JP2001247476A - Use of antibody against nef protein for decreasing chemotaxis which is induced with retrovirus-infected astrocyte - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a medicine for treating a neuropathogenesis, especially encephalopathy, caused by decreasing chemotaxis which is induced with retrovirus-infected astrocyte. SOLUTION: The use of antibodies against the Nef protein, of functionally active parts of these antibodies is mentioned which antibodies include enzymically truncated antibodies, and/or of nucleic acid sequences which encode these antibodies for decreasing, preferably preventing, especially treating neuropathogenesis, preferably encephalopathy, the chemotaxis which is induced by retrovirus-induced astrocytes.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the Nef protein for reducing, preferably preventing, chemotaxis induced by astrocytes infected with retroviruses, in particular for treating neuropathies associated therewith, preferably encephalopathy. The present invention relates to the use of antibodies, functionally effective portions of these antibodies, and / or nucleic acid sequences encoding these antibodies.

[0002] Retroviruses are viruses that infect humans and animals, and are capable of converting single-stranded RNA, and the so-called reverse transcriptase, the viral RNA that has entered the host cell, to D.
It also contains a polymerase that transcribes to NA. This DNA is then integrated into the chromosomal DNA of the host cell along with additional repetitive sequences (long terminal repeats, or LTRs) and replicated with the cell genome as a so-called provirus. Transcription of viral protein mRNA
Is produced, and from this RNA viral proteins are produced as polyproteins that depend on proteases for their maturation. Generally, retroviruses do not kill their host cells. However, some representatives are highly pathogenic. These latter include, inter alia, HIV-1 and 2 (AIDS pathogens).

[0003] A representative of the best known and most important retroviruses in this context with respect to the present invention is the HIV-1 virus (human immunodeficiency virus), which is AIDS (acquired immune virus). It causes a relatively large number of syndromes known as deficiency syndromes). The HIV-1 virus is an enveloped virus with a relatively small capsid protein (MW2
4,000) and its reverse transcriptase is activated by magnesium ions.

AIDS has been known in the United States since 1981 and refers to a syndrome based on defective cells associated with an immune defense in which HIV virus infection has been demonstrated. AIDS is a contagious disease, with an estimated over 10 million cases worldwide. The main symptom of HIV infection is immunopathogenesis, the development of pathological changes in the immune system. In this regard, HIV is at least 3
Infecting cells of the species, the primary targets are helper T lymphocytes, as well as monocytes / macrophages and dendritic cells that carry the CD4 receptor. These cells, which play a central role in the immune system, infect and then infect other cells, and in some cases fuse with uninfected cells and / or die. This significantly impairs the interactions of cells involved in the immune system and impairs the intrinsic resistance to pathogens. This in turn results in a weakening of the immune system, ie, immunodeficiency, which means that the infected patient's cellular immune response is weaker than in a healthy state. As a result, infections have a serious and life-threatening course. It is also known that tumors of lymphoid tissues and tumors in the blood vessels of the skin and internal organs (Kaposi's sarcoma) can form in connection with HIV infection.

[0005] After infection with HIV, and after infection with other retroviruses, the cells of the central nervous system (CNS) can be infected separately, but often simultaneously, and usually simultaneously.
Also cause pathological changes, ie, neuropathogenesis. This infection results in an impairment in brain function, ie encephalopathy. Encephalopathy is understood to be a syndrome in which neurons are particularly damaged and destroyed in the long term. In particular, encephalopathy is understood to indicate almost systemic nerve damage. This damage can be accompanied by neurological symptoms, dementia and severe loss of brain function.

The HIV virus is also known to invade the central nervous system shortly after infection and infect mononuclear cells (macrophages and microglia) that carry replicating viruses (Michaels et al., 1988, Immunodefic). . Re
v. 1, 71; Wiley et al. (1996) AIDS 10, 843). In this regard, virus entry into the brain is caused by HIV-associated cells in monocytes and T cells that cross the blood / brain barrier.
It is clear that this is due to No. 1 (Nottet (1996)
J. Immunol. 156, 1284). For central nervous system infection, two weeks after viral infection, rhesus monkey cerebrospinal fluid (C
In SF), the SIV virus, monkey-specific immunodeficiency virus I and its components have already been detected (Sharer et al. (1996), Hum. Pathol. 27, 614).
In addition to demonstrating a proliferative infection of microglia, this study also showed that astrocytes, the most frequently occurring cell type in the brain, were affected. Astrocytes show a phenotype of latent infection, which is characterized by low levels of virus production and high levels of expression of Nef regulatory proteins.

Astrocytes are cells of the brain, most of which have a stellate appearance. They belong to macroglia as well as oligodendrocytes and ependymal cells. Wherever a neuron is not surrounded by oligodendrocytes,
As its surface is covered by cell processes from astrocytes, the localization of astrocytes gives suggestions for their potential function. The endothelial cells of the capillaries that form the blood / brain barrier also belong to these latter cells, and are associated with astrocytes through a layered structure called nerve fiber terminals. In this regard, it is highly likely that many substances that leave or become incorporated into neurons must undergo cell permeation transport through astrocytes (Ceballos and Rubio (1995) J. Neur
ochem. 64, 991-995). Astrocytes participate in the supply of neurons with essential metabolites and in the regulation of ion and neurotransmitter concentrations. Thus, astrocytes participate in maintaining the microenvironment of neurons.

Astrocytes are persistently infected (Ward et al. (19)
91) Am. J. Pathol. 127, 199), in particular, in addition to expressing the regulatory proteins of the virus, macrophages and microglia also become proliferatively infected, which infection leads to HIV replication in the central nervous system. Is important for (Tak
ahashi et al. (1996), Ann. Neurol. 39, 705). However, the exact mechanism and course of HIV encephalopathy is less known than having a mechanism of immune pathogenesis and consequent immunodeficiency.

[0009] Various mechanisms are thought to contribute to the dysfunction or disruption of the immune system as a whole. Based on the induction of dysfunction of individual cells and the destruction of various cell types, such as HIV-infected T helper cells, by fusion or direct apoptosis via secretion of various immunosuppressive cytokines such as IL-10 Although various mechanisms have been described, these mechanisms individually cannot adequately explain the entire course of HIV-mediated immunopathogenesis or are not frequently observed in patients in vivo.

[0010] Reliable in vivo signs of the development of AIDS are high levels of detectable viral replication and spread.
Disease manifests more rapidly as the abundance of the virus is increased. Disruption of viral replication by nucleoside analogues and proteinase inhibitors and / or combinations thereof (HAART, highly active antiretroviral therapy) and
Such as the improvement of the survival rate, as a result cause AIDS
Improvement in the patient's symptoms depends on the level of viral replication and AI
Demonstrates a direct relationship that exists during the development of DS disease. The problem with this treatment is the consequence of strong side effects and the tight regulation of required intake. In addition, the HIV virus, like other retroviruses, is more resistant to chemotherapy, which means that new forms of treatment that use new points of attack against the virus must be found urgently. Is what you do.

Although the function of the Nef protein is not exactly known, it appears to be involved in maintaining high viral titers in vivo and is a potential target for such novel therapeutic approaches Is a molecule. Usually, retroviruses carry only three genes encoding the proteins Gag, Env and Pol. However, the retroviruses that are involved in the development of AIDS and that, like HIV, belong to the most complex class of all retroviruses known to date, have a complementary gene, ie, whose protein is in the HIV genome. It also retains the tat gene, which provides for strong expression, the rev gene whose protein regulates translocation of unspliced mRNA to the cytoplasm, and also the Nef gene.

[0012] It is known that the Nef gene or Nef protein can exert an effect on the expression of various cell molecules and can exert an effect on a specific cell stage. Perhaps the most obvious finding regarding the role of Nef in E. coli is from in vivo studies. N in rhesus monkeys and humans
These studies using ef-defective SIV and HIV viruses show that nef-defective viruses replicate slower in vivo, and therefore have lower abundance of the virus than in nef non-defective viruses. Was done. A series of studies performed on humans demonstrated that modification of the Nef gene could indeed significantly delay the onset of AIDS disease. In these studies, HIV-1 sequence analysis was used to detect deletions in the Nef gene of long-lived patients. These patients were donated by HIV-1 positive donors and had stable and normal CDs for 10-14 years.
Consisting of 6 individuals with 4-positive T lymphocyte levels (Deacon
et al. (1995) Science 270, 988-991).

In monkeys infected with SIV, HIV-
The 1Nef protein is expressed early in viral infection and participates in active viral replication and AIDS pathogens (Klotm
an et al. (1991), Proc. Natl. Acad. Sci. USA 88, 5
011). Especially in animal models, Nef has been shown to be involved in maintaining high viral titers (Kestler et al.
al. (1991), Cell 65, 651; Skowronski et al., (19
93) EMBO J. 12, 703-708; Jamieson et al. (1994) J.
Virol. 68 (6), 3478-3485). Normally, the Nef protein is localized in infected cells, but can be detected extracellularly in HIV-seropositive individuals as well as in Nef-specific antibodies (Bahraoui et al., (1990), AIDS Res. Hum. Retrovi).
ruses, 6, 1780; Ameisen (1989) AIDS Res.Hum.Retr
oviruses, 5, 279). In addition, in vitro studies show that HI
V-infected cells have been shown to be able to secrete Nef protein into vesicles (Guy, (1990), Virology, 17
6,413; kienzle et al., (1992), Arch.Virol., 124,
one two Three). Furthermore, the Nef protein can bind to cell surface receptors (Fujinaga, (1995), J. Immunol., 155, 5289),
For example, MHC-II molecules and CD4 T cells, CD8
Can bind to T cells, B lymphocytes, macrophages and neutrophils (Torres and Johnson (1994) Biochem. Biop.
hys. Res. Commun. 200, 1059; Okada et al., (1997)
FEBS Lett. 414, 603).

[0014] The Nef protein can also be detected in the central nervous system when none of the other structural proteins of the virus can be detected, or when only a few such proteins can be detected, in which case the Nef protein is also detected.
ef mRNA and Nef protein are also observed in astrocytes (Brack-Werner et al., (1992) AIDS 6,
273; Neumann et al., (1995) J. Virol. 69, 2159; To
rnatore (1994) J. Virol. 68, 93; Tornatore (1994)
Neurology 44, 481). Interestingly, the Nef protein shows additional structural and functional similarities to the scorpion neurotoxin, especially with respect to its interaction with potassium channels (Werner et al. (1991) AIDS, 5, 1301).

Recently, the number of infected macrophages and HIV
A clear correlation was found between associated dementias, suggesting that macrophage infiltration plays a role in neuropathogenesis.

Within the scope of the present invention, surprisingly, the fact that the Nef protein has a chemotactic effect on leukocytes (granulocytes and monocytes) when expressed by astrocytes. Was found. Nef
It could also be demonstrated that specific antibodies could reduce this chemotactic effect of astrocytes expressing Nef on leukocytes. Leukocytes, especially monocytes, are retroviruses, especially H
It is an important target cell for IV. Because HIV is generally carried from one infected cell to the next target cell by cell-to-cell contact, attracting target cells, eg, monocytes, using infected CNS cells, especially astrocytes, requires C
Increase the probability of virus spread within the NS. These experiments were also the first to show potential for preventing retrovirus-related neuropathogens, especially encephalopathy, or slowing the progression of these symptoms.

It is therefore an object of the present invention to reduce or preferably prevent the chemotactic effect of Nef, thereby opening new possibilities for treating neuropathogens, preferably encephalopathies, caused by retroviruses, in particular HIV. Was to make it available.

The present invention provides for reducing, preferably preventing, the chemotaxis induced by astrocytes infected with a retrovirus, in particular for treating neuropathogens associated therewith, preferably encephalopathy. The use of functionally effective portions of these antibodies, including antibodies against the Nef protein, enzymatically truncated antibodies, and / or nucleic acid sequences encoding these antibodies. However, the invention also encompasses prophylaxis, for example in the form of passive vaccination.

One use according to the invention is, in particular, HIV-
1. Use for the treatment of neuropathogens associated with HIV-2 and / or SIV (Simian Immunodeficiency Virus), in particular encephalopathy.

In a particularly preferred use, the antibodies are directed against the C-terminus of the Nef protein. On the other hand, it is thought that the C-terminal of the Nef protein participates in binding Nef to the cell surface (Okada, (1998) Med. Mikr
186, 201), on the other hand, epitope studies indicate that the carboxy terminus of Nef is preferably exposed on the cell surface of HIV-1 infected T cell lines and monocyte cells (PBMC). (Ota
ke K. (1994) J. Immunol. 153, 5826). It is known from the literature that the carboxy-terminal domain of Nef elicited the highest immunity in chimpanzees and rodents (Estaqueir et al., (1992), Mol. Immunol. 2
9, 1337). The carboxy terminus of the Nef protein also
In the formation of syncytia between HIV infected and uninfected CD4 T cells, and in CD4 T
Play an important role in the specific induction of cell proliferation of cells (Otake (1994), J. Immunol. 153, 5826; Fujii et a
l. (1996) FEBS LETT 393, 105).

Furthermore, in particular, the Nef of the HIV-1 virus
Antibodies directed against amino acid residues 171-190 of the protein, structures with corresponding antigenic effects, and / or
Alternatively, the use of nucleic acids encoding these structures is specifically claimed. In this regard, the method of Ovod et al. (1992; AIDS
6, 25-34), the use of the corresponding monocyte antibody 2H12, especially also the subtype IgG1, is particularly preferred.

Within the meaning of the present invention, antibodies are understood to be so-called antigens, in the present invention proteins and peptides such as glycoproteins which participate in an antigen-antibody reaction with an epitope of the Nef protein. Physiologically, antibodies together with the so-called complement system underlie the humoral immune response and are thought to belong to the immunoglobulins. Accordingly, within the meaning of the present invention, antibodies to the Nef protein are understood to be all structures which can participate in an antigen-antibody reaction with the Nef protein.

In particular, the antibodies used within the scope of the present invention are selected from the group consisting of polyclonal, monoclonal, chimeric, humanized, single chain or synthetic antibodies or antibodies isolated from human gene libraries. Antibodies are of all subclasses, including IgA (related to secretion) and especially IgG and its subclass IgG1. Monoclonal antibodies are antibodies produced only from a single clone of plasma cells (eg, EP 380).
443 B1), and in particular within the meaning of the present invention are understood to be antibodies produced from rat, mouse or human cells. These antibodies are very specific because they are directed against only certain epitopes. On the other hand,
Polyclonal antibodies are generally obtained by immunizing laboratory animals, usually hare and rabbits and goats, as well as primates, which are normal natural immune responses and have different affinities for individual antigenic determinants And specificity, usually belonging to various subclasses. A chimeric antibody is understood to be an antibody that is derived from two different organisms or sources but is still bound. A humanized antibody is an antibody that has been inserted into a human antibody using methods known to those of skill in the art to avoid an immune response and improve patient resistance, for example, based on recombinant DNA technology. A so-called single-chain antibody is, for example, an antibody as described in WO 88/01649 and usually shows the same binding characteristics as a normal antibody consisting of a light chain and a heavy chain, but only one polypeptide single chain. Consists of This allows them to be expressed, especially in the area of gene therapy, for example using vectors having nucleic acids encoding these single-chain antibodies that have been cloned into the vector. Obtaining an antibody from a human gene library is understood to mean that an FV fragment is constructed from a repertoire of variable gene regions described, for example, in EP 0 368 684 B1. In this regard, the nucleic acids encoding the light and heavy chain variable domains are linked and expressed in mammalian cells. For example, a human monoclonal antibody can be prepared in this manner.

The use of the present invention also includes the functionally effective parts of the above-mentioned antibodies, whereby these structures are necessary for the corresponding antibody-antigen-antibody reaction and have components which trigger this reaction. It can be understood that. These functionally effective moieties also include enzymatically truncated antibody forms, ie Fab or Fab ₂ synthesized from Fab or Fab ₂ fragments or gene fragments.
₂ fragments. The use of a particular form within the meaning of the present invention also encompasses the use of a nucleic acid sequence encoding an antibody of the present invention and a functionally effective part for treating a neuropathogen caused by a retrovirus. In particular, this use means, for example, in the form of an expression vector into which a nucleic acid encoding a single-chain antibody or a part thereof has been inserted, and these vectors are introduced into cells, for example, in the scope of gene therapy. It is understood to cause expression within.

In a further use of the present invention, the antibody or corresponding functionally effective moiety comprises a portion of the Nef protein exposed on the surface of the astrocytes and / or an astrocyte binding portion of the Nef protein. Join.

Another important aspect of the present invention is also to reduce, preferably prevent, the chemotaxis induced by astrocytes infected with the retrovirus, in particular by this chemotaxis. Neuropathogens caused,
HIV-1, HIV-, preferably for treating encephalopathy
2 or SIV Nef protein, an immunogenic portion of the protein, and / or the use of a nucleic acid sequence encoding the protein or an immunogenic portion thereof. Therefore, in particular, prophylactically in the form of vaccinations,
Or, for example, uses wherein endogenous antibody formation is stimulated, either immediately or early in the infection.

Furthermore, the use according to the invention can be carried out orally or parenterally, in particular epidurally or intranasally or intradermally, for example in the form of infusions, injections, sprays and or tablets.

The present invention also provides antibodies to the Nef protein, functionally effective portions of these antibodies and / or nucleic acid sequences encoding these antibodies, and / or HIV-1, HIV-2 or SIV Nef proteins, In addition to the immunogenic part of the protein and / or the nucleic acid sequence encoding this protein and these immunogenic parts, other active compounds and / or, where appropriate, suitable additives and / or auxiliary substances are used. About use. Other active compounds are understood in particular to be therapeutically effective compounds, such as antibiotics, anti-inflammatory agents, etc., which have previously been used individually or in combination in therapy, and also nucleoside analogues and protease inhibitors (HAART). . This also includes so-called combination preparations,
There are also several different antibodies for use in the present invention,
Particular mention may be made of the nucleic acids coding for a part thereof or correspondingly and / or the Nef proteins, the immunogenic parts of these proteins or the nucleic acids correspondingly coding for them.

Within the meaning of the invention, the expression further additives and / or auxiliary substances refers to antioxidants,
Preservatives, stabilizers, adjuvants, bulking agents, buffers, media, and lubricants and vectors, especially eukaryotic, viral and bacterial vectors, proteinase inhibitors, D
It is to be understood that it implies structures required for gene therapy, such as Nase inhibitors and RNase inhibitors.

In particular, administration can also be carried out with virosomes, liposomes, liposome structures, artificial or natural membranes or membrane fragments,
It can be performed using synthetic or natural cationic, anionic or neutral lipids or mixtures thereof.

Another part of the present subject matter is to reduce or preferably prevent chemotaxis induced by astrocytes infected with retroviruses in gene therapy, in particular the neuropathogens caused thereby, Encoding an antibody against the Nef protein, or a functional part thereof, preferably for treating encephalopathy;
Use of a nucleic acid sequence encoding an ef protein or immunogenic portion thereof. Within the meaning of the present invention, the main task of gene therapy is to introduce nucleic acids into cells and express effector genes. This will, within the meaning of the invention, express the antibody of the invention. In this connection, in principle, a distinction is made in gene therapy between in vitro and in vivo methods. in virto
In the method, cells are removed from an organism, transfected ex vivo with a vector, and then reintroduced into the same or another organism. In in vivo gene therapy, the vector is administered systemically (eg, by blood flow). However, it is also possible to carry out local administration, in which a vector having a gene therapy effect is administered locally into the tissue.

Another part of the present subject matter is that the chemotaxis induced by astrocytes infected with retroviruses,
A method for the manufacture of a medicament according to the invention for reducing, preferably preventing, in particular for treating a neuropathogen caused thereby, preferably encephalopathy.

[0033]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in more detail with reference to the drawings and embodiments, but is not limited thereto.

Table 1 Chemotaxis of HIV-1 Nef protein on human polymorphonuclear and mononuclear leukocytes, determined by the modified Boyden chamber method ^* : 100 ng of HIV-1 Nef protein / ml
P as compared to <0.05 ^†: HIV of 100ng using Mab _Nef 3E6
P <0.1 when compared to -1 Nef protein / ml.
05 ^‡ : p <0.05 n.n compared to 10 ng of HIV-1 Nef protein / ml. d. : Did not perform the experiment

[0035]

[Table 1]

Table 2 CSF leukocyte counts and CSF IFN- _γ and M at 24 hours after intracisternal injection into experimental rat models
Effect of HIV-1 Nef protein on CP-1 concentration ^* : p <0.05 when compared to rats injected with 100 ng of HIV-1 Nef protein in cisternal.

[0037]

[Table 2]

TABLE 3 C 6 hours after injection in the cisterna magna into the experimental rat model
SF IL-6, TNF-α, IFN- _γ and MCP
Effect of HIV-1 Nef protein on -1 concentration ^a : The number of rats used for the experiment ^b : TNF- _α concentration in CSF was below the detection limit (30 μ / ml) in 2 out of 6 rats ^* : P <0.05 when compared to rats injected with 100 ng of HIV-1 Nef protein in the cisternal.

[0039]

[Table 3]

Example 1 Preparation of Recombinant Nef Protein E. coli K12 strain was transfected with plasmid pTG1166 containing HIV-1-LAV-Bru-Nef, and expression of the recombinant Nef protein was determined by Guy et al.
(1987) Induced as described in Nature 330,266. Lyse the bacterial pellet and transfer the recombinant Nef protein to Kohlei
Purified as described in sen et al. (1996) J. Virol. Methods 60, 98. In this case, after removing the supernatant by centrifugation, the pellet containing Nef was solubilized with 8M urea and 1% β-mercaptoethanol and then subjected to affinity chromatography on heparin sepharose. The fractionated Nef fraction was tested for purity using a step gradient of sodium chloride for elution.

Example 2 Preparation of Independent Recombinant Nef Protein As in Example 1, HIV-1-Bru and HIV
TH-derived Nef gene (myristilizati
on) deficient Nef) was cloned into the vector pQE30, with six histidine residues at the Nef terminus and Factor Xa
It was expressed as a recombinant fusion protein containing a cleavage site. Nef was purified from the bacterial pellet by metal chelate affinity chromatography under non-denaturing conditions, followed by affinity chromatography on a heparin Sepharose column.

Example 3 Monoclonal Antibodies Against Recombinant Nef Protein
Preparation B according to the method of Ovod et al. (1992; AIDS 6, 25-34).
In ALB / c mice, a monoclonal antibody against recombinant Nef protein described in Examples 1 and 2 (A
b) was prepared. Stable clones were obtained, including clones producing antibodies (mainly IgG1 subtype). In particular,
Amino acid residues 171 to 19 of Nef protein, respectively
Antibody 2HI directed against 0 and 168-175
2 and 3E6 were used.

Example 4 Isolation of peripheral, especially polymorphonuclear leukocytes (PMN) Venous blood was collected from healthy volunteers and immediately mixed with sodium heparin. Polymorphonuclear cells (PMN) in 1: 1 solution containing 6% dextran and Histopa
ck1.119 (Sigma Chemicals Inc.). Then, Histopack1.
A 077 density gradient centrifugation was performed. After hypotonic lysis of the remaining erythrocytes, the PMN was washed twice in Hanks' salt solution and resuspended in RPMI / FCS.

Example 5 Isolation of peripheral, especially mononuclear leukocytes (PBMC) Venous blood was collected from healthy volunteers and immediately mixed with sodium heparin. Histopack 1.07
After centrifugation at 900 g by a 7 (Sigma Chemicals Inc.) density gradient for 30 minutes to collect mononuclear cells having a cell layer on Histopak 1.077, mononuclear leukocytes (PBMCs) were isolated. After the washing step, PBMC is RP
Resuspended in MI / FCS.

Example 6 Cells expressing the Nef gene The Nef gene of HIV-1-Bru isolate or TH isolate (astrocyte-specific isolate containing the myristylation-defective Nef gene) was identified. It was cloned into a nuclear expression vector. Human astroglioma cell line (U251 M
To stably transfect G), these vectors were used with a vector carrying the neomycin resistance gene, and individual clones were isolated under selective conditions. Nef expression was detected by immunoperoxidase staining and Western blot. Cell lines transfected with the Neo construct alone and reversely with the construct containing the Nef gene (pSGK) were used as controls. Neomycin resistant clones expressing Nef were isolated (Bru or TH clones, respectively).

Example 7 In vitro chemotaxis test using a soluble chemotactic substance Leukocyte transfection was performed according to the method of Seebach (1995) (J. Immunol.
155, 5367) using the modified Boyden chamber method. For this, chemoattractants such as Nef protein and other samples and controls were also diluted to various concentrations in RPMI / FCS and 25 μl volumes were placed in the lower sample chamber. Then, RMN
5 containing (Example 4) or PBMC (Example 5)
0 μl of the cell suspension was pipetted into the upper chamber (the upper chamber was isolated from the lower chamber by a polycarbonate porous filter without polyvinylpyrrolidone). Pore width is 3 μm for PMN chemotaxis experiments and 5 μm for PBMC chemotaxis experiments. Chamber at 37 ° C. in 5% CO ₂ (60 min for PMN and 90 min for PMBC)
After incubation, the filter was removed, the top was emptied, fixed with methanol and stained. Chemotaxis was measured by counting cells that had completely migrated through the filter under a microscope. As observed in this experiment (Table 1), the number of PMNs and PBMCs was N
258% and 34% of control, respectively, depending on ef concentration
Increase by 4%. On the other hand, when the sample is treated with an antibody directed against the Nef protein (eg, especially 2H12), a significant decrease in the chemotactic effect of the Nef protein can be observed, which decrease is almost absent in the case of PBMC. It has dropped to the control level.

Example 8 In Vitro Chemotaxis Assay Using Cells Expressing Nef Gene
In an experimental arrangement similar to that described in Example 7, with the astroglioma cells expressing the Nef gene (astro-4 / 4.2; see Example 6) in the lower chamber, The Boyden chamber method was used to demonstrate that the cells had a chemotactic effect. It was observed that PBMC migration was more than twice as high in chambers containing cells expressing the Nef gene as in chambers containing control cells (U251 MG, pSGK).

Example 9 Chemotaxis induced by astrocytes expressing Nef
Of monoclonal Nef-specific antibody 2H12 at 10 μg /
When added to the in vitro test system described in Example 8 at a concentration of ml, the chemotaxis of the astroglioma line expressing Nef against PBMC was reduced from 23.3 to 14.4.

Example 10 In Vivo on Chemotaxis in Rat Muscle and Skin
Experiments such as Pfister et al. (1992) J. Exp. Med. 176, 265;
Kodel et al. (1995) Ann. Neurol 37, 313; Koedel et
al. (1996) J. Immunol. 157, 5185,
Male Wistar rats were anesthetized while measuring blood pressure using a catheter, tracheostomized, and artificially ventilated with lungs. 100 ng of heat-treated Nef protein as a negative control 100 ng of Nef as a sample and 100 μg of carrageenan as a positive control on the skin or muscle tissue on the back of the animal as a 100 μl solution in each case Injected. Six hours later, rats were sacrificed by whole blood draw, after which tissue samples were taken and frozen.

Cuzzocrea et al. (1997; J. Immunol. 15
9, 5089-5097), the specific activity of myeloperoxidase (MPO) in tissue samples was measured. This enzyme is a marker for leukocyte infiltration.
Compared to the negative control, a significant increase in MPO activity could be measured in both the dorsal skin (FIG. 1) and the dorsal muscle tissue (FIG. 2).

Example 11 : An experimental rat model for chemotaxis in the CNS
Bei 176 animals in male Wistar rats known sufficiently, and tests have been described based (Pfister et al. (1992) loc. Ci
t .; Koedel et al. (1995) loc.cit .; Koedelet al.
(1996) loc. Cit.). In principle, this test system simulates meningitis. Rats,
Anesthesia was performed, tracheostomy was performed, and lung ventilation was performed artificially while monitoring physiological data such as blood pressure and body temperature and pH using blood samples. Following treatment, 30 minutes after the condition returns to normal, 100 μl through the catheter
Cerebrospinal fluid (CSF) was collected. Then 100 μl
Samples were administered to the animals. Thereafter, a CSF sample was collected from the large tank, and leukocytes and monocyte chemoattractant protein 1 (MCP-1), interleukin 6 (I
L-6), other substances such as tumor necrosis factor α (TNF-α) and interferon-γ (IFN-γ).

Example 12 In Vivo Experiment on Chemotaxis in CNS 100 μl of rats prepared as described in Example 10
The following substances were administered to the large tank in various groups.

[0053]

[Table 4]

Six hours after the administration of the samples, CSF samples were taken from the cisterna magna and their leukocyte content was examined. As can be seen from the figures (FIGS. 3-5), when administering 100 and 1000 ng of Nef protein,
Regardless of the origin of the Nef protein, the white blood cell count is significantly and significantly increased compared to the negative control (FIG. 3). On the other hand, since the treatment of the present invention is carried out with the antibody (13
Group), it can be seen from FIG. 4 that this heat treatment results in a significant reduction in leukocyte translocation. In this regard, HIV
The antibody (2H12) directed against the carboxyl-terminal portion of -1Nef protein is particularly effective in suppressing the chemotactic effect of Nef protein (Group 15; FIG. 5). On the other hand,
In particular, artificial peptides (groups 17-21) that also contain a corresponding linear epitope site to which the 2H12 antibody is directed have no effect.

Example 13 In one experiment, rats were treated as described in Examples 10 and 11, then awake after treatment with the sample, and then 24 hours later they were anesthetized and further treated with CSF.
Was collected. As can be seen from Table 2, the leukocyte count increased to over 1000% compared to the negative control.

Example 14 In one experiment, rats were treated according to Examples 10 and 11, CSF was harvested after 6 hours, and then IL-6, TNF-α, IFN-γ and MCP-1 in CSF.
Was measured for activity or concentration. As can be seen from Table 3,
The activity or concentration of IL-6, TNF-α, and IFN-γ was significantly increased compared to the negative control.

[Brief description of the drawings]

FIG. 1 shows specific myeloperoxidase activity (which is a leukocyte infiltration marker) in dorsal skin after treatment with Nef protein and controls.

FIG. 2 shows specific myeloperoxidase activity (which is a leukocyte infiltration marker) in dorsal muscle tissue after treatment with Nef protein and controls.

FIG. 3 shows leukocyte counts in cerebrospinal fluid after in vivo treatment with Nef protein and other samples and controls.

FIG. 4. In vivo with Nef protein (with and without antibody) and other samples and controls
o Shows the number of leukocytes in cerebrospinal fluid after treatment.

FIG. 5. In vivo with Nef protein (with and without antibody) and other samples and controls
o Shows the number of leukocytes in cerebrospinal fluid after treatment.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) C12N 15/09 C07K 16/10 // C07K 16/10 C12P 21/08 C12P 21/08 C12N 15/00 A (72) Inventors Birgit, Koraisen Unterschleissheim, Federal Republic of Germany, Pegasusstrasse, 14 (72) Inventor Bernd, Sporer Munich, Federal Republic of Germany, Rotsummtstrasse, 6 (72) Inventor Fritz, Raltz Correction (72) Inventors Vladimir, Obodo Tanipelle, Finland, Methsanitín Katsu, 1 Gy 24 (72) Inventors Adriano, Fontana Zumikon, Switzerland, Rettenstrasse, 12 (72) Inventors Volker, Elfre Munich, Germany, Kolberger Strasse, 7 (72) 72) Inventor Hans-Walter, Pfister Germany Federal Republic of Noirito, Mangufo Le Strasse, 16

Claims (16)

[Claims] 1. Antibodies to the Nef protein for reducing, preferably preventing chemotaxis induced by astrocytes infected with retrovirus, these antibodies including enzymatically truncated antibodies Use of functionally effective portions of and / or nucleic acid sequences encoding these antibodies. 2. Use according to claim 1, for treating neuropathogens. 3. Use according to claim 1, for treating encephalopathy. 4. The method according to claim 1, wherein the retrovirus is HIV-1, HIV-2.
The use according to claim 1, which is selected from and / or SIV. 5. Use according to claim 1, wherein the antibody is directed against the C-terminus of the Nef protein. 6. The antibody of claim 1, wherein the antibody is directed against amino acid residues 171-190 of the HIV-1 Nef protein.
Use of the description. 7. The method according to claim 1, wherein the antibody is polyclonal, monoclonal,
The use according to claim 1, wherein the use is selected from the group consisting of an antibody consisting of chimeric, humanized, single chain, synthetic antibodies, and an antibody isolated from a human gene library. 8. The method according to claim 8, wherein the antibody comprises a part of the Nef protein exposed on the cell surface and / or a soluble extracellular Nef.
The use according to claim 1, which binds to the f protein. 9. Use according to claim 1, wherein the antibody binds to the cell binding portion of the Nef protein. 10. For reducing or preferably preventing chemotaxis induced by astrocytes infected with a retrovirus, for treating or preventing a neuropathogen caused thereby, preferably encephalopathy. HI
Use of a V-1, HIV-2 or SIV Nef protein, an immunogenic portion of the protein, and / or a nucleic acid sequence encoding the protein or an immunogenic portion thereof. 11. The use according to claim 1, which is carried out in the form of an infusion, injection, spray and / or tablet. 12. Oral and / or parenteral, especially transdermal,
The use according to claim 1, which is performed intranasally or intradermally. 13. Use according to claim 1, wherein other active compounds and / or, if desired, suitable additives and / or auxiliary substances are additionally used. 14. Antioxidants, preservatives, stabilizers, adjuvants, bulking agents, buffers, media and lubricants, and / or vectors required for gene therapy, especially for eukaryotic, viral and bacterial vectors. Structures, proteinase inhibitors, DNase inhibitors and / or RNase inhibitors, and particularly preferably virosomes, liposomes,
A transfection system selected from liposome-like structures, artificial or natural membranes or membrane fragments, or synthetic or natural cationic, anionic or neutral lipids, or mixtures thereof, is used as auxiliary substance. Use according to 1. 15. Gene therapy for reducing, preferably preventing, and especially for treating the neuropathogens caused thereby, preferably encephalopathy, induced by retrovirus-infected astrocytes. Use of a nucleic acid sequence encoding an antibody against a Nef protein or a functional part thereof, or encoding a Nef protein or an immunogenic part thereof in the above. 16. A method for the manufacture of a medicament for reducing, preferably preventing, and in particular for treating a neuropathogen, preferably encephalopathy, induced by a retrovirus-infected astrocytes induced by astrocytes.