins were produced.

Western blot of monoclonal antibody against p24 (Biogenesis, Cat. No. 4999-8607) in FIG. 14C detected Pr55 and p24 precursors of Gag. It is thought that the precursor of Gag can be processed or denatured even if the protease of HIV-1 is not expressed in COS-1 cells.

<Example 9>

Construction of pSFV / env-gag and pSFV / env-gag-pro Expression Vectors

PSFV / pro was constructed to clone the pro (2268-2606 bp) gene with the 26S subpromoter. 4 μl of 2.5 mM dNTP (Boehringer Mannheim, Germany), 1 μl of sense primer (100 pmol / μl), antisense into pUHD (100 ng / μl) with HIV-1 clade E full-length gene as template using primer 10 and primer 11 1 μl of primer (100 pmol / μl), 5 μl of Taq polymerase 10X buffer, 37 μl of DW to prepare a mixed solution. After 5 min at 98 °, 1 μl of Taq polymerase was added, followed by 1 min at 94 ° C. and 1 min at 55 ° C. 40 series of reactions were repeated for 30 seconds at 72 ° C.

Primer 10: Sense (SEQ ID NO: 10)

5'-CCAAGATCTATGACAGCCTCCTCCTTAGTTTC-3 '

Primer 11: Antisense (SEQ ID NO: 11)

5'-CAACCCGGGTCGCGATTAAGTGTCAATAGGACTAAT-3 '

Genes amplified in the above reaction were inserted into the SmaI site, which is a multicloning site of pSFV, using the EcoRV and SmaI sites attached to both ends of the primer (FIG. 15).

The pSFV / env-gag vector amplified the 26S gag gene using primers 12 (sense) for the 26S subgenomic promoter and primers 2 (antisense) for the gag with the pSFV / gag prepared in Example 1 as a template. It was prepared by insertion into the SmaI site of pSFV / env (FIG. 16).

Primer 12: Sense (SEQ ID NO: 12)

5'-CCGGATATCACCTCTACGGCGGTCCTA-3 '

Primer 13: Antisense (SEQ ID NO: 13)

5'-CGCCCGGGTTACTGTGACAAGGGGTCGTTGCCAAA-3 '

Using the above-prepared pSFV / pro as a template, the 26Spro gene was amplified using a subgenomic promoter primer (SEQ ID NO: 12) and an antisense primer of SEQ (SEQ ID NO: 11), and inserted into the SmaI site of pSFV / env-gag. pSFV / env-gag-pro was constructed (FIG. 17). Herein, the amplified pro gene has a smaller N-terminal sequence than the gagpro pro gene of Example 1 and further includes a C-terminal sequence. This is an amplification of the pro gene, including the sequence required for self-processing (Viviane V. et al., J. Gen. Virol. 73, 639-651 (1992)). The pSFV / env-gag-pro vector of the present invention was deposited with the accession number KCCM-10233 to the Korean Culture Center of Microorganisms, a depository institution under the Budapest Treaty.

<Example 10>

Production of VLPs by Expression of pSFV / env-gag

Cells were infected with the expression vector pSFV / env-gag of Example 9, in which two structural proteins of HIV were cloned successively into one vector under each promoter to make a VLP with Env in Gag. After 48 hours of transfection of RNA transcripts of pSFV / env-gag to BHK-21 cells, VLPs obtained from the cell lysates or media supernatants were obtained from Western blot using ECL (enhanced chemiluminescent substrate) method with serum of AIDS patients. (FIG. 18) (lane 1: negative control; lane 2,3: cell lysate; lane 4,5: cell supernatant). Both proteins, Gag and Env, were found in the VLP. When two clones were cloned by connecting the env and gag genes under two subgenomic promoters, both proteins were expressed in the cells and mutually formed to form viral particles. Able to know. In Fig. 18, the Gag protein size of the medium supernatant is small because it is electrophoresed by the albumin of 65 kDa as a serum component.

<Example 11>

Preparation of VLPs by Expression of pSFV / env-gag-pro

The pSFV / env-gag-pro vector prepared in Example 9 was transfected into a host cell to express each protein. 48 hours after transfecting the RNA transcript of pSFV / env-gag-pro to BHK-21 cells, the cells were subjected to AIDS patient serum, monoclonal antibodies against gp160, polyclonal antibodies and proteases to p24. Immunocytochemical staining with polyclonal antibody (positive) against (FIG. 19) (A: negative control; B: patient serum; C: monoclonal antibody against gp160; D: polyclonal antibody against p24 E: polyclonal antibody to protease). Since all of these antibodies recognized each antigen, it can be seen that both Gag, Env and Pro were expressed from each 26S subgenomic promoter. The experiment further shows that all three or more foreign genes linked to each of the three subgenomic promoters of the alpha viral expression vector can be expressed.

<Example 12>

Preparation of pSFV / env-gag-gagpro-CTE Expression Vectors

To prepare a pSFV / CTE vector, pGEM 7fz (−) / MPMV was amplified using primers 14 and 15 below as a template, and inserted into BamHI and SmaI sites of pSFV (FIG. 20). PSFV / gagpro-CTE was prepared by inserting the gagpro of pSFV / gagpro prepared in Example 1 into the BamHI site of pSFV / CTE in the vector (FIG. 21). The pSFV / gagpro-CTE vector inserts the CTE gene of the Mason-Pyja monkey virus after the pSFV / gagpro sequence.

Using the pSFV / gagpro-CTE as a template, the gagpro full-length gene including the 26S subgenomic promoter and the CTE gene were amplified by PCR, and inserted into the SmaI site of the pSFV / env-gag plasmid prepared in Example 9, and then inserted into pSFV / env. A -gag-gagpro-CTE expression vector was prepared (FIG. 22). The amplification reaction was carried out using primer 12 and primer 15, 4 μl of 2.5 mM dNTP (Boehringer Mannheim, Germany), 1 μl of sense primer (100 pmol / μl), 1 μl of antisense primer (100 pmol / μl) in 1 μl of template pSFV / gagpro-CTE. 5 μl of Taq polymerase 10X buffer and 37 μl of DW were added to form a mixed solution. After 5 minutes at 98 ° C., 1 μl of Taq polymerase was added, followed by 1 minute at 94 ° C., 1 minute at 55 ° C., and 2 minutes at 72 ° C. The series of reactions maintained was repeated 40 times. The pSFV / env-gag-gagpro-CTE vector of the present invention was deposited with the accession number KCCM-10234 to the Korean Culture Center of Microorganisms, an international depository organization under the Budapest Treaty.

Primer 14 Sense (SEQ ID NO: 14)

5'-AATGGATCCCCTCCCCTGTGAGCTAGACT-3 '

Primer 15 Antisense (SEQ ID NO: 15)

5'-AATGATATCAGATCTCCAAGACATCATCCGGGCAA-3 '

<Example 13>

Preparation of pSFV / env-gag-gagΔpro-CTE Expression Vectors

gagΔpro removes five Ts, a sequence conserved in gagpro's ribosomal frame shift signal. In order to make gagΔpro, first, pSFV / gag of Example 1 was used as a template, and the gag gene 832-2113 immediately before the 26S subgenomic promoter and frame shift was used as primer 12 and primer 16 below. After amplification by PCR was inserted into pGEMT vector (Promega).

Amplification was carried out with 1 μl of pSFV / gag, 4 μl of 2.5 mM dNTP (Boehringer Mannheim, Germany), 1 μl of sense primer (100 pmol / μl), 1 μl of antisense primer (100 pmol / μl), 5 μl of Taq polymerase 10X buffer, DW After adding 37 μl to form a mixed solution, after 5 minutes at 98 °, add 1 μl of Taq polymerase, and repeat a series of reactions for 40 minutes at 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 1 minute. It was. In addition, the full-length gene of pro excluding the preservation sequence of the frame shift signal using pSFV / gagpro-CTE of Example 12 as a template was amplified by PCR using primers 17 and 15 and then inserted into the pGEMT vector.

Primer 16 antisense (SEQ ID NO: 16)

5'-AATAGGCCTGTCTTTCAGTGCAGTCTT-3 '

Primer 17 Sense (SEQ ID NO: 17)

5'-CACAGGCCTATAGGGAAAATCTGGCCTTC-3 '

The two genes were linked by treatment with StuI restriction enzymes. During this process, the amino acid Asn (Asparagine) was replaced with Tyr (Tyrosine), and two Phe (Phenylalanine) amino acids were removed by removing 5 T. The completed gagΔpro was treated with EcoRV restriction enzyme and inserted into the SmaI site of pSFV / env-gag to prepare a pSFV / env-gag-gagΔpro-CTE expression vector (FIG. 23).

(SEQ ID NO: 18) F F R E

Frame Shift Site of CAG GCT AATTT TTT AGG GAA Gag-pol

Q A N

(SEQ ID NO: 19)

Deformation Sites of CAG GCC TAT AGG GAA GagΔpro

Q A Y R E

<Example 14>

Preparation of pSFV / gagpol Expression Vectors

In the present invention, an expression vector expressing Gagpol polyprotein was prepared by inserting a gagpol gene of HIV-1 into alpha viral replicon.

To make pSFV / gagpol, the nucleotide sequence of HIV-1 (832-5132bp) corresponding to Gagpol's full-length gene was amplified by PCR using primers 18 and 19 below. 4 μl of 2.5 mM dNTP (Boehringer Mannheim, Germany), 1 μl of sense primer (100 pmol / μl), 1 μl of antisense primer (100 pmol / μl) in pUHD (100 ng / μl) with HIV-1 clade E full-length gene as template 5 μl of Taq polymerase 10X buffer and 37 μl of DW were added to form a mixed solution. The mixture was denatured at 98 ° C. for 5 minutes, and then 1 μl of Taq polymerase was added, followed by 1 minute at 94 ° C., 1 minute at 55 ° C., and 72 ° C. The series of reactions was repeated 40 times, maintaining at 3 minutes at.

Primer 18: Sense (SEQ ID NO: 20)

5'-TTAGGATCCATGGGTGCGAGAGCGTCA-3 '

Primer 19: antisense (SEQ ID NO: 21)

5'-CGCGGATCCCTAATCCTCATTCTGTCTACC-3 '

The amplified gene was inserted into the BamH1 site of pSFV to make pSFV / gagpol (FIG. 24).

<Example 15>

Production of VLPs with pSFV / gagpol

To express the VLP consisting of the processed HIV-1 core, the expression vector pSFV / gagpol constructed in Example 14 was transcribed in vitro to BHK-21 cells without pSFV-helper RNA transcript, Each was transfected by the method of 2. Transfected cells were fixed for 4-6 minutes with cold 100% MtOH at 4 ° C. and −20 ° C. and blocked for 1 hour in 1% gelatin before the cells were subjected to AIDS patient serum, polyclonal antibodies against p24 and protease. Immunocytochemical staining with polyclonal antibodies (FIG. 25) (A: negative control; B: patient serum, C: polyclonal antibody against p24p; D: polyclonal antibody against protease). As a result of the experiment, it was confirmed that not only Gag protein but also Gagpol polyprotein were expressed in the transformed BHK-21 cells.

RNA transcripts of pSFV / gagpol were transfected into BHK-21 cells, and 48 hours later, VLPs were isolated from the cell lysates or media supernatants and Western blotted using polyclonal antibodies against p24 using the ECL method (FIG. 26) (lane 1,4: negative control; lane 2,5: sample obtained from pSFV / gag; lane 3,6: sample obtained from pSFV / gagpol; cell lysate (lane 1,2,3); VLP (lane 4,5,6)).

Only Pr55 was expressed in BHK-21 cells transfected with pSFV / gag, and immature VLP was detected in supernatant, but most of Pr55 was processed in BHK-21 cells transfected with pSFV / gagpol to express large amounts of p24. And processed supernatant VLPs in the supernatant.

<Example 16>

Preparation of pSFV / gag-envMCTE and pSFV / gagpol-envMCTE Expression Vectors

In the present invention, pSFV / gpol-envMCTE was prepared by amplifying the env gene including the 26S subgenomic promoter and MCTE and inserting it into the SmaI site of PSFV / gpol to produce mature HIVLP having Env. In addition, pSFV / gag-envMCTE was prepared for comparison with this construct.

In order to make pSFV / gag-envMCTE, we first built a new pSFV / gag. Since the gag gene of pSFV / gag made in Example 1 contains the SmaI site, primer 18 and primer 2 from which the SmaI site was removed were used to sequence the base sequence of HIV-1 corresponding to the full-length gene of Gag (832-2113bp). Amplification by PCR prepared in the same manner as in Example 1.

pSFV / gag-envMCTE is a primer 12 (sense) for 26S subgenomic promoter with pSFV / envMCTE (FIG. 27) prepared in the same manner as in Example 4 in the BamHI site of pSFV / MCTE prepared in Example 12. ) And primers 15 for MCTE (antisense) were prepared by amplifying the 26SenvMCTE gene and inserting it into the SmaI site of pSFV / gag (FIG. 28).

pSFV / gagpol-envMCTE was prepared by amplifying the 26SenvMCTE gene and inserting it into the SmaI site of pSFV / gagpol by the above method (FIG. 29). The pSFV / gagpol-env-MCTE vector of the present invention was deposited in the Korean Culture Center of Microorganisms, an international depository under the Budapest Treaty, under accession number KCCM-10348.

<Example 17>

Production of VLPs with pSFV / gag-envMCTE and pSFV / gagpol-envMCTE

pSFV / gag-envMCTE was transcribed in vitro and transfected into BHK-21 cells without pSFV-helper RNA transcript by the method of Example 2 above. Transfected cells were fixed for 4-6 minutes with cold 100% MtOH from 4 ?? to -20 ?? and blocked for 1 hour in 1% gelatin, then cells were treated with AIDS patient serum, polyclonal antibody against p24, protease Immunocytochemical staining with polyclonal antibody against and monoclonal antibody against env (FIG. 30) (A: negative control; B: patient serum; C: polyclonal antibody to p24p; D: protease Polyclonal antibodies against Ise). As a result of the experiment, it was confirmed that not only Gag protein but also Env protein was expressed in the transformed BHK-21 cells.

pSFV / gagpol-envMCTE was also transfected by the method of Example 2 above (FIG. 31) (A: negative control; B: patient serum; C: polyclonal antibody against p24; D: poly to protease Clonal antibody; E; monoclonal antibody to env). As a result of the experiment, it was confirmed that not only Gagpol protein but also Env protein were expressed in each transformed BHK-21 cell.

RNA transcripts of pSFV / gag-envMCTE and pSFV / gagpol-envMCTE were transfected into BHK-21 cells, and after 48 hours, VLPs were isolated from the media supernatant to polyclonal antibodies against p24, monoclonal against env. Western blot was used as raw antibody using the ECL method (FIG. 32) (A: patient serum; B: monoclonal antibody against env; VLP obtained from lanes 1,4: pSFV / gag; lanes 2,5: pSFV / VLP obtained from gag-envMCTE; lane 3,6: VLP obtained from pSFV / gagpol-envMCTE. As a result, expression of pSFV / gagpol-envMCTE was able to produce mature VLP containing Env and processed.

Provided are methods for preparing mature HIV-like particles having a form similar to the original HIV antigen using an alpha virus system. The HIV-like particles of the present invention can be usefully used to prepare antigen or vaccine compositions of diagnostic kits for diagnosing HIV infection.

Claims (29)

The env gene (6264-8879 bp) of HIV-1 is operably linked under a first subgenomic promoter and the gag gene (832-2328 bp) of HIV-1 is operable under a second subgenomic promoter Alpha virus-based expression vectors linked to one another. The pSFV / env-gag expression vector of claim 1. The gag gene (832-2328 bp) of HIV-1 is operably linked under the first subgenomic promoter, and the env gene (6264-8879 bp) of HIV-1 is operable under the second subgenomic promoter Alpha virus-based expression vectors linked to one another. The pSFV / gag-env expression vector of claim 3. The env gene (6264-8879 bp) of HIV-1 is operably linked under a first subgenomic promoter and the gag gene (832-2328 bp) of HIV-1 is operable under a second subgenomic promoter Alpha virus-based expression vector, wherein the pro gene of HIV-1 (2268-2606 bp) is operably linked under a third subgenomic promoter. The pSFV / env-gag-pro expression vector of claim 5. The env gene of HIV-1 (6264- 8879 bp) is operably linked under the first subgenomic promoter, and the gag gene (832-2328 bp) of HIV-1 is operable under the second subgenomic promoter Alpha virus-based expression vector, wherein the gagpro gene of HIV-1 (832-2577 bp) is operably linked under a third subgenomic promoter. 8. The pSFV / env-gag-gagpro expression vector of claim 7. pSFV / env-gag-gagΔpro expression vector. The gagpol gene (832-5132 bp) of HIV-1 is operably linked under the first subgenomic promoter, and the env gene (6264-8879 bp) of HIV-1 is operable under the second subgenomic promoter Alpha virus-based expression vectors linked to one another. The pSFV / gagpol-env expression vector of claim 10. The expression vector of claim 1, further comprising a constitutive transport element (CTE) gene. The pSFV / env-gag-gagpro-CTE expression vector according to claim 12. The pSFV / env-gag-gag Δpro-CTE expression vector according to claim 12. The pSFV / gagpol-env-MCTE expression vector according to claim 12. The pSFV / gag-env-MCTE expression vector according to claim 12. Expression vector deposited with accession number KCCM-1O233. Expression vector deposited with accession number KCCM-1O234. Expression vector deposited with accession number KCCM-10348. 20. A method for producing HIV-like particles (HIVLP), wherein the expression vector or RNA transcript thereof according to any one of claims 1 to 19 is introduced into an animal cell for expression. The method of claim 20, wherein the mature HIV-like particles are prepared. The method of claim 21, wherein the mature HIV-like particles are infectious. 20. A host cell transformed with the vector of any one of claims 1 to 19. 24. The host cell of claim 23, wherein the animal cell is stably transformed. 20. An AIDS vaccine composition comprising an expression vector according to claims 1 to 19 or an RNA transcript thereof sufficient to elicit an immune response. An AIDS vaccine composition comprising mature HIV-like particles produced by the method of claim 21 in an amount sufficient to elicit an immune response. 27. An AIDS prophylaxis method comprising administering the vaccine composition of claim 25 or the vaccine composition of claim 26 to a human. A diagnostic kit for diagnosing HIV-infection, comprising mature HIV-like particles prepared by the method of claim 21. (a) taking a sample that is expected to contain HIV specific antibodies, and (b) using the sample taken from step (a) as an antigen, a mature HIV-like particle prepared by the method of claim 21, an antigen and an antibody. Contacting under conditions that allow binding of the antigen to form an antigen-antibody immune complex, and (c) detecting the formation of the immune complex, specifically binding to the HIV retroviral antigen in the sample. A method of determining the presence of reacting antibodies.